The Reactome Book

The Reactome Book
A textbook of biological pathways
MP Morrow; J Tschopp; L Van Aelst; M Bukrinsky; JA Borowiec; EH

Blackburn; S Ozturk; K Khanna; RH Scheuermann; G Bosco; J Conaway; AP
Rice; L Comai; C.E. Rudd; M Aladjem; J Scott; B Jassal; RA Bambara; HE
Johansson; F Gebauer; MW Hentze; NJ Proudfoot; A Castro; AR Krainer; B
de Bono; N Watanabe; S Krupa; CH Heldin; GA Kassavetis; T Hunter; WF
Marzluff; Y Tsujimoto; MM Falk; PA Ortiz; JJ Zwaginga; T Lorca; EE Schmidt;
T Yen; R Ulloque; JH Hoeijmakers; P D'Eustachio; E Birney; K
Schulze-Osthoff; L Thompson; A Moustakas; G Gopinathrao; R Farndale; M
Hastings; S Tom; F Luo; J Hemish; M Catlett; M O'Connell; BK Tye; S
Lees-Miller; S Nasi; H. T. M. Timmers; R Horwitz; S Buratowski; TJ Yen; B
Squires; S Iordanskiy; A Pegg; R Conaway; D Reinberg; WC Merrick; C
Gustafsson; W.H. Ouwehand; ML Hammarskjold; M O'Donnell; R Tatoud; N
Walworth; SS Mahajan; D Bedwell; G Joshi-Tope; E Wahle; J Ferrer; A
Garcia-Sastre; ME Gillespie; E Alnemri; E Bortz; G Marsh; EP Geiduschek; I
Hoffmann; BA Balar; N-G Larsson; M Hengartner; MJ Davey; Phani Vija
Garapati; L Matthews; M Pagano; AR Kornblihtt; J MÃ©ndez; N Hernandez;
R Jakobi; E Geiduschek; AP Bevan; DM Bedwell; D Kimelman; B Geiger; JM
Hardwick; TG Kinzy; R Schultz; D Annibali; J Seidel; N.P. Pace; L
Huminiecki; P Hu; L Castagnoli; J Steel; M Anand; M Charalambous; S
Forsburg; D Segretain; GG Carmichael; NJ Gay; B May; S Gross; YR
Pittman; Z Zhang; Jr Gale M; KS Lee; Y Sanchez; J Gilleron
European Bioinformatics Institute & Cold Spring Harbor Laboratory
The contents of this book may be freely copied and distributed in any media, provided the authors, plus the
European Bioinformatics Institute and the Cold Spring Harbor Laboratory, are credited.
The Reactome Book: A textbook of biological pathways 3
Table of Contents
Table of Contents 3
Preface 64
Acknowledgements 65
1 Apoptosis 66
1.1 Extrinsic Pathway for Apoptosis 68
1.1.1 Death Receptor Signalling 69
1.1.1.1 FasL/ CD95L signaling 70
1.1.1.1.1 FASL binds FAS Receptor 71
1.1.1.1.2 Trimerization of the FASL:FAS receptor complex 72
1.1.1.1.3 FasL:Fas binds FADD 73
1.1.1.1.4 FASL:FAS Receptor Trimer:FADD complex binds pro-Caspase-8 75
1.1.1.1.5 FASL:FAS Receptor Trimer:FADD complex binds pro-Caspase-10 76
1.1.1.2 TNF signaling 77
1.1.1.2.1 TNF Binds TNF-R1 77
1.1.1.2.2 TNF:TNF-R1 binds TRADD, TRAF2 and RIP Complex 78
1.1.1.2.3 TRADD:TRAF2:RIP1 complex dissociates from the TNF-alpha:TNF-R1 complex. 79
1.1.1.2.4 TRADD:TRAF2:RIP1 complex binds FADD 79
1.1.1.2.5 TRADD:TRAF2:RIP1:FADD complex binds Pro-Caspase 8 80
1.1.1.3 TRAIL signaling 81
1.1.1.3.1 TRAIL Binds TRAIL-Receptor2 81
1.1.1.3.2 Trimerization of TRAIL: TRAIL receptor-2 complex 82
1.1.1.3.3 TRAIL:TRAIL receptor-2 Trimer Binds FADD 82
1.1.1.3.4 TRAIL:TRAIL-Receptor2 Trimer:FADD complex binds Caspase-8 83
1.1.1.3.5 TRAIL:TRAIL-Receptor2 Trimer:FADD complex binds Caspase-10 83
1.1.2 Caspase-8 is formed from procaspase-8 84
1.1.2.1 Activation of Pro-Caspase 8 84
1.1.2.1.1 FAS Mediated Activation of Pro-caspase 8 84
1.1.2.1.2 TRAIL Mediated Activation of Pro-caspase 8 86
1.1.2.1.3 TNF Mediated Activation of Pro-caspase 8 86
1.1.2.2 Formation of Caspase-8 dimer 87
1.2 Intrinsic Pathway for Apoptosis 87
1.2.1 Activation, myristolyation of BID and translocation to mitochondria 89
1.2.1.1 Caspase-8 activates BID by cleavage 91
1.2.1.2 Granzyme-B activates BID by cleavage 92
1.2.1.3 Myristoylation of tBID by NMT1 93
1.2.1.4 Translocation of tBID to mitochondria 93
1.2.2 Activation of BH3-only proteins 94
1.2.2.1 Activation of BAD and translocation to mitochondria 95
1.2.2.1.1 Akt1 phosphorylates BAD protein 96
1.2.2.1.2 Sequestration of BAD protein by 14-3-3 97
1.2.2.1.3 Activation of BAD by calcineurin 97
1.2.2.1.4 Translocation of activated BAD protein to mitochondria 98
1.2.2.1.5 BAD displaces tBID from BCL-2 sequestration 98
1.2.2.2 Activation of NOXA and translocation to mitochondria 99
1.2.2.2.1 Transactivation of NOXA by p53 99
1.2.2.2.2 Transactivation of NOXA by E2F1 100
1.2.2.2.3 Translocation of NOXA to mitochondria 100
1.2.2.3 Activation of PUMA and translocation to mitochondria 101
1.2.2.3.1 Transactivation of PUMA by p53 101
1.2.2.3.2 Transactivation of PUMA by E2F1 102
1.2.2.3.3 Translocation of PUMA protein to mitochondria 102
1.2.2.4 Activation of BIM and translocation to mitochondria 103
1.2.2.4.1 Phosphorylation of DLC1 by MAPK 8 104
1.2.2.4.2 Translocation of BIM to mitochondria 104
1.2.2.5 Activation of BMF and translocation to mitochondria 105
1.2.2.5.1 Phosphorylation of DLC2 by MAPK-8 106
1.2.2.5.2 Translocation of BMF to mitochondria 106
1.2.3 BH3-only proteins associate with and inactivate anti-apoptotic BCL-2 members 107
1.2.3.1 Interaction of BAD with BCL2 107
1.2.3.2 Interaction of BAD with BCL-xl 108
1.2.3.3 Sequestration of tBID by BCL-2 108
1.2.3.4 Interaction of tBID with BCL-xl 109
1.2.3.5 Interaction of BIM with BCL2 109
1.2.3.6 Interaction of BIM with BCL-xl 110

1.2.3.7 Interaction of NOXA with BCL2 110
1.2.3.8 Interaction of PUMA and Bcl-2 111
1.2.3.9 Interaction of PUMA and Bcl-XL 111
1.2.3.10 Interaction of NOXA with BCL-xl 112
1.2.4 Activation, translocation and oligomerization of BAX 113
1.2.4.1 tBID activates BAX protein 113
1.2.4.2 Translocation of activated BAX to the mitochondria 114
1.2.4.3 Oligomerization of BAX at the mitochondrial membrane 114
1.2.4.4 tBID binds to inactive BAX protein 115
1.2.5 Activation and oligomerization of BAK protein 115
1.2.5.1 tBID activates BAK protein 115
1.2.5.2 Oligomerization of BAK at the mitochondrial membrane 116
1.2.5.3 tBID binds to inactive BAK protein 117
1.2.6 Permeabilization of mitochondria 117
1.2.7 Release of apoptotic factors from the mitochondria 118
1.2.7.1 Release of Cytochrome c from mitochondria 118
1.2.7.2 Release of SMAC from mitochondria 119
1.2.8 Apoptotic factor-mediated response 120
1.2.8.1 Cytochrome c-mediated apoptotic response 121
1.2.8.1.1 Formation of apoptosome 123
1.2.8.1.1.1 Cytochrome C Binds to Apaf-1 123
1.2.8.1.1.2 Cytochrome C:Apaf-1 binds Procaspase-9 124
1.2.8.1.1.3 Cleavage of Procaspase-9 to Caspase-9 124
1.2.8.1.2 Activation of caspases through apoptosome-mediated cleavage 125
1.2.8.1.2.1 Cleavage of Â Procaspase-3 by the apoptosome 125
1.2.8.1.2.2 Cleavage of Procaspase-7 by the apoptosome 127
1.2.8.2 SMAC-mediated apoptotic response 128
1.2.8.2.1 SMAC binds to IAPs 130
1.2.8.2.1.1 SMAC binds XIAP:Caspase-3 130
1.2.8.2.2 SMAC-mediated dissociation of IAP:caspase complexes 132
1.2.8.2.2.1 Dissociation of Caspase-3 from SMAC:XIAP:Caspase-3 133
1.3 Activation of Effector Caspases 136
1.4 Apoptotic execution phase 136
1.4.1 Apoptotic cleavage of cellular proteins 137
1.4.1.1 Caspase-mediated cleavage of cytoskeletal proteins 137
1.4.1.1.1 Caspase-mediated cleavage of alpha adducin 138
1.4.1.1.2 Caspase mediated cleavage of alpha-II-Fodrin 139
1.4.1.1.3 Caspase-mediated cleavage of GAS2 140
1.4.1.1.4 Caspase mediated cleavage of HIP-55 141
1.4.1.1.5 Caspase-mediated cleavage of vimentin at DSVD (85) 142
1.4.1.1.6 Caspase mediated cleavage of vimentin at IDVD (259) 143
1.4.1.1.7 Caspase-mediated cleavage of vimentin at TNLD (429) 144
1.4.1.1.8 Caspase-mediated cleavage of gelsolin 145
1.4.1.1.9 Caspase-mediated cleavage of plectin-1 146
1.4.1.1.10 Caspase-mediated cleavage of Tau 147
1.4.1.2 Apoptotic cleavage of cell adhesion proteins 148
1.4.1.2.1 Caspase-mediated cleavage of E-Cadherin 149
1.4.1.2.2 Caspase mediated cleavage of beta-catenin 149
1.4.1.2.3 Caspase-mediated cleavage of Desmoglein 3 150
1.4.1.2.6 Caspase-mediated cleavage of Desmoplakin 153
1.4.1.2.7 Caspase-mediated cleavage of plakophilin-1 154
1.4.1.2.8 Caspase-mediated cleavage of Z0-1 155
1.4.1.2.9 Caspase-mediated cleavage of Z0-2 156
1.4.1.2.10 Caspase-mediated cleavage of occludin 157
1.4.1.3 Breakdown of the nuclear lamina 158
1.4.1.3.1 Caspase-mediated cleavage of Lamin A 159
1.4.1.3.2 Caspase-mediated cleavage of Lamin B1 160
1.4.1.4 Caspase mediated cleavage of APC 161
1.4.1.5 Caspase mediated cleavage of C-IAP1 162
1.4.1.6 Caspase-mediated cleavage of FADK 1 163
1.4.1.7 Caspase 3-mediated cleavage of PKC delta 164
1.4.1.8 Caspase-mediated cleavage of PKC theta 165

1.4.1.9 Caspase-mediated cleavage of Acinus 166
1.4.1.10 Caspase-mediated cleavage of Rock-1 167
1.4.1.11 Caspase-mediated cleavage of farnesyltransferase/geranylgeranyltransferase subunit alpha 168
1.4.1.12 Caspase mediated cleavage of BAP31 169
1.4.1.13 Caspase-mediated cleavage of Etk 170
1.4.1.14 Caspase-mediated cleavage of MASK 171
1.4.1.15 Caspase-mediated cleavage of MST3 171
1.4.2 Apoptosis induced DNA fragmentation 172
1.4.2.1 Activation of DNA fragmentation factor 173
1.4.2.1.1 Association of DFF40 with DFF45 174
1.4.2.1.2 Association of DFF with alpha:beta importin 175
1.4.2.1.3 Translocation of DFF to the nucleus 176
1.4.2.1.4 Caspase 3-mediated cleavage of DFF45 (117) 176
1.4.2.1.5 Cleavage of DFF45 (224) by caspase-3 177
1.4.2.1.6 Cleaved fragments of DFF45 dissociate from DFF40 178
1.4.2.1.7 Homodimerization of DFF40 179
1.4.2.1.8 Association of DFF40 with chromatin 180
1.4.2.1.9 Cleavage of DNA by DFF40 181
1.4.3 Stimulation of the cell death response by PAK-2p34 182
1.4.3.1 Cleavage of PAK-2 at 212 182
1.4.3.2 Autophosphorylation of PAK-2p34 in the activation loop 184
1.4.3.3 Proteolytic PAK-2p34 fragment translocates to the nucleus 185
1.5 Regulation of Apoptosis 186
1.5.1 Regulation of activated PAK-2p34 by proteasome mediated degradation 186
1.5.1.1 Ubiquitination of PAK-2p34 187
1.5.1.2 Proteasome mediated degradation of PAK-2p34 188
1.5.2 Regulation of PAK-2p34 activity by PS-GAP/RHG10 190
1.5.2.1 RHG10 interacts with caspase-activated PAK-2p34 190
1.5.2.2 Interaction of PAK-2p34 with RGH10/ PS-GAP results in accumulation of PAK-2p34 in the perinuclear region 191
2 Biological oxidations 193
2.1 Phase 1 - Functionalization of compounds 194
2.1.1 Cytochrome P450 - arranged by substrate type 195
2.1.1.1 Endogenous sterols 197
2.1.1.1.1 CYP1B1 4-hydroxylates estradiol-17beta 198
2.1.1.1.2 Cholesterol is hydroxylated to 7alpha-hydroxycholesterol by CYP7A1 199
2.1.1.1.3 25-hydroxycholesterol is 7alpha-hydroxylated by CYP7B1 200
2.1.1.1.4 Sterols are 12-hydroxylated by CYP8B1 201
2.1.1.1.4.1 4-Cholesten-7alpha,24(S)-diol-3-one is 12alpha-hydroxylated to 4-cholesten-7-alpha,12-alpha,24(S)-triol-3-one 202
2.1.1.1.4.2 4-Cholesten-7alpha,27-diol-3-one is 12alpha-hydroxylated to 4-Cholesten-7alpha,12alpha,27-triol-3-one 203
2.1.1.1.4.3 4-Cholesten-7alpha-ol-3-one is 12alpha-hydroxylated to 7-alpha,12-alpha-dihydroxycholest-4-en-3-one 204
2.1.1.1.5 20alpha,22beta-hydroxycholesterol is cleaved by CYP11A1 to yield pregnenolone and isocaproaldehyde 205
2.1.1.1.6 11-deoxycortisol is oxidised to cortisol by CYP11B1 205
2.1.1.1.7 11-deoxycorticosterone is oxidised to corticosterone by CYP11B2 206
2.1.1.1.8 Hydroxylation of pregnenolone to form 17alpha-hydroxypregnenolone 207
2.1.1.1.9 Adrostenedione is converted to estrone by Aromatase (CYP19A1) 208
2.1.1.1.10 21-hydroxylation of progesterone to form 11-deoxycorticosterone 209
2.1.1.1.11 Cholesterol is hydroxylated to 27-hydroxycholesterol by CYP27 210
2.1.1.1.12 24-hydroxycholesterol is 7alpha-hydroxylated to yield cholest-5-ene-3beta,7alpha,24-triol 211
2.1.1.1.13 Cholesterol is hydroxylated to 24-hydroxycholesterol by CYP46A1 212
2.1.1.1.14 Lanosterol is oxidatively demethylated to 4,4-dimethylcholesta-8(9),14,24-trien-3beta-ol 213
2.1.1.2 Xenobiotics 214
2.1.1.2.1 Ethylene is oxidized to Ethylene oxide by CYP1A1 215
2.1.1.2.2 Aromatic amines can be N-hydroxylated or N-dealkylated by CYP1A2 216
2.1.1.2.2.1 N-atom dealkylation of caffeine 216
2.1.1.2.2.2 N-hydroxylation of 4-aminobiphenyl 217
2.1.1.2.3 Coumarin is 7-hydroxylated by CYP2A6 218
2.1.1.2.4 Coumarin is 7-hydroxylated by CYP2A13 219
2.1.1.2.5 Cyclophosphamide is 4-hydroxylated by CYP2B6 220
2.1.1.2.6 CYP2C8 inactivates paclitaxel by 6alpha-hydroxylation 221
2.1.1.2.7 CYP2C9 inactivates tolbutamide by 4methyl-hydroxylation 222
2.1.1.2.8 CYP2C18 initiates bioactivation of phenytoin by 4-hydroxylation 223
2.1.1.2.9 CYP2C19 5-hydroxylates omeprazole 224
2.1.1.2.10 CYP2E1 reactions 225
2.1.1.2.10.1 Acetaminophen oxidised to N-acetylbenzoquinoneimine (NAPQI) 226
2.1.1.2.10.2 Benzene is hydroxylated to phenol 226
2.1.1.2.10.3 Dehalogenation of carbon tetrachloride to form a free radical 227
2.1.1.2.10.4 Dehalogenation of the poly-halogenated hydrocarbon Halothane to form the acylhalide Trifluoroacetlychloride and
hydrogen bromide
2.1.1.2.10.5 MEOS oxidizes ethanol to acetaldehyde 229
2.1.1.2.10.6 Vinyl chloride is oxidized to 2-Chloroethylene oxide 231
2.1.1.2.11 CYP2D6 4-hydroxylates debrisoquine 232
2.1.1.2.12 CYP2F1 dehydrogenates 3-methylindole 233
2.1.1.2.13 CYP3A4 can N-demethylate loperaminde 233
2.1.1.2.14 CYP3A5 oxidises aflatoxin B1 to aflatoxin-8,9-oxide 234
2.1.1.2.15 CYP3A7 can 6beta-hydroxylate testosterone 236
2.1.1.3 Fatty acids 236
2.1.1.3.1 CYP2J2 epoxygenates arachidonic acid 237
2.1.1.3.2 CYP4A11 omega-hydroxylates laurate 238
2.1.1.3.3 CYP4B1 can 12-hydroxylate arachidonic acid 239
2.1.1.3.4 CYP4F12 hydroxylates arachidonic acid 240
2.1.1.4 Eicosanoids 241
2.1.1.4.1 CYP4F2 omega-hydroxylate leukotriene B4, thus inactivating it 242
2.1.1.4.2 CYP4F3 omega-hydroxylate leukotriene B4, thus inactivating it 243
2.1.1.4.3 CYP4F8 hydroxylates prostaglandin H2 244
2.1.1.4.4 Thromboxane synthase (CYP5A1) mediates the isomerization of prostaglandin H2 to thromboxane A2 245
2.1.1.4.5 Prostacyclin synthase (CYP8A1) mediates the isomerization of prostaglandin H2 to prostaglandin I2 246
2.1.1.5 Vitamins 247
2.1.1.5.1 25-Hydroxylation of vitamin D3 in liver 247
2.1.1.5.2 CYP24A1 catalyzes tjhe initial step in the deactivation of the hormonally active form of vitamin D3 248
2.1.1.5.3 CYP26A1 breaks down all-trans-retinoic acid by 4-hydroxylation 249
2.1.1.5.4 CYP26B1 also deactivates all-trans-retinoic acid by 4-hydroxylation 250
2.1.1.5.5 CYP26C1 deactivates 9-cis-retinoic acid by 4-hydroxylation 251
2.1.1.5.6 Further hydroxylation of calcidiol in kidney to form calcitriol 252
2.1.1.6 Unknown 253
2.1.1.6.1 CYP2S1 can deactivate all-trans-retinoic acid 254
2.1.1.6.2 CYP2U1 can omega-hydroxylate arachidonate 255
2.1.1.6.3 CYP2W1 can oxidize indole 256
2.1.1.6.4 CYP3A43 catalyzes the 6beta-hydroxylation of testosterone 257
2.1.1.6.5 CYP4F11 omega-hydroxylates 3-hydroxypalmitate 258
2.1.2 FMO oxidizes nucleophiles 259
2.1.2.1 FMO1 N-oxidizes the anti-cancer drug tamoxifen 261
2.1.2.2 FMO2 S-oxidizes the antithyroid drug methimazole 262
2.1.2.3 FMO3 N-oxidizes the tertiary amine trimethylamine 262
2.1.3 COX reactions 263
2.1.3.1 Arachidonic acid oxidised to PGG2 265
2.1.3.2 Peroxidative reduction of PGG2 to PGH2 266
2.1.4 Amine Oxidase reactions 267
2.1.4.1 Monoamines are oxidized to aldehydes by MAOA and MAOB, producing NH3 and H2O2 268
2.1.4.1.1 Dietary tyramine is oxidatively deaminated to an aldehyde by MAOB 269
2.1.4.1.2 Oxidative deamination of 5-Hydroxytryptamine by MAOA 270
2.1.4.1.3 Oxidative deamination of Phenyethylamine by MAOB 271
2.1.4.2 Polyamines are oxidized to amines, aldehydes and H2O2 by PAOs 272
2.1.4.2.1 Spermine is oxidized to spermidine 272
2.1.4.2.2 N-acetylspermidine is oxidized to putrescine 273
2.1.4.2.3 N-acetylspermine is oxidised to spermidine 274
2.1.5 Ethanol catabolism 275
2.1.5.1 Alcohol Dehydrogenase 276
2.1.5.1.1 Ethanol is oxidized by NAD+ to form acetaldehyde, NADH, and H+ 277
2.1.5.1.1.1 Ethanol + NAD+ <=> Acetaldehyde + NADH + H+ [class I, alpha/alpha] 278
2.1.5.1.1.2 Ethanol + NAD+ <=> Acetaldehyde + NADH + H+ [class I, alpha/beta] 279
2.1.5.1.1.3 Ethanol + NAD+ <=> Acetaldehyde + NADH + H+ [class I, alpha/gamma] 280
2.1.5.1.1.4 Ethanol + NAD+ <=> Acetaldehyde + NADH + H+ [class I, beta/beta] 281
2.1.5.1.1.5 Ethanol + NAD+ <=> Acetaldehyde + NADH + H+ [class I, beta/gamma] 282
2.1.5.1.1.6 Ethanol + NAD+ <=> Acetaldehyde + NADH + H+ [class I, gamma/gamma] 283
2.1.5.1.1.7 Ethanol + NAD+ <=> Acetaldehyde + NADH + H+ [class II, pi/pi] 284
2.1.5.1.1.8 Ethanol + NAD+ <=> Acetaldehyde + NADH + H+ [class IV, mu or sigma] 285
2.1.5.1.1.9 Ethanol + NAD+ <=> Acetaldehyde + NADH + H+ [class V] 286
2.1.5.2 Aldehyde Dehydrogenase 287
2.1.5.2.1 Acetaldehyde is oxidized by NAD+ to form acetate, NADH, and H+ 288
2.1.5.2.1.1 Acetaldehyde + NAD+ <=> Acetate + NADH + H+ [cytosolic] 288
2.1.5.2.1.2 Acetaldehyde + NAD+ <=> Acetate + NADH + H+ [mitochondrial] 289
2.1.5.3 Acetate, ATP, and coenzyme A react to form acetyl CoA, AMP, and pyrophosphate 291
2.1.5.3.1 ATP + Acetate + CoA <=> AMP + Pyrophosphate + Acetyl-CoA 291
2.2 Phase II conjugation 292
2.2.1 Glucuronidation 228
293
2.2.1.1 Formation of the active cofactor, UDP-glucuronate 294

2.2.1.1.1 UTP + D-glucose 1-phosphate <=> pyrophosphate + UDP-glucose [liver] 295
2.2.1.1.2 UDP-glucose is oxidised to UDP-glucuronate 296
2.2.1.1.3 UDP-glucuronate transport from the cytosol to ER lumen 297
2.2.1.2 Formation of O-glucuronides 298
2.2.1.3 Formation of N-glucuronides 298
2.2.2 Cytosolic sulfonation of small molecules 299
2.2.2.1 Acetaminophen can form an O- sulfate conjugate 300
2.2.2.2 N-hydroxy-4-aminobiphenyl can form a sulfate conjugate 301
2.2.2.3 Phenol can form a sulfate conjugate 302
2.2.2.4 Dopamine can form an O-sulfate conjugate 303
2.2.2.5 3,3'-diiodothyronine + PAPS => 3,3'-diiodothyronine 4-sulfate + PAP 304
2.2.2.6 3,5,3'-triiodothyronine + PAPS => 3,5,3'-triiodothyronine 4-sulfate + PAP 305
2.2.2.7 p-nitrophenol + PAPS => p-nitrophenol sulfate + PAP 306
2.2.2.8 N-hydroxy-2-acetylaminofluorene + PAPS => 2-acetylaminofluorene-N-sulfate + PAP 307
2.2.2.9 cholesterol + PAPS => cholesterol sulfate + PAP 307
2.2.2.10 27-hydroxycholesterol + PAPS => 27-hydroxycholesterol 3-sulfate + PAP 308
2.2.2.11 pregnenolone + PAPS => pregnenolone sulfate + PAP 309
2.2.2.12 estrone + PAPS => estrone 3-sulfate + PAP 310
2.2.2.13 dehydroepiandrosterone (DHEA) + PAPS => DHEA sulfate + PAP 311
2.2.2.14 beta-estradiol + PAPS => beta-estradiol 3-sulfate + PAP 312
2.2.2.15 lithocholate + PAPS => lithocholate sulfate + PAP 312
2.2.2.16 taurolithocholate + PAPS => taurolithocholate sulfate + PAP 313
2.2.3 Acetylation 314
2.2.3.1 The acetyl group from acetyl-CoA is transferred to the NAT1 315
2.2.3.2 The acetyl group from acetyl-CoA is transferred to the NAT2 315
2.2.3.3 NAT1 acetylation 316
2.2.3.4 NAT2 acetylation 316
2.2.4 Methylation 317
2.2.4.1 SAM is sythesized from methionine's reaction with ATP 318
2.2.4.2 6-mercaptopurine can be S-methylated 319
2.2.4.3 2-mercaptoethanol can be S-methylated 320
2.2.4.4 Pyridine can be N-methylated 320
2.2.4.5 3,4-dihydroxybenzoate can be O-methylated 321
2.2.4.6 S-adenoylhomocysteine is hydrolyzed 322
2.2.4.7 Methionine is reformed 322
2.2.5 Glutathione conjugation 323
2.2.5.1 Glutathione synthesis 324
2.2.5.1.1 Glutamate and cysteine combine 324
2.2.5.1.2 gamma-glutamylcysteine combines with glycine to form glutathione 325
2.2.5.2 Glutathione conjugation of luminal substrates 326
2.2.5.3 Glutathione conjugation of cytosolic substrates 326
2.2.6 Amino Acid conjugation 327
2.2.6.1 Conjugation of carboxylic acids 327
2.2.6.1.1 Conjugation of benzoate with glycine 328
2.2.6.1.1.1 benzoate + Coenzyme A + ATP => benzoyl-CoA + AMP + pyrophosphate 328
2.2.6.1.1.2 benzoyl-CoA + glycine => benzoyl glycine (hippuric acid) + Coenzyme A 329
2.2.6.1.2 Conjugation of phenylacetate with glutamine 330
2.2.6.1.2.1 phenylacetate + Coenzyme A + ATP => phenylacetyl-CoA + AMP + pyrophosphate 330
2.2.6.1.2.2 phenylacetyl-CoA + glutamine => phenylacetyl glutamine + Coenzyme A 331
2.2.6.1.3 Conjugation of salicylate with glycine 332
2.2.6.1.3.1 salicylic acid + Coenzyme A + ATP => salicylate-CoA + AMP + pyrophosphate 333
2.2.6.1.3.2 salicylate-CoA + glycine => salicyluric acid + Coenzyme A 334
3 Botulinum neurotoxicity 335
3.1 Binding of BoNT toxins to gut epithelial membrane 337
3.2 Transcytosis and internalization of BoNT 339
3.3 Translocation of BoNT Light chain 340
3.3.1 Conformational change in BoNT induced by pH 341
3.3.2 Pore or channel formation in endosomal membrane 342
3.3.3 Transportation of BoNT Light chain to cytosol 343
3.4 Proteolytic cleavage of SNARE complex proteins 345
3.4.1 BoNT Light Chain Types B, D, and F cleave VAMP/Synaptobrevin 346
3.4.1.1 BoNT Light Chain Type B cleaves VAMP-2 346
3.4.1.2 BoNT Light Chain Type D cleaves VAMP 348
3.4.1.3 BoNT Light Chain Type F cleaves VAMP 349
3.4.2 BoNT Light Chain Types A, C1, E cleave SNAP-25 350
3.4.2.1 BoNT Light Chain Type A cleaves SNAP-25 350
3.4.2.2 BoNT Light Chain Type C1 cleaves SNAP-25 351
3.4.2.3 BoNT Light Chain Type E cleaves SNAP-25 353

3.4.3 BoNT Light Chain Type C1 cleaves Syntaxin 354
4 Cell Cycle Checkpoints 355
4.1 G1/S DNA Damage Checkpoints 356
4.1.1 p53-Dependent G1/S DNA damage checkpoint 356
4.1.1.1 Detection of DNA Damage 357
4.1.1.2 p53-Dependent G1 DNA Damage Response 357
4.1.1.2.1 Stabilization of p53 358
4.1.1.2.1.1 Phosphorylation of p53 at ser-15 by ATM kinase 359
4.1.1.2.1.2 Phosphorylation of MDM2 at serine-395 by ATM kinase 360
4.1.1.2.2 Transcriptional activation of p53 responsive genes 362
4.1.1.2.2.1 Transcriptional activation of cell cycle inhibitor p21 362
4.1.1.2.2.1.1 Transcriptional activation of p21 by p53 after DNA damage 362
4.1.1.2.3 Inactivation of Cyclin E:Cdk2 complexes by p27/p21 363
4.1.1.2.3.1 Inactivation of Cyclin E1:Cdk2 complex by p27/p21 365
4.1.1.2.3.2 Inactivation of Cyclin E2:Cdk2 complex by p27/p21 365
4.1.2 p53-Independent G1/S DNA damage checkpoint 366
4.1.2.2 p53-Independent DNA Damage Response 366
4.1.2.2.1 Phosphorylation of Cdc25A at Ser-123 in response to DNA damage 367
4.1.2.2.2 Ubiquitin Mediated Degradation of Phosphorylated Cdc25A 368
4.1.2.2.2.1 Ubiquitination of phosphorylated Cdc25A 369
4.1.2.2.2.2 Proteolytic degradation of ubiquitinated-Cdc25A 370
4.2 G2/M Checkpoints 370
4.2.1 G2/M DNA damage checkpoint 370
4.2.1.2 Recruitment and activation of Chk1 372
4.2.1.3 Phosphorylation and activation of CHK2 by ATM 373
4.2.1.4 Chk1/Chk2(Cds1) mediated inactivation of Cyclin B:Cdk1 complex 373
4.2.1.4.1 Phosphorylation of Cdc25C at Ser216 374
4.2.1.4.2 Association of phospho-Cdc25C(Ser 216) with 14-3-3 proteins 375
4.2.1.4.3 Retention of phospho-Cdc25C:14-3-3 complexes within the cytoplasm 375
4.2.1.4.4 Phosphorylation of Wee1 kinase by Chk1 376
4.2.1.4.5 Wee1- mediated phosphorylation of Cyclin B1:phospho-Cdc2 complexes 377
4.2.2 G2/M DNA replication checkpoint 378
4.2.2.1 Myt-1 mediated phosphorylation of Cyclin B:Cdc2 complexes 379
4.2.2.2 Wee1- mediated phosphorylation of Cyclin B1:phospho-Cdc2 complexes 380
4.2.3 Activation of ATR in response to replication stress 381
4.2.3.1 Stalling of DNA replication fork and RPA binding 383
4.2.3.2 Binding of ATR-ATRIP to the RPA-ssDNA complex 384
4.2.3.3 Recruitment of Rad17-RFC complex to DNA 385
4.2.3.4 Recruitment of the Rad9-Hus1-Rad1 complex to DNA 386
4.2.3.5 Loading of claspin onto DNA during replication origin firing 387
4.2.3.6 Activation of claspin 388
4.2.3.7 Recruitment and activation of Chk1 389
4.2.3.8 Phosphorylation of Cdc25A at Ser-123 by Chk1 390
4.2.3.9 Phosphorylation of Cdc25C at Ser 216 by Chk1 391
4.3 Mitotic Spindle Checkpoint 392
4.3.1 Initiation of checkpoint signal from defective kinetochores 392
4.3.1.1 Detection of kinetochores lacking bipolar connections to spindle microtubules 393
4.3.1.1.1 Detection of insufficient microtubule occupancy at kinetochore 393
4.3.1.1.2 Detection of insufficient tension at kinetochore 394
4.3.2 Amplification of signal from the kinetochores 394
4.3.2.1 Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal 394
4.3.2.1.1 Mad1 binds kinetochore 395
4.3.2.1.2 MAD2 associates with the Mad1 kinetochore complex 396
4.3.2.1.3 MAD2 converted to an inhibitory state via interaction with Mad1 396
4.3.2.1.4 Release of activated MAD2 from kinetochores 397
4.3.2.2 Amplification of signal from unattached kinetochore via a checkpoint kinase cascade 397
4.3.3 Inhibition of the proteolytic activity of APC/C required for the onset of anaphase by mitotic spindle checkpoint components 398
4.3.3.1 Inactivation of APC/C via CDC20 sequestration 399
4.3.3.2 Inactivation of APC/C via direct inhibition of the APC/C complex 400
4.3.3.2.1 Formation of the MCC complex 401
4.3.3.2.2 Binding of the MCC complex to the APC/C complex 402
5 Cell Cycle, Mitotic 403
5.1 G1 Phase 404
5.1.1 Cyclin D associated events in G1 404
5.1.1.1 Formation of Cyclin D:Cdk4/6 complexes 405
5.1.1.2 Translocation of Cyclin D:Cdk4/6 complexes from the cytoplasm to the nucleus 407
5.1.1.3 Phosphorylation of Cyclin D:Cdk4/6 complexes 407
5.1.1.4 Cyclin D:Cdk4/6 mediated phosphorylation of Rb and dissociation of Rb from the Rb:E2F:DP-1 complexes 409
5.1.2 G1-Specific Transcription 410
5.2 G1/S Transition 411
5.2.1 Cyclin E associated events during G1/S transition 411
5.2.1.1 Formation of Cyclin E:Cdk2 complexes 412
5.2.1.1.1 Formation of Cyclin E1:Cdk2 complexes 413
5.2.1.1.2 Formation of Cyclin E2:Cdk2 complexes 414
5.2.1.2 Translocation of Cyclin E:Cdk2 complex to the nucleus 415
5.2.1.2.1 Translocation of cyclin E1:Cdk2 complexes to the nucleus 415
5.2.1.2.2 Translocation of cyclin E2:Cdk2 complexes to the nucleus 416
5.2.1.3 Inactivation of Cyclin E:Cdk2 complexes by p27/p21 416
5.2.1.3.1 Inactivation of Cyclin E1:Cdk2 complex by p27/p21 418
5.2.1.3.2 Inactivation of Cyclin E2:Cdk2 complex by p27/p21 418
5.2.1.4 SCF(Skp2)-mediated degradation of p27/p21 419
5.2.1.4.1 Cyclin E/A:Cdk2-mediated phosphorylation of p27/p21 421
5.2.1.4.1.1 Cyclin A:Cdk2 mediated phosphorylation of p27/p21 422
5.2.1.4.1.2 Cyclin E:Cdk2- mediated phosphorylation of p27/p21 423
5.2.1.4.2 Association of Cks1 with SCF(Skp2) complex 424
5.2.1.4.3 Binding of phospho-p27/p21:Cdk2:Cyclin E/A to the SCF(Skp2):Cks1 complex 424
5.2.1.4.4 Ubiquitination of phospho-p27/p21 425
5.2.1.4.5 Degradation of ubiquitinated p27/p21 by the 26S proteasome 426
5.2.1.5 Phosphorylation of Cyclin E:Cdk2 complexes 427
5.2.1.5.1 Phosphorylation of Cyclin E1:Cdk2 complexes by Wee1 429
5.2.1.5.2 Phosphorylation of Cyclin E2:Cdk2 complexes by Wee1 429
5.2.1.6 Dephosphorylation of Cyclin E:Cdk2 complexes by Cdc25A 429
5.2.1.6.1 Dephosphorylation of Cyclin E1:Cdk2 complexes by Cdc25A 431
5.2.1.6.2 Dephosphorylation of Cyclin E2:Cdk2 complexes by Cdc25A 432
5.2.1.7 CAK-mediated phosphorylation of Cyclin E:Cdk2 432
5.2.1.8 Phosphorylation of proteins involved in G1/S transition by active Cyclin E:Cdk2 complexes 433
5.2.1.8.1 Cyclin E:Cdk2-mediated phosphorylation of Rb 434
5.2.1.9 Ubiquitin-Dependent Degradation of Cyclin E 435
5.2.1.9.1 Ubiquitin-Dependent Degradation of Cyclin E1 435
5.2.1.9.2 Ubiquitin-Dependent Degradation of Cyclin E2 436
5.2.2 G1/S-Specific Transcription 436
5.2.3 Activation of the pre-replicative complex 436
5.2.3.1 Mcm10 associates with the pre-replicative complex, stabilizing Mcm2-7 437
5.2.3.2 Cdt1 is displaced from the pre-replicative complex. 438
5.2.3.3 CDK and DDK associate with the Mcm10:pre-replicative complex 438
5.2.3.4 Mcm2-7 is phosphorylated by DDK 439
5.2.3.5 Cdc45 associates with the pre-replicative complex at the origin 440
5.2.3.6 DNA Replication Factor A (RPA) associates with the pre-replicative complex at the origin 441
5.2.3.7 DNA polymerase alpha:primase binds at the origin 442
5.2.3.8 DNA polymerase epsilon binds at the origin 443
5.2.4 E2F mediated regulation of DNA replication 443
5.2.4.1 E2F transcriptional targets at G1/S 444
5.2.4.1.1 Activation of Cdc45 by E2F1 444
5.2.4.1.2 Activation of Cdt1 by E2F1 445
5.2.4.1.3 Activation of Cyclin E by E2F1 445
5.2.4.1.4 Activation of PCNA by E2F1 446
5.2.4.1.5 Activation of PolA catalytic subunit by E2F1 446
5.2.4.1.6 Activation of Rir2 by E2F1 447
5.2.4.1.7 Activation of Cdc25a (string) by E2F1 447
5.2.4.1.8 Activation of ORC1 by E2F1 448
5.2.4.1.9 Activation of TK2 (Dnk1) by E2F1 449
5.2.4.1.10 Activation of Thymidylate synthetase by E2F1 450
5.2.4.1.11 Activation of Dihydrofoloate reductase by E2F1 450
5.2.4.1.12 Activation of Cdc2 by E2F1 451
5.2.4.1.13 Activation of Cyclin A1 by E2F1 451
5.2.4.1.14 CDC6 protein is synthesized under the control of E2F transcription factors 452
5.2.4.1.15 Activation of Emi1 by E2F1 453
5.2.4.2 Inhibition of replication initiation of damaged DNA by Rb/E2F1 453
5.2.4.2.1 Detection of damage during initiation of DNA synthesis in S-phase 453
5.2.4.2.2 Pp2a mediated localization of Rb protein in chromatin 454
5.2.4.2.3 Replication initiation regulation by Rb1/E2F1 454
5.2.4.3 E2F-enabled inhibition of pre-replication complex formation 455
5.2.4.3.1 Activation of Cyclin B/Cdk1 by Cdc25a (string) protein 455
5.2.4.3.2 Association of Cyclin B/Cdk1 with replicative origin inhibits pre-RC formation 456
5.3 S Phase 457
5.3.1 Cyclin A:Cdk2-associated events at S phase entry 458
5.3.1.1 Formation of Cyclin A:Cdk2 complexes 459
5.3.1.2 Translocation of Cyclin A:Cdk2 complexes to the nucleus 459
5.3.1.3 Inactivation of Cyclin A:Cdk2 complexes by p27/p21 460
5.3.1.4 SCF(Skp2)-mediated degradation of p27/p21 461
5.3.1.4.1 Cyclin E/A:Cdk2-mediated phosphorylation of p27/p21 463
5.3.1.4.1.1 Cyclin A:Cdk2 mediated phosphorylation of p27/p21 464
5.3.1.4.1.2 Cyclin E:Cdk2- mediated phosphorylation of p27/p21 465
5.3.1.4.2 Association of Cks1 with SCF(Skp2) complex 466
5.3.1.4.3 Binding of phospho-p27/p21:Cdk2:Cyclin E/A to the SCF(Skp2):Cks1 complex 466
5.3.1.4.4 Ubiquitination of phospho-p27/p21 467
5.3.1.4.5 Degradation of ubiquitinated p27/p21 by the 26S proteasome 468
5.3.1.5 Phosphorylation of Cyclin A:Cdk2 at Tyr 15 469
5.3.1.6 Cdc25A mediated dephosphorylation of Cyclin A:phospho-Cdk2 469
5.3.1.7 CAK-mediated phosphorylation of Cyclin A:Cdk2 470
5.3.1.8 Phosphorylation of proteins involved in the G1/S transition by Cyclin A:Cdk2 471
5.3.2 Synthesis of DNA 472
5.3.2.1 DNA replication initiation 472
5.3.2.1.1 The primase component of DNA polymerase:primase synthesizes a 6-10 nucleotide RNA primer at the origin 473
5.3.2.1.2 The polymerase component of DNA polymerase alpha:primase synthesizes a 20-nucleotide primer at the origin 474
5.3.2.2 Switching of origins to a post-replicative state 474
5.3.2.2.1 Orc1 removal from chromatin 474
5.3.2.2.1.1 Orc1 is phosphorylated by cyclin A/CDK2 474
5.3.2.2.1.2 Phosphorylated Orc1 is ubiquitinated while still associated with chromatin 475
5.3.2.2.1.3 Ubiquitinated Orc1 enters the cytosol 476
5.3.2.2.1.4 Ubiquitinated Orc1 is degraded by the proteasome 476
5.3.2.2.2 CDK-mediated phosphorylation and removal of Cdc6 477
5.3.2.2.2.1 Cdc6 protein is phosphorylated by CDK 477
5.3.2.2.2.2 Phosphorylated Cdc6 is exported from the nucleus 478
5.3.2.2.2.3 Cytoplasmic phosphorylated Cdc6 is ubiquitinated by the anaphase-promoting complex 478
5.3.2.2.2.4 Ubiquitinated Cdc6 is degraded by the proteasome 479
5.3.2.2.3 Mcm4,6,7 trimer forms and associates with the replication fork 480
5.3.2.3 DNA strand elongation 481
5.3.2.3.1 Unwinding of DNA 482
5.3.2.3.1.1 MCM2-7 mediated fork unwinding 482
5.3.2.3.1.2 MCM8 mediated fork unwinding 483
5.3.2.3.1.3 Formation of GINS complex 484
5.3.2.3.1.4 Multiple proteins are localized at replication fork 485
5.3.2.3.2 Leading Strand Synthesis 486
5.3.2.3.2.1 Polymerase switching 486
5.3.2.3.2.1.1 RFC binding displaces Pol Alpha 488
5.3.2.3.2.1.2 Loading of PCNA - Sliding Clamp Formation 488
5.3.2.3.2.1.3 RFC dissociates after sliding clamp formation 489
5.3.2.3.2.1.4 Formation of Processive Complex 489
5.3.2.3.2.2 Processive synthesis on the leading strand 490
5.3.2.3.3 Lagging Strand Synthesis 491
5.3.2.3.3.1 Polymerase switching 491
5.3.2.3.3.1.1 RFC binding displaces Pol Alpha 493
5.3.2.3.3.1.2 Loading of PCNA - Sliding Clamp Formation 493
5.3.2.3.3.1.3 RFC dissociates after sliding clamp formation 494
5.3.2.3.3.1.4 Formation of Processive Complex 494
5.3.2.3.3.2 Processive synthesis on the lagging strand 495
5.3.2.3.3.2.1 Formation of Okazaki fragments 496
5.3.2.3.3.2.2 Formation of the Flap Intermediate 496
5.3.2.3.3.2.3 Removal of the Flap Intermediate 497
5.3.2.3.3.2.3.1 RPA binds to the Flap 497
5.3.2.3.3.2.3.2 Recruitment of Dna2 endonuclease 498
5.3.2.3.3.2.3.3 Removal of RNA primer and dissociation of RPA and Dna2 498
5.3.2.3.3.2.3.4 Removal of remaining Flap 499
5.3.2.3.3.2.4 Joining of adjacent Okazaki fragments 500
5.3.3 Ubiquitin-dependent degradation of Cyclin D 501
5.3.3.1 Ubiquitin-dependent degradation of Cyclin D1 501
5.3.3.1.1 Phosphorylation of Cyclin D1 at T286 by glycogen synthase kinase-3 beta 501
5.3.3.1.2 Relocalization of nuclearly localized phospho-(T286):cyclin D1:Cdk4 to cytoplasm 502
5.3.3.1.3 Relocalization of nuclearly localized Cyclin D1 to the cytoplasm 502
5.3.3.1.4 Ubiquitination of Cyclin D1 503
5.3.3.1.5 Proteasome mediated degradation of Cyclin D1 504

5.3.4 S-specific transcription in mitotic cell cycle 504
5.4 G2 Phase 505
5.4.1 G2-specific transcription in mitotic cell cycle 505
5.4.2 Phosphorylation of E2F1/E2F3 by Cyclin A:phosph-Cdk2(Thr 160) 505
5.5 G2/M Transition 506
5.5.1 Cyclin A/B1 associated events during G2/M transition 507
5.5.1.1 Formation of Cyclin A:Cdc2 complexes 508
5.5.1.2 Myt-1 mediated phosphorylation of Cyclin A:Cdc2 510
5.5.1.3 Translocation of Cyclin A:phospho-Cdc2 (Thr 14) to the nucleus 511
5.5.1.4 CAK-mediated phosphorylation of Cyclin A:Cdc2 complexes 511
5.5.1.5 Wee1-mediated phosphorylation of Cyclin A:phospho-Cdc2 complexes 512
5.5.1.6 Dephosphorylation of nuclear Cyclin A:phosph-Cdc2 complexes 513
5.5.1.7 Phosphorylation of proteins involved in the G2/M transition by Cyclin A:Cdc2 complexes 515
5.5.1.7.1 Phosphorylation of proteins involved in G2/M transition by active Cyclin A1:Cdc2 complexes 516
5.5.1.7.2 Phosphorylation of proteins involved in G2/M transition by active Cyclin A2:Cdc2 complexes 516
5.5.1.8 Formation of Cyclin B:Cdc2 complexes 517
5.5.1.9 Myt-1 mediated phosphorylation of Cyclin B:Cdc2 complexes 518
5.5.1.10 Translocation of Cyclin B1:phospho-Cdc2 complexes to the nucleus 519
5.5.1.11 CAK-mediated phosphorylation of Cyclin B1:Cdc2 complexes 520
5.5.1.12 Wee1- mediated phosphorylation of Cyclin B1:phospho-Cdc2 complexes 520
5.5.1.13 Translocation of Cyclin B1:phospho-Cdc2 to the cytoplasm 521
5.5.1.14 Translocation of Cdc25B to the cytoplasm 522
5.5.1.15 Dephosphorylation of cytoplasmic Cyclin B1:phospho-Cdc2 (Thr 14, Tyr 15) complexes by Cdc25 phosphatases 523
5.5.1.16 Phosphorylation of Cyclin B1 in the CRS domain 524
5.5.1.17 Translocation of CRS phosphorylated Cyclin B1:Cdc2 complexes 525
5.5.1.18 Translocation of Cdc25 to the nucleus 525
5.5.1.19 Dephosphorylation of nuclear Cyclin B1:phospho-Cdc2 (Thr 14, Tyr15) complexes by Cdc25 phosphatases 526
5.5.1.20 Phosphorylation of M phase proteins by active Cyclin B1:Cdc2 complexes 527
5.5.2 Cyclin B2 mediated events 528
5.5.2.1 Dephosphorylation of cyclin B2:phospho-Cdc2 (Thr 14) by Cdc25 528
5.5.2.2 Phosphorylation of proteins involved in G2/M transition by active Cyclin B2:Cdc2 complexes 530
5.5.3 G2/M-specific transcription in mitotic cell cycle 531
5.5.4 Polo-like kinase mediated events 532
5.5.4.1 Inactivation of Wee1 kinase 533
5.5.4.2 Activation of Cdc25C 534
5.5.4.3 Inactivation of Myt1 kinase 536
5.6 M Phase 537
5.6.1 Mitotic Prophase 537
5.6.1.1 Golgi Cisternae Pericentriolar Stack Reorganization 537
5.6.1.1.1 Activation of the Anaphase Promoting Complex (APC) by Plk1 538
5.6.2 Mitotic Prometaphase 539
5.6.2.1 Phosphorylation of the SA2 Cohesion Complex 539
5.6.3 Mitotic Metaphase 541
5.6.4 Mitotic Metaphase/Anaphase Transition 541
5.6.4.1 Down Regulation of Emi1 through Phosphorylation of Emi1 541
5.6.4.2 Phosphorylation of the Scc1:Cohesion Complex 543
5.6.5 Mitotic Anaphase 544
5.6.6 Mitotic Telophase /Cytokinesis 544
5.6.6.1 Regulation of MKLP-1 by phosphorylation 544
5.6.6.2 Regulation of MKLP-2 by phosphorylation 545
5.6.6.3 Regulation of NudC by phosphorylation 546
5.7 M/G1 Transition 547
5.7.1 Assembly of the pre-replicative complex 547
5.7.1.1 Assembly of the ORC complex at the origin of replication 549
5.7.1.1.1 Orc3 associates with Orc2 constitutively bound at origins of replication 550
5.7.1.1.2 Orc5 associates with Orc3:Orc2:origin complexes 550
5.7.1.1.3 Orc4 associates with Orc5:Orc3:Orc2:origin complexes 551
5.7.1.1.4 Orc1 associates with Orc4:Orc5:Orc3:Orc2:origin complexes 552
5.7.1.1.5 Orc6 associates with Orc1:Orc4:Orc5:Orc3:Orc2:origin complexes, forming ORC:origin complexes 552
5.7.1.2 Association of MCM8 with ORC:origin complex 553
5.7.1.3 CDC6 association with the ORC:origin complex 553
5.7.1.3.2 CDC6 association with ORC:origin complexes mediated by MCM8 555
5.7.1.4 CDT1 association with the CDC6:ORC:origin complex 556
5.7.1.4.1 The geminin component of geminin:Cdt1 complexes is ubiquitinated, releasing Cdt1 557
5.7.1.4.2 Ubiquitinated geminin is degraded by the proteasome 558
5.7.1.4.3 Free CDT1 associates with CDC6:ORC:origin complexes 558
5.7.1.5 Mcm2-7 associates with the Cdt1:CDC6:ORC:origin complex, forming the pre-replicative complex (preRC) 559
5.8 APC/C-mediated degradation of cell cycle proteins 560
5.8.1 Regulation of APC/C activators between G1/S and early anaphase 563
5.8.1.1 Association of Cyclin A:Cdk2 with Cdh1 565
5.8.1.2 Phosphorylation of Cdh1 by Cyclin A:Cdk2 565
5.8.1.3 Dissociation of phospho-Cdh1 from the APC/C complex 566
5.8.1.4 Association of Emi1 with Cdh1 567
5.8.1.5 Association of Emi1 with Cdc20 568
5.8.1.6 Phosphorylation of Emi1 569
5.8.1.6.1 Phosphorylation of the Emi1 DSGxxS degron by Cyclin B:Cdc2 569
5.8.1.6.2 Phosphorylation of the Emi1 DSGxxS degron by Plk1 570
5.8.1.7 SCF-beta-TrCP mediated degradation of Emi1 571
5.8.1.7.1 Phosphorylated Emi1 binds the beta-TrCP in the SCF complex 572
5.8.1.7.2 Ubiquitination of Emi1 by SCF-beta-TrCP 572
5.8.1.7.3 SCF-mediated degradation of Emi1 573
5.8.1.8 Phosphorylation of Cdh1 by Cyclin B1:Cdc2 574
5.8.1.9 Inhibition of the proteolytic activity of APC/C required for the onset of anaphase by mitotic spindle checkpoint components 575
5.8.1.9.1 Inactivation of APC/C via CDC20 sequestration 576
5.8.1.9.2 Inactivation of APC/C via direct inhibition of the APC/C complex 577
5.8.1.9.2.1 Formation of the MCC complex 578
5.8.1.9.2.2 Binding of the MCC complex to the APC/C complex 579
5.8.2 Activation of APC/C and APC/C:Cdc20 mediated degradation of mitotic proteins 579
5.8.2.1 Phosphorylation of the APC/C 580
5.8.2.1.1 Free APC/C phosphorylated by Plk1 580
5.8.2.1.2 Free APC/C phosphorylated by Cyclin B:Cdc2 581
5.8.2.2 APC/C:Cdc20 mediated degradation of mitotic proteins 582
5.8.2.2.1 Activation of APC/C:Cdc20 by dissociation of Cdc20:phospho-APC/C from Cdc20:phospho-APC/C:Mad2:Bub3:BubR1 583
5.8.2.2.2 APC/C:Cdc20 mediated degradation of Cyclin B 584
5.8.2.2.2.1 Association of Cyclin B:Cdc2 with Cdc20:APC/C complex 585
5.8.2.2.2.2 Ubiquitination of Cyclin B by phospho-APC/C:Cdc20 complex 586
5.8.2.2.2.3 Degradation of multiubiquitinated Cyclin B 586
5.8.2.2.3 APC/C:Cdc20 mediated degradation of Securin 587
5.8.2.2.3.1 Association of Securin with Cdc20:APC/C complex 588
5.8.2.2.3.2 Ubiquitination of Securin by phospho-APC/C:Cdc20 complex 589
5.8.2.2.3.3 Degradation of multiubiquitinated Securin 590
5.8.3 Conversion from APC/C:Cdc20 to APC/C:Cdh1 in late anaphase 591
5.8.3.1 Dephosphorylation of phospho-Cdh1 592
5.8.3.2 Dissociation of Cdc20 from APC/C complex 592
5.8.3.3 Association of Cdh1 with the APC/C 593
5.8.4 APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1 594
5.8.4.1 Association of cell cycle proteins with the APC/C:Cdh1 complex 595
5.8.4.2 Ubiquitination of cell cycle proteins targeted by the APC/C:Cdh1complex 596
5.8.4.3 Degradation of multiubiquitinated cell cycle proteins 597
5.8.5 Autodegradation of Cdh1 by Cdh1:APC/C 598
5.8.5.1 Multiubiquitination of APC/C-associated Cdh1 598
5.8.5.2 Degradation of multiubiquitinated Cdh1 599
6 DNA Repair 601
6.1 Base Excision Repair 601
6.1.1 Base-Excision Repair, AP Site Formation 602
6.1.1.1 Depurination 602
6.1.1.1.1 Recognition and association of DNA glycosylase with site containing an affected purine 603
6.1.1.1.1.1 MPG glycosylase mediated recognition and binding of 3-methyladenine 603
6.1.1.1.1.2 MPG glycosylase mediated recognition and binding of ethenoadenine 603
6.1.1.1.1.3 MPG glycosylase mediated recognition and binding of hypoxanthine 604
6.1.1.1.1.4 MYH glycosylase mediated recognition and binding of an adenine opposite to an 8-oxo guanine 604
6.1.1.1.1.5 hOGG1 glycosylase mediated recognition and binding of a formamidopyrimidine 605
6.1.1.1.1.6 hOGG1 glycosylase mediated recognition and binding of an 8-oxo guanine opposite to a cytosine 605
6.1.1.1.2 Cleavage of the damaged purine 606
6.1.1.1.2.1 Cleavage 8-oxo guanine by MYH glycosylase 606
6.1.1.1.2.2 Cleavage of 3-methyladenine by MPG glycosylase 606
6.1.1.1.2.3 Cleavage of 8-oxo guanine by hOGG1 glycosylase 607
6.1.1.1.2.4 Cleavage of ethenoadenine by MPG glycosylase 608
6.1.1.1.2.5 Cleavage of formamidopyrimidine by hOGG1 glycosylase 608
6.1.1.1.2.6 Cleavage of hypoxanthine by MPG glycosylase 609
6.1.1.2 Depyrimidination 609
6.1.1.2.1 Recognition and association of DNA glycosylase with site containing an affected pyrimidine 610
6.1.1.2.1.1 UNG glycosylase mediated recognition and binding of a 5-hydroxyuracil opposite to a guanine 610
6.1.1.2.1.2 UNG glycosylase mediated recognition and binding of an uracil opposite to a guanine 610
6.1.1.2.1.3 TDG glycosylase mediated recognition and binding of an ethenocytosine 611

6.1.1.2.1.4 TDG glycosylase mediated recognition and binding of an thymine opposite to a guanine 611
6.1.1.2.1.5 TDG glycosylase mediated recognition and binding of an uracil opposite to a guanine 612
6.1.1.2.1.6 hSMUG1 glycosylase mediated recognition and binding of uracil within single-stranded DNA 612
6.1.1.2.1.7 MBD4 glycosylase mediated recognition and binding of a thymine opposite to a guanine at CpG sequences 613
6.1.1.2.1.8 MBD4 glycosylase mediated recognition and binding of an uracil opposite to a guanine at CpG sequences 613
6.1.1.2.1.9 hNTH1 glycosylase mediated recognition and binding of cytosine glycol 613
6.1.1.2.1.10 hNTH1 glycosylase mediated recognition and binding of dihydrouracil 614
6.1.1.2.1.11 hNTH1 glycosylase mediated recognition and binding of formamidopyrimidine 614
6.1.1.2.1.12 hNTH1 glycosylase mediated recognition and binding of thymine glycol 615
6.1.1.2.2 Cleavage of the damaged pyrimidine 615
6.1.1.2.2.1 Cleavage of uracil by UNG2 glycosylase 615
6.1.1.2.2.2 Cleavage of 5-hydroxyluracil by UNG2 glycosylase 616
6.1.1.2.2.3 Cleavage of uracil by TDG glycosylase 617
6.1.1.2.2.4 Cleavage of thymine by TDG glycosylase 617
6.1.1.2.2.5 Cleavage of uracil by hSMUG1 glycosylase 618
6.1.1.2.2.6 Cleavage of thymine glycol by hNTH1 glycosylase 618
6.1.1.2.2.7 Cleavage of cytosine glycol by hNTH1 glycosylase 619
6.1.1.2.2.8 Cleavage of dihydrouracil by hNTH1 glycosylase 619
6.1.1.2.2.9 Cleavage of formamidopyrimidine by hNTH1 glycosylase 620
6.1.1.2.2.10 Cleavage of uracil by MBD4 glycosylase 620
6.1.1.2.2.11 Cleavage of thymine by MBD4 glycosylase 620
6.1.1.2.2.12 Cleavage of ethenocytosine by TDG glycosylase 621
6.1.2 Resolution of Abasic Sites (AP sites) 621
6.1.2.1 Resolution of AP sites via the single-nucleotide replacement pathway 622
6.1.2.1.1 Base-free sugar-phosphate removal via the single-nucleotide replacement pathway 624
6.1.2.1.1.1 Displacement of DNA glycosylase by APE1 624
6.1.2.1.1.1.1 Displacement of UNG2 glycosylase by APE1 at the AP site 624
6.1.2.1.1.1.2 Displacement of TDG glycosylase by APE1 at the AP site 625
6.1.2.1.1.1.3 Displacement of hSMUG1 glycosylase by APE1 at the AP site 625
6.1.2.1.1.1.4 Displacement of hNTH1 glycosylase by APE1 at the AP site 626
6.1.2.1.1.1.5 Displacement of MBD4 glycosylase by APE1 at the AP site 626
6.1.2.1.1.1.6 Displacement of hOGG1 glycosylase by APE1 at the AP site 627
6.1.2.1.1.1.7 Displacement of MYH glycosylase by APE1 at the AP site 627
6.1.2.1.1.1.8 Displacement of MPG glycosylase by APE1 at the AP site 627
6.1.2.1.1.2 APE1 mediated endonucleolytic cleavage at the 5' side of the base-free deoxyribose residue 628
6.1.2.1.1.3 Recruitment of POL Beta to the AP site 628
6.1.2.1.1.4 Excision of the abasic sugar phosphate (dRP) residue at the strand break 629
6.1.2.1.2 Recruitment of LIG3:XRRC1 complex to the site of repair by POL Beta 630
6.1.2.1.3 Resynthesis of excised residue 630
6.1.2.1.4 DNA ligation via the single-nucleotide replacement pathway 631
6.1.2.1.5 Dissociation of LIG3:XRCC1 complex from site of BER 631
6.1.2.2 Resolution of AP sites via the multiple-nucleotide patch replacement pathway 632
6.1.2.2.1 Removal of DNA patch containing abasic residue 634
6.1.2.2.1.1 Displacement of DNA glycosylase by APE1 634
6.1.2.2.1.1.1 Displacement of UNG2 glycosylase by APE1 at the AP site 634
6.1.2.2.1.1.2 Displacement of TDG glycosylase by APE1 at the AP site 635
6.1.2.2.1.1.3 Displacement of hSMUG1 glycosylase by APE1 at the AP site 635
6.1.2.2.1.1.4 Displacement of hNTH1 glycosylase by APE1 at the AP site 635
6.1.2.2.1.1.5 Displacement of MBD4 glycosylase by APE1 at the AP site 636
6.1.2.2.1.1.6 Displacement of hOGG1 glycosylase by APE1 at the AP site 636
6.1.2.2.1.1.7 Displacement of MYH glycosylase by APE1 at the AP site 636
6.1.2.2.1.1.8 Displacement of MPG glycosylase by APE1 at the AP site 637
6.1.2.2.1.2 APE1 mediated endonucleolytic cleavage at the 5' side of the base-free deoxyribose residue 637
6.1.2.2.1.3 Recruitment of POL Beta to the AP site 637
6.1.2.2.1.4 POL Beta mediated incorporation of the first replacement nucleotide 638
6.1.2.2.1.5 POL delta associates with AP site displacing POL Beta 639
6.1.2.2.1.6 DNA strand displacement synthesis 639
6.1.2.2.1.7 Cleavage of flap structures 640
6.1.2.2.2 Ligation of DNA at sites of patch replacement 641
6.2 DNA Damage Bypass 641
6.2.1 Translesion synthesis by DNA polymerases bypassing lesion on DNA template 642
6.2.1.1 Translesion synthesis by HREV1 642
6.2.1.1.1 Binding of HREV1 to lesioned DNA template 643
6.2.1.1.2 Misinsertion of bases opposite to the lesion by HREV1 643
6.2.1.1.3 Elongation by HREV1 protein 643
6.2.1.2 Translesion synthesis by Pol eta 644
6.2.1.2.1 Binding of Pol eta to lesioned DNA template 644
6.2.1.2.2 Insertion of correct bases opposite to the lesion by Pol eta 645
6.2.1.2.3 Elongation by Pol eta 645
6.2.1.3 Translesion synthesis by Pol zeta 646
6.2.1.3.1 Binding of Pol zeta to lesioned DNA template 646
6.2.1.3.2 Elongation by Pol zeta complex 647
6.3 DNA Damage Reversal 647
6.3.1 MGMT/hAGT mediated DNA Damage Reversal 647
6.3.2 Reversal of Alkylation Damage By DNA Dioxygenases 648
6.3.2.1 ABH2 mediated Reversal of Alkylation Damage 649
6.3.2.1.1 Oxidative demethylation of 1-MeA damaged DNA By ABH2 650
6.3.2.1.2 Oxidative demethylation of 1-EtA damaged DNA By ABH2 650
6.3.2.2 ABH3 mediated Reversal of Alkylation Damage 651
6.3.2.2.1 Oxidative demethylation of 1-MeA damaged DNA By ABH3 651
6.3.2.2.2 Oxidative demethylation of 3-MeC damaged DNA By ABH3 652
6.3.2.2.3 Oxidative demethylation of 1-EtA damaged DNA By ABH3 653
6.4 Double-Strand Break Repair 653
6.4.1 Homologous Recombination Repair 656
6.4.1.1 Homologous recombination repair of replication-independent double-strand breaks 658
6.4.1.1.1 ATM mediated response to DNA double-strand break 658
6.4.1.1.1.1 Intermolecular autophosphorylation of ATM within dimeric ATM complexes 658
6.4.1.1.1.2 Dissociation of dimeric phospho-ATM complexes 659
6.4.1.1.1.3 ATM mediated phosphorylation of repair proteins 660
6.4.1.1.1.3.1 Phosphorylation of histone H2AX at Serine-139 by ATM at the site of DSB 660
6.4.1.1.1.3.2 Phosphorylation of BRCA1 at multiple sites by ATM 661
6.4.1.1.1.3.3 Phosphorylation of NBS1 by ATM 662
6.4.1.1.1.3.4 Phosphorylation of MDC1/NFBD1 by ATM (within 2 c-term BRCT domains) 663
6.4.1.1.2 Recruitment of repair and signaling proteins to double-strand breaks 663
6.4.1.1.2.1 MDC1/NFBD1associates with gamma H2AX at nuclear foci 665
6.4.1.1.2.2 phospho-ATM (Serine 1981) associates with DNA at the site of double-strand breaks 665
6.4.1.1.2.3 53BP1 associates with gamma H2AX at nuclear foci 666
6.4.1.1.2.4 MRN complex relocalizes to nuclear foci 667
6.4.1.1.2.4.1 Association of gamma-H2AX with NBS1 667
6.4.1.1.2.4.2 Assembly of the RAD50-MRE11-NBS1 complex at DNA double-strand breaks 668
6.4.1.1.2.4.2.1 Formation of RAD50:MRE11 complex 668
6.4.1.1.2.4.2.2 Association of RAD50:MRE11 complex with NBS1 via MRE11 interaction 668
6.4.1.1.2.4.2.3 Association of MRN with sites of DSB 669
6.4.1.1.2.5 BRCA1 associates with 53BP1at the site of DNA double-strand break 669
6.4.1.1.3 Processing of DNA double-strand break ends 670
6.4.1.1.3.1 Resection of double-strand break ends 670
6.4.1.1.3.2 Association of RPA complexes with ssDNA 672
6.4.1.1.4 Homologous DNA pairing and strand exchange 672
6.4.1.1.4.1 Presynaptic phase of homologous DNA pairing and strand exchange 673
6.4.1.1.4.1.1 Displacement of RPA from ssDNA 673
6.4.1.1.4.1.2 Association of RAD51 with BRCA2 674
6.4.1.1.4.1.3 Formation of RAD52 heptameric ring structure complexes on ssDNA 674
6.4.1.1.4.1.4 Association of RAD52 with the RPA complex 675
6.4.1.1.4.1.5 Association of RAD52 with ssDNA at resected ends of double-strand break 675
6.4.1.1.4.1.6 Assembly of the RAD51-ssDNA nucleoprotein complex 676
6.4.1.1.4.1.6.1 Association of RAD51 with RAD52:DNA double-strand break ends 678
6.4.1.1.4.1.6.2 Association of RAD51 with the RPA complex 678
6.4.1.1.4.1.6.3 Association of RAD51 with the resected ends of the DNA double-strand break 679
6.4.1.1.4.2 Synaptic stable pairing/D-loop structure formation between recombining DNA molecules 680
6.4.1.1.4.2.1 Strand invasion 681
6.4.1.1.4.2.2 Heteroduplex formation 683
6.4.1.1.4.3 Strand exchange/Branch migration 685
6.4.1.1.5 DNA repair synthesis 686
6.4.1.1.6 Resolution of D-loop structures 688
6.4.1.1.6.1 Resolution of D-loop structures through Holliday junction intermediates 690
6.4.1.1.6.1.1 Ligation of DNA and formation of Holliday structures following repair synthesis 691
6.4.1.1.6.1.2 Cleavage of Holliday junctions 692
6.4.1.1.6.2 Resolution of D-loop structures through synthesis-dependent strand-annealing 694
6.4.1.1.6.2.1 Dissociation of the extended strands 695
6.4.1.1.6.2.2 Fill-in DNA synthesis 696
6.4.1.2 Homologous recombination repair of replication-dependent double-strand breaks 698
6.4.2 Nonhomologous End-joining (NHEJ) 698
6.4.2.1 Association of Ku heterodimer with ends of DNA double-strand break 700
6.4.2.2 Autophosphorylation of DNA-PKcs 701
6.4.2.3 Association of DNA-PKcs with Ku-bound ends of DNA double-strand breaks 701
6.4.2.4 Synapsis, or interaction between two DNA-PK:DNA complexes at opposing ends of DNA DSB 702
6.4.2.5 Processing of DNA ends prior to end rejoining 703
6.4.2.5.1 Removal of 3'-phosphoglycolate (PG) moiety from DSB ends 703
6.4.2.5.2 Removal of 3'-phosphate moiety from DSB ends 704
6.4.2.6 Association of the XRCC4:DNA ligase IV complex with the DNA-PK:DNA synaptic complex 705
6.4.2.7 Removal of repair proteins and ligation of the processed ends of the DNA double-strand break 705
6.5 Nucleotide Excision Repair 707
6.5.1 Global Genomic NER (GG-NER) 707
6.5.1.1 DNA Damage Recognition in GG-NER 708
6.5.1.1.1 XPC binds to HR23B forming a heterodimeric complex 709
6.5.1.1.2 XPC:HR23B complex binds to damaged DNA site with lesion 709
6.5.1.2 Formation of incision complex in GG-NER 710
6.5.1.2.1 Recruitment of repair factors to form preincision complex 710
6.5.1.2.2 Formation of open bubble structure in DNA by helicases 711
6.5.1.2.3 Binding of ERCC1-XPF to preincision complex 712
6.5.1.3 Dual incision reaction in GG-NER 713
6.5.1.3.1 3'- incision of DNA by XPG in GG-NER 713
6.5.1.3.2 5'-incision of DNA by ERCC1-XPF in GG-NER 714
6.5.1.4 Gap-filling DNA repair synthesis and ligation in GG-NER 715
6.5.1.4.1 Repair synthesis of patch ~27-30 bases long by DNA polymerase 716
6.5.1.4.1.1 Repair synthesis of ~27-30 bases long patch by DNA Pol Epsilon 716
6.5.1.4.1.2 Repair synthesis of patch ~27-30 bases long by DNA Pol Delta 717
6.5.1.4.2 Ligation of newly synthesized repair patch to incised DNA in GG-NER 717
6.5.2 Transcription-coupled NER (TC-NER) 718
6.5.2.1 Formation of transcription-coupled NER (TC-NER) repair complex 719
6.5.2.1.1 Formation of active Pol II complex with lesioned DNA template 719
6.5.2.1.2 RNA Pol II is blocked by the lesion leading to reduced transcription 720
6.5.2.1.3 Assembly of repair proteins at the site of Pol II blockage 721
6.5.2.2 Dual incision reaction in TC-NER 722
6.5.2.2.1 Displacement of stalled Pol II from the lesion site 722
6.5.2.2.2 3' incision of the lesioned strand of DNA in TC-NER 723
6.5.2.2.3 5' incision leading to excision of DNA fragment with lesion in TC-NER 723
6.5.2.3 Gap-filling DNA repair synthesis and ligation in TC-NER 724
6.5.2.3.1 Repair synthesis for gap-filling by DNA polymerase in TC-NER 724
6.5.2.3.1.1 Repair synthesis for gap-filling by DNA pol delta in TC-NER 724
6.5.2.3.1.2 Repair synthesis for gap-filling by DNA pol epsilon in TC-NER 725
6.5.2.3.2 Ligation of newly synthesized repair patch to incised DNA in TC-NER 725
6.5.2.4 Resumption of transcription after TC-NER 726
7 DNA Replication 728
7.1 DNA Replication Pre-Initiation 729
7.1.1 Assembly of the pre-replicative complex 729
7.1.1.1 Assembly of the ORC complex at the origin of replication 731
7.1.1.1.1 Orc3 associates with Orc2 constitutively bound at origins of replication 732
7.1.1.1.2 Orc5 associates with Orc3:Orc2:origin complexes 732
7.1.1.1.3 Orc4 associates with Orc5:Orc3:Orc2:origin complexes 733
7.1.1.1.4 Orc1 associates with Orc4:Orc5:Orc3:Orc2:origin complexes 734
7.1.1.1.5 Orc6 associates with Orc1:Orc4:Orc5:Orc3:Orc2:origin complexes, forming ORC:origin complexes 734
7.1.1.2 Association of MCM8 with ORC:origin complex 735
7.1.1.3 CDC6 association with the ORC:origin complex 735
7.1.1.3.2 CDC6 association with ORC:origin complexes mediated by MCM8 737
7.1.1.4 CDT1 association with the CDC6:ORC:origin complex 738
7.1.1.4.1 The geminin component of geminin:Cdt1 complexes is ubiquitinated, releasing Cdt1 739
7.1.1.4.2 Ubiquitinated geminin is degraded by the proteasome 740
7.1.1.4.3 Free CDT1 associates with CDC6:ORC:origin complexes 741
7.1.1.5 Mcm2-7 associates with the Cdt1:CDC6:ORC:origin complex, forming the pre-replicative complex (preRC) 741
7.1.2 Activation of the pre-replicative complex 743
7.1.2.1 Mcm10 associates with the pre-replicative complex, stabilizing Mcm2-7 744
7.1.2.3 CDK and DDK associate with the Mcm10:pre-replicative complex 745
7.1.2.4 Mcm2-7 is phosphorylated by DDK 746
7.1.2.5 Cdc45 associates with the pre-replicative complex at the origin 746
7.1.2.6 DNA Replication Factor A (RPA) associates with the pre-replicative complex at the origin 747
7.1.2.7 DNA polymerase alpha:primase binds at the origin 748
7.1.2.8 DNA polymerase epsilon binds at the origin 750
7.2 DNA replication initiation 750
7.2.1 The primase component of DNA polymerase:primase synthesizes a 6-10 nucleotide RNA primer at the origin 751
7.2.2 The polymerase component of DNA polymerase alpha:primase synthesizes a 20-nucleotide primer at the origin 751
7.3 Switching of origins to a post-replicative state 752

7.3.1 Orc1 removal from chromatin 752
7.3.1.1 Orc1 is phosphorylated by cyclin A/CDK2 752
7.3.1.2 Phosphorylated Orc1 is ubiquitinated while still associated with chromatin 753
7.3.1.3 Ubiquitinated Orc1 enters the cytosol 754
7.3.1.4 Ubiquitinated Orc1 is degraded by the proteasome 754
7.3.2 CDK-mediated phosphorylation and removal of Cdc6 755
7.3.2.1 Cdc6 protein is phosphorylated by CDK 755
7.3.2.2 Phosphorylated Cdc6 is exported from the nucleus 756
7.3.2.3 Cytoplasmic phosphorylated Cdc6 is ubiquitinated by the anaphase-promoting complex 756
7.3.2.4 Ubiquitinated Cdc6 is degraded by the proteasome 757
7.3.3 Mcm4,6,7 trimer forms and associates with the replication fork 758
7.4 DNA strand elongation 759
7.4.1 Unwinding of DNA 760
7.4.1.1 MCM2-7 mediated fork unwinding 760
7.4.1.2 MCM8 mediated fork unwinding 761
7.4.1.3 Formation of GINS complex 762
7.4.1.4 Multiple proteins are localized at replication fork 763
7.4.2 Leading Strand Synthesis 764
7.4.2.1 Polymerase switching 764
7.4.2.1.1 RFC binding displaces Pol Alpha 766
7.4.2.1.2 Loading of PCNA - Sliding Clamp Formation 766
7.4.2.1.3 RFC dissociates after sliding clamp formation 767
7.4.2.1.4 Formation of Processive Complex 767
7.4.2.2 Processive synthesis on the leading strand 768
7.4.3 Lagging Strand Synthesis 769
7.4.3.1 Polymerase switching 769
7.4.3.1.1 RFC binding displaces Pol Alpha 771
7.4.3.1.2 Loading of PCNA - Sliding Clamp Formation 771
7.4.3.1.3 RFC dissociates after sliding clamp formation 772
7.4.3.1.4 Formation of Processive Complex 772
7.4.3.2 Processive synthesis on the lagging strand 773
7.4.3.2.1 Formation of Okazaki fragments 774
7.4.3.2.2 Formation of the Flap Intermediate 774
7.4.3.2.3 Removal of the Flap Intermediate 775
7.4.3.2.3.1 RPA binds to the Flap 775
7.4.3.2.3.2 Recruitment of Dna2 endonuclease 776
7.4.3.2.3.3 Removal of RNA primer and dissociation of RPA and Dna2 776
7.4.3.2.3.4 Removal of remaining Flap 777
7.4.3.2.4 Joining of adjacent Okazaki fragments 778
7.5 Regulation of DNA replication 779
7.5.1 Association of licensing factors with the pre-replicative complex 779
7.5.1.1 Orc1 associates with Orc4:Orc5:Orc3:Orc2:origin complexes 779
7.5.1.2 CDC6 protein is synthesized under the control of E2F transcription factors 780
7.5.1.3 The geminin component of geminin:Cdt1 complexes is ubiquitinated, releasing Cdt1 780
7.5.2 Removal of licensing factors from origins 781
7.5.2.1 Orc1 removal from chromatin 781
7.5.2.1.1 Orc1 is phosphorylated by cyclin A/CDK2 781
7.5.2.1.2 Phosphorylated Orc1 is ubiquitinated while still associated with chromatin 782
7.5.2.1.3 Ubiquitinated Orc1 enters the cytosol 782
7.5.2.1.4 Ubiquitinated Orc1 is degraded by the proteasome 782
7.5.2.3 Cdt1 associates with geminin 783
7.5.2.4 CDK-mediated phosphorylation and removal of Cdc6 784
7.5.2.4.1 Cdc6 protein is phosphorylated by CDK 784
7.5.2.4.2 Phosphorylated Cdc6 is exported from the nucleus 785
7.5.2.4.3 Cytoplasmic phosphorylated Cdc6 is ubiquitinated by the anaphase-promoting complex 785
7.5.2.4.4 Ubiquitinated Cdc6 is degraded by the proteasome 785
7.5.2.5 Mcm4,6,7 trimer forms and associates with the replication fork 786
8 Electron Transport Chain 788
8.1 NADH enters the respiratory chain at Complex I 790
8.2 Transfer of electrons through the succinate dehydrogenase complex 791
8.3 Reducing equivalents from beta-oxidation of fatty acids transfer to ETF 792
8.4 Transfer of electrons from ETF to ubiquinone by ETF-QO 793
8.5 Electron transfer from ubiquinol to cytochrome c of complex III 793
8.6 Electron transfer from reduced cytochrome c to molecular oxygen 794
9 Gap junction trafficking and regulation 796
9.1 Gap junction trafficking 797
9.1.1 Gap junction assembly 798

9.1.1.1 Connexin synthesis 800
9.1.1.2 Transport of connexins along the secretory pathway 801
9.1.1.2.1 Transport of connexins to the ER-Golgi intermediate compartment 802
9.1.1.2.2 Transport of connexins to the Trans-Golgi Network (TGN) 803
9.1.1.3 Oligomerization of connexins into connexons 804
9.1.1.3.1 Connexin oligomerization in endoplasmic reticulum membrane 806
9.1.1.3.2 Connexin oligomerization in ER-Golgi-Intermediate Compartment 806
9.1.1.3.3 Connexin oligomerization in Trans-Golgi Network (TGN) 807
9.1.1.4 Transport of connexons to the plasma membrane 808
9.1.1.4.1 Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane 809
9.1.1.4.1.1 Budding of connexon-containing transport vesicles from the Golgi 810
9.1.1.4.1.2 Association of Golgi transport vesicles with microtubules and transport to the plasma membrane 810
9.1.1.4.2 Microtubule-independent trafficking of connexons to the plasma membrane 811
9.1.1.5 Docking of connexons into junctional, double-membrane spanning channels 812
9.1.1.6 Insertion of connexons into the plasma membrane resulting in the formation of hemi-channels 813
9.1.1.7 Formation of junctional channels 815
9.1.1.8 Assembly of gap junction plaques 817
9.1.2 Gap junction degradation 818
9.1.2.1 Formation of annular gap junctions 819
9.1.2.1.1 Dab2 is recruited to the junctional plaques 820
9.1.2.1.2 Dab2 interacts with clathrin 821
9.1.2.1.3 Dynamin is recruited to the gap junction plaque 822
9.1.2.2 Internalization of gap junction plaques 822
9.1.2.3 Lysosomal degradation of gap junction plaques 824
9.2 Regulation of gap junction activity 825
9.2.1 c-src mediated regulation of Cx43 function and closure of gap junctions 825
9.2.1.1 Association of Cx43 with ZO-1 826
9.2.1.2 c-src associates with Cx43 in gap junctions 827
9.2.1.3 Phosphorylation of Cx43 by c-src 827
9.2.1.4 Closure of gap junction 828
10 Gene Expression 829
10.1 Generic Transcription Pathway 829
10.1.1 Formation of ARC coactivator complex 832
10.1.2 Formation of DRIP coactivator complex 834
10.1.3 Formation of TRAP coactivator complex 835
10.2 Formation and Maturation of mRNA Transcript 837
10.2.1 RNA Polymerase II Transcription Pre-Initiation 837
10.2.1.1 Recognition and Binding of Core Promoter Elements by TFIID 838
10.2.1.2 Binding of TFIIA and TFIIB to the pol II promoter:TFIID complex 838
10.2.1.3 Recruitment of RNA Polymerase II Holoenzyme by TFIIF to the pol II promoter:TFIID:TFIIA:TFIIB complex 839
10.2.1.4 Binding of TFIIE to the growing preinitiation complex 840
10.2.1.5 Formation of the closed pre-initiation complex 841
10.2.2 RNA Polymerase II Transcription Initiation And Promoter Clearance 841
10.2.2.1 RNA Polymerase II Promoter Opening: First Transition 841
10.2.2.2 RNA Polymerase II Transcription Initiation 843
10.2.2.2.1 NTP Binds Active Site of RNA Polymerase II 843
10.2.2.2.2 Nucleophillic Attack by 3'-hydroxyl Oxygen of nascent transcript on the Alpha Phosphate of NTP 844
10.2.2.2.3 Newly Formed Phosphodiester Bond Stabilized and PPi Released 844
10.2.2.3 RNA Polymerase II Promoter Escape 845
10.2.2.3.1 Addition of the third nucleotide on the nascent transcript 845
10.2.2.3.2 Addition of the fourth nucleotide on the Nascent Transcript: Second Transition 846
10.2.2.3.3 Addition of Nucleotides 5 through 9 on the growing Transcript 847
10.2.2.3.4 Addition of nucleotides 10 and 11 on the growing transcript: Third Transition 848
10.2.2.3.5 Addition of nucleotides between position +11 and +30 849
10.2.3 mRNA Capping 850
10.2.3.1 Capping complex formation 852
10.2.3.2 Hydrolysis of the 5'-end of the nascent transcript by the capping enzyme 852
10.2.3.3 Formation of the CE:GMP intermediate complex 854
10.2.3.4 Transfer of GMP from the capping enzyme GT site to 5'-end of mRNA 854
10.2.3.5 Dissociation of transcript with 5'-GMP from GT 855
10.2.3.6 Methylation of GMP-cap by RNA Methyltransferase 856
10.2.3.7 Recognition and binding of the mRNA cap by the cap-binding complex 857
10.2.4 Elongation and Processing of Capped Transcripts 858
10.2.4.1 Elongation of Intron-Containing Transcripts and co-transcriptional mRNA splicing 858
10.2.4.1.1 RNA Polymerase II Transcription Elongation 858
10.2.4.1.1.1 Formation of the Early Elongation Complex 860
10.2.4.1.1.1.1 Hypophosphorylation of RNA Pol II CTD by FCP1P protein 860
10.2.4.1.1.1.2 DSIF complex binds to RNA Pol II (hypophosphorylated) 861

10.2.4.1.1.1.3 Formation of DSIF:NELF:early elongation complex 861
10.2.4.1.1.2 Formation of RNA Pol II elongation complex 862
10.2.4.1.1.2.1 Hyperphosphorylation (Ser2) of RNA Pol II CTD by P-TEFb complex 863
10.2.4.1.1.2.2 Recruitment of elongation factors to form elongation complex 864
10.2.4.1.1.3 Addition of nucleotides leads to transcript elongation 864
10.2.4.1.1.4 Pol II elongation complex moves on the template as transcript elongates 865
10.2.4.1.1.5 Separation of elongating transcript from template 866
10.2.4.1.2 Internal Methylation of mRNA 866
10.2.4.1.3 Formation of pre-mRNPs 867
10.2.4.1.4 mRNA Splicing 868
10.2.4.1.4.1 mRNA Splicing - Major Pathway 868
10.2.4.1.4.1.1 Formation of the Spliceosomal E complex 869
10.2.4.1.4.1.2 Formation of the Spliceosomal A Complex 871
10.2.4.1.4.1.3 Formation of the Spliceosomal B Complex 871
10.2.4.1.4.1.4 Formation of an intermediate Spliceosomal C complex 873
10.2.4.1.4.1.5 Formation of the active Spliceosomal C complex 874
10.2.4.1.4.1.6 Lariat Formation and 5'-Splice Site Cleavage 874
10.2.4.1.4.1.7 Formation of Exon Junction Complex 876
10.2.4.1.4.1.8 Cleavage at the 3'-Splice Site and Exon Ligation 876
10.2.4.1.4.2 mRNA Splicing - Minor Pathway 877
10.2.4.1.4.2.1 Formation of AT-AC A complex 879
10.2.4.1.4.2.2 Formation of AT-AC B Complex 881
10.2.4.1.4.2.3 Formation of AT-AC C complex 883
10.2.4.1.4.2.4 ATAC spliceosome mediated Lariat formation,5' splice site cleavage 885
10.2.4.1.4.2.5 ATAC spliceosome mediated 3' splice site cleavage, exon ligation 887
10.2.4.2 Elongation of Intronless Transcripts 889
10.2.5 Post-Elongation Processing of the Transcript 889
10.2.5.1 Post-Elongation Processing of Intron-Containing pre-mRNA 890
10.2.5.1.1 mRNA 3'-end processing 890
10.2.5.1.1.1 Cleavage of mRNA at the 3'-end 890
10.2.5.1.1.2 mRNA polyadenylation 891
10.2.5.2 Post-Elongation Processing of Intronless pre-mRNA 892
10.2.5.2.1 Processing of Capped Intronless Pre-mRNA 892
10.2.5.2.1.1 SLBP Dependent Processing of Replication-Dependent Histone Pre-mRNAs 893
10.2.5.2.1.1.1 Binding of SLBP to Replication-Dependent Histone Pre-mRNA 894
10.2.5.2.1.1.2 Recruitment of U7 snRNP:ZFP100 complex to the SLBP Bound Pre-mRNA 895
10.2.5.2.1.1.3 Cleavage of the 3'-end of Replication Dependent Histone Pre-mRNA 897
10.2.5.2.1.2 SLBP independent Processing of Histone Pre-mRNAs 898
10.2.5.2.1.2.1 Recruitment of U7 snRNP:ZFP100 complex to the Histone Pre-mRNA 898
10.2.5.2.1.2.2 Cleavage of the 3'-end of the Histone Pre-mRNA 899
10.2.5.2.1.3 Processing of Intronless Pre-mRNAs 900
10.2.5.2.1.3.1 Recognition of AAUAAA sequence by CPSF 900
10.2.5.2.1.3.2 Recruitment of CstF to the CPSF Bound Pre-mRNA 901
10.2.5.2.1.3.3 Binding of Cleavage factors and Poly(A)Polymerase to the CstF:CPSF:Pre-mRNA Complex 902
10.2.5.2.1.3.4 Cleavage and polyadenylation of Intronless Pre-mRNA 902
10.3 Transport of Mature Transcript to Cytoplasm 903
10.3.1 Transport of Mature mRNA derived from an Intron-Containing Transcript 904
10.3.1.1 Recruitment of TAP to the EJC 904
10.3.1.2 Docking of the TAP:EJC Complex with the NPC 904
10.3.1.3 Transport of the export-competent complex through the NPC 905
10.3.1.4 Release from the NPC and Disassembly of the mRNP 906
10.3.2 Transport of Mature mRNAs Derived from Intronless Transcripts 906
10.3.2.1 Transport of the SLBP Dependant Mature mRNA 906
10.3.2.1.1 Docking of Mature Replication Dependent Histone mRNA with the NPC 907
10.3.2.1.2 Transport of the Mature IntronlessTranscript Derived Histone mRNA:SLBP:TAP:Aly/Ref complex through the NPC 908
10.3.2.1.3 Release of the Mature intronless transcript derived Histone mRNA:SLBP:eIF4E Complex 908
10.3.2.2 Transport of the SLBP independent Mature mRNA 909
10.3.2.2.1 Docking of Mature Histone mRNA complex:TAP at the NPC 909
10.3.2.2.2 Transport of the Mature Intronless Transcript Derived Histone mRNA:TAP:Aly/Ref Complex through the NPC 910
10.3.2.2.3 Release of the SLBP independent Histone mRNA from the NPC 910
10.3.2.3 Transport of Mature mRNA Derived from an Intronless Transcript 911
10.3.2.3.1 Docking of the Mature intronless derived transcript derived mRNA, TAP and Aly/Ref at the NPC 911
10.3.2.3.2 Transport of the Mature intronless transcript derived mRNA:TAP:Aly/Ref Complex through the NPC 912
10.3.2.3.3 Release of the Mature intronless derived mRNA, TAP, and Aly/Ref from the NPC 912
10.4 Translation 913
10.4.1 Eukaryotic Translation Initiation 913
10.4.1.1 Cap-dependent Translation Initiation 913
10.4.1.1.1 Formation of a pool of free 40S subunits 916

10.4.1.1.1.1 Release of 40S and 60S subunits from the 80S ribosome 916
10.4.1.1.1.2 eIF3 and eIF1A bind to the 40S subunit 916
10.4.1.1.2 Formation of the ternary complex, and subsequently, the 43S complex 917
10.4.1.1.2.1 De novo formation of eIF2:GTP 917
10.4.1.1.2.2 Met-tRNAi binds to eIF2:GTP to form the ternary complex 918
10.4.1.1.2.3 Formation of the 43S pre-initiation complex 918
10.4.1.1.3 Activation of the mRNA upon binding of the cap-binding complex and eIFs, and subsequent binding to 43S 919
10.4.1.1.3.1 Release of eIF4E from the inactive eIF4E:4E-BP complex 920
10.4.1.1.3.2 Formation of the cap-binding eIF4F complex 920
10.4.1.1.3.3 eIF4F binds to mRNP 921
10.4.1.1.3.4 Cap-bound mRNA is activated by helicases 921
10.4.1.1.3.5 Translation initiation complex formation 922
10.4.1.1.3.5.1 Formation of translation initiation complexes containing mRNA that does not circularize 922
10.4.1.1.3.5.2 Formation of translation initiation complexes yielding circularized Ceruloplasmin mRNA in a 'closed-loop' 923
conformation
10.4.1.1.4 Ribosomal scanning and start codon recognition 924
10.4.1.1.4.1 Ribosomal scanning 924
10.4.1.1.4.2 Start codon recognition 925
10.4.1.1.5 GTP hydrolysis and joining of the 60S ribosomal subunit 926
10.4.1.1.5.1 eIF2:GTP is hydrolyzed, eIFs are released 926
10.4.1.1.5.2 eIF5B:GTP is hydrolyzed and released 928
10.4.1.1.5.3 The 60S subunit joins the translation initiation complex 928
10.4.1.1.6 Recycling of eIF2:GDP 929
10.4.1.1.6.1 Formation of eIF2:GDP:eIF2B intermediate 929
10.4.1.1.6.2 eIF2 activation 930
10.4.1.2 Cap-independent Translation Initiation 930
10.4.2 Eukaryotic Translation Elongation 931
10.4.2.1 eEF1A complexes with GTP 932
10.4.2.2 eEF1A:GTP:aminoacyl tRNA ternary complex formation. 934
10.4.2.3 Peptide chain elongation 935
10.4.2.3.1 Aminoacyl-tRNA binds to the ribosome at the A-site 935
10.4.2.3.2 Hydrolysis of eEF1A:GTP 937
10.4.2.3.3 Peptide transfer from P-site tRNA to the A-site tRNA 938
10.4.2.3.4 Translocation of ribosome by 3 bases in the 3' direction 939
10.4.2.4 Regeneration of eEF1A:GTP by eEF1B activity 941
10.4.3 Eukaryotic Translation Termination 942
10.4.3.1 GTP bound eRF3:eRF1 complex binds the peptidyl tRNA:mRNA:80S Ribosome complex 943
10.4.3.2 GTP Hydrolysis by eRF3 bound to the eRF1:mRNA:polypeptide:80S Ribosome complex 944
10.4.3.3 Polypeptide release from the eRF3-GDP:eRF1:mRNA:80S Ribosome complex 945
11 HIV Infection 947
11.1 HIV Life Cycle 948
11.1.1 Early Phase of HIV Life Cycle 949
11.1.1.1 Binding and entry of HIV virion 950
11.1.1.1.1 Binding of gp120 of ENV oligomer to the host CD4 951
11.1.1.1.2 Conformational change in gp120 of Env oligomer 953
11.1.1.1.3 CD4:gp120 binds to chemokine co-receptor CCR5/CXCR4 954
11.1.1.1.4 Conformational changes in gp120 exposes gp41 955
11.1.1.1.5 Fusogenic activation of gp41 956
11.1.1.1.6 Insertion of gp41 fusion peptide into the target membrane 957
11.1.1.1.7 N and C terminal heptad repeat helices of gp41 form six-helix bundle 958
11.1.1.1.8 Fusion of viral membrane with host cell membrane 960
11.1.1.2 Uncoating of the HIV Virion 961
11.1.1.2.1 Disintegration of matrix layer 962
11.1.1.2.2 Disassembly of viral capsid 962
11.1.1.3 Formation of RTC (Reverse Transcription Complex) 964
11.1.1.4 Annealing of 3'-end of unwound transfer RNA primer with genomic RNA 965
11.1.1.5 Reverse Transcription of HIV RNA 967
11.1.1.5.1 Minus-strand DNA synthesis 969
11.1.1.5.1.1 Synthesis of minus strand strong stop DNA (-sssDNA) 971
11.1.1.5.1.2 RNase H-mediated cleavage of the RNA strand of the -sssDNA:RNA duplex 973
11.1.1.5.1.3 RNase H-mediated degradation of the RNA strand of the -sssDNA:RNA duplex 975
11.1.1.5.1.4 First strand transfer mediated by Repeated (R) sequence 977
11.1.1.5.1.5 Minus strand DNA synthesis resumes 979
11.1.1.5.1.6 RNase H-mediated cleavage of the template strand 981
11.1.1.5.1.7 RNase H-mediated degradation of the template strand 983
11.1.1.5.2 Plus-strand DNA synthesis 985
11.1.1.5.2.1 3' PPT-primed initiation of plus-strand DNA synthesis 987
11.1.1.5.2.2 RNase H-mediated digestion of tRNA, 3'PPT and cPPT RNA primers 989
11.1.1.5.2.3 Second strand transfer by annealing complementary PBS sequences 991
11.1.1.5.2.4 Synthesis of full-length duplex viral DNA with a discontinuous plus strand 993
11.1.1.6 Removal of plus-strand flap and gap closure complete synthesis of linear duplex viral DNA 995
11.1.1.7 Integration of provirus 996
11.1.1.7.1 Formation of Pre-Integration Complex (PIC) 999
11.1.1.7.2 Integrase binds viral DNA ends 1000
11.1.1.7.3 Terminal (3' end) cleavage of viral DNA 1001
11.1.1.7.4 Import of PIC to the Host Nucleus 1002
11.1.1.7.5 Integration of viral DNA into host genomic DNA 1004
11.1.1.7.5.1 Target DNA binding 1005
11.1.1.7.5.2 Transesterification to connect viral DNA 3' ends to host DNA 5' ends 1007
11.1.1.7.5.3 Gap repair completes provirus integration 1008
11.1.1.7.6 1-LTR circle formation 1009
11.1.1.7.7 2-LTR circle formation 1010
11.1.1.7.7.1 Association of Ku heterodimer with viral DNA ends 1011
11.1.1.7.7.2 Association of XRCC4:DNA ligase IV complex with viral DNA ends 1012
11.1.1.7.7.3 2-LTR formation due to circularization of viral DNA 1013
11.1.1.7.8 Autointegration results in viral DNA circles 1014
11.1.1.7.8.1 Suicidal integration leading to inverted circle formation 1015
11.1.1.7.8.2 Suicidal integration leading to smaller circles of viral DNA 1016
11.1.2 Late Phase of HIV Life Cycle 1017
11.1.2.1 Transcription of the HIV genome 1018
11.1.2.1.1 HIV-1 Transcription Pre-Initiation 1020
11.1.2.1.1.1 Recognition and Binding of Core HIV-1 Promoter Elements by TFIID 1020
11.1.2.1.1.2 Binding of TFIIA and TFIIB to the HIV-1 promoter:TFIID complex 1021
11.1.2.1.1.3 Recruitment of RNA Polymerase II Holoenzyme by TFIIF to the HIV-1promoter:TFIID:TFIIA:TFIIB complex 1022
11.1.2.1.1.4 Binding of TFIIE to the growing HIV-1 preinitiation complex 1023
11.1.2.1.1.5 Formation of the HIV-1 closed pre-initiation complex 1023
11.1.2.1.2 HIV-1 Promoter Opening: First Transition 1024
11.1.2.1.3 HIV-1 Transcription Initiation 1025
11.1.2.1.3.1 NTP binds active site of RNA Polymerase II in HIV-1 open pre-initiation complex 1026
11.1.2.1.3.2 Nucleophillic attack by 3'-hydroxyl oxygen of nascent HIV-1 transcript on the Alpha phosphate of NTP 1027
11.1.2.1.3.3 Newly formed phosphodiester bond stabilized and PPi released 1028
11.1.2.1.4 RNA Polymerase II HIV-1 Promoter Escape 1028
11.1.2.1.4.1 Addition of the third nucleotide on the nascent HIV-1 transcript 1029
11.1.2.1.4.2 Addition of the fourth nucleotide on the nascent HIV-1 transcript: Second Transition 1030
11.1.2.1.4.3 Addition of nucleotides 5 through 9 on the growing HIV-1 transcript 1031
11.1.2.1.4.4 Addition of nucleotides 10 and 11 on the growing HIV-1 transcript: Third Transition 1032
11.1.2.1.4.5 Addition of nucleotides between position +11 and +30 on HIV-1 transcript 1033
11.1.2.1.5 RNA Pol II CTD phosphorylation and interaction with CE 1034
11.1.2.1.5.1 Extrusion of 5'-end of 30 nt long HIV-1 transcript through the pore in Pol II complex 1034
11.1.2.1.5.2 Phosphorylation (Ser5) of RNA pol II CTD 1035
11.1.2.1.5.3 RNA Polymerase II CTD (phosphorylated) binds to CE 1036
11.1.2.1.5.4 Activation of GT 1037
11.1.2.1.5.5 SPT5 subunit of Pol II binds the RNA triphosphatase (RTP) 1037
11.1.2.1.6 HIV-1 Transcription Elongation 1038
11.1.2.1.6.1 Formation of the HIV-1 Early Elongation Complex 1039
11.1.2.1.6.1.1 Hypophosphorylation of RNA Pol II CTD by FCP1P protein 1040
11.1.2.1.6.1.2 DSIF complex binds to RNA Pol II (hypophosphorylated) 1041
11.1.2.1.6.1.3 Formation of DSIF:NELF:HIV-1 early elongation complex 1042
11.1.2.1.6.2 Tat-mediated elongation of the HIV-1 transcript 1043
11.1.2.1.6.2.1 Formation of HIV-1 elongation complex containing HIV-1 Tat 1045
11.1.2.1.6.2.1.1 Association of Tat with P-TEFb(Cyclin T1:Cdk9) 1046
11.1.2.1.6.2.1.2 Hyperphosphorylation (Ser2) of RNA Pol II CTD by the P-TEFb(Cyclin T1:Cdk9) complex 1046
11.1.2.1.6.2.1.3 Phosphorylation of NEFL by the P-TEFb(Cyclin T1:Cdk9) complex 1047
11.1.2.1.6.2.1.4 Phosphorylation of DSIF by the P-TEFb(Cyclin T1:Cdk9) complex 1048
11.1.2.1.6.2.1.5 Recruitment of elongation factors to form HIV-1 elongation complex 1049
11.1.2.1.6.2.2 Addition of nucleotides leads to HIV-1 transcript elongation 1049
11.1.2.1.6.2.3 Pol II elongation complex moves on the HIV-1 template as transcript elongates 1051
11.1.2.1.6.2.4 Separation of elongating HIV-1 transcript from template 1052
11.1.2.1.6.3 Abortive elongation of HIV-1 transcript in the absence of Tat 1052
11.1.2.1.6.3.1 Limited elongation of the HIV-1 transcript 1053
11.1.2.1.6.3.2 Separation of abortive HIV-1 transcript from template 1054
11.1.2.1.7 HIV-1 Transcription Termination 1055
11.1.2.2 Rev-mediated nuclear export of HIV-1 RNA 1055
11.1.2.2.1 Rev molecules assemble onto the RRE RNA sequence through their ARM sequence 1057
11.1.2.2.2 Multimerization of Rev 1057
11.1.2.2.3 Rev multimer-bound HIV-1 mRNA associates with Crm1 1058

11.1.2.2.4 Rev multimer-bound HIV-1 mRNA:CRM1 complex associates with Ran:GTP 1059
11.1.2.2.5 Rev multimer-bound HIV-1 mRNA:Crm1:Ran:GTP complex associates with the NPC 1060
11.1.2.2.6 Translocation of nuclear RNA transport complex to cytoplasm 1060
11.1.2.2.7 Association of RanBP1 with Ran-GTP:CRM1:Rev:mRNA complex 1061
11.1.2.2.8 Release of the HIV-1 mRNA and Crm1 from Rev in the cytoplasm 1062
11.1.2.2.9 Hydrolysis of Ran:GTP to Ran:GDP 1063
11.1.2.3 Assembly of HIV virion 1064
11.1.2.4 Budding and maturation of HIV-1 virion 1064
11.2 Host Interactions of HIV factors 1064
11.2.1 Interactions of Vpr with host cellular proteins 1065
11.2.1.1 Vpr-mediated induction of apoptosis by mitochondrial outer membrane permeabilization 1066
11.2.1.1.1 Translocation of Vpr to the mitochondria 1067
11.2.1.1.2 Association of Vpr with ANT1 1068
11.2.1.2 Vpr-mediated nuclear import of PICs 1068
11.2.1.2.1 Vpr binds nucleoporins 1069
11.2.1.2.2 Interaction of Vpr with importin alpha 1070
11.2.2 Interactions of Rev with host cellular proteins 1071
11.2.2.1 Nuclear import of Rev protein 1071
11.2.2.1.1 Association of multimerized Rev with beta-importin 1072
11.2.2.1.2 Rev associates with B23 1073
11.2.2.1.3 Rev:importin beta:B23 recruited to the nuclear pore 1073
11.2.2.1.4 Translocation of Rev:importin-beta:B23 to the nucleus 1074
11.2.2.1.5 Association of Ran-GTP with importin-beta 1075
11.2.2.1.6 Disassembly of the Rev-importin beta-B23:Ran-GTP complex 1076
11.2.2.2 Rev-mediated nuclear export of HIV-1 RNA 1077
11.2.2.2.1 Rev molecules assemble onto the RRE RNA sequence through their ARM sequence 1079
11.2.2.2.2 Multimerization of Rev 1079
11.2.2.2.3 Rev multimer-bound HIV-1 mRNA associates with Crm1 1080
11.2.2.2.4 Rev multimer-bound HIV-1 mRNA:CRM1 complex associates with Ran:GTP 1081
11.2.2.2.5 Rev multimer-bound HIV-1 mRNA:Crm1:Ran:GTP complex associates with the NPC 1081
11.2.2.2.6 Translocation of nuclear RNA transport complex to cytoplasm 1082
11.2.2.2.7 Association of RanBP1 with Ran-GTP:CRM1:Rev:mRNA complex 1083
11.2.2.2.8 Release of the HIV-1 mRNA and Crm1 from Rev in the cytoplasm 1083
11.2.2.2.9 Hydrolysis of Ran:GTP to Ran:GDP 1084
11.2.3 APOBEC3G mediated resistance to HIV-1 infection 1085
11.2.3.1 Association of APOBEC3G with Gag 1085
11.2.3.2 Association of APOBEC3G with single-stranded region of forming HIV-1 minus strand 1087
11.2.3.3 Deamination of C residues during synthesis of HIV-1 reverse transcript minus-strand 1088
11.2.4 Vif-mediated degradation of APOBEC3G 1089
11.2.4.1 Association of Vif with APOBEC3G 1090
11.2.4.2 Association of APOBEC3G:Vif with the Cul5-SCF complex 1091
11.2.4.3 Multi-ubiquitination of APOBEC3G 1092
11.2.4.4 Proteosome-mediated degradation of APOBEC3G 1093
11.2.5 Interactions of Tat with host cellular proteins 1094
11.2.5.1 Association of Tat with P-TEFb(Cyclin T1:Cdk9) 1094
11.2.6 The role of Nef in HIV-1 replication and disease pathogenesis 1095
11.2.6.1 Nef and signal transduction 1096
11.2.6.1.1 Nef mediated activation of the T-cell receptor 1096
11.2.6.1.2 Nef Binds and activates the Src-family tyrosine kinase Hck 1097
11.2.6.1.3 Nef Binds and activates the Src-family tyrosine kinase Fyn 1098
11.2.6.1.4 Nef Binds and activates the Src-family tyrosine kinase Lck 1099
11.2.6.1.5 Nef binds a ternary complex comprising DOCK2 guanine nucleotide exchange factor for small Rho-family GTPase Rac, its1100
cofactor ELMO1, and Rac, and activates Rac through this interaction
11.2.6.2 Nef-mediates down modulation of cell surface receptors by recruiting them to clathrin adapters 1101
11.2.6.2.1 Nef mediated downregulation of CD28 cell surface expression 1102
11.2.6.2.1.1 Formation of Nef CD28 cytoplasmic tail complex 1102
11.2.6.2.1.2 Formation of Nef:Cd28:Clathrin-coated Pit Adapter Protein complex 1103
11.2.6.2.1.3 Internalization of Nef:CD28:Clathrin-Coated Pit Adapter Protein Complex 1104
11.2.6.2.2 Nef mediated downregulation of MHC class I complex cell surface expression 1104
11.2.6.2.2.1 Formation of MHC I:Nef Complex 1105
11.2.6.2.2.2 Formation of MHC I:Nef:AP-1:PACS-1 Complex 1106
11.2.6.2.2.3 Transport of MHC I:Nef:AP-1:PACS-1 Complex 1106
11.2.6.2.2.4 Degradation of MHC I Complex 1107
11.2.6.2.3 Nef Mediated CD4 Down-regulation 1108
11.2.6.2.3.1 Nef mediated disruption of CD4:Lck Complex 1109
11.2.6.2.3.2 Formation of CD4:Nef:AP-2 Complex:v-ATPase Complex 1109
11.2.6.2.3.3 Internalization of the CD4:Nef:AP-2 Complex:v-ATPase Complex 1110
11.2.6.2.3.4 Formation of a Nef:ARF1:CD4 complex 1111

11.2.6.2.3.5 Degradation of CD4 1111
11.2.6.2.4 Nef Mediated CD8 Down-regulation 1112
11.2.6.2.4.1 Formation of CD8:Nef:AP-2 Complex:v-ATPase Complex 1113
11.2.6.2.4.2 Internalization of the CD8:Nef:AP-2 Complex:v-ATPase Complex 1114
11.2.6.2.4.3 Degradation of CD8 1114
11.2.7 Vpu mediated degradation of CD4 1115
11.2.7.1 Association of Vpu with CD4 1116
11.2.7.2 Vpu:CD4 associates with beta-TrCP 1117
11.2.7.3 The Vpu:CD4:beta-TrCP complex recruits SKP1 1118
11.2.7.4 Ubiquitination of CD4 by Vpu:CD4:beta-TrCP:SKP1 complex 1119
11.2.7.5 Degradation of ubiquitinated CD4 1120
12 Hemostasis 1121
12.1 Formation of Platelet plug 1121
12.1.1 Platelet Adhesion to exposed collagen 1122
12.1.1.1 Collagen adhesion via alpha 2 beta 1 glycoprotein 1123
12.1.1.1.1 Adhesion via alpha 2 beta 1 glycoprotein 1124
12.1.1.2 Collagen adhesion via Gp IV 1125
12.1.1.2.1 Platelet glycoprotein IV [plasma membrane] GP IV : Collagen IV complex formation 1125
12.1.1.3 vWF interaction with collagen 1125
12.1.1.3.1 vWF binds to collagen 1126
12.1.1.3.2 GP Ib-IX-V binds to [vWF:Collagen] complex 1126
12.1.2 Platelet Activation 1127
12.1.2.1 Platelet activation triggers 1128
12.1.2.1.1 Collagen-mediated activation cascade 1128
12.1.2.1.1.1 Binding of GP VI:Fc Epsilon R1 gamma receptor complex with collagen 1128
12.1.2.1.1.2 Src-mediated phoshorylation of FcR1 gamma 1128
12.1.2.1.1.3 Syk-mediated phosphorylation of Phospholipase C gamma 2 1129
12.1.2.1.1.4 Binding of SRC tyrosine kinase 1130
12.1.2.1.1.5 Binding of Syk tyrosine kinase 1130
12.1.2.1.2 Thrombin-activated activation cascade 1131
12.1.2.1.2.1 Thrombin-mediated activation of PARs 1131
12.1.2.1.2.1.1 Thrombin-mediated activation of PAR1 1131
12.1.2.1.2.2 G-protein cascades 1133
12.1.2.1.2.2.1 G alpha 12 cascade 1133
12.1.2.1.2.2.1.1 GTP loading of G alpha 12 1133
12.1.2.1.2.2.1.2 Activation of Rho guanine nucleotide exchange factor 1 (p115-RhoGEF) 1134
12.1.2.1.2.2.1.3 Activation of RAC1 1134
12.1.2.1.2.2.1.4 Activation of PI3K 1135
12.1.2.1.2.2.2 G alpha Q cascade 1136
12.1.2.1.2.2.2.1 GTP loading of G alpha Q 1136
12.1.2.1.2.2.2.2 Activation of PLC beta 1 1136
12.1.2.2 Phospholipase-mediated signalling 1137
12.1.2.2.1 PLC-mediated hydrolysis 1137
12.1.2.2.1.1 PLC beta 1-mediated hydrolysis 1137
12.1.2.2.1.2 PLC gamma 2-mediated hydrolysis 1138
12.1.2.2.2 Effects of IP3 1138
12.1.2.2.2.1 Effects on Ca++ levels 1138
12.1.2.2.2.1.1 Binding of IP3 to IP3 receptor 1138
12.1.2.3 Elevation of cytosolic Ca++ levels 1139
12.1.2.3.1 Entry of Ca++ from platelet dense tubular system 1139
12.1.2.3.2 Entry of Ca++ from plasma 1140
12.1.2.3.3 Response to elevated platelet cytosolic Ca++ 1140
12.1.2.3.3.1 Activation of PKC 1141
12.1.2.3.3.2 Disinhibition of SNARE formation 1142
12.1.2.3.3.2.1 Phosphorylation of Platelet Sec-1 1142
12.1.2.3.3.2.2 Phosphorylation of Syntaxin-4 1143
12.1.2.3.3.3 Platelet degranulation 1143
12.1.2.3.3.3.1 Exocytosis of Dense granule 1143
12.1.2.3.3.3.1.1 Exocytosis of ADP 1144
12.1.2.3.3.3.1.2 Exocytosis of ATP 1144
12.1.2.3.3.3.1.3 Exocytosis of Ca++ 1144
12.1.2.3.3.3.1.4 Exocytosis of GDP 1145
12.1.2.3.3.3.1.5 Exocytosis of GTP 1145
12.1.2.3.3.3.1.6 Exocytosis of Mg++ 1146
12.1.2.3.3.3.1.7 Exocytosis of orthophosphate 1146
12.1.2.3.3.3.1.8 Exocytosis of pyrophosphate 1147

12.1.2.3.3.3.1.9 Exocytosis of serotonin 1147
12.1.2.3.3.3.1.10 Surface deployment of CD63 1147
12.1.2.3.3.3.1.11 Surface deployment of LAMP2 1148
12.1.2.3.3.3.2 Exocytosis of Alpha granule 1148
12.1.2.3.3.3.2.1 Surface deployment of alpha IIb beta 3 integrin 1148
12.1.2.3.3.3.2.2 Exocytosis of albumin 1149
12.1.2.3.3.3.2.3 Exocytosis of beta-thromboglobulin 1150
12.1.2.3.3.3.2.4 Exocytosis of factor V 1150
12.1.2.3.3.3.2.5 Exocytosis of factor VIII 1151
12.1.2.3.3.3.2.6 Exocytosis of fibrinogen 1151
12.1.2.3.3.3.2.7 Exocytosis of fibronectin 1152
12.1.2.3.3.3.2.8 Exocytosis of low-affinity platelet factor IV 1152
12.1.2.3.3.3.2.9 Exocytosis of multimerin 1152
12.1.2.3.3.3.2.10 Exocytosis of neutrophil-activating peptide 2 1153
12.1.2.3.3.3.2.11 Exocytosis of osteonectin 1153
12.1.2.3.3.3.2.12 Surface deployment of P-selectin 1154
12.1.2.3.3.3.2.13 Exocytosis of plasminogen 1154
12.1.2.3.3.3.2.14 Exocytosis of plasminogen activator inhibitor 1155
12.1.2.3.3.3.2.15 Exocytosis of Platelet factor 4 1155
12.1.2.3.3.3.2.16 Exocytosis of TGF beta 1156
12.1.2.3.3.3.2.17 Exocytosis of thrombospondin 1156
12.1.2.3.3.3.2.18 Exocytosis of vWF 1157
12.1.2.3.3.3.2.19 Surface deployment of GpIV 1157
12.1.2.3.3.3.2.20 Surface deployment of CD9 1157
12.1.2.3.3.3.2.21 Surface deployment of GP Ib-IX-V complex 1158
12.1.2.3.3.3.2.22 Surface deployment of PECAM-1 1159
12.1.2.3.3.3.2.23 Exocytosis of protease nexin II APP 1159
12.1.2.3.3.3.2.24 Exocytosis of histidine rich glycoprotein 1160
12.1.2.3.3.3.2.25 Exocytosis of alpha 2 macroglobulin 1160
12.1.2.3.3.3.2.26 Exocytosis of alpha 1 antitrypsin 1161
12.1.2.3.3.3.2.27 Exocytosis of complement factor D 1161
12.1.2.3.3.3.2.28 Exocytosis of kininogen 1162
12.1.2.3.3.3.2.29 Exocytosis of hepatocyte growth factor 1162
12.1.2.3.3.3.2.30 Exocytosis of C1 inhibitor 1162
12.1.2.3.3.3.2.31 Exocytosis of PDGF 1163
12.1.2.3.3.3.2.32 Exocytosis of EGF 1163
12.1.2.3.3.3.2.33 Exocytosis of Insulin-like growth factor 1164
12.1.2.3.3.3.2.34 Exocytosis of VEGF 1164
12.1.2.3.3.3.2.35 Exocytosis of alpha 2 antiplasmin 1165
12.1.2.3.3.3.2.36 Exocytosis of Alpha Actinins 1166
12.1.2.3.3.3.2.37 Exocytosis of SRGN 1166
12.1.2.3.3.3.2.38 Exocytosis of Clusterin 1167
12.1.2.3.3.3.2.39 Exocytosis of Coagulation factor XIIIA 1168
12.1.2.3.3.3.2.40 Exocytosis of Metalloproteinase inhibitor 1 1169
12.1.2.3.3.3.2.41 Exocytosis of Protein S 1170
12.1.2.3.3.3.2.42 Exocytosis of Thymosin beta-4 1170
12.1.2.3.3.3.2.43 Exocytosis of Fructose-bisphosphate aldolase 1171
12.1.3 Plug Formation 1172
12.1.3.1 Adhesion of alpha IIb beta 3 integrin to fibrin network 1173
12.2 Formation of Fibrin Clot (Clotting Cascade) 1173
12.2.1 Extrinsic Pathway 1174
12.2.1.1 sequestered tissue factor -> tissue factor 1175
12.2.1.2 tissue factor (TF) + factor VII (F7) -> TF:F7 complex 1175
12.2.1.3 factor X -> factor Xa + factor X activation peptide (TF:F7 catalyst) 1176
12.2.1.4 factor VII -> factor VIIa 1177
12.2.1.5 tissue factor (TF) + activated factor VII (F7a) -> TF:F7a complex 1177
12.2.1.6 factor X -> factor Xa + factor X activation peptide (TF:F7a catalyst) 1178
12.2.1.7 factor IX -> factor IXa + factor IX activation peptide (TF:F7a catalyst) 1179
12.2.1.8 TFPI + TF:F7a + factor Xa -> TFPI:TF:F7a:factor Xa 1180
12.2.2 Intrinsic Pathway 1180
12.2.2.1 kininogen + C1q binding protein tetramer -> kininogen:C1q binding protein tetramer 1182
12.2.2.2 prekallikrein + kininogen:C1q binding protein tetramer -> prekallikrein:kininogen:C1q binding protein tetramer 1183
12.2.2.3 prekallikrein:kininogen:C1q binding protein tetramer -> kallikrein:kininogen:C1q binding protein tetramer 1183
12.2.2.4 kallikrein:kininogen:C1q binding protein tetramer -> kallikrein + activated kininogen:C1q binding protein tetramer + bradykinin 1184
12.2.2.5 factor XII -> factor XIIa 1185
12.2.2.6 factor XI + platelet glycoprotein (GP) Ib:IX:V complex -> factor XI:platelet glycoprotein (GP) Ib:IX:V complex 1186
12.2.2.7 factor XI:platelet glycoprotein (GP) Ib:IX:V complex -> factor XIa:platelet glycoprotein (GP) Ib:IX:V complex (XIIa catalyst) 1187
12.2.2.8 factor XI:platelet glycoprotein (GP) Ib:IX:V complex -> factor XIa:platelet glycoprotein (GP) Ib:IX:V complex (thrombin catalyst)1189
12.2.2.9 factor IX -> factor IXa + factor IX activation peptide (factor XIa catalyst) 1190
12.2.2.10 factor VIII + von Willebrand factor multimer -> factor VIII:von Willibrand factor multimer 1190
12.2.2.11 factor VIII:von Willibrand factor multimer -> factor VIIIa + factor VIIIa B A3 acidic polypeptide + von Willibrand factor multimer1191
12.2.2.12 factor VIIIa + factor IXa -> factor VIIIa:factor IXa 1192
12.2.2.13 factor X -> factor Xa + factor X activation peptide (VIIIa:IXa catalyst) 1193
12.2.2.14 kallikrein + C1Inh -> kallikrein:C1Inh 1194
12.2.2.15 kallikrein + alpha2-macroglobulin -> kallikrein:alpha2-macrogloulin 1195
12.2.2.16 factor XIIa + C1Inh -> factor XIIa:C1Inh 1195
12.2.3 Common Pathway 1196
12.2.3.1 prothrombin -> activated thrombin (factor IIa) + thrombin activation peptide (Xa catalyst) 1197
12.2.3.2 factor V -> factor Va + factor V activation peptide 1198
12.2.3.3 factor Va + factor Xa -> Va:Xa complex (prothrombinase) 1199
12.2.3.4 prothrombin -> activated thrombin (factor IIa) + thrombin activation peptide (prothrombinase catalyst) 1199
12.2.3.5 fibrinogen -> fibrin monomer + 2 fibrinopeptide A + 2 fibrinopeptide B 1200
12.2.3.6 n fibrin monomers -> fibrin multimer 1201
12.2.3.7 factor XIII -> factor XIII cleaved tetramer + 2 factor XIII A activation peptides 1202
12.2.3.8 factor XIII cleaved tetramer + 2 Ca++ -> factor XIIIa + 2 factor XIII B chain 1202
12.2.3.9 fibrin multimer -> fibrin multimer, crosslinked + NH4+ 1203
12.2.3.10 antithrombin III + heparin -> antithrombin III:heparin 1204
12.2.3.11 activated thrombin (factor IIa) + antithrombin III:heparin -> thrombin:antithrombin III:heparin 1204
12.2.3.12 thrombin:antithrombin III:heparin -> thrombin:cleaved antithrombin III:heparin 1205
12.2.3.13 thrombin:cleaved antithrombin III:heparin -> thrombin:cleaved antithrombin III + heparin 1205
12.2.3.14 activated thrombin (factor IIa) + thrombomodulin -> activated thrombin:thrombomodulin 1206
12.2.3.15 protein C -> activated protein C + protein C heavy chain activation peptide 1207
12.2.3.16 factor Va -> factor Vi 1207
12.3 Dissolution of Fibrin Clot 1208
12.3.1 histidine-rich glycoprotein + plasminogen <-> histidine-rich glycoprotein:plasminogen 1210
12.3.2 histidine-rich glycoprotein:plasminogen <-> histidine-rich glycoprotein + plasminogen 1210
12.3.3 crosslinked fibrin multimer + tissue plasminogen activator (one-chain) -> crosslinked fibrin multimer:tissue plasminogen activator 1211
(one-chain)
12.3.4 crosslinked fibrin multimer:tissue plasminogen activator (one-chain) + plasminogen -> crosslinked fibrin multimer:tissue 1212
plasminogen activator (one-chain):plasminogen
12.3.5 crosslinked fibrin multimer:tissue plasminogen activator (one-chain):plasminogen -> crosslinked fibrin multimer:tissue plasminogen 1213
activator (one-chain) + plasmin
12.3.6 fibrin multimer, crosslinked -> fibrin digestion products (plasmin) 1214
12.3.7 crosslinked fibrin multimer:tissue plasminogen activator (one-chain) -> crosslinked fibrin multimer:tissue plasminogen activator 1215
(two-chain)
12.3.8 crosslinked fibrin multimer:tissue plasminogen activator (two-chain) + plasminogen -> crosslinked fibrin multimer:tissue 1215
plasminogen activator (two-chain):plasminogen
12.3.9 crosslinked fibrin multimer:tissue plasminogen activator (two-chain):plasminogen -> crosslinked fibrin multimer:tissue plasminogen1216
activator (two-chain) + plasmin
12.3.10 fibrin multimer, crosslinked:tissue plasminogen activator (two-chain) + plasminogen activator inhibitor 1 -> fibrin multimer, 1217
crosslinked:tissue plasminogen activator (two-chain):plasminogen activator inhibitor 1
12.3.11 fibrin multimer, crosslinked:tissue plasminogen activator (one-chain) + plasminogen activator inhibitor 1 -> fibrin multimer, 1218
crosslinked:tissue plasminogen activator (one-chain):plasminogen activator inhibitor 1
12.3.12 alpha-2-antiplasmin + plasmin -> alpha-2-antiplasmin:plasmin 1218
12.3.13 urokinase plasminogen activator + urokinase plasminogen activator receptor (uPAR) -> urokinase plasminogen activator:uPAR 1219
12.3.14 plasminogen + histidine-rich glycoprotein -> plasminogen:histidine-rich glycoprotein 1220
12.3.15 plasminogen:histidine-rich glycoprotein -> plasmin + histidine-rich glycoprotein (uPA [one-chain] catalyst) 1221
12.3.16 urokinase plasminogen activator (one-chain):uPAR -> urokinase plasminogen activator (two-chain):uPAR 1222
12.3.17 plasminogen:histidine-rich glycoprotein -> plasmin + histidine-rich glycoprotein (uPA [two-chain] catalyst) 1223
12.3.18 urokinase plasminogen activator (two-chain):uPAR + plasminogen activator inhibitor 1 (PAI-1) -> PAI-1:urokinase plasminogen 1223
activator (two-chain):uPAR
12.3.19 urokinase plasminogen activator (two-chain):uPAR + plasminogen activator inhibitor 2 (PAI-2) -> PAI-2:urokinase plasminogen 1224
activator (two-chain):uPAR
12.4 Cell surface interactions at the vascular wall 1225
12.4.1 Binding of GP VI:Fc Epsilon R1 gamma receptor complex with collagen 1227
12.4.2 MAC1 binds JAM-C 1227
12.4.3 JAM-B binds JAM-C 1228
12.4.4 VLA-4 binds JAM-B 1229
12.4.5 JAM-A homodimerises 1230
12.4.6 LFA1 binds JAM-A 1230
12.4.7 Integrin alphaX beta2 binds JAM-C 1231
12.4.8 MERTK receptor binds ligands (Gas6 or Protein S) 1232
12.4.9 CD84 homodimerises 1233
12.4.10 P-selectin binds P-selectin ligand 1234
12.4.11 JAM-B homodimerises 1235
12.4.12 JAM-C homodimerises 1236

12.4.13 CD177 binds PECAM-1 1236
12.4.14 CD47 binds SIRP 1237
12.4.15 CD48 binds CD244 1238
12.4.16 CD58 binds CD2 1239
12.4.17 Integrin alpha 5 beta 1 binds fibronectin 1240
12.4.18 CXADR binds to AMICA1 1240
12.4.19 protein C -> activated protein C + protein C heavy chain activation peptide 1241
12.4.20 OLR1 binds to oxidized LDL 1242
12.4.21 Platelet-derived TREM-1 ligand binds to TREM-1 1243
12.4.22 Tie2 Signaling 1243
12.4.22.1 Interaction of Tie2 with Ang1 1244
12.4.22.2 Dimerization of Tie2/Ang1 complex 1245
12.4.22.3 Trans-phosphorylation of Tie2 1246
12.4.22.4 Interaction of Tie2 and p85 of PI3K 1247
12.4.22.5 Interaction of Tie2 and Grb2 1248
12.4.22.6 Interaction of SOS-1 to Tie2 bound Grb2 1249
12.4.22.7 Sos-mediated nucleotide exchange of Ras (Tie2 receptor:Grb2:Sos) 1250
12.4.22.8 Interaction of Tie2 and Shc1 1250
12.4.22.9 Interaction of Tie2 and Dok-2 1251
12.4.22.11 Interaction of Tie2 and Ang4 1253
12.4.23 PECAM1 interactions 1254
12.4.23.1 Trans-homophilic interaction of PECAM-1 1254
12.4.23.2 Heterophilic interaction of PECAM-1 and Integrin alpha-v beta-3 1255
12.4.23.3 Phoshorylation of PECAM-1 by Fyn or Lyn or c-Src 1256
12.4.23.4 Interaction of PECAM-1 and SHIP 1257
12.4.23.5 Interaction of PECAM-1 and SHP-2 1258
12.4.23.6 Interaction of PECAM-1 and PLC gamma1 1258
12.4.24 Basigin interactions 1260
12.4.24.1 CyP60 chaperones Basigin 1261
12.4.24.2 Basigin homodimerises 1261
12.4.24.3 Caveolin-1 binds Basigin 1262
12.4.24.4 Basigin binds CyPA 1263
12.5 Further platelet releasate 1264
12.5.1 Release of Serotransferrin 1264
12.5.2 Release of Tubulin alpha-4A chain 1265
12.5.3 Release of Apolipoprotein A-I 1266
12.5.4 Release of Calmodulin 1267
12.5.5 Release of Pleckstrin 1268
12.5.6 Release of Vinculin 1269
12.5.7 Release of WD repeat-containing protein 1 1270
12.5.8 Release of Superoxide dismutase [Cu-Zn] 1271
12.5.9 Release of Bromodomain and PHD finger-containing protein 3 1271
12.5.10 Release of Titin 1272
12.5.11 Release of Kappa-actin 1273
12.5.12 Release of Intracellular hyaluronan-binding protein 4 1274
12.5.13 Release of Filamin-A 1275
12.5.14 Release of Talin-1 1276
12.5.15 Release of Peptidyl-prolyl cis-trans isomerase A 1277
12.5.16 Release of Calumenin 1278
12.5.17 Release of Adenylyl cyclase-associated protein 1 1279
12.5.18 Release of Cofilin-1 1280
12.5.19 Release of Profilin-1 1281
12.5.20 Release of Secretogranin-3 1282
12.5.21 Release of 78 kDa glucose-regulated protein 1283
12.5.22 Exocytosis of Proactivator polypeptide 1285
13 Influenza Infection 1286
13.1 Influenza Life Cycle 1287
13.1.1 Binding of the influenza virion to the host cell 1288
13.1.2 Entry of Influenza Virion into Host Cell via Endocytosis 1290
13.1.2.1 Clathrin-Mediated Pit Formation And Endocytosis Of The Influenza Virion 1291
13.1.3 Fusion and Uncoating of the Influenza Virion 1291
13.1.3.1 Fusion of the Influenza Virion to the Host Cell Endosome 1293
13.1.3.1.1 Conformation change in hemagglutinin freeing the fusion peptide of HA2 1293
13.1.3.1.2 Fusion of the influenza virion HA2 protein transmembrane domain to the host cell endosome membrane 1294
13.1.3.1.3 Concerted hemagglutinin pore formation 1294
13.1.3.2 Uncoating of the Influenza Virion 1295

13.1.3.2.1 Virion-associated M2 protein mediated ion infusion 1295
13.1.3.2.2 Ribonucleoprotein release from M1 proteins 1296
13.1.4 Transport of Ribonucleoproteins into the Host Nucleus 1296
13.1.4.1 Recognition of the Nuclear Localization Signal (NLS) by a Karyopherin Alpha Family Protein 1298
13.1.4.2 Recruitment of Karyopherin Beta to form a Trimeric Complex 1299
13.1.4.3 Docking and transport of the RNP:Karyopherin complex through the nuclear pore 1299
13.1.4.4 Release of the RNP into the host cell nucleus 1300
13.1.5 Influenza Viral RNA Transcription and Replication 1301
13.1.5.1 vRNP Assembly 1303
13.1.5.1.1 Viral Polymerase Assembly 1303
13.1.5.1.2 NP binds vRNA 1305
13.1.5.2 Viral Messenger RNA Synthesis 1306
13.1.5.2.1 Assembly of an Active Transcription Complex 1306
13.1.5.2.2 Priming and Initiation of Transcription 1308
13.1.5.2.3 Elongation of viral mRNA 1309
13.1.5.2.4 Polyadenylation and Termination 1310
13.1.5.2.5 Viral mRNA Splicing (M, NS segments) 1311
13.1.5.2.6 Export of Spliced Viral mRNA 1312
13.1.5.2.7 Viral mRNA Export 1313
13.1.5.3 Viral mRNA Translation 1314
13.1.5.3.1 Synthesis of PB1-F2 1315
13.1.5.3.2 Viral Protein Synthesis 1316
13.1.5.4 cRNA Synthesis 1317
13.1.5.4.1 Initiation of cRNA Synthesis 1317
13.1.5.4.2 cRNA Extension 1318
13.1.5.5 vRNA Synthesis 1319
13.1.5.5.1 Initiation of vRNA Synthesis 1320
13.1.5.5.2 vRNA Extension 1321
13.1.6 Export of Viral Ribonucleoproteins from Nucleus 1322
13.1.6.1 Viral RNP Complexes in the Host Cell Nucleus 1323
13.1.6.1.1 Newly synthesized vRNP for export 1324
13.1.6.1.2 Binding of M1 to vRNP 1325
13.1.6.1.3 Binding of NEP/NS2 to vRNP:M1 1326
13.1.6.2 NEP/NS2 Interacts with the Cellular Export Machinery 1327
13.1.6.2.1 Binding of vRNP:M1:NEP complex to CRM1 export receptor 1327
13.1.6.2.2 vRNP Export through the nuclear pore 1328
13.1.7 Virus Assembly and Release 1329
13.1.7.1 Assembly of Viral Components at the Budding Site 1331
13.1.7.1.1 Entry of M2 into the endoplasmic reticulum 1331
13.1.7.1.2 Assembly of M2 tetramers 1332
13.1.7.1.3 Entry of NA into the endoplasmic reticulum 1333
13.1.7.1.4 Glycosylation of NA 1333
13.1.7.1.5 Assembly of NA tetramers 1334
13.1.7.1.6 Entry of HA into the endoplasmic reticulum 1335
13.1.7.1.7 Glycosylation and Folding of HA 1336
13.1.7.1.8 Trimerization of HA 1337
13.1.7.1.9 Transport of HA trimer, NA tetramer and M2 tetramer from the endoplasmic reticulum to the Golgi Apparatus 1338
13.1.7.1.9.1 Budding of vesicle with the HA trimer, NA tetramer and M2 tetramer from the endoplasmic reticulum into the cytosol 1338
13.1.7.1.9.2 Fusion of vesicle containing the HA trimer, NA tetramer and M2 tetramer to the Golgi apparatus 1339
13.1.7.1.10 Palmitoylation of cysteine residues on HA in the cis-Golgi network 1340
13.1.7.1.11 Palmitoylation of cysteine residues on M2 in the cis-golgi network 1341
13.1.7.1.12 Association of HA into rafts 1342
13.1.7.1.13 Association of NP into rafts 1343
13.1.7.1.14 Association of NA into rafts 1343
13.1.7.1.15 Transport of processed viral proteins to the cell membrane 1344
13.1.7.1.16 Accumulation of M1 at the inner leaflet of the lipid bilayer 1345
13.1.7.2 Packaging of Eight RNA Segments 1346
13.1.7.2.1 RNP association 1347
13.1.7.2.2 Association with M1 at cell membrane 1348
13.1.7.3 Budding 1349
13.1.7.3.1 Membrane fusion 1350
13.1.7.4 Release 1351
13.1.7.4.1 Neuraminidase enzymatic release from sialic acid 1351
13.2 Host Interactions with Influenza Factors 1353
13.2.1 NS1 Mediated Effects on Host Pathways 1353
13.2.1.1 Inhibition of Host mRNA Processing and RNA Silencing 1353
13.2.1.1.1 Binding of NS1 to cleavage and host polyadenylation specificity factor (CPSF) 1354
13.2.1.1.2 Binding of NS1 to poly(A)-binding protein II (PABII) 1355

13.2.1.2 Inhibition of Interferon Synthesis 1355
13.2.1.2.1 Inhibition of IFN-beta 1355
13.2.1.2.1.1 Binding of NS1 to dsRNA 1356
13.2.1.3 Inhibition of PKR 1356
13.2.1.3.1 Binding of NS1 to dsRNA 1356
13.2.1.3.2 Binding of NS1 to PKR 1357
13.2.2 Influenza Virus Induced Apoptosis 1357
13.2.2.1 NA activation of TGF-beta 1358
13.2.2.2 PB1-F2 binds to the mitochondrial adenine nucleotide translocator 3 ANT3, inducing apoptosis 1358
14 Integration of energy metabolism 1360
14.1 Glucagon signaling in metabolic regulation 1362
14.1.1 G(s)-alpha mediated events in glucagon signalling 1363
14.1.1.1 Glucagon binds to Glucagon receptor 1364
14.1.1.2 Glucagon:GCGR mediates GTP-GDP exchange 1365
14.1.1.3 G(s)-alpha:GTP binds to Adenylate cyclase 1365
14.1.2 PKA activation in glucagon signalling 1366
14.1.2.1 Activated Adenylate cyclase catalyses cAMP synthesis 1366
14.1.2.2 cAMP induces dissociation of inactive PKA tetramers 1366
14.2 PKA-mediated phosphorylation of key metabolic factors 1367
14.2.1 PKA catalytic subunit translocates to the nucleus 1368
14.2.2 Phosphorylation of ChREBP at Thr(666) by PKA 1368
14.2.3 PhosphoChREBP (Thr 666) is exported to cytosol 1369
14.2.4 Phosphorylation of pChREBP (Thr 666) at Ser(196) by PKA 1369
14.2.5 Nuclear transport of pChREBP (Thr 666) protein 1370
14.2.6 Phosphorylation of PF2K-Pase by PKA catalytic subunit 1371
14.2.7 D-fructose 2,6-bisphosphate + H2O => D-fructose 6-phosphate + orthophosphate [liver + muscle] 1372
14.3 Insulin effects increased synthesis of Xylulose-5-Phosphate 1372
14.3.1 D-fructose 6-phosphate + D-erythrose 4-phosphate <=> sedoheptulose 7-phosphate + D-glyceraldehyde 3-phosphate 1373
14.3.2 D-glyceraldehyde 3-phosphate + D-fructose 6-phosphate <=> xylulose 5-phosphate + D-erythrose 4-phosphate 1373
14.3.3 D-glyceraldehyde 3-phosphate + sedoheptulose 7-phosphate<=> xylulose 5-phosphate+ribose 5-phosphate 1374
14.4 Activation of PP2A by Xylulose-5-phosphate 1375
14.5 AMPK inhibits chREBP transcriptional activation activity 1376
14.5.1 AMP binds to gamma subunit of AMP kinase heterotrimer 1377
14.5.2 LKB1 phosphorylates the alpha subunit of AMPK heterotrimer 1378
14.5.3 Phosphorylation of ChREBP at Serine 568 by AMPK 1379
14.6 PP2A-mediated dephosphorylation of key metabolic factors 1380
14.6.1 Dephosphorylation of pChREBP (Ser 196) by PP2A 1380
14.6.2 Dephosphorylation of pChREBP (Ser 568) by PP2A 1381
14.6.3 Dephosphorylation of pChREBP (Thr 666) by PP2A 1382
14.6.4 ATP + D-fructose 6-phosphate => ADP + D-fructose 2,6-bisphosphate [liver + muscle] 1382
14.6.5 Dephosphorylation of PF2K-Pase by PP2A complex 1383
14.7 ChREBP activates metabolic gene expression 1384
14.7.1 Formation of ChREBP:MLX heterodimer 1385
14.7.2 Transcriptional activation of glucose metabolism genes by ChREBP:MLX 1385
14.7.2.1 Transcriptional activation of pyruvate kinase gene by ChREBP:MLX 1386
14.7.3 Transcriptional activation of lipogenesis genes by ChREBP:MLX 1386
14.7.3.1 Transcriptional activation of Citrate lyase monomer gene by ChREBP:MLX 1387
14.7.3.2 Transcriptional activation of FAS monomer gene by ChREBP:MLX 1387
14.7.3.3 Transcriptional activation of Acetyl-CoA carboxylase by ChREBP:MLX 1387
14.7.3.4 Transcriptional activation of GP-acyl transferase gene by ChREBP:MLX 1388
14.8 Activated AMPK stimulates fatty-acid oxidation in muscle 1388
14.8.1 Formation of Malonyl-CoA from Acetyl-CoA (muscle) 1390
14.8.2 Activation of cytosolic AMPK by phosphorylation 1391
14.8.3 pAMPK inactivates ACC2 inhibiting malonyl-CoA synthesis 1392
14.8.4 Conversion of palmitic acid to palmitoyl-CoA 1392
14.8.5 Import of palmitoyl-CoA into the mitochondrial matrix 1393
14.8.5.1 CPT1 converts palmitoyl-CoA to palmitoyl carnitine 1394
14.8.5.2 Transport of palmitoyl carnitine into mitochondria 1395
14.8.5.3 CPT2 converts palmitoyl carnitine to palmitoyl-CoA 1396
14.9 Regulation of pyruvate dehydrogenase complex (PDC) 1397
14.9.1 Inactivation of PDC by phosphorylation of PDC E1 alpha component 1398
14.9.2 Activation of PDC by dephosphorylation of phospho-E1 alpha component 1399
15 Lipid and lipoprotein metabolism 1400
15.1 Digestion of dietary lipid 1400
15.1.1 Digestion of cholesterol esters by extracellular CEL (bile salt-dependent lipase) 1401
15.1.2 Digestion of monoacylglycerols by extracellular CEL (bile salt-dependent lipase) 1402
15.1.3 Digestion of diacylglycerols by extracellular PTL:colipase 1403
15.1.4 Digestion of triacylglycerols by extracellular CEL (bile salt-dependent lipase) 1403

15.1.5 Digestion of triacylglycerols by extracellular PTL:colipase 1404
15.1.6 Digestion of triacylglycerols by extracellular pancreatic lipase-related protein 2 1405
15.2 Trafficking of dietary sterols 1406
15.2.1 NPC1L1-mediated cholesterol uptake 1407
15.2.2 NPC1L1-mediated phytosterol uptake 1408
15.2.3 NPC1L1 inactivation by ezetimibe 1409
15.2.4 ABCG5:ABCG8-mediated export of cholesterol and phytosterols 1410
15.3 Triacylglyceride Biosynthesis 1411
15.3.1 Fatty Acyl-CoA Biosynthesis 1412
15.3.1.1 Palmitate synthesis 1412
15.3.1.1.1 Butyryl-ACP biosynthesis 1414
15.3.1.1.1.1 Generation of Cytoplasmic Acetyl CoA from Citrate 1415
15.3.1.1.1.2 Formation of Acetyl-FAS on one FAS monomer 1416
15.3.1.1.1.2.1 Formation of Acetyl-ACP(intermediate) 1416
15.3.1.1.1.2.2 Acetyl-ACP(intermediate) => Acetyl-synthase 1417
15.3.1.1.1.3 Formation of Malonyl-ACP on the other FAS monomer 1418
15.3.1.1.1.3.1 Formation of Malonyl-CoA from Acetyl-CoA (liver) 1418
15.3.1.1.1.3.2 Conversion of Malonyl-CoA to Malonyl-ACP 1418
15.3.1.1.1.4 Condensation of Malonyl-ACP and Acetate on different monomers of FAS dimer to Acetoacetyl-ACP 1419
15.3.1.1.1.5 Reduction of Acetoacetyl-ACP to beta-Hydroxybutyryl-ACP 1420
15.3.1.1.1.6 Dehydration of beta-Hydroxybutyryl-ACP to Crotonoyl-ACP 1420
15.3.1.1.1.7 Reduction of Crotonoyl-ACP to Butyrl-ACP 1421
15.3.1.1.2 Conversion of Butyryl-ACP to Palmitoyl-ACP 1422
15.3.1.1.3 Hydrolysis of Palmitoyl-ACP to Palmitic Acid 1423
15.3.1.1.4 Formation of fatty acid synthase (FAS) dimer 1424
15.3.1.2 Conversion of Palmitic Acid to Long Chain Fatty Acids 1424
15.3.1.3 Conversion of Long Chain Fatty Acids to Fatty Acyl-CoAs 1425
15.3.2 Conversion of Fatty Acyl-CoA to Phosphatidic Acid 1425
15.3.2.1 Synthesis of lysophosphatidic acid from glycerol-3-phosphate 1425
15.3.2.1.1 Formation of Cytosolic Glycerol-3-phosphate 1426
15.3.2.1.1.1 Conversion of Dihydroxyacetone Phosphate to Glycerol -3- phosphate 1426
15.3.2.1.1.2 Conversion of Glycerol to Glycerol-3-phosphate 1426
15.3.2.1.2 Conversion of glycerol-3-phosphate to lysophosphatidic acid 1427
15.3.2.2 Synthesis of phosphatidic acid from lysophosphatidic acid 1428
15.3.2.2.1 lysophosphatic acid + fatty acyl CoA => phosphatidic acid + CoA (1) 1428
15.3.2.2.2 lysophosphatidic acid + fatty acyl CoA => phosphatidic acid + CoA (2) 1429
15.3.3 Conversion of Phosphatidic Acid to Diacylglycerol 1432
15.3.3.1 Phosphatidic acid + H2O <=> Diacylglycerol + phosphate (1) 1432
15.3.4 Conversion of Diacylglycerol to Triacylglycerol 1434
15.4 Hormone-sensitive lipase (HSL)-mediated triacylglycerol hydrolysis 1435
15.4.1 hormone-sensitive lipase (HSL) + 2 ATP -> phosphorylated HSL + 2 ADP 1437
15.4.2 2 phosphorylated HSL monomers -> phosphorylated HSL dimer 1438
15.4.3 perilipin:CGI-58 complex -> perilipin + CGI-58 1439
15.4.4 perilipin + 2 ATP -> phosphorylated perilipin + 2 ADP 1440
15.4.5 Phosphorylated HSL dimer translocates from the cytosol to the lipid particle 1442
15.4.6 phosphorylated HSL dimer + FABP4 -> phosphorylated HSL dimer:FABP4 complex 1443
15.4.7 cholesterol ester + H2O -> cholesterol + fatty acid 1444
15.4.8 triacylglycerol + H2O -> diacylglycerol + fatty acid 1445
15.4.9 diacylglycerol + H2O -> 2-acylglycerol + fatty acid 1446
15.4.10 2-acylglycerol + H2O -> glycerol + fatty acid 1447
15.4.11 phosphorylated HSL + H2O -> HSL + orthophosphate 1448
15.4.12 phosphorylated perilipin + H2O -> perilipin + orthophosphate 1449
15.5 Import of palmitoyl-CoA into the mitochondrial matrix 1450
15.5.1 CPT1 converts palmitoyl-CoA to palmitoyl carnitine 1451
15.5.2 Transport of palmitoyl carnitine into mitochondria 1452
15.5.3 CPT2 converts palmitoyl carnitine to palmitoyl-CoA 1453
15.6 Mitochondrial Fatty Acid Beta-Oxidation 1454
15.6.1 mitochondrial fatty acid beta-oxidation of saturated fatty acids 1455
15.6.1.1 Beta oxidation of palmitoyl-CoA to myristoyl-CoA 1456
15.6.1.1.1 palmitoyl-CoA+FAD<=>trans-Hexadec-2-enoyl-CoA+FADH2 1457
15.6.1.1.2 trans-Hexadec-2-enoyl-CoA+H2O<=>(S)-3-Hydroxyhexadecanoyl-CoA 1457
15.6.1.1.3 (S)-3-Hydroxyhexadecanoyl-CoA+NAD<=>3-Oxopalmitoyl-CoA+NADH+H 1458
15.6.1.1.4 3-Oxopalmitoyl-CoA+CoA-SH<=>myristoyl-CoA 1459

15.6.1.2 Beta oxidation of myristoyl-CoA to lauroyl-CoA 1459
15.6.1.2.1 myristoyl-CoA+FAD<=>trans-Tetradec-2-enoyl-CoA+FADH2 1460
15.6.1.2.2 trans-Tetradec-2-enoyl-CoA+H2O<=>(S)-3-Hydroxytetradecanoyl-CoA 1460
15.6.1.2.3 (S)-3-Hydroxytetradecanoyl-CoA+NAD<=>3-Oxotetradecanoyl-CoA+NADH+H 1461
15.6.1.2.4 3-Oxotetradecanoyl-CoA+CoA-SH<=>Lauroyl-CoA 1462
15.6.1.3 Beta oxidation of lauroyl-CoA to decanoyl-CoA-CoA 1462
15.6.1.3.1 lauroyl-CoA+FAD<=>2-trans-Dodecenoyl-CoA+FADH2 1463
15.6.1.3.2 2-trans-Dodecenoyl-CoA+H2O<=>(S)-3-Hydroxydodecanoyl-CoA 1463
15.6.1.3.3 (S)-3-Hydroxydodecanoyl-CoA+NAD<=>3-Oxododecanoyl-CoA+NADH+H 1464
15.6.1.3.4 3-Oxododecanoyl-CoA+CoA-SH<=>Decanoyl-CoA 1465
15.6.1.4 Beta oxidation of decanoyl-CoA to octanoyl-CoA-CoA 1465
15.6.1.4.1 Decanoyl-CoA+FAD<=>trans-Dec-2-enoyl-CoA+FADH2 1466
15.6.1.4.2 trans-Dec-2-enoyl-CoA+H2O<=>(S)-Hydroxydecanoyl-CoA 1466
15.6.1.4.3 (S)-Hydroxydecanoyl-CoA+NAD<=>3-Oxodecanoyl-CoA+NADH+H 1467
15.6.1.4.4 3-Oxodecanoyl-CoA+CoA-SH<=>Octanoyl-CoA 1468
15.6.1.5 Beta oxidation of octanoyl-CoA to hexanoyl-CoA 1468
15.6.1.5.1 Octanoyl-CoA+FAD<=>trans-Oct-2-enoyl-CoA+FADH2 1469
15.6.1.5.2 trans-Oct-2-enoyl-CoA+H2O<=>(S)-Hydroxyoctanoyl-CoA 1469
15.6.1.5.3 (S)-Hydroxyoctanoyl-CoA+NAD<=>3-Oxooctanoyl-CoA+NADH+H 1470
15.6.1.5.4 3-Oxooctanoyl-CoA+CoA-SH<=>Hexanoyl-CoA 1471
15.6.1.6 Beta oxidation of hexanoyl-CoA to butanoyl-CoA 1471
15.6.1.6.1 Hexanoyl-CoA+FAD<=>trans-Hex-2-enoyl-CoA+FADH2 1472
15.6.1.6.2 trans-Hex-2-enoyl-CoA+H2O<=>(S)-Hydroxyhexanoyl-CoA 1472
15.6.1.6.3 (S)-Hydroxyhexanoyl-CoA+NAD<=>3-Oxohexanoyl-CoA+NADH+H 1473
15.6.1.6.4 3-Oxohexanoyl-CoA+CoA-SH<=>Butanoyl-CoA 1474
15.6.1.7 Beta oxidation of butanoyl-CoA to acetyl-CoA 1474
15.6.1.7.1 Butanoyl-CoA+FAD<=>Crotonoyl-CoA+FADH2 1475
15.6.1.7.2 Crotonoyl-CoA+H2O<=>(S)-3-Hydroxybutanoyl-CoA 1475
15.6.1.7.3 (S)-Hydroxybutanoyl-CoA+NAD<=>Acetoacetyl-CoA+NADH+H 1476
15.6.2 mitochondrial fatty acid beta-oxidation of unsaturated fatty acids 1477
15.6.2.1 Removal of six carbons from Linoleoyl-CoA to form cis,cis-3,6- Dodecadienoyl-CoA 1478
15.6.2.2 Isomerization of cis,cis-3,6-Dodecadienoyl-CoA to form trans,cis-Lauro-2,6-dienoyl-CoA 1480
15.6.2.3 Removal of 2 Carbon atoms from trans,cis-Lauro-2,6-dienoyl-CoA to form 4-cis-decenoyl-CoA 1482
15.6.2.4 dehydrogenation of 4-cis-decenoyl-CoA to form 2-trans-4-cis-decadienoyl-CoA 1483
15.6.2.5 Reduction of 2-trans-4-cis-decadienoyl-CoA to form 3-trans-decenoyl-CoA 1485
15.6.2.6 Isomerization of 3-trans-decenoyl-CoA to form trans-dec-2-enoyl-CoA 1486
15.7 Ketone body metabolism 1488
15.7.1 Synthesis of Ketone Bodies 1488
15.7.1.1 Formation of Acetoacetic Acid 1488
15.7.1.1.1 2 acetyl-CoA <=> acetoacetyl-CoA+CoA 1489
15.7.1.1.2 acetoacetyl-CoA+acetyl-CoA => HMG-CoA 1489
15.7.1.1.3 HMG CoA => acetoacetic acid+ acetyl CoA 1490
15.7.1.2 Reduction of Acetoacetate to beta-Hydroxybutyrate 1491
15.7.2 Utilization of Ketone Bodies 1491
15.7.2.1 D-beta hydroxybutyrate+NAD+ <=> acetoacetate+NADH+H+ 1492
15.7.2.2 acetoacetate+succinyl-CoA <=> acetoacetyl-CoA+succinate 1492
15.7.2.3 acetoacetyl-CoA+CoA <=> 2 acetyl-CoA 1493
15.8 Steroid metabolism 1494
15.8.1 Cholesterol biosynthesis 1494
15.8.1.1 Condensation of acetyl CoA with acetoacetyl CoA to form HMG-CoA 1496
15.8.1.2 Reduction of HMG-CoA produces mevalonate 1496
15.8.1.3 Mevalonate is phosphorylated to mevalonate-5-phosphate 1497
15.8.1.4 Mevalonate-5-phosphate is further phosphorylated. 1498
15.8.1.5 Mevalonate-5-pyrophosphate is decarboxylated 1499
15.8.1.6 Isopentenyl pyrophosphate rearranges to dimethylallyl pyrophosphate 1500
15.8.1.7 Addition of isopentenyl pyrophosphate to DMAPP 1501
15.8.1.8 Another isopentenyl pyrophosphate is added to geranyl pyrophosphate 1501
15.8.1.9 Two FPP molecules dimerize to form presqualene diphosphate 1502
15.8.1.10 Reduction of presqualene diphosphate to form squalene 1503
15.8.1.11 Squalene is oxidized to its epoxide 1504
15.8.1.12 Squalene 2,3-epoxide cyclizes, forming lanosterol 1505
15.8.1.13 Transformation of lanosterol to cholesterol 1505
15.8.1.13.1 Lanosterol is oxidatively demethylated to 4,4-dimethylcholesta-8(9),14,24-trien-3beta-ol 1506
15.8.1.13.2 4,4-dimethylcholesta-8(9),14,24-trien-3beta-ol is reduced to 4,4-dimethylcholesta-8(9),24-dien-3beta-ol [LBR] 1507
15.8.1.13.3 4,4-dimethylcholesta-8(9),14,24-trien-3beta-ol is reduced to 4,4-dimethylcholesta-8(9),24-dien-3beta-ol [TM7SF2] 1508
15.8.1.13.4 4,4-dimethylcholesta-8(9),24-dien-3beta-ol is oxidized to 4-methyl,4-carboxycholesta-8(9),24-dien-3beta-ol 1509
15.8.1.13.5 4-methyl,4-carboxycholesta-8(9),24-dien-3beta-ol is decarboxylated and oxidized to form
4-methylcholesta-8(9),24-dien-3-one
15.8.1.13.6 4-methylcholesta-8(9),24-dien-3-one is reduced to 4-methylcholesta-8(9),24-dien-3beta-ol 1511
15.8.1.13.7 4-methylcholesta-8(9),24-dien-3beta-ol is oxidized to 4-carboxycholesta-8(9),24-dien-3beta-ol 1512
15.8.1.13.8 4-carboxycholesta-8(9),24-dien-3beta-ol is decarboxylated and oxidized to form cholesta-8(9),24-dien-3-one 1513
(zymosterone)
15.8.1.13.9 Zymosterone (cholesta-8(9),24-dien-3-one) is reduced to zymosterol (cholesta-8(9),24-dien-3beta-ol) 1514
15.8.1.13.10 Zymosterol is isomerized to cholesta-7,24-dien-3beta-ol 1515
15.8.1.13.11 Cholesta-7,24-dien-3beta-ol is desaturated to form cholesta-5,7,24-trien-3beta-ol 1516
15.8.1.13.12 Cholesta-5,7,24-trien-3beta-ol is reduced to desmosterol 1517
15.8.1.13.13 Reduction of desmosterol to cholesterol 1518
15.8.2 Metabolism of bile acids and bile salts 1519
15.8.2.1 Synthesis of bile acids and bile salts 1520
15.8.2.1.1 Synthesis of bile acids and bile salts via 7alpha-hydroxycholesterol 1520
15.8.2.1.1.1 Cholesterol is hydroxylated to 7alpha-hydroxycholesterol by CYP7A1 1522
15.8.2.1.1.2 7alpha-hydroxycholesterol is oxidized and isomerized to 4-cholesten-7alpha-ol-3-one 1523
15.8.2.1.1.3 4-Cholesten-7alpha-ol-3-one is 12alpha-hydroxylated to 7-alpha,12-alpha-dihydroxycholest-4-en-3-one 1524
15.8.2.1.1.4 4-cholesten-7alpha, 12alpha-diol-3-one is reduced to 5beta-cholesten-7alpha, 12alpha-diol-3-one 1525
15.8.2.1.1.5 4-cholesten-7alpha-ol-3-one is reduced to 5beta-cholestan-7alpha-ol-3-one 1526
15.8.2.1.1.6 5Beta-cholesten-7alpha, 12alpha-diol-3-one is reduced to 5beta-cholestan-3alpha, 7alpha, 12alpha-triol 1527
15.8.2.1.1.7 5beta-cholestan-7alpha-ol-3-one is reduced to 5beta-cholestan-3alpha, 7alpha-diol 1528
15.8.2.1.1.8 5beta-cholestan-3alpha, 7alpha, 12alpha-triol is translocated from the cytosol to the mitochondrial matrix 1529
15.8.2.1.1.9 5beta-cholestan-3alpha, 7alpha-diol is translocated from the cytosol to the mitochondrial matrix 1530
15.8.2.1.1.10 5beta-cholestan-3alpha, 7alpha, 12alpha-triol is hydroxylated to 5beta-cholestan-3alpha, 7alpha, 12alpha, 27-tetrol 1530
15.8.2.1.1.11 5beta-cholestan-3alpha, 7alpha-diol is hydroxylated to 5beta-cholestan-3alpha, 7alpha, 26-triol 1531
15.8.2.1.1.12 5beta-cholestan-3alpha,7alpha,12alpha,27-tetrol is oxidized to 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestan-27-al
1532
15.8.2.1.1.13 5beta-cholestan-3alpha, 7alpha, 26-triol is oxidized to 3alpha, 7alpha-dihydroxy-5beta-cholestan-26-al 1533
15.8.2.1.1.14 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestan-27-al is oxidized to 1534
3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoate (THCA)
15.8.2.1.1.15 3alpha, 7alpha-dihydroxy-5beta-cholestan-26-al is oxidized to 3alpha, 7alpha-dihydroxy-5beta-cholestanoate (DHCA)1535
15.8.2.1.1.16 THCA is translocated from the mitochondrial matrix to the cytosol 1536
15.8.2.1.1.17 DHCA is translocated from the mitochondrial matrix to the cytosol 1537
15.8.2.1.1.18 THCA is conjugated with Coenzyme A (SLC27A2 VLCS) 1538
15.8.2.1.1.19 DHCA is conjugated with Coenzyme A (SLC27A2 VLCS) 1539
15.8.2.1.1.20 THCA is conjugated with Coenzyme A (SLC27A5 BACS) 1540
15.8.2.1.1.21 DHCA is conjugated with Coenzyme A (SLC27A5 BACS) 1541
15.8.2.1.1.22 25(R) THCA-CoA is translocated from the cytosol to the peroxisome 1542
15.8.2.1.1.23 25(R) DHCA-CoA is translocated from the cytosol to the peroxisome 1543
15.8.2.1.1.24 Isomerization of 25(R) THCA-CoA to 25(S) THCA-CoA 1543
15.8.2.1.1.25 Isomerization of 25(R) DHCA-CoA to 25(S) DHCA-CoA 1544
15.8.2.1.1.26 25(S) THCA-CoA is dehydrogenated to 25(S) 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-24-enoyl-CoA 1545
15.8.2.1.1.27 25(S) DHCA-CoA is dehydrogenated to 25(S) 3alpha,7alpha-dihydroxy-5beta-cholest-24-enoyl-CoA 1546
15.8.2.1.1.28 25(S) 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-24-enoyl-CoA is hydrated to (24R, 25R) 1547
3alpha,7alpha,12alpha,24-tetrahydroxy-5beta-cholestanoyl-CoA
15.8.2.1.1.29 25(S) 3alpha,7alpha-dihydroxy-5beta-cholest-24-enoyl-CoA is hydrated to (24R, 25R) 1548
3alpha,7alpha,24-trihydroxy-5beta-cholestanoyl-CoA
15.8.2.1.1.30 (24R, 25R) 3alpha,7alpha,12alpha,24-tetrahydroxy-5beta-cholestanoyl-CoA is oxidized to 1549
3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-24-one-CoA
15.8.2.1.1.31 (24R, 25R) 3alpha,7alpha,24-trihydroxy-5beta-cholestanoyl-CoA is oxidized to 1550
3alpha,7alpha-dihydroxy-5beta-cholest-24-one-CoA
15.8.2.1.1.32 Thiolysis of 3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-one-CoA yields choloyl-CoA 1551
(3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-one-CoA) and propionyl CoA
15.8.2.1.1.33 Thiolysis of 3alpha,7alpha-dihydroxy-5beta-cholan-24-one-CoA yields chenodeoxycholoyl-CoA 1552
(3alpha,7alpha-dihydroxy-5beta-cholan-24-one-CoA) and propionyl CoA
15.8.2.1.1.34 Hydrolysis of choloyl-CoA to cholate and CoASH 1553
15.8.2.1.1.35 Cholate is translocated from the peroxisomal matrix to the cytosol 1554
15.8.2.1.1.36 Choloyl CoA reacts with glycine or taurine to form glycocholate or taurocholate 1555
15.8.2.1.1.37 Chenodeoxycholoyl CoA reacts with glycine or taurine to form glycochenodeoxycholate or taurochenodeoxycholate 1556
15.8.2.1.1.38 Bile salts are translocated from the peroxisomal matrix to the cytosol 1557
15.8.2.1.1.39 Transport (efflux) of bile salts by ABCB11 (bile salt export pump) 1558
15.8.2.1.2 Synthesis of bile acids and bile salts via 24-hydroxycholesterol 1559
15.8.2.1.2.1 Cholesterol is hydroxylated to 24-hydroxycholesterol by CYP46A1 1560
15.8.2.1.2.2 Efflux of 24-hydroxycholesterol 1561
15.8.2.1.2.3 Influx of 24-hydroxycholesterol 1562
15.8.2.1.2.4 24-hydroxycholesterol is 7alpha-hydroxylated to yield cholest-5-ene-3beta,7alpha,24-triol 1563
15.8.2.1.2.5 Cholest-5-ene-3beta,7alpha,24(S)-triol is oxidized and isomerized to 4-cholesten-7alpha,24(S)-diol-3-one 1564
15.8.2.1.2.6 4-Cholesten-7alpha,24(S)-diol-3-one is 12alpha-hydroxylated to 4-cholesten-7-alpha,12-alpha,24(S)-triol-3-one 1565
15.8.2.1.2.7 4-cholesten-7alpha,12alpha,24(S)-triol-3-one is reduced to 5beta-cholestan-7alpha,12alpha,24(S)-triol-3-one 1566
15.8.2.1.2.8 4-cholesten-7alpha,24(S)-diol-3-one is reduced to 5beta-cholestan-7alpha,24(S)-diol-3-one 1510
1567
15.8.2.1.2.9 5Beta-cholestan-7alpha,12alpha,24(S)-triol-3-one is reduced to 5beta-cholestan-3alpha,7alpha,12alpha,24(S)-tetrol 1568

15.8.2.1.2.10 5beta-cholestan-7alpha,24(S)-diol-3-one is reduced to 5beta-cholestan-3alpha,7alpha,24(S)-triol 1569
15.8.2.1.2.11 5beta-cholestan-3alpha,7alpha,12alpha,24(S)-tetrol is translocated from the cytosol to the mitochondrial matrix 1570
15.8.2.1.2.12 5beta-cholestan-3alpha,7alpha,24(S)-triol is translocated from the cytosol to the mitochondrial matrix 1570
15.8.2.1.2.13 5beta-cholestan-3alpha,7alpha,12alpha,24(S)-tetrol is hydroxylated to 5beta-cholestan-3alpha,7alpha,12alpha,24(S),1571
27-pentol
15.8.2.1.2.14 5beta-cholestan-3alpha,7alpha,24(S)-triol is hydroxylated to 5beta-cholestan-3alpha,7alpha,24(S), 27-tetrol 1572
15.8.2.1.2.15 5beta-cholestan-3alpha,7alpha,12alpha,24(S),27-pentol is oxidized to 1573
3alpha,7alpha,12alpha,24(S)-tetrahydroxy-5beta-cholestan-27-al
15.8.2.1.2.16 5beta-cholestan-3alpha,7alpha,24(S),27-tetrol is oxidized to 3alpha,7alpha,24(S)-trihydroxy-5beta-cholestan-27-al 1574
15.8.2.1.2.17 3alpha,7alpha,12alpha,24(S)-tetrahydroxy-5beta-cholestan-27-al is oxidized to 1575
3alpha,7alpha,12alpha,24(S)-tetrahydroxy-5beta-cholestanoate (TetraHCA)
15.8.2.1.2.18 3alpha,7alpha,24(S)-trihydroxy-5beta-cholestan-27-al is oxidized to 1576
3alpha,7alpha,24(S)-trihydroxy-5beta-cholestanoate (3,7,24THCA)
15.8.2.1.2.19 TetraHCA is translocated from the mitochondrial matrix to the cytosol 1577
15.8.2.1.2.20 3,7,24THCA is translocated from the mitochondrial matrix to the cytosol 1577
15.8.2.1.2.21 TetraHCA is conjugated with Coenzyme A (SLC27A5 BACS) 1578
15.8.2.1.2.22 3,7,24THCA is conjugated with Coenzyme A (SLC27A5 BACS) 1579
15.8.2.1.2.23 TetraHCA is conjugated with Coenzyme A (SLC27A2 VLCS) 1580
15.8.2.1.2.24 3,7,24THCA is conjugated with Coenzyme A (SLC27A2 VLCS) 1581
15.8.2.1.2.25 25(R) TetraHCA-CoA is translocated from the cytosol to the peroxisome 1582
15.8.2.1.2.26 3,7,24THCA-CoA is translocated from the cytosol to the peroxisome 1583
15.8.2.1.2.27 Isomerization of 25(R) TetraHCA-CoA to (24R, 25R) 3alpha,7alpha,12alpha,24-tetrahydroxy-5beta-cholestanoyl-CoA1584
15.8.2.1.2.28 Isomerization of 3,7,24THCA-CoA to (24R, 25R) 3alpha,7alpha,24-trihydroxy-5beta-cholestanoyl-CoA 1585
15.8.2.1.3 Synthesis of bile acids and bile salts via 27-hydroxycholesterol 1586
15.8.2.1.3.1 Cholesterol is hydroxylated to 27-hydroxycholesterol by CYP27 1587
15.8.2.1.3.2 Efflux of 27-hydroxycholesterol 1588
15.8.2.1.3.3 Influx of 27-hydroxycholesterol 1589
15.8.2.1.3.4 27-hydroxycholesterol is 7alpha-hydroxylated 1590
15.8.2.1.3.5 Cholest-5-ene-3beta,7alpha,27-triol is oxidized and isomerized to 4-cholesten-7alpha,27-diol-3-one 1591
15.8.2.1.3.6 4-Cholesten-7alpha,27-diol-3-one is 12alpha-hydroxylated to 4-Cholesten-7alpha,12alpha,27-triol-3-one 1592
15.8.2.1.3.7 4-cholesten-7alpha,12alpha,27-triol-3-one is reduced to 5beta-cholestan-7alpha,12alpha,27-triol-3-one 1593
15.8.2.1.3.8 4-cholesten-7alpha,27-diol-3-one is reduced to 5beta-cholestan-7alpha,27-diol-3-one 1594
15.8.2.1.3.9 5Beta-cholestan-7alpha,12alpha,27-triol-3-one is reduced to 5beta-cholestan-3alpha,7alpha,12alpha,27-tetrol 1595
15.8.2.1.3.10 5beta-cholestan-7alpha,27-diol-3-one is reduced to 5beta-cholestan-3alpha,7alpha,27-triol 1596
15.8.2.1.3.11 5beta-cholestan-3alpha,7alpha,12alpha,27-tetrol is translocated from the cytosol to the mitochondrial matrix 1597
15.8.2.1.3.12 5beta-cholestan-3alpha,7alpha,27-triol is translocated from the cytosol to the mitochondrial matrix 1598
15.8.2.1.4 Cholesterol is hydroxylated to 25-hydroxycholesterol 1598
15.8.2.1.5 25-hydroxycholesterol is 7alpha-hydroxylated by CYP7B1 1599
15.8.2.2 Recycling of bile acids and salts 1600
15.8.2.2.1 Co-transport (influx) of bile salts and acids and sodium ions by ASBT 1601
15.8.2.2.2 Transport (efflux) of bile salts by ABCC3 (MRP3) 1602
15.8.2.2.3 Co-transport (influx) of bile salts and sodium ions by NTCP 1603
15.8.2.2.4 Transport (influx) of bile salts and acids by OATP-A 1604
15.8.2.2.5 Transport (influx) of glycocholate and taurocholate by OATP-C 1605
15.8.2.2.6 Transport (influx) of glycocholate and taurocholate by OATP-8 1607
15.8.2.2.7 Cytosolic cholate and chenodeoxycholate are conjugated with Coenzyme A (SLC27A5 BACS) 1608
15.8.2.2.8 Cytosolic chenodeoxycholoyl-CoA or choloyl-CoA are conjugated with glycine or taurine 1608
15.8.2.2.9 Transport (efflux) of bile salts by ABCB11 (bile salt export pump) 1609
15.8.3 Steroid hormone biosynthesis 1610
15.8.3.1 Pregnenolone biosynthesis 1612
15.8.3.1.1 Cholesterol translocates to the inner mitochondrial membrane 1612
15.8.3.1.2 Cholesterol is released into the inner mitochondrial membrane 1614
15.8.3.1.3 Oxidation of cholesterol to 22beta-hydroxycholesterol 1614
15.8.3.1.4 Oxidation of 22beta-hydroxycholesterol to 20alpha,22beta-hydroxycholesterol 1615
15.8.3.1.5 20alpha,22beta-hydroxycholesterol is cleaved by CYP11A1 to yield pregnenolone and isocaproaldehyde 1616
15.8.3.1.6 Pregnenolone translocates from the mitochondrial matrix to the cytosol 1617
15.8.3.2 Glucocorticoid biosynthesis 1618
15.8.3.2.2 17-Hydroxypregnenolone is dehydrogenated to form pregn-5-ene-3,20-dione-17-ol 1619
15.8.3.2.3 Pregn-5-ene-3,20-dione-17-ol isomerizes to 17-hydroxyprogesterone 1620
15.8.3.2.4 Hydroxylation of 17-hydroxyprogesterone to form 11-deoxycortisol 1621
15.8.3.2.5 11-deoxycortisol translocates to the mitochondrion 1622
15.8.3.2.6 11-deoxycortisol is oxidised to cortisol by CYP11B1 1623
15.8.3.2.7 Cortisol translocates from the mitochondrial matrix to the cytosol 1624
15.8.3.2.8 Oxidation of cortisol to yield cortisone 1625
15.8.3.3 Mineralocorticoid biosynthesis 1626
15.8.3.3.1 Pregnenolone is dehydrogenated to form pregn-5-ene-3,20-dione 1626
15.8.3.3.2 Pregn-5-ene-3,20-dione isomerizes to progesterone 1627

15.8.3.3.3 21-hydroxylation of progesterone to form 11-deoxycorticosterone 1628
15.8.3.3.4 11-deoxycorticosterone translocates from the cytosol to the mitochondrial matrix 1629
15.8.3.3.5 Hydroxylation of 11-deoxycorticosterone to form corticosterone 1630
15.8.3.3.6 Hydroxylation of corticosterone to form 18-hydroxycorticosterone 1631
15.8.3.3.7 Conversion of 18-hydroxycorticosterone to aldosterone 1632
15.8.3.4 Androgen biosynthesis 1633
15.8.3.4.2 Side chain cleavage of 17alpha-hydroxyprenenolone to yield DHA 1634
15.8.3.4.3 DHA isomerizes to 4-Androstene3,17-dione 1635
15.8.3.4.4 Hydroxylation of progesterone to form 17alpha-hydroxyprogesterone 1635
15.8.3.4.5 Side chain cleavage of 17alpha-hydroxyprogesterone to form 4-Androstene-3, 17-dione 1636
15.8.3.4.6 Reduction of androstenedione to testosterone 1637
15.8.3.5 Estrogen biosynthesis 1638
15.8.3.5.1 Testosterone is converted to estradiol 1639
15.8.3.5.2 Adrostenedione is converted to estrone by Aromatase (CYP19A1) 1639
15.9 Lipoprotein metabolism 1641
15.9.1 Chylomicron-mediated lipid transport 1641
15.9.1.1 ApoB-48 + 40 triacylglycerol + 60 phospholipid => ApoB-48:TG:PL complex 1642
15.9.1.2 Degradation of newly synthesized ApoB-48 1643
15.9.1.3 ApoB-48:TG:PL complex + 100 triacylglycerols + ApoA-I + ApoA-IV => nascent chylomicron 1644
15.9.1.4 nascent chylomicron [endoplasmic reticulum lumen] => nascent chylomicron [extracellular] 1645
15.9.1.5 nascent chylomicron + spherical HDL:apoC-II:apoC-III:apoE =>spherical HDL + chylomicron 1645
15.9.1.6 chylomicron => TG-depleted chylomicron + 50 long-chain fatty acids + 50 diacylglycerols 1646
15.9.1.7 TG-depleted chylomicron + spherical HDL => chylomicron remnant + spherical HDL:apoA-I:apoA-II:apoA-IV:apoC-II:apoC-III 1647
15.9.1.8 chylomicron remnant + apoE => chylomicron remnant:apoE complex 1648
15.9.1.9 chylomicron remnant:apoE complex + LDLR => chylomicron remnant:apoE:LDLR complex 1649
15.9.1.10 chylomicron remnant:apoE:LDLR complex [plasma membrane] => chylomicron remnant:apoE:LDLR complex [clathrin-coated 1650
vesicle] (LDLRAP1-dependent)
15.9.1.11 chylomicron remnant:apoE:LDLR complex [coated vesicle membrane] => chylomicron remnant:apoE:LDLR complex 1651
[endosome membrane]
15.9.1.12 chylomicron remnant:apoE:LDLR complex => chylomicron remnant:apoE + LDLR 1651
15.9.2 HDL-mediated lipid transport 1652
15.9.2.1 Conversion of pro-apoA-I to apoA-I 1653
15.9.2.2 ABCA1 tetramer binds apoA-I 1654
15.9.2.3 ABCA1-mediated transport of intracellular cholesterol to the cell surface 1655
15.9.2.4 ABCA1-mediated transport of intracellular phospholipid to the cell surface 1656
15.9.2.5 Apolipoprotein A-I binds membrane-associated cholesterol and phospholipid to form a discoidal HDL particle 1657
15.9.2.6 pre-beta HDL binds membrane-associated cholesterol and phospholipid to form a discoidal HDL particle 1658
15.9.2.7 ABCG1-mediated transport of intracellular cholesterol to the cell surface 1659
15.9.2.8 Transformation of discoidal HDL to spherical HDL 1660
15.9.2.8.1 Discoidal HDL binds membrane-associated free cholesterol 1661
15.9.2.8.2 LCAT + discoidal HDL <=> LCAT:discoidal HDL complex 1662
15.9.2.8.3 cholesterol + phosphatidylcholine (lecithin) => cholesterol ester + 2-lysophosphatidylcholine (lysolecithin) 1663
15.9.2.9 LCAT:discoidal HDL complex <=> LCAT + discoidal HDL 1664
15.9.2.10 Remodeling of spherical HDL 1664
15.9.2.10.1 TG-depleted chylomicron + spherical HDL => chylomicron remnant + spherical 1665
HDL:apoA-I:apoA-II:apoA-IV:apoC-II:apoC-III
15.9.2.10.2 nascent chylomicron + spherical HDL:apoC-II:apoC-III:apoE =>spherical HDL + chylomicron 1666
15.9.2.10.3 Spherical HDL binds C and E apolipoproteins 1667
15.9.2.10.4 Spherical HDL binds membrane-associated free cholesterol and phospholipids 1668
15.9.2.10.5 cholesterol + phosphatidylcholine (lecithin) => cholesterol ester + 2-lysophosphatidylcholine (lysolecithin) 1669
15.9.2.10.6 LCAT + spherical HDL <=> LCAT:spherical HDL complex 1669
15.9.2.10.7 CETP-mediated lipid exchange: spherical HDL gains triacylglycerol 1670
15.9.2.11 CETP + spherical HDL + torcetrapib => CETP:spherical HDL:torcetrapib complex 1671
15.9.2.12 LCAT:spherical HDL complex <=> LCAT + spherical HDL 1672
15.9.2.13 spherical HDL and SR-BI receptor form a complex at the cell surface 1673
15.9.2.14 Disassembly of SR-BI-bound spherical HDL 1674
15.9.2.15 Endocytosis and degradation of apoA-I 1675
15.9.2.16 Serum albumin binds 2-lysophosphatidylcholine 1676
15.9.2.17 apoA-I binds to CUBN:AMN 1676
15.9.3 LDL endocytosis 1677
15.9.3.1 LDL + LDLR => LDL:LDLR complex 1679
15.9.3.2 LDL:LDLR complex [plasma membrane] => LDL:LDLR complex [clathrin-coated vesicle] (LDLRAP1-dependent) 1679
15.9.3.3 LDL:LDLR complex [plasma membrane] => LDL:LDLR complex [clathrin-coated vesicle] (LDLRAP1-independent) 1680
15.9.3.4 LDLR:LDL complex [coated vesicle membrane] => LDLR:LDL complex [endosome membrane] 1681
15.9.3.5 LDLR:LDL complex => LDLR + LDL 1682
15.9.3.6 LDLR [endosome membrane] => LDLR [plasma membrane] 1683
16 Membrane Trafficking 1684

16.1 ER to Golgi Transport 1685
16.1.1 COPII (Coat Protein 2) Mediated Vesicle Transport 1685
16.1.1.1 Sar1p Activation And Membrane Binding 1686
16.1.1.2 Coat Assembly 1687
16.1.1.3 Loss of Sar1p GTPase 1688
16.1.1.4 Cargo, Sec31p:Sec13p, and v-SNARE recruitment 1688
16.1.1.5 Vesicle Budding 1689
16.1.1.6 Vesicle Uncoating 1690
16.2 Golgi to ER Retrograde Transport 1691
16.2.1 COPI Mediated Transport 1691
16.2.1.1 Arf1 Activation by GBF1 1692
16.2.1.2 Coat Complex Formation 1693
16.2.1.3 GAP Recruitment to the Coatomer:Arf1-GTP Complex 1694
16.2.1.4 Coatomer:Arf1-GTP:GAP lattice formation on golgi membrane 1695
16.2.1.5 Sculpting and pinching-off of Golgi vessicle 1695
16.2.1.6 Hydrolysis of Arf1-GTP to Arf1-GDP 1696
16.2.1.7 Diffusion of inactive Arf1-GDP from membrane 1697
16.2.1.8 Golgi vesicle lattice disassociation 1698
17 Metabolism of amino acids 1699
17.1 Amino acid uptake across the plasma membrane 1699
17.1.1 SLC16A10-mediated uptake of aromatic amino acids 1700
17.1.2 SLC38A1 (ATA1)-mediated uptake of neutral amino acids 1701
17.1.3 SLC38A2 (ATA2)-mediated uptake of neutral amino acids 1702
17.1.4 SLC38A3-mediated uptake of glutamine, histidine, asparagine, and alanine 1703
17.1.5 SLC38A4 (ATA3)-mediated uptake of arginine and lysine 1704
17.1.6 SLC38A5-mediated uptake of glutamine, histidine, asparagine, and serine 1705
17.1.7 SLC43A1 (LAT3)-mediated uptake of large neutral amino acids 1706
17.1.8 SLC43A2 (LAT4)-mediated uptake of large neutral amino acids 1707
17.1.9 SLC6A12 (BGT-1)-mediated uptake of GABA and betaine 1708
17.1.10 SLC6A15-mediated amino acid uptake 1709
17.1.11 SLC6A18-mediated glycine uptake 1710
17.1.12 SLC6A20-mediated uptake of proline 1711
17.1.13 SLC6A6-mediated uptake of taurine and beta-alanine 1712
17.1.14 SLC7A5-mediated uptake of neutral amino acids 1713
17.1.15 SLC7A8-mediated uptake of neutral amino acids 1714
17.1.16 SLC1A4-mediated exchange of alanine, serine, threonine, and cysteine across the plasma membrane 1715
17.1.17 SLC1A5-mediated exchange of alanine and glutamine across the plasma membrane 1716
17.2 Urea synthesis 1717
17.2.1 glutamate + acetyl CoA => N-acetyl glutamate + CoA 1718
17.2.2 2 ATP + NH4+ + HCO3- => 2 ADP + orthophosphate + carbamoyl phosphate [mitochondrial] 1718
17.2.3 carbamoyl phosphate + ornithine => citrulline + orthophosphate 1719
17.2.4 ATP + aspartate + citrulline <=> argininosuccinate + AMP + pyrophosphate 1720
17.2.5 argininosuccinate <=> fumarate + arginine 1720
17.2.6 arginine => ornithine + urea 1721
17.2.7 ornithine (cytosolic) + citrulline (mitochondrial) => ornithine (mitochondrial) + citrulline (cytosolic) 1722
17.3 Alanine metabolism 1722
17.3.1 alanine + alpha-ketoglutarate <=> pyruvate + glutamate 1723
17.3.2 pyruvate + glutamate <=> alanine + alpha-ketoglutarate 1723
17.4 Aspartate, asparagine, glutamate, and glutamine metabolism 1724
17.4.1 alpha-ketoglutarate + NH4+ + NADPH + H+ => glutamate + NADP+ 1725
17.4.2 aspartate + alpha-ketoglutarate <=> oxaloacetate + glutamate [cytosolic] 1725
17.4.3 aspartate + alpha-ketoglutarate <=> oxaloacetate + glutamate [mitochondrial] 1726
17.4.4 aspartate + glutamine + ATP <=> asparagine + glutamate + AMP + pyrophosphate 1727
17.4.5 glutamate + NAD+ => alpha-ketoglutarate + NH4+ + NADH + H+ 1728
17.4.6 glutamate + NH4+ + ATP => glutamine + ADP + orthophosphate 1728
17.4.7 glutamine + H2O => glutamate + NH4+ [kidney] 1729
17.4.8 glutamine + H2O => glutamate + NH4+ [liver] 1730
17.4.9 oxaloacetate + glutamate <=> aspartate + alpha-ketoglutarate [cytosolic] 1731
17.4.10 oxaloacetate + glutamate <=> aspartate + alpha-ketoglutarate [mitochondrial] 1731
17.5 Ornithine and proline metabolism 1732
17.5.1 Ornithine metabolism 1734
17.5.1.1 argininosuccinate <=> fumarate + arginine 1734
17.5.1.2 arginine => ornithine + urea 1734
17.5.1.3 ornithine => putrescine + CO2 1735
17.5.1.4 glutamate + L-glutamate gamma-semialdehyde <=> ornithine + alpha-ketoglutarate 1735
17.5.1.5 carbamoyl phosphate + ornithine => citrulline + orthophosphate 1736
17.5.1.6 ATP + aspartate + citrulline <=> argininosuccinate + AMP + pyrophosphate 1736
17.5.1.7 ornithine (cytosolic) + citrulline (mitochondrial) => ornithine (mitochondrial) + citrulline (cytosolic) 1737
17.5.1.8 ornithine + alpha-ketoglutarate <=> glutamate + L-glutamate gamma-semialdehyde 1737
17.5.1.9 L-glutamate gamma-semialdehyde <=> L-1-pyrroline-5-carboxylate 1738
17.5.1.10 Regulation of ornithine decarboxylase (ODC) 1739
17.5.1.10.1 ornithine => putrescine + CO2 1740
17.5.1.10.2 Antizyme OAZ binds to Ornithine decarboxylase 1740
17.5.1.10.3 26S proteosome degrades ODC holoenzyme complex 1741
17.5.1.10.4 Antizyme inhibitor binds to OAZ and stablizes ODC complex 1742
17.5.1.10.5 NQO1 interaction with ODC 1743
17.5.2 Proline synthesis 1744
17.5.2.1 ornithine + alpha-ketoglutarate <=> glutamate + L-glutamate gamma-semialdehyde 1744
17.5.2.2 L-glutamate gamma-semialdehyde <=> L-1-pyrroline-5-carboxylate 1745
17.5.2.3 L-1-pyrroline-5-carboxylate + NADPH + H+ => proline + NADP+ 1745
17.5.3 Proline catabolism 1746
17.5.3.1 proline => L-1-pyrroline-5-carboxylate 1746
17.5.3.2 L-1-pyrroline-5-carboxylate <=> L-glutamate gamma-semialdehyde 1747
17.5.3.3 glutamate + L-glutamate gamma-semialdehyde <=> ornithine + alpha-ketoglutarate 1747
17.5.3.4 L-glutamate gamma-semialdehyde + NAD+ => glutamate + NADH + H+ 1748
17.6 Branched-chain amino acid catabolism 1749
17.6.1 Leucine catabolism 1750
17.6.1.1 L-Glutamate [cytosolic] from leucine catabolism 1750
17.6.1.2 L-Glutamate [mitochondrial] from leucine catabolism 1751
17.6.1.3 Oxidative decarboxylation of alpha-ketoisocaproate to isovaleryl-CoA by branched-chain alpha-ketoacid dehydrogenase 1751
17.6.1.3.1 alpha-ketoisocaproate + TPP => 3-methyl-1-hydroxybutyl-TPP + CO2 1752
17.6.1.3.2 3-methyl-1-hydroxybutyl-TPP + lipoamide => S-isovaleryldihydrolipoamide + TPP 1753
17.6.1.3.3 S-isovaleryldihydrolipoamide + CoA => isovaleryl-CoA + dihydrolipoamide 1753
17.6.1.3.4 dihydrolipoamide + FAD => lipoamide + FADH2 [branched-chain ketoacid dehydrogenase] 1754
17.6.1.3.5 FADH2 + NAD+ => FAD + NADH + H+ [branched-chain ketoacid dehydrogenase] 1755
17.6.1.4 isovaleryl-CoA + FAD => beta-methylcrotonyl-CoA + FADH2 1755
17.6.1.5 beta-methylcrotonyl-CoA + ATP + CO2 <=> beta-methylglutaconyl-CoA + ADP + orthophosphate + H2O 1756
17.6.1.6 beta-methylglutaconyl-CoA + H2O <=> beta-hydroxy-beta-methylglutaryl-CoA 1757
17.6.2 Isoleucine catabolism 1757
17.6.2.1 isoleucine + alpha-ketoglutarate <=> alpha-keto-beta-methylvalerate + glutamate [cytosolic] 1758
17.6.2.2 isoleucine + alpha-ketoglutarate <=> alpha-keto-beta-methylvalerate + glutamate [mitochondrial] 1758
17.6.2.3 Oxidative decarboxylation of alpha-keto-beta-methylvalerate to alpha-methylbutyryl-CoA by branched-chain alpha-ketoacid 1759
dehydrogenase
17.6.2.3.1 alpha-keto-beta-methylvalerate + TPP => 2-methyl-1-hydroxybutyl-TPP + CO2 1760
17.6.2.3.2 2-methyl-1-hydroxybutyl-TPP + lipoamide => S-(2-methylbutanoyl)-dihydrolipoamide + TPP 1760
17.6.2.3.3 S-(2-methylbutanoyl)-dihydrolipoamide + CoA => alpha-methylbutyryl-CoA + dihydrolipoamide 1761
17.6.2.4 alpha-methylbutyryl-CoA + FAD => tiglyl-CoA + FADH2 1762
17.6.2.5 tiglyl-CoA + H2O <=> alpha-methyl-beta-hydroxybutyryl-CoA 1763
17.6.2.6 alpha-methyl-beta-hydroxybutyryl-CoA + NAD+ <=> alpha-methylacetoacetyl-CoA + NADH + H+ 1764
17.6.2.7 alpha-methyl-acetoacetyl-CoA + CoA <=> propionyl-CoA + acetyl-CoA 1765
17.6.3 Valine catabolism 1765
17.6.3.1 valine + alpha-ketoglutarate <=> alpha-ketoisovalerate + glutamate [cytosolic] 1766
17.6.3.2 valine + alpha-ketoglutarate <=> alpha-ketoisovalerate + glutamate [mitochondrial] 1766
17.6.3.3 Oxidative decarboxylation of alpha-ketoisovalerate to isobutyryl-CoA by branched-chain alpha-ketoacid dehydrogenase 1767
17.6.3.3.1 alpha-ketoisovalerate + TPP => 2-methyl-1-hydroxypropyl-TPP + CO2 1768
17.6.3.3.2 2-methyl-1-hydroxypropyl-TPP + lipoamide => S-(isobutyryl)-dihydrolipoamide + TPP 1768
17.6.3.3.3 S-(isobutyryl)-dihydrolipoamide + CoA => isobutyryl-CoA + dihydrolipoamide 1769
17.6.3.4 isobutyryl-CoA + FAD => methacrylyl-CoA + FADH2 1770
17.6.3.5 methacrylyl-CoA + H2O <=> beta-hydroxyisobutyryl-CoA 1771
17.6.3.6 beta-hydroxyisobutyryl-CoA + H2O => beta-hydroxyisobutyrate + CoA 1772
17.6.3.7 beta-hydroxyisobutyrate + NAD+ <=> methylmalonyl semialdehyde + NADH + H+ 1773
17.6.3.8 methylmalonate semialdehyde + NAD+ + CoA => propionyl-CoA + CO2 + NADH + H+ 1773
17.7 Histidine catabolism 1774
17.7.1 histidine => urocanate + NH4+ 1774
17.7.2 urocanate + H2O => 4-imidazolone-5-propionate 1775
17.7.3 4-imidazolone-5-propionate + H2O => N-formiminoglutamate 1776
17.7.4 N-formiminoglutamate + tetrahydrofolate => glutamate + 5-formiminotetrahydrofolate 1776
17.8 Lysine catabolism 1777
17.8.1 lysine + alpha-ketoglutarate +NADPH + H+ => saccharopine + NADP+ + H2O 1778
17.8.2 saccharopine + NAD+ + H2O => alpha-aminoadipic semialdehyde + glutamate + NADH + H+ 1778
17.8.3 alpha-aminoadipic semialdehyde + NAD+ => alpha-aminoadipate + NADH + H+ 1779
17.8.4 alpha-aminoadipate + alpha-ketoglutarate => alpha-ketoadipate + glutamate 1780

17.8.5 Oxidative decarboxylation of alpha-ketoadipate to glutaryl CoA by alpha-ketoglutarate dehydrogenase 1780
17.8.5.1 alpha-ketoadipate + TPP => 4-carboxy-1-hydroxybutyl-TPP + CO2 1781
17.8.5.2 4-carboxy-1-hydroxybutyl-TPP + lipoamide => S-glutaryldihydrolipoamide + TPP 1782
17.8.5.3 S-glutaryldihydrolipoamide + CoA => glutaryl-CoA + dihydrolipoamide 1782
17.8.5.4 dihydrolipoamide + FAD => lipoamide + FADH2 [alpha-ketoglutarate dehydrogenase] 1783
17.8.5.5 FADH2 + NAD+ => FAD + NADH + H+ [alpha-ketoglutarate dehydrogenase] 1784
17.8.6 glutaryl-CoA + FAD => crotonyl-CoA + FADH2 + CO2 1784
17.8.7 Crotonoyl-CoA+H2O<=>(S)-3-Hydroxybutanoyl-CoA 1785
17.8.8 (S)-Hydroxybutanoyl-CoA+NAD<=>Acetoacetyl-CoA+NADH+H 1786
17.9 Phenylalanine and tyrosine catabolism 1786
17.9.1 phenylalanine + tetrahydrobiopterin + O2 => tyrosine + 4a-hydroxytetrahydrobiopterin + H2O 1787
17.9.2 4a-hydroxytetrahydrobiopterin => q-dihydrobiopterin + H2O 1788
17.9.3 q-dihydrobiopterin + NADH + H+ => tetrahydrobiopterin + NAD+ 1788
17.9.4 tyrosine + alpha-ketoglutarate => p-hydroxyphenylpyruvate + glutamate 1789
17.9.5 p-hydroxyphenylpyruvate + O2 => homogentisate + CO2 1789
17.9.6 homogentisate + O2 => maleylacetoacetate 1790
17.9.7 maleylacetoacetate => fumarylacetoacetate 1791
17.9.8 fumarylacetoacetate => fumarate + acetoacetate 1792
17.10 Tryptophan catabolism 1792
17.10.1 tryptophan + O2 => N-formylkynurenine [TDO] 1792
17.10.2 N-formylkynurenine + H2O => kynurenine + formate 1793
17.10.3 kynurenine + O2 + NADPH + H+ => 3-hydroxykynurenine + NADP+ + H2O 1794
17.10.4 3-hydroxykynurenine + H2O => 3-hydroxyanthranilate + alanine 1795
17.10.5 3-hydroxyanthranilate + O2 => 2-amino-3-carboxymuconate semialdehyde 1795
17.10.6 2-amino-3-carboxymuconate semialdehyde => 2-aminomuconate semialdehyde + CO2 1796
17.10.7 2-aminomuconate semialdehyde + NAD+ => aminomuconate + NADH + H+ 1797
17.11 Carnitine synthesis 1798
17.11.1 trimethyllysine + alpha-ketoglutarate + O2 => beta-hydroxy-trimethyllysine + succinate + CO2 1798
17.11.2 beta-hydroxy-trimethyllysine => gamma-butyrobetaine aldehyde + glycine 1799
17.11.3 gamma-butyrobetaine semialdehyde + NAD+ => gamma-butyrobetaine + NADH + H+ 1799
17.11.4 gamma-butyrobetaine + alpha-ketoglutarate + O2 => carnitine + succinate + CO2 1800
17.12 Creatine metabolism 1801
17.12.1 arginine + glycine => ornithine + guanidoacetate 1801
17.12.2 guanidinoacetate + S-adenosylmethionine => creatine + S-adenosylhomocysteine 1802
17.12.3 creatine + ATP => phosphocreatine + ADP [CKB,CKM] 1803
17.12.4 creatine + ATP => phosphocreatine + ADP [CK octamer] 1804
17.12.5 phosphocreatine + H2O => creatinine + orthophosphate 1804
18 Metabolism of carbohydrates 1806
18.1 Digestion of dietary carbohydrate 1806
18.1.1 Digestion of linear starch (amylose) by extracellular amylase 1807
18.1.2 Digestion of branched starch (amylopectin) by extracellular amylase 1808
18.1.3 Digestion of 1-6 linkages of limit dextrins to yield maltose, maltotriose, longer maltosides, and glucose 1810
18.1.4 maltotriose + H2O => maltose + D-glucose (maltase-glucoamylase) 1811
18.1.5 maltotriose + H2O => maltose + D-glucose (sucrase-isomaltase) 1812
18.1.6 maltose + H2O => 2 D-glucose (maltase-glucoamylase) 1813
18.1.7 maltose + H2O => 2 D-glucose (sucrase-isomaltase) 1814
18.1.8 lactose + H2O => D-glucose + D-galactose 1815
18.1.9 sucrose + H2O => glucose + fructose 1816
18.1.10 trehalose + H2O => 2 D-glucose 1817
18.2 Hexose uptake 1818
18.2.1 Transport (influx) of fructose by GLUT5 1819
18.2.2 Transport (influx) of glucose, galactose, and sodium ions by SGLT1 1820
18.2.3 Transport (efflux) of fructose, galactose, and glucose by GLUT2 1821
18.3 Glucose metabolism 1822
18.3.1 Glucose uptake 1823
18.3.1.1 Glucose is carried across the plasma membrane by a glucose transport protein (GLUT) 1825
18.3.1.1.1 alpha-D-glucose (extracellular) <=> alpha-D-glucose (cytosol) [GLUT1] 1825
18.3.1.2 Glucose + ATP => glucose-6-phosphate + ADP 1828
18.3.1.2.1 ATP + alpha-D-Glucose => ADP + alpha-D-glucose 6-phosphate [glucokinase] 1828
18.3.1.2.2 ATP + alpha-D-Glucose => ADP + alpha-D-glucose 6-phosphate [hexokinase 1] 1829
18.3.1.3 Negative Regulation of Glucokinase by Glucokinase Regulatory Protein 1830
18.3.1.3.1 glucokinase (GK) + glucokinase regulatory protein (GKRP) <=> GK:GKRP complex 1831
18.3.1.3.2 cytosolic GK:GKRP complex <=> glucokinase (GK) + glucokinase regulatory protein (GKRP) 1831
18.3.1.3.3 GK:GKRP [cytosol] => GK:GKRP [nucleoplasm] 1832
18.3.1.3.4 nucleoplasmic GK:GKRP complex => glucokinase (GK) + glucokinase regulatory protein (GKRP) 1833
18.3.1.3.5 glucokinase [nucleoplasm] => glucokinase [cytosol] 1833
18.3.2 Glycolysis 1834
18.3.2.1 Glucose 6-phosphate is isomerized to form fructose-6-phosphate 1834
18.3.2.1.1 alpha-D-glucose 6-phosphate <=> D-fructose 6-phosphate 1835
18.3.2.2 Fructose 6-phosphate and ATP react to form fructose 2,6-bisphosphate and ADP 1835
18.3.2.2.1 ATP + D-fructose 6-phosphate => ADP + D-fructose 2,6-bisphosphate [brain + placenta] 1836
18.3.2.2.2 ATP + D-fructose 6-phosphate => ADP + D-fructose 2,6-bisphosphate [heart] 1837
18.3.2.2.3 ATP + D-fructose 6-phosphate => ADP + D-fructose 2,6-bisphosphate [liver + muscle] 1837
18.3.2.2.4 ATP + D-fructose 6-phosphate => ADP + D-fructose 2,6-bisphosphate [testis] 1838
18.3.2.3 ATP + D-fructose 6-phosphate => ADP + D-fructose 1,6-bisphosphate 1839
18.3.2.4 D-fructose 1,6-bisphosphate <=> dihydroxyacetone phosphate + D-glyceraldehyde 3-phosphate 1839
18.3.2.5 Dihydroxyacetone phosphate is isomerized to form glyceraldehyde-3-phosphate 1840
18.3.2.5.1 dihydroxyacetone phosphate <=> D-glyceraldehyde 3-phosphate 1840
18.3.2.6 Glyceraldehyde 3-phosphate, NAD+, and orthophosphate react to form 1,3-bisphosphoglycerate, NADH, and H+ 1841
18.3.2.6.1 D-glyceraldehyde 3-phosphate + orthophosphate + NAD+ <=> 1,3-bisphospho-D-glycerate + NADH + H+ 1841
18.3.2.7 1,3-bisphosphoglycerate and ADP react to form 3-phosphoglycerate and ATP 1842
18.3.2.7.1 ADP + 3-Phospho-D-glyceroyl phosphate <=> ATP + 3-Phospho-D-glycerate 1842
18.3.2.8 3-Phospho-D-glycerate <=> 2-Phospho-D-glycerate 1843
18.3.2.9 2-Phospho-D-glycerate <=> Phosphoenolpyruvate + H2O 1844
18.3.2.10 Phosphoenolpyruvate and ADP react to form pyruvate and ATP 1845
18.3.2.10.1 ADP + Phosphoenolpyruvate => ATP + Pyruvate (pyruvate kinase M2) 1845
18.3.2.10.2 ADP + Phosphoenolpyruvate => ATP + Pyruvate (pyruvate kinase R/L) 1846
18.3.3 Gluconeogenesis 1846
18.3.3.1 Pyruvate, CO2, and ATP react to form oxaloacetate, ADP, and orthophosphate 1847
18.3.3.2 Oxaloacetate + NADH + H+ <=> (S)-Malate + NAD+ 1847
18.3.3.3 Exchange of malate and alpha-ketoglutarate (2-oxoglutarate) across the inner mitochondrial membrane 1848
18.3.3.4 malate + NAD+ <=> oxaloacetate + NADH + H+ 1849
18.3.3.5 Oxaloacetate and GTP react to form phosphoenolpyruvate, CO2, and GDP 1850
18.3.3.6 Phosphoenolpyruvate + H2O <=> 2-Phospho-D-glycerate 1850
18.3.3.7 2-Phospho-D-glycerate <=> 3-Phospho-D-glycerate 1851
18.3.3.8 ATP + 3-Phospho-D-glycerate <=> ADP + 1,3-bisphospho-D-glycerate 1852
18.3.3.9 1,3-bisphospho-D-glycerate + NADH + H+ <=> D-glyceraldehyde 3-phosphate + Orthophosphate + NAD+ 1853
18.3.3.10 D-glyceraldehyde 3-phosphate <=> dihydroxyacetone phosphate 1853
18.3.3.11 dihydroxyacetone phosphate + D-glyceraldehyde 3-phosphate <=> D-fructose 1,6-bisphosphate 1854
18.3.3.12 Fructose 1,6-bisphosphate is hydrolyzed to form fructose 6-phosphate and orthophosphate 1855
18.3.3.12.1 D-fructose 1,6-bisphosphate + H2O => D-fructose 6-phosphate + orthophosphate 1855
18.3.3.12.2 D-fructose 1,6-bisphosphate + H2O => D-fructose 6-phosphate + orthophosphate 1855
18.3.3.13 D-fructose 6-phosphate <=> alpha-D-Glucose 6-phosphate 1856
18.3.3.14 alpha-D-glucose 6-phosphate [cytosol] => alpha-D-glucose 6-phosphate [endoplasmic reticulum lumen] 1857
18.3.3.15 alpha-D-Glucose 6-phosphate + H2O => alpha-D-Glucose + Orthophosphate 1857
18.3.3.16 Efflux of glucose from the endoplasmic reticulum 1858
18.3.3.17 Fructose 2,6-bisphosphate is hydrolyzed to form fructose-6-phosphate and orthophosphate 1859
18.3.4 Cori Cycle (interconversion of glucose and lactate) 1859
18.3.4.1 Glycolysis 1860
18.3.4.1.1 Glucose 6-phosphate is isomerized to form fructose-6-phosphate 1860
18.3.4.1.1.1 alpha-D-glucose 6-phosphate <=> D-fructose 6-phosphate 1861
18.3.4.1.2 Fructose 6-phosphate and ATP react to form fructose 2,6-bisphosphate and ADP 1861
18.3.4.1.2.1 ATP + D-fructose 6-phosphate => ADP + D-fructose 2,6-bisphosphate [brain + placenta] 1862
18.3.4.1.2.2 ATP + D-fructose 6-phosphate => ADP + D-fructose 2,6-bisphosphate [heart] 1862
18.3.4.1.2.3 ATP + D-fructose 6-phosphate => ADP + D-fructose 2,6-bisphosphate [liver + muscle] 1862
18.3.4.1.2.4 ATP + D-fructose 6-phosphate => ADP + D-fructose 2,6-bisphosphate [testis] 1863
18.3.4.1.3 ATP + D-fructose 6-phosphate => ADP + D-fructose 1,6-bisphosphate 1863
18.3.4.1.4 D-fructose 1,6-bisphosphate <=> dihydroxyacetone phosphate + D-glyceraldehyde 3-phosphate 1864
18.3.4.1.5 Dihydroxyacetone phosphate is isomerized to form glyceraldehyde-3-phosphate 1864
18.3.4.1.5.1 dihydroxyacetone phosphate <=> D-glyceraldehyde 3-phosphate 1865
18.3.4.1.6 Glyceraldehyde 3-phosphate, NAD+, and orthophosphate react to form 1,3-bisphosphoglycerate, NADH, and H+ 1865
18.3.4.1.6.1 D-glyceraldehyde 3-phosphate + orthophosphate + NAD+ <=> 1,3-bisphospho-D-glycerate + NADH + H+ 1865
18.3.4.1.7 1,3-bisphosphoglycerate and ADP react to form 3-phosphoglycerate and ATP 1866
18.3.4.1.7.1 ADP + 3-Phospho-D-glyceroyl phosphate <=> ATP + 3-Phospho-D-glycerate 1866
18.3.4.1.8 3-Phospho-D-glycerate <=> 2-Phospho-D-glycerate 1867
18.3.4.1.9 2-Phospho-D-glycerate <=> Phosphoenolpyruvate + H2O 1867
18.3.4.1.10 Phosphoenolpyruvate and ADP react to form pyruvate and ATP 1868
18.3.4.1.10.1 ADP + Phosphoenolpyruvate => ATP + Pyruvate (pyruvate kinase M2) 1868
18.3.4.1.10.2 ADP + Phosphoenolpyruvate => ATP + Pyruvate (pyruvate kinase R/L) 1869
18.3.4.2 Gluconeogenesis 1869
18.3.4.2.1 Pyruvate, CO2, and ATP react to form oxaloacetate, ADP, and orthophosphate 1870
18.3.4.2.2 Oxaloacetate + NADH + H+ <=> (S)-Malate + NAD+ 1870
18.3.4.2.3 Exchange of malate and alpha-ketoglutarate (2-oxoglutarate) across the inner mitochondrial membrane 1870
18.3.4.2.4 malate + NAD+ <=> oxaloacetate + NADH + H+ 1871
18.3.4.2.5 Oxaloacetate and GTP react to form phosphoenolpyruvate, CO2, and GDP 1872
18.3.4.2.6 Phosphoenolpyruvate + H2O <=> 2-Phospho-D-glycerate 1872
18.3.4.2.7 2-Phospho-D-glycerate <=> 3-Phospho-D-glycerate 1873
18.3.4.2.8 ATP + 3-Phospho-D-glycerate <=> ADP + 1,3-bisphospho-D-glycerate 1873
18.3.4.2.9 1,3-bisphospho-D-glycerate + NADH + H+ <=> D-glyceraldehyde 3-phosphate + Orthophosphate + NAD+ 1874
18.3.4.2.10 D-glyceraldehyde 3-phosphate <=> dihydroxyacetone phosphate 1874
18.3.4.2.11 dihydroxyacetone phosphate + D-glyceraldehyde 3-phosphate <=> D-fructose 1,6-bisphosphate 1875
18.3.4.2.12 Fructose 1,6-bisphosphate is hydrolyzed to form fructose 6-phosphate and orthophosphate 1875
18.3.4.2.12.1 D-fructose 1,6-bisphosphate + H2O => D-fructose 6-phosphate + orthophosphate 1875
18.3.4.2.12.2 D-fructose 1,6-bisphosphate + H2O => D-fructose 6-phosphate + orthophosphate 1876
18.3.4.2.13 D-fructose 6-phosphate <=> alpha-D-Glucose 6-phosphate 1876
18.3.4.2.14 alpha-D-glucose 6-phosphate [cytosol] => alpha-D-glucose 6-phosphate [endoplasmic reticulum lumen] 1877
18.3.4.2.15 alpha-D-Glucose 6-phosphate + H2O => alpha-D-Glucose + Orthophosphate 1877
18.3.4.2.16 Efflux of glucose from the endoplasmic reticulum 1878
18.3.4.2.17 Fructose 2,6-bisphosphate is hydrolyzed to form fructose-6-phosphate and orthophosphate 1878
18.3.5 Glycogen synthesis 1879
18.3.5.1 alpha-D-Glucose 6-phosphate <=> D-Glucose 1-phosphate 1881
18.3.5.2 UTP and glucose 1-phosphate react to form UDP-glucose and pyrophosphate 1881
18.3.5.2.1 UTP + D-glucose 1-phosphate <=> pyrophosphate + UDP-glucose [liver] 1882
18.3.5.2.2 UTP + D-glucose 1-phosphate <=> pyrophosphate + UDP-glucose [muscle] 1883
18.3.5.3 Glycogenin catalyzes the synthesis of an oligo(1,4)glucose moiety covalently attached to itself 1883
18.3.5.3.1 n UDP-glucose + glycogenin-2 => n UDP + {(1,4)-alpha-D-glucosyl}n glycogenin-2 1883
18.3.5.3.2 n UDP-glucose + glycogenin-1 => n UDP + {(1,4)-alpha-D-glucosyl}n glycogenin-1 1884
18.3.5.4 Glycogen synthase catalyzes the addition of glucose residues to the non-reducing end of a (1,4)-alpha-D-glucose multimer 1885
18.3.5.4.1 m UDP-glucose + {(1,4)-alpha-D-glucosyl}n glycogenin-1 => m UDP + {(1,4)-alpha-D-glucosyl}m+n glycogenin-1 [muscle, 1885 I
form]
18.3.5.4.2 m UDP-glucose + {(1,4)-alpha-D-glucosyl}n glycogenin-2 => m UDP + {(1,4)-alpha-D-glucosyl}m+n glycogenin-2 [liver, I 1885
form]
18.3.5.5 Phosphorylated glycogen synthase catalyzes the addition of glucose residues to the non-reducing end of a 1886
(1,4)-alpha-D-glucose multimer when activated by glucose-6-phosphate
18.3.5.5.1 m UDP-glucose + {(1,4)-alpha-D-glucosyl}n glycogenin-1 => m UDP + {(1,4)-alpha-D-glucosyl}m+n glycogenin-1 [muscle, 1886 D
form]
18.3.5.5.2 m UDP-glucose + {(1,4)-alpha-D-glucosyl}n glycogenin-2 => m UDP + {(1,4)-alpha-D-glucosyl}m+n glycogenin-2 [liver, D 1887
form]
18.3.5.6 Glycogen branching enzyme transfers terminal alpha(1,4)glucose blocks to form alpha(1,6) branches 1887
18.3.5.6.1 poly{(1,4)-alpha-D-glucosyl} glycogenin-1 => glycogen-glycogenin-1 1888
18.3.5.6.2 poly{(1,4)-alpha-D-glucosyl} glycogenin-2 => glycogen-glycogenin-2 1888
18.3.6 Glycogen breakdown (glycogenolysis) 1889
18.3.6.1 Phosphorylase kinase activates glycogen phosphorylase 1890
18.3.6.1.1 glycogen phosphorylase, liver form, tetramer b + ATP => glycogen phosphorylase, liver form, tetramer a + ADP 1891
18.3.6.1.2 glycogen phosphorylase, muscle form, tetramer b + ATP => glycogen phosphorylase, muscle form, tetramer a + ADP 1891
18.3.6.2 Glycogen-glycogenin reacts with n orthophosphates, yielding n glucose 1-phosphates and limit dextrin-glycogenin 1892
18.3.6.2.1 glycogen-glycogenin-1 + n orthophosphate => limit dextrin-glycogenin-1 + n D-glucose 1-phosphate [muscle a form] 1892
18.3.6.2.2 glycogen-glycogenin-1 + n orthophosphate => limit dextrin-glycogenin-1 + n D-glucose 1-phosphate [muscle b form] 1893
18.3.6.2.3 glycogen-glycogenin-2 + n orthophosphate => limit dextrin-glycogenin-2 + n D-glucose 1-phosphate [liver a form] 1894
18.3.6.2.4 glycogen-glycogenin-2 + n orthophosphate => limit dextrin-glycogenin-2 + n D-glucose 1-phosphate [liver b form] 1894
18.3.6.3 Debranching enzyme transfers 3-glucose blocks from branches in limit dextrin 1895
18.3.6.3.1 limit dextrin-glycogenin-1 => {(1,6)-alpha-glucosyl}poly{(1,4)-alpha-glucosyl} glycogenin-1 1895
18.3.6.3.2 limit dextrin-glycogenin-2 => {(1,6)-alpha-glucosyl}poly{(1,4)-alpha-glucosyl} glycogenin-2 1896
18.3.6.4 (1,6)-alpha-glucose residues are removed, as D-glucose, from limit dextrin by debranching enzyme 1896
18.3.6.4.1 {(1,6)-alpha-glucosyl}poly{(1,4)-alpha-glucosyl}glycogenin-1 => poly{(1,4)-alpha-glucosyl} glycogenin-1 + alpha-D-glucose1897
18.3.6.4.2 {(1,6)-alpha-glucosyl}poly{(1,4)-alpha-glucosyl}glycogenin-2 => poly{(1,4)-alpha-glucosyl} glycogenin-2 + alpha-D-glucose1897
18.3.6.5 Poly{(1,4)-alpha-glucosyl} glycogenin reacts with n orthophosphates to form glycogenin and n D-glucose 1-phosphates 1898
18.3.6.5.1 poly{(1,4)-alpha-glucosyl} glycogenin-1 + n orthophosphate => glycogenin-1 + n D-glucose 1-phosphate [muscle a form] 1898
18.3.6.5.2 poly{(1,4)-alpha-glucosyl} glycogenin-1 + n orthophosphate => glycogenin-1 + n D-glucose 1-phosphate [muscle b form] 1899
18.3.6.5.3 poly{(1,4)-alpha-glucosyl} glycogenin-2 + n orthophosphate => glycogenin-2 + n D-glucose 1-phosphate [liver a form] 1900
18.3.6.5.4 poly{(1,4)-alpha-glucosyl} glycogenin-2 + n orthophosphate => glycogenin-2 + n D-glucose 1-phosphate [liver b form] 1900
18.3.6.6 D-Glucose 1-phosphate <=> alpha-D-Glucose 6-phosphate 1901
18.4 Fructose catabolism 1902
18.4.1 ATP + beta-D-fructose => ADP + D-fructose 1-phosphate 1903
18.4.2 D-fructose 1-phosphate <=> D-glyceraldehyde + dihydroxyacetone phosphate 1904
18.4.3 ATP + D-glyceraldehyde => ADP + D-glyceraldehyde 3-phosphate 1905
18.5 Galactose catabolism 1905
18.5.1 ATP + D-galactose => ADP + D-galactose 1-phosphate 1906
18.5.2 D-galactose 1-phosphate + UDP-glucose <=> D-glucose 1-phosphate + UDP-galactose 1907

18.5.3 UDP-galactose <=> UDP-glucose 1908
18.6 Pentose phosphate pathway (hexose monophosphate shunt) 1909
18.6.1 alpha-D-glucose 6-phosphate + NADP+ => D-glucono-1,5-lactone 6-phosphate + NADPH + H+ [G6PD dimer] 1909
18.6.2 alpha-D-glucose 6-phosphate + NADP+ => D-glucono-1,5-lactone 6-phosphate + NADPH + H+ [G6PD tetramer] 1910
18.6.3 D-glucono-1,5-lactone 6-phosphate + H2O => 6-phospho-D-gluconate 1911
18.6.4 6-phospho-D-gluconate + NADP+ => D-ribulose 5-phosphate + CO2 + NADPH + H+ 1912
18.6.5 D-ribulose 5-phosphate <=> xylulose 5-phosphate 1912
18.6.6 xylulose 5-phosphate <=> D-ribulose 5-phosphate 1913
18.6.7 ribose 5-phosphate + xylulose 5-phosphate <=> sedoheptulose 7-phosphate + D-glyceraldehyde 3-phosphate 1914
18.6.8 D-glyceraldehyde 3-phosphate + sedoheptulose 7-phosphate<=> xylulose 5-phosphate+ribose 5-phosphate 1914
18.6.9 sedoheptulose 7-phosphate + D-glyceraldehyde 3-phosphate <=> D-erythrose 4-phosphate + D-fructose 6-phosphate 1915
18.6.10 D-fructose 6-phosphate + D-erythrose 4-phosphate <=> sedoheptulose 7-phosphate + D-glyceraldehyde 3-phosphate 1916
18.6.11 xylulose 5-phosphate + D-erythrose 4-phosphate <=> D-glyceraldehyde 3-phosphate + D-fructose 6-phosphate 1916
18.6.12 D-glyceraldehyde 3-phosphate + D-fructose 6-phosphate <=> xylulose 5-phosphate + D-erythrose 4-phosphate 1917
18.6.13 D-ribulose 5-phosphate <=> ribose 5-phosphate 1918
18.6.14 ribose 5-phosphate <=> D-ribulose 5-phosphate 1918
18.7 5-Phosphoribose 1-diphosphate biosynthesis 1919
18.7.1 D-ribose 5-phosphate + 2'-deoxyadenosine 5'-triphosphate (dATP) => 5-Phospho-alpha-D-ribose 1-diphosphate (PRPP) + 1920
2'-deoxyadenosine 5'-monophosphate
18.7.2 D-ribose 5-phosphate + ATP => 5-phospho-alpha-D-ribose 1-diphosphate (PRPP) + adenosine 5'-monophosphate 1921
19 Metabolism of nitric oxide 1922
19.1 eNOS activation and regulation 1922
19.1.1 eNOS activation 1923
19.1.1.1 N-myristoylation of eNOS 1924
19.1.1.2 palmitoylation of eNOS 1925
19.1.1.3 eNOS translocation from Golgi to Caveolae 1925
19.1.1.4 Calcium binds calmodulin 1926
19.1.1.5 eNOS associates with Caveolin-1 1927
19.1.1.6 eNOS:Caveolin-1 complex binds to CaM 1927
19.1.1.7 HSP90 binds eNOS:Caveolin-1:CaM complex 1928
19.1.1.8 Caveolin-1 dissociates from eNOS:CaM:HSP90 complex 1929
19.1.1.9 Akt binds eNOS complex via HSP90 1930
19.1.1.10 Akt phosphorylates eNOS 1931
19.1.1.11 NO biosynthesis 1931
19.1.2 eNOS acylation cycle 1932
19.1.2.1 palmitoylation of eNOS 1933
19.1.2.2 eNOS translocation from Golgi to Caveolae 1933
19.1.2.3 depalmitoylated eNOS translocates from plasma membrane 1934
19.1.2.4 depalmitoylation of eNOS 1935
19.1.3 NOSIP mediated eNOS trafficking 1936
19.1.3.1 eNOS binds NOSIP 1936
19.1.3.2 eNOS:NOSIP translocation from caveolae to intracellular compartments 1937
19.1.4 NOSTRIN mediated eNOS trafficking 1938
19.1.4.1 eNOS associates with Caveolin-1 1938
19.1.4.2 eNOS:Caveolin-1 complex binds to Nostrin 1939
19.1.4.3 eNOS:Caveolin-1:NOSTRIN complex binds dynamin-2 1940
19.1.4.4 eNOS:Caveolin-1:NOSTRIN:dynamin-2 complex binds N-WASP 1940
19.1.4.5 NOSTRIN mediated translocation of eNOS from plasma membrane 1941
20 Metabolism of non-coding RNA 1942
20.1 snRNP Assembly 1942
20.1.1 Nuclear export of snRNA transcripts 1944
20.1.2 Loading and methylation of Sm proteins onto SMN Complexes 1945
20.1.3 snRNP complex assembly 1947
20.1.4 snRNA Cap hypermethylation 1949
20.1.5 snRNP:Snurportin complex formation 1950
20.1.6 snRNP nuclear import and release 1951
21 Metabolism of vitamins and cofactors 1954
21.1 Metabolism of water-soluble vitamins and cofactors 1954
21.1.1 Vitamin C (ascorbate) metabolism 1954
21.1.1.1 Dehydroascorbate transport across the plasma membrane 1955
21.1.1.2 Ascorbate transport across the plasma membrane 1956
21.1.1.3 Reduction of semidehydroascorbate to ascorbate 1956
21.1.1.4 Reduction of dehydroascorbate to ascorbate 1957
21.1.2 Vitamin B1 (thiamin) metabolism 1958
21.1.2.1 Thiamin transport across the plasma membrane 1958
21.1.2.2 Thiamin is pyrophosphorylated 1959
21.1.3 Vitamin B2 (riboflavin) metabolism 1960
21.1.3.1 Riboflavin is phosphorylated to FMN 1960

21.1.3.2 FMN is futher phosphorylated to FAD 1961
21.1.3.3 FAD can be hydrolyzed back to FMN 1961
21.1.3.4 FMN can be hydrolyzed back to riboflavin 1962
21.1.4 Vitamin B5 (pantothenate) metabolism 1963
21.1.4.1 Pantothenate transport across the plasma membrane 1963
21.1.4.2 Coenzyme A biosynthesis 1964
21.1.4.2.1 Pantothenate is phosphorylated [PANK2] 1964
21.1.4.2.2 Pantothenate is phosphorylated [PANK1;3;4] 1965
21.1.4.2.3 Condensation of phosphopantothenate with cysteine 1966
21.1.4.2.4 Phosphopantothenoylcysteine is decarboxylated 1967
21.1.4.2.5 Adenylation of phosphopantetheine 1967
21.1.4.2.6 Phosphorylation of dephospho-CoA to produce CoA 1968
21.1.4.2.7 CoA transport across the inner mitochondrial membrane 1969
21.1.4.3 Phosphopantetheine conjugation of the ACP domain of FAS 1970
21.1.5 Vitamin B12 (cobalamin) metabolism 1970
21.1.6 Biotin metabolism 1971
21.1.6.1 Biotin transport across the plasma membrane 1971
21.1.7 Nicotinate metabolism 1972
21.1.7.1 tryptophan + O2 => N-formylkynurenine [TDO] 1972
21.1.7.2 tryptophan + O2 => N-formylkynurenine [IDO] 1973
21.1.7.3 N-formylkynurenine + H2O => kynurenine + formate 1973
21.1.7.4 kynurenine + O2 + NADPH + H+ => 3-hydroxykynurenine + NADP+ + H2O 1974
21.1.7.5 3-hydroxykynurenine + H2O => 3-hydroxyanthranilate + alanine 1975
21.1.7.6 3-hydroxyanthranilate + O2 => 2-amino-3-carboxymuconate semialdehyde 1975
21.1.7.7 2-amino 3-carboxymuconate semialdehyde transforms non-enzymatically to quinolinate 1976
21.1.7.8 A phospho-ribosyl group is added to quinolinate 1976
21.1.7.9 Nicotinate D-ribonucleotide + ATP => deamino-NAD+ + pyrophosphate [NMNAT1] 1977
21.1.7.12 Amidation of deamino-NAD+ to NAD+ 1979
21.1.7.13 NAD+ is phosphorylated to NADP+ 1980
21.1.7.14 Nicotinamide salvaging 1980
21.1.7.14.1 Deamination of nicotinamide to nicotinate 1980
21.1.7.14.2 Condensation of nicotinamide to nicotinamide D-ribonucleotide (NMN) 1981
21.1.7.14.3 A phosphoribosyl group is added to nicotinate to form nicotinate mononucleotide (NaMN) 1982
21.1.8 Metabolism of folate and pterines 1983
21.1.8.1 Extracellular folate import across the plasma membrane 1983
21.1.8.2 Cytosolic folate export across the plasma membrane 1984
21.1.8.3 Extracellular 5-methyltetrahydrofolate import across the plasma membrane 1984
21.1.8.4 Folate is reduced to dihydrofolate (DHF) 1985
21.1.8.5 DHF is reduced to tetrahydrofolate (THF) 1985
21.1.8.6 Conversion of cytosolic THF to THF-polyglutamate 1986
21.1.8.7 Conversion of cytosolic 5-methyltetrahydrofolate (5-methylTHF) to 5-methylTHF-polyglutamate 1987
21.1.8.8 Cytosolic tetrahydrofolate import across the inner mitochondrial membrane 1987
21.1.8.9 Mitochondrial tetrahydrofolate export across the inner mitochondrial membrane 1988
21.1.8.10 Conversion of mitochondrial THF to THF-polyglutamate 1989
21.1.8.11 THF polyglutamate + formate + ATP => 10-formylTHF polyglutamate + ADP + orthophosphate 1990
21.1.8.12 10-formylTHF polyglutamate <=> 5,10-methenylTHF polyglutamate + H2O 1990
21.1.8.13 5,10-methenylTHF polyglutamate + NADPH + H+ <=> 5,10-methyleneTHF polyglutamate + NADP+ 1991
21.1.8.14 Tetrahydrofolate polyglutamate (THF polyglutamate) + serine <=> 5,10-methyleneTHF polyglutamate + glycine 1991
21.1.8.15 5,10-methyleneTHF polyglutamate + NADPH + H+ => 5-methylTHF polyglutamate + NADP+ 1992
21.1.8.16 5,10-methyleneTHF polyglutamate + glycine <=> tetrahydrofolate polyglutamate (THF polyglutamate) + serine 1993
21.1.8.17 5,10-methyleneTHF polyglutamate + NADP+ <=> 5,10-methenylTHF polyglutamate + NADPH + H+ 1993
21.1.8.18 5,10-methenylTHF polyglutamate + H2O <=> 10-formylTHF polyglutamate 1994
22 Nucleotide metabolism 1995
22.1 Transport of nucleosides and free purine and pyrimidine bases across the plasma membrane 1997
22.1.1 Concentrative transport (import) of a nucleoside and a sodium ions by solute carrier family 28 (sodium-coupled nucleoside 1998
transporter), member 1
22.1.2 Concentrative transport (import) of a nucleoside and two sodium ions by solute carrier family 28 (sodium-coupled nucleoside 1999
22.1.3 Concentrative transport (import) of nucleosides plus sodium ions by solute carrier family 28 (sodium-coupled nucleoside 1999
22.1.4 Equilibrative transport (export) of nucleosides and free bases by solute carrier family 29 (nucleoside transporters), member 1 2000
22.1.5 Equilibrative transport (export) of nucleosides and free bases by solute carrier family 29 (nucleoside transporters), member 2 2000
22.1.6 Equilibrative transport (import) of nucleosides and free bases by solute carrier family 29 (nucleoside transporters), member 1 2001
22.1.7 Equilibrative transport (import) of nucleosides and free bases by solute carrier family 29 (nucleoside transporters), member 2 2002
22.2 Purine metabolism 2002
22.2.1 De novo synthesis of IMP 2003

22.2.1.1 5-phospho-alpha-D-ribose 1-diphosphate (PRPP) + H2O + L-glutamine <=> 5-phosphoribosylamine + L-glutamate 2005
+pyrophosphate
22.2.1.2 5-phosphoribosylamine + glycine + ATP <=> 5-phosphoribosylglycinamide (GAR) + adenosine 5'-diphosphate + 2006
orthophosphate
22.2.1.3 5-phosphoribosylglycinamide (GAR) + 10-formyl-tetrahydrofolate => 5'-phosphoribosylformylglycinamide (FGAR) + 2007
tetrahydrofolate
22.2.1.4 5'-phosphoribosylformylglycinamide (FGAR) + L-glutamine + ATP + H2O => 5'-phosphoribosylformylglycinamidine (FGAM) + 2008
L-glutamate + adenosine 5'-diphosphate + orthophosphate
22.2.1.5 5'-phosphoribosylformylglycinamidine (FGAM) + ATP => 5'-phosphoribosyl-5-aminoimidazole (AIR) + ADP + orthophosphate 2009
22.2.1.6 5'-phosphoribosyl-5-aminoimidazole (AIR) + CO2 <=> 5'-phosphoribosyl-5-aminoimidazole-4-carboxylate (CAIR) 2010
22.2.1.7 5'-phosphoribosyl-5-aminoimidazole-4-carboxylate (CAIR) + L-aspartate + ATP <=> 2010
5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide (SAICAR) + adenosine 5'-diphosphate + orthophosphate
22.2.1.8 5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide (SAICAR) <=> 2011
5'-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR) + fumarate
22.2.1.9 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR) + 10-formyltetrahydrofolate => 2012
5'-phosphoribosyl-5-formaminoimidazole-4-carboxamide (FAICAR) + tetrahydrofolate
22.2.1.10 5'-phosphoribosyl-5-formaminoimidazole-4-carboxamide (FAICAR) <=> inosine 5'-monophosphate + H2O 2013
22.2.2 Purine biosynthesis 2014
22.2.2.1 dGTP formation 2015
22.2.2.1.1 guanosine 5'-diphosphate + thioredoxin (reduced) => 2'-deoxyguanosine 5'-diphosphate + thioredoxin (oxidized) + H2O 2015
22.2.2.1.2 thioredoxin, oxidized + NADPH + H+ => thioredoxin, reduced + NADP+ 2016
22.2.2.1.3 2'-deoxyguanosine 5'-diphosphate (dGDP) + ATP <=> dGTP + adenosine 5'-diphosphate (ADP) 2017
22.2.2.2 ATP formation 2017
22.2.2.2.1 Conversion of cytosolic inosine 5'-monophosphate, L-aspartate, and GTP to adenylosuccinate, guanosine 5'-diphosphate,2018
and orthophosphate
22.2.2.2.1.1 inosine 5'-monophosphate + L-aspartate + GTP => adenylosuccinate + guanosine 5'-diphosphate + orthophosphate 2019
22.2.2.2.1.2 inosine 5'-monophosphate + L-aspartate + GTP => adenylosuccinate + guanosine 5'-diphosphate + orthophosphate 2020
22.2.2.2.2 adenylosuccinate => adenosine 5'-monophosphate + fumarate 2021
22.2.2.2.3 adenosine 5'-monophosphate (AMP) + ATP <=> adenosine 5'-diphosphate (ADP) + ADP 2021
22.2.2.2.4 ADP-ATP translocase maintains a high ADP:ATP ratio in the matrix 2022
22.2.2.2.5 Formation of ATP by chemiosmotic coupling 2023
22.2.2.2.5.1 ADP and Pi bind to ATPase 2024
22.2.2.2.5.2 ATP is synthesized from ADP and Pi by ATPase 2024
22.2.2.2.5.3 Enzyme-bound ATP is released 2025
22.2.2.3 dATP formation 2026
22.2.2.3.1 adenosine 5'-diphosphate + thioredoxin (reduced) => 2'-deoxyadenosine 5'-diphosphate + thioredoxin (oxidized) + H2O 2026
22.2.2.3.2 thioredoxin, oxidized + NADPH + H+ => thioredoxin, reduced + NADP+ 2027
22.2.2.3.3 2'-deoxyadenosine 5'-diphosphate (dADP) + ATP <=> dATP + adenosine 5'-diphosphate (ADP) 2027
22.2.2.4 2'-deoxyadenosine 5'-diphosphate (dADP) + ATP <=> dATP + adenosine 5'-diphosphate (ADP) 2028
22.2.2.5 dAMP + ATP <=> dADP + ADP 2028
22.2.2.6 2'-deoxyguanosine 5'-diphosphate (dGDP) + ATP <=> dGTP + adenosine 5'-diphosphate (ADP) 2029
22.2.2.7 2'-deoxyguanosine 5'-monophosphate (dGMP) + ATP <=> 2'-deoxyguanosine 5'-diphosphate (dGDP) + ADP 2029
22.2.2.8 guanosine 5'-monophosphate (GMP) + ATP <=> guanosine 5'-diphosphate (GDP) + ADP 2030
22.2.2.9 Conversion of cytosolic inosine 5'-monophosphate (IMP), NAD+, and H2O to xanthosine 5'-monophosphate (XMP) and NADH2030
+ H+
22.2.2.9.1 inosine 5'-monophosphate (IMP) + NAD+ + H2O => xanthosine 5'-monophosphate (XMP) + NADH + H+ 2031
22.2.2.9.2 inosine 5'-monophosphate (IMP) + NAD+ + H2O => xanthosine 5'-monophosphate (XMP) + NADH + H+ 2032
22.2.2.10 xanthosine 5'-monophosphate (XMP) + L-glutamine + ATP + H2O => guanosine 5'-monophosphate (GMP) + L-glutamate + 2033
adenosine 5'-monophosphate (AMP) + pyrophosphate
22.2.3 Purine salvage reactions 2034
22.2.3.1 Deoxyribonucleotide salvage 2035
22.2.3.1.1 2'-deoxyguanosine 5'-monophosphate (dGMP) + H2O => 2'-deoxyguanosine + orthophosphate 2036
22.2.3.1.2 2'-deoxyguanosine + orthophosphate <=> guanine + 2-deoxy-D-ribose 1-phosphate 2036
22.2.3.1.3 2'-deoxyguanosine + ATP => 2'-deoxyguanosine 5'-monophosphate + ADP 2037
22.2.3.1.4 2'-Deoxyadenosine + H2O => 2'-Deoxyinosine + NH3 2038
22.2.3.1.5 2'-deoxyinosine + orthophosphate <=> hypoxanthine + 2-deoxy-D-ribose 1-phosphate 2038
22.2.3.1.6 2'-deoxyadenosine + ATP => 2'-deoxyadenosine 5'-monophosphate + ADP 2039
22.2.3.2 Ribonucleotide salvage 2040
22.2.3.2.1 guanosine 5'-monophosphate (GMP) + H2O => guanosine + orthophosphate 2040
22.2.3.2.2 guanosine + orthophosphate <=> guanine + D-ribose 1-phosphate 2040
22.2.3.2.3 Guanine + PRPP => GMP + PPi 2041
22.2.3.2.4 Adenine + PRPP => AMP + PPi 2042
22.2.3.2.5 adenosine + ATP => adenosine 5'-monophosphate (AMP) + ADP 2042
22.2.3.2.6 adenosine 5'-monophosphate (AMP) + H2O => inosine 5'-monophosphate (IMP) + NH4+ (L isoform) 2043
22.2.3.2.7 adenosine 5'-monophosphate (AMP) + H2O => inosine 5'-monophosphate (IMP) + NH4+ (M isoform) 2044
22.2.3.2.8 adenosine 5'-monophosphate (AMP) + H2O => inosine 5'-monophosphate (IMP) + NH4+ (E isoform) 2044
22.2.3.2.9 Adenosine + H2O => Inosine + NH3 2045
22.2.3.2.10 inosine 5'-monophosphate (IMP) + H2O => inosine + orthophosphate 2046

22.2.3.2.11 inosine + orthophosphate <=> hypoxanthine + D-ribose 1-phosphate 2046
22.2.3.2.12 Hypoxanthine + PRPP => IMP + PPi 2047
22.2.4 Purine catabolism 2047
22.2.4.1 Guanine + H2O => Xanthine + NH3 2048
22.2.4.2 Hypoxanthine + H2O + O2 => Xanthine + H2O2 2049
22.2.4.3 Xanthine + H2O + O2 => Urate + H2O2 2049
22.2.4.4 Xanthine formation 2050
22.2.4.4.1 Inosine formation 2050
22.2.4.4.1.1 Adenosine + H2O => Inosine + NH3 2051
22.2.4.4.1.2 inosine 5'-monophosphate (IMP) + H2O => inosine + orthophosphate 2051
22.2.4.4.2 inosine + orthophosphate <=> hypoxanthine + D-ribose 1-phosphate 2051
22.2.4.4.3 Hypoxanthine formation 2052
22.2.4.4.3.1 2'-Deoxyadenosine + H2O => 2'-Deoxyinosine + NH3 2052
22.2.4.4.3.2 2'-deoxyinosine + orthophosphate <=> hypoxanthine + 2-deoxy-D-ribose 1-phosphate 2052
22.2.4.4.4 Hypoxanthine + H2O + O2 => Xanthine + H2O2 2053
22.2.4.4.5 Guanine formation 2053
22.2.4.4.5.1 2'-deoxyguanosine 5'-monophosphate (dGMP) + H2O => 2'-deoxyguanosine + orthophosphate 2053
22.2.4.4.5.2 2'-deoxyguanosine + orthophosphate <=> guanine + 2-deoxy-D-ribose 1-phosphate 2054
22.2.4.4.5.3 guanosine 5'-monophosphate (GMP) + H2O => guanosine + orthophosphate 2054
22.2.4.4.5.4 guanosine + orthophosphate <=> guanine + D-ribose 1-phosphate 2055
22.2.4.4.6 Guanine + H2O => Xanthine + NH3 2055
22.3 Pyrimidine metabolism 2056
22.3.1 De novo synthesis of the pyrimidine ring and conversion to UMP 2056
22.3.1.1 L-glutamine + 2 ATP + HCO3- + H2O => carbamoyl phosphate + L-glutamate + 2 adenosine 5'-diphosphate + orthophosphate2058
22.3.1.2 carbamoyl phosphate + L-aspartate <=> N-carbamoyl L-aspartate + orthophosphate 2059
22.3.1.3 N-carbamoyl L-aspartate + H+ <=> (S)-dihydroorotate + H2O 2060
22.3.1.4 (S)-dihydroorotate + ubiquinone => orotate + ubiquinol 2060
22.3.1.5 orotate + 5-phospho-alpha-D-ribose 1-diphosphate (PRPP) <=> orotidine 5'-monophosphate (OMP) + pyrophosphate 2062
22.3.1.6 orotidine 5'-monophosphate => uridine 5'-monophosphate + CO2 2063
22.3.2 Pyrimidine biosynthesis (interconversion) 2064
22.3.2.1 uridine 5'-monophosphate (UMP) + ATP <=> uridine 5'-diphosphate (UDP) + ADP 2065
22.3.2.2 uridine 5'-diphosphate (UDP) + ATP <=> uridine 5'-triphosphate (UTP) + ADP 2065
22.3.2.3 uridine 5'-triphosphate + glutamine + ATP + H2O => cytidine 5'-triphosphate + glutamate + ADP + orthophosphate 2066
22.3.2.4 adenosine 5'-diphosphate (ADP) + CTP <=> ATP + cytidine 5'-diphosphate (CDP) 2066
22.3.2.5 cytidine 5'-diphosphate + thioredoxin (reduced) => 2'-deoxycytidine 5'-diphosphate + thioredoxin (oxidized) + H2O 2067
22.3.2.6 2'-deoxycytidine 5'-diphosphate (dCDP) + ATP <=> dCTP + adenosine 5'-diphosphate (ADP) 2068
22.3.2.7 adenosine 5'-diphosphate (ADP) + dCTP <=> ATP + 2'-deoxycytidine 5'-diphosphate (dCDP) 2069
22.3.2.8 2'-deoxycytidine 5'-diphosphate (dCDP) + ADP <=> deoxycytidine 5'-monophosphate (dCMP) + ATP 2069
22.3.2.9 2'-deoxycytidine 5'-monophosphate (dCMP) + H2O => 2'-deoxyuridine 5'-monophosphate (dUMP) + NH4+ 2070
22.3.2.10 2'-deoxycytidine 5'-monophosphate (dCMP) + ATP <=> deoxycytidine 5'-diphosphate (dCDP) + ADP 2071
22.3.2.11 cytidine 5'-diphosphate (CDP) + ADP <=> cytidine 5'-monophosphate (CMP) + ATP 2071
22.3.2.12 cytidine 5'-monophosphate (CMP) + ATP <=> cytidine 5'-diphosphate (CDP) + ADP 2072
22.3.2.13 cytidine 5'-diphosphate + ATP <=> cytidine 5'-triphosphate + ADP 2073
22.3.2.14 adenosine 5'-diphosphate (ADP) + UTP <=> ATP + uridine 5'-diphosphate (UDP) 2073
22.3.2.15 uridine 5'-diphosphate (UDP) + ADP <=> uridine 5'-monophosphate (UMP) + ATP 2074
22.3.2.16 uridine 5'-diphosphate + thioredoxin (reduced) => 2'-deoxyuridine 5'-diphosphate + thioredoxin (oxidized) + H2O 2075
22.3.2.17 2'-deoxyuridine 5'-monophosphate (dUMP) + ATP <=> 2'-deoxyuridine 5'-diphosphate (dUDP) + ADP 2076
22.3.2.18 2'-deoxyuridine 5'-diphosphate (dUDP) + ATP <=> deoxyuridine 5'-triphosphate (dUTP) + ADP 2076
22.3.2.19 2'-deoxyuridine 5'-triphosphate (dUTP) + H2O <=> deoxyuridine 5'-monophosphate (dUMP) + pyrophosphate 2077
22.3.2.20 2'-deoxyuridine 5'-monophosphate (dUMP) + N5,N10-methylene tetrahydrofolate => thymidine 5'-monophosphate (TMP) + 2077
dihydrofolate
22.3.2.21 thymidine 5'-monophosphate (TMP) + ATP <=> thymidine 5'-diphosphate (TDP) + ADP 2078
22.3.2.22 thymidine 5'-diphosphate (TDP) + ATP <=> thymidine 5'-triphosphate (TTP) + ADP 2079
22.3.2.23 adenosine 5'-diphosphate (ADP) + TTP <=> ATP + thymidine 5'-diphosphate (TDP) 2079
22.3.2.24 thymidine 5'-diphosphate (TDP) + ADP <=> thymidine 5'-monophosphate (TMP) + ATP 2080
22.3.2.25 2'-deoxyuridine 5'-triphosphate (dUTP) + ADP <=> 2'-deoxyuridine 5'-diphosphate (dUDP) + ATP 2081
22.3.2.26 2'-deoxyuridine 5'-diphosphate (dUDP) + ADP <=> 2'deoxyuridine 5'-monophosphate (dUMP) + ATP 2082
22.3.3 Pyrimidine salvage reactions 2082
22.3.3.1 uracil + D-ribose 1-phosphate <=> uridine + orthophosphate 2083
22.3.3.2 uridine + orthophosphate <=> uracil + D-ribose 1-phosphate 2084
22.3.3.3 cytidine + H2O => uridine + NH4+ 2084
22.3.3.4 uridine + ATP => uridine 5'-monophosphate (UMP) + ADP 2085
22.3.3.5 uridine 5'-monophosphate + H2O => uridine + orthophosphate 2086
22.3.3.6 uracil + 5-phospho-alpha-D-ribose 1-diphosphate (PRPP) <=> uridine 5'-monophosphate (UMP) + pyrophosphate 2086
22.3.3.7 cytidine + ATP => cytidine 5'-monophosphate (CMP) + ADP 2087
22.3.3.8 cytidine 5'-monophosphate + H2O => cytidine + orthophosphate 2087
22.3.3.9 2'-deoxycytidine + ATP => 2'-deoxycytidine 5'-monophosphate (dCMP) + ADP 2088
22.3.3.10 2'-deoxycytidine 5'-monophosphate + H2O => 2'-deoxycytidine + orthophosphate 2089

22.3.3.11 2'-deoxycytidine + H2O => 2'-deoxyuridine + NH4+ 2090
22.3.3.12 2'-deoxyuridine 5'-monophosphate + H2O => 2'-deoxyuridine + orthophosphate 2090
22.3.3.13 2'-deoxyuridine + orthophosphate <=> uracil + 2-deoxy-D-ribose 1-phosphate 2091
22.3.3.14 uracil + 2-deoxy-D-ribose 1-phosphate <=> 2'-deoxyuridine + orthophosphate 2091
22.3.3.15 thymine + 2-deoxy-D-ribose 1-phosphate <=> thymidine + orthophosphate 2092
22.3.3.16 thymidine + orthophosphate <=> thymine + 2-deoxy-D-ribose 1-phosphate 2093
22.3.3.17 thymidine + ATP => thymidine 5'-monophosphate (dTMP) + ADP 2093
22.3.3.18 thymidine 5'-monophosphate + H2O => thymidine + orthophosphate 2094
22.3.4 Reversible phosphorolysis of pyrimidine nucleosides 2095
22.3.4.1 Reversible phosphorolysis of pyrimidine nucleosides by thymidine phosphorylase 2096
22.3.4.1.1 thymidine + orthophosphate <=> thymine + 2-deoxy-D-ribose 1-phosphate 2096
22.3.4.1.2 thymine + 2-deoxy-D-ribose 1-phosphate <=> thymidine + orthophosphate 2096
22.3.4.1.3 2'-deoxyuridine + orthophosphate <=> uracil + 2-deoxy-D-ribose 1-phosphate 2097
22.3.4.1.4 uracil + 2-deoxy-D-ribose 1-phosphate <=> 2'-deoxyuridine + orthophosphate 2097
22.3.4.2 Reversible phosphorolysis of pyrimidine nucleosides by uridine phosphorylase 1 2098
22.3.4.2.1 uridine + orthophosphate <=> uracil + D-ribose 1-phosphate 2098
22.3.4.2.2 uracil + D-ribose 1-phosphate <=> uridine + orthophosphate 2099
22.3.4.2.3 uracil + 2-deoxy-D-ribose 1-phosphate <=> 2'-deoxyuridine + orthophosphate 2099
22.3.4.2.4 2'-deoxyuridine + orthophosphate <=> uracil + 2-deoxy-D-ribose 1-phosphate 2100
22.3.5 Pyrimidine catabolism 2100
22.3.5.1 uracil + NADPH + H+ => 5,6-dihydrouracil + NADP+ 2101
22.3.5.2 5,6-dihydrouracil + H2O => beta-ureidopropionate 2102
22.3.5.3 beta-ureidopropionate + H2O => beta-alanine + NH4+ + CO2 2103
22.3.5.4 thymine + NADPH + H+ => 5,6-dihydrothymine + NADP+ 2103
22.3.5.5 5,6-dihydrothymine + H2O => beta-ureidoisobutyrate 2104
22.3.5.6 beta-ureidoisobutyrate + H2O => beta-aminoisobutyrate + NH4+ + CO2 2104
22.4 Synthesis of deoxyribonucleoside 5'-diphosphates from ribonucleoside 5'-diphosphates 2105
22.4.1 Reduction of cytosolic ribonucleoside 5'-diphosphates to deoxyribonucleoside 5'-diphosphates by ribonucleotide reductase and 2107
glutaredoxin
22.4.1.1 Reduction of cytosolic ribonucleoside 5'-diphosphates to deoxyribonucleoside 5'-diphosphates (glutaredoxin) 2107
22.4.1.1.1 adenosine 5'-diphosphate + glutaredoxin (reduced) => 2'-deoxyadenosine 5'-diphosphate + glutaredoxin (oxidized) + H2O2107
22.4.1.1.2 cytidine 5'-diphosphate + glutaredoxin (reduced) => 2'-deoxycytidine 5'-diphosphate + glutaredoxin (oxidized) + H2O 2108
22.4.1.1.3 guanosine 5'-diphosphate + glutaredoxin (reduced) => 2'-deoxyguanosine 5'-diphosphate + glutaredoxin (oxidized) + H2O2109
22.4.1.1.4 uridine 5'-diphosphate + glutaredoxin (reduced) => 2'-deoxyuridine 5'-diphosphate + glutaredoxin (oxidized) + H2O 2109
22.4.1.2 glutaredoxin (oxidized) + glutathione (reduced) => glutaredoxin (reduced) + glutathione (oxidized) 2110
22.4.1.3 glutathione (oxidized) + NADPH + H+ => 2 glutathione (reduced) + NADP+ 2110
22.4.2 Reduction of cytosolic ribonucleoside 5'-diphosphates to deoxyribonucleoside 5'-diphosphates by ribonucleotide reductase and 2111
thioredoxin
22.4.2.1 Reduction of cytosolic ribonucleoside 5'-diphosphates to deoxyribonucleoside 5'-diphosphates (thioredoxin) 2111
22.4.2.1.1 uridine 5'-diphosphate + thioredoxin (reduced) => 2'-deoxyuridine 5'-diphosphate + thioredoxin (oxidized) + H2O 2112
22.4.2.1.2 cytidine 5'-diphosphate + thioredoxin (reduced) => 2'-deoxycytidine 5'-diphosphate + thioredoxin (oxidized) + H2O 2112
22.4.2.2 thioredoxin, oxidized + NADPH + H+ => thioredoxin, reduced + NADP+ 2114
22.4.3 Reduction of nuclear ribonucleoside 5'-diphosphates to deoxyribonucleoside 5'-diphosphates by ribonucleotide reductase and 2114
thioredoxin
22.4.3.1 thioredoxin, reduced (cytosol) <=> thioredoxin, reduced (nucleus) 2115
22.4.3.2 Reduction of nuclear ribonucleoside 5'-diphosphates to deoxyribonucleoside 5'-diphosphates (thioredoxin) 2115
22.4.3.2.2 cytidine 5'-diphosphate + thioredoxin (reduced) => 2'-deoxycytidine 5'-diphosphate + thioredoxin (oxidized) + H2O 2116
22.4.3.2.4 uridine 5'-diphosphate + thioredoxin (reduced) => 2'-deoxyuridine 5'-diphosphate + thioredoxin (oxidized) + H2O 2118
22.4.3.3 thioredoxin, oxidized (nucleus) <=> thioredoxin, oxidized (cytosol) 2119
22.4.3.4 thioredoxin, oxidized + NADPH + H+ => thioredoxin, reduced + NADP+ 2119
22.5 Hydrolysis of nucleoside 5'-monophosphates to nucleosides plus orthophosphate 2120
22.5.1 Hydrolysis of extracellular nucleoside 5'-monophosphates by plasma membrane-bound 5'-nucleotidase 2121
22.5.1.1 adenosine 5'-monophosphate (AMP) + H2O => adenosine + orthophosphate 2122
22.5.1.2 uridine 5'-monophosphate (UMP) + H2O => uridine + orthophosphate 2122
22.5.1.3 guanosine 5'-monophosphate (GMP) + H2O => guanosine + orthophosphate 2123
22.5.1.4 inosine 5'-monophosphate (IMP) + H2O => inosine + orthophosphate 2124
22.5.1.5 cytidine 5'-monophosphate (CMP) + H2O => cytidine + orthophosphate 2124
22.5.1.6 thymidine 5'-monophosphate (TMP) + H2O => thymidine + orthophosphate 2125
22.5.1.7 2'-deoxyadenosine 5'-monophosphate (dAMP) + H2O => deoxyadenosine + orthophosphate 2126
22.5.2 Hydrolysis of cytosolic nucleoside 5'-monophosphates by 5'-nucleotidase cytosolic II 2127
22.5.2.2 2'-deoxyguanosine 5'-monophosphate (dGMP) + H2O => 2'-deoxyguanosine + orthophosphate 2128
22.5.2.4 2'-deoxyinosine 5'-monophosphate (dIMP) + H2O => 2'-deoxyinosine + orthophosphate 2129

22.5.3 Hydrolysis of cytosolic nucleoside 5'-monophosphates by 5'-nucleotidase cytosolic IA 2129
22.5.3.2 2'-deoxycytidine 5'-monophosphate (dCMP) + H2O => 2'-deoxycytidine + orthophosphate 2131
22.5.3.3 2'-deoxyuridine 5'-monophosphate (dUMP) + H2O => 2'-deoxyuridine + orthophosphate 2132
22.5.3.4 2'-deoxyadenosine 5'-monophosphate (dAMP) + H2O => 2'-deoxyadenosine + orthophosphate 2132
22.5.3.6 2'-deoxyinosine 5'-monophosphate (dIMP) + H2O => 2'deoxyinosine + orthophosphate 2134
22.5.4 Hydrolysis of cytosolic nucleoside 5'-monophosphates by 5'-nucleotidase cytosolic IB 2138
22.5.5 Hydrolysis of cytosolic nucleoside 5'-monophosphates by 5'-nucleotidase, cytosolic III 2139
22.5.5.3 2'-deoxycytidine 5'-monophosphate (dCMP) + H2O => 2'-deoxycytidine + orthophosphate 2141
22.5.6 Hydrolysis of cytosolic nucleoside 5'- and 3'-monophosphates by 5',3'-nucleotidase, cytosolic 2143
22.5.6.2 2'-deoxyinosine 5'-monophosphate (dIMP) + H2O => 2'-deoxyinosine + orthophosphate 2144
22.5.6.3 2'-deoxyuridine 3'-monophosphate + H2O => 2'-deoxyuridine + orthophosphate 2145
22.5.7 Hydrolysis of mitochondrial nucleoside 5'- and 3'-monophosphates by 5',3'-nucleotidase, mitochondrial 2150
22.6 Phosphorylation of nucleosides to form nucleoside 5'-monophosphates 2155
22.6.1 Phosphorylation of cytosolic nucleosides by adenosine kinase 2156
22.6.1.1 adenosine + ATP => adenosine 5'-monophosphate (AMP) + ADP 2157
22.6.1.2 2'-deoxyadenosine + ATP => 2'-deoxyadenosine 5'-monophosphate + ADP 2157
22.6.2 Phosphorylation of cytosolic nucleosides by deoxycytidine kinase 2158
22.6.2.1 2'-deoxycytidine + ATP => 2'-deoxycytidine 5'-monophosphate (dCMP) + ADP 2159
22.6.2.3 2'-deoxyguanosine + ATP => 2'-deoxyguanosine 5'-monophosphate + ADP 2160
22.6.3 Phosphorylation of mitochondrial nucleosides by deoxyguanosine kinase 2162
22.6.3.1 2'-deoxyguanosine + ATP => 2'-deoxyguanosine 5'-monophosphate + ADP 2162
22.6.3.2 2'-deoxyinosine + ATP => 2'-deoxyinosine 5'-monophosphate + ADP 2163
22.6.4 Phosphorylation of mitochondrial nucleosides by thymidine kinase 2, mitochondrial 2164
22.6.4.1 thymidine + ATP => thymidine 5'-monophosphate + ADP 2165
22.6.4.2 2'-deoxyuridine + ATP => 2'-deoxyuridine 5'-monophosphate + ADP 2166
22.6.4.3 2'-deoxycytidine + ATP => 2'-deoxycytidine 5'-monophosphate + ADP 2167
22.6.5 Phosphorylation of cytosolic nucleosides by thymidine kinase 1, soluble 2167
22.6.5.1 thymidine + ATP => thymidine 5'-monophosphate (dTMP) + ADP 2168
22.6.6 Phosphorylation of cytosolic nucleosides by uridine-cytidine kinase 1 2169
22.6.7 Phosphorylation of cytosolic nucleosides by uridine-cytidine kinase 2 2170
22.7 Reversible phosphorylation of nucleoside monophosphates 2172
22.7.1 Reversible phosphorylation of cytosolic nucleoside monophosphates by deoxythymidylate kinase (thymidylate kinase) 2173
22.7.1.1 thymidine 5'-monophosphate (TMP) + ATP <=> thymidine 5'-diphosphate (TDP) + ADP 2174
22.7.1.2 2'-deoxyuridine 5'-monophosphate (dUMP) + ATP <=> 2'-deoxyuridine 5'-diphosphate (dUDP) + ADP 2174
22.7.1.3 2'-deoxyuridine 5'-diphosphate (dUDP) + ADP <=> 2'deoxyuridine 5'-monophosphate (dUMP) + ATP 2175
22.7.1.4 thymidine 5'-diphosphate (TDP) + ADP <=> thymidine 5'-monophosphate (TMP) + ATP 2175
22.7.2 Reversible phosphorylation of cytosolic nucleoside monophosphates by UMP-CMP kinase 2176
22.7.2.2 uridine 5'-monophosphate (UMP) + ATP <=> uridine 5'-diphosphate (UDP) + ADP 2177
22.7.2.3 2'-deoxycytidine 5'-monophosphate (dCMP) + ATP <=> deoxycytidine 5'-diphosphate (dCDP) + ADP 2177
22.7.2.4 uridine 5'-diphosphate (UDP) + ADP <=> uridine 5'-monophosphate (UMP) + ATP 2178
22.7.2.5 2'-deoxycytidine 5'-diphosphate (dCDP) + ADP <=> deoxycytidine 5'-monophosphate (dCMP) + ATP 2178
22.7.3 Reversible phosphorylation of cytosolic nucleoside monophosphates by guanylate kinase 1 2179
22.7.3.1 guanosine 5'-monophosphate (GMP) + ATP <=> guanosine 5'-diphosphate (GDP) + ADP 2180
22.7.3.2 2'-deoxyguanosine 5'-monophosphate (dGMP) + ATP <=> 2'-deoxyguanosine 5'-diphosphate (dGDP) + ADP 2180
22.7.3.3 guanosine 5'-diphosphate (GDP) + ADP <=> guanosine 5'-monophosphate (GMP) + ATP 2181
22.7.3.4 2'-deoxyguanosine 5'-diphosphate (dGDP) + ADP <=> 2'-deoxyguanosine 5'-monophosphate (dGMP) + ATP 2182
22.7.4 Reversible phosphorylation of cytosolic nucleoside monophosphates by adenylate kinase 1 2182
22.7.4.1 adenosine 5'-monophosphate (AMP) + ATP <=> adenosine 5'-diphosphate (ADP) + ADP 2183
22.7.4.2 2'-deoxyadenosine 5'-diphosphate (dADP) + ADP <=> 2'-deoxyadenosine 5'-monophosphate (dAMP) + ATP 2183
22.7.4.3 2'-deoxyadenosine 5'-monophosphate (dAMP) + ATP <=> 2'-deoxyadenosine 5'-diphosphate (dADP) + ADP 2184
22.7.4.4 adenosine 5'-diphosphate (ADP) + ADP <=> adenosine 5'-monophosphate (AMP) + ATP 2185
22.7.5 Reversible phosphorylation of cytosolic nucleoside monophosphates by adenylate kinase 5 2185
22.7.5.1 2'-deoxyadenosine 5'-diphosphate (dADP) + ADP <=> 2'-deoxyadenosine 5'-monophosphate (dAMP) + ATP 2186
22.7.5.2 2'-deoxyadenosine 5'-monophosphate (dAMP) + ATP <=> 2'-deoxyadenosine 5'-diphosphate (dADP) + ADP 2187
22.7.5.3 2'-deoxycytidine 5'-diphosphate (dCDP) + ADP <=> 2'-deoxycytidine 5'-monophosphate (dCMP) + ATP 2187
22.7.5.4 2'-deoxycytidine 5'-monophosphate (dCMP) + ATP <=> 2'-deoxycytidine 5'-diphosphate (dCDP) + ADP 2188
22.7.6 Reversible phosphorylation of mitochondrial nucleoside monophosphates by adenylate kinase 2 2191
22.8 Reversible phosphorylation of nucleoside diphosphates 2193
22.8.1 Reversible phosphorylation of cytosolic nucleoside diphosphates by nucleoside diphosphate kinase A hexamer 2194
22.8.1.1 cytidine 5'-diphosphate (CDP) + ATP <=> CTP + adenosine 5'-diphosphate (ADP) 2195
22.8.1.4 adenosine 5'-diphosphate (ADP) + dATP <=> ATP + 2'-deoxyadenosine 5'-diphosphate (dADP) 2198
22.8.1.8 adenosine 5'-diphosphate (ADP) + dGTP <=> ATP + 2'-deoxyguanosine 5'-diphosphate (dGDP) 2201
22.8.1.9 2'-deoxyuridine 5'-diphosphate (dUDP) + ATP <=> dUTP + adenosine 5'-diphosphate (ADP) 2202
22.8.1.10 adenosine 5'-diphosphate (ADP) + dUTP <=> ATP + 2'-deoxyuridine 5'-diphosphate (dUDP) 2202
22.8.1.11 adenosine 5'-diphosphate (ADP) + GTP <=> ATP + guanosine 5'-diphosphate (GDP) 2203
22.8.1.12 guanosine 5'-diphosphate (GDP) + ATP <=> GTP + adenosine 5'-diphosphate (ADP) 2204
22.8.1.14 thymidine 5'-diphosphate (TDP) + ATP <=> TTP + adenosine 5'-diphosphate (ADP) 2206
22.8.1.16 uridine 5'-diphosphate (UDP) + ATP <=> UTP + adenosine 5'-diphosphate (ADP) 2207
22.8.2 Reversible phosphorylation of cytosolic nucleoside diphosphates by nucleoside diphosphate kinase B hexamer 2208
22.8.3 Reversible phosphorylation of cytosolic nucleoside diphosphates by nucleoside diphosphate kinase C hexamer 2222
22.8.4 Reversible phosphorylation of mitochondrial nucleoside diphosphates by nucleoside diphosphate kinase D, mitochondrial hexamer
2234
22.8.5 Reversible phosphorylation of cytosolic nucleoside diphosphates by nucleoside diphosphate kinase A,B heterohexamer 2247
22.8.5.1 cytidine 5'-diphosphate + ATP <=> cytidine 5'-triphosphate + ADP 2248
22.8.5.2 uridine 5'-diphosphate (UDP) + ATP <=> uridine 5'-triphosphate (UTP) + ADP 2248
22.8.5.4 thymidine 5'-diphosphate (TDP) + ATP <=> thymidine 5'-triphosphate (TTP) + ADP 2249
22.8.5.5 2'-deoxyuridine 5'-diphosphate (dUDP) + ATP <=> deoxyuridine 5'-triphosphate (dUTP) + ADP 2250
22.8.5.11 2'-deoxyuridine 5'-triphosphate (dUTP) + ADP <=> 2'-deoxyuridine 5'-diphosphate (dUDP) + ATP 2253
23 Porphyrin metabolism 2258
23.1 Heme biosynthesis 2258
23.1.1 Succinyl CoA and glycine condense to form 5-aminolevulinate (ALA) 2260
23.1.2 ALA is transported from the mitochondrial matrix to the cytosol 2261
23.1.3 ALAD octamer associates with Pb++, forming a catalytically inactive complex 2261
23.1.4 Two molecules of ALA condense to form porphobilinogen (PBG) 2262
23.1.5 Four PBGs combine through deamination to form hydroxymethylbilane (HMB) 2263
23.1.6 Conversion of HMB to uroporphyrinogen III 2264
23.1.7 Spontaneous conversion of HMB to uroporphyrinogen I 2265
23.1.8 Uroporphyrinogen III is decarboxylated to form coproporphyrinogen III 2266
23.1.9 Uroporphyrinogen I is decarboxylated to form coproporphyrinogen I 2267
23.1.10 Translocation of coproporphyrinogen III from the cytosol to the mitochondrial intermembrane space 2268
23.1.11 Conversion of coproporphyrinogen III to protoporphyrinogen IX 2269
23.1.12 Oxidation of protoporphyrinogen IX to protoporphyrin IX 2270
23.1.13 Protoporphyrin IX is transported from the mitochondrial intermembrane space into the mitochondrial matrix 2271
23.1.14 Ferrous iron is inserted into protoporphyrin IX to form heme 2271
24 Pyruvate metabolism and TCA cycle 2273
24.1 Oxidative decarboxylation of pyruvate to acetyl CoA by pyruvate dehydrogenase 2274
24.1.1 pyruvate + TPP => 2-(alpha-hydroxyethyl)-TPP + CO2 2275
24.1.2 2-(alpha-hydroxyethyl)-TPP + lipoamide => S-acetyldihydrolipoamide + TPP 2275
24.1.3 S-acetyldihydrolipoamide + CoA => acetyl-CoA + dihydrolipoamide 2276

24.1.4 dihydrolipoamide + FAD => lipoamide + FADH2 [pyruvate dehydrogenase] 2276
24.1.5 FADH2 + NAD+ => FAD + NADH + H+ [pyruvate dehydrogenase] 2277
24.2 Citric acid cycle (TCA cycle) 2278
24.2.1 Acetyl-CoA + H2O + Oxaloacetate => Citrate + CoA 2278
24.2.2 Citrate <=> cis-Aconitate + H2O 2279
24.2.3 cis-Aconitate + H2O <=> Isocitrate 2280
24.2.4 Isocitrate + NAD+ => alpha-ketoglutarate + CO2 + NADH + H+ 2280
24.2.5 Oxidative decarboxylation of alpha-ketoglutarate to succinyl CoA by alpha-ketoglutarate dehydrogenase 2281
24.2.5.1 alpha-ketoglutarate + TPP => 3-carboxy-1-hydroxypropyl-TPP + CO2 2282
24.2.5.2 3-carboxy-1-hydroxypropyl-TPP + lipoamide => S-succinyldihydrolipoamide + TPP 2282
24.2.5.3 S-succinyldihydrolipoamide + CoA => succinyl-CoA + dihydrolipoamide 2283
24.2.5.4 dihydrolipoamide + FAD => lipoamide + FADH2 [alpha-ketoglutarate dehydrogenase] 2284
24.2.5.5 FADH2 + NAD+ => FAD + NADH + H+ [alpha-ketoglutarate dehydrogenase] 2284
24.2.6 GDP + Orthophosphate + Succinyl-CoA <=> GTP + Succinate + CoA 2285
24.2.7 ADP + Orthophosphate + Succinyl-CoA <=> ATP + Succinate + CoA 2286
24.2.8 Succinate <=> Fumarate (with FAD redox reaction on enzyme) 2287
24.2.9 Fumarate + H2O <=> (S)-Malate 2288
24.2.10 (S)-Malate + NAD+ <=> Oxaloacetate + NADH + H+ 2288
25 Post-translational protein modification 2290
25.1 Gamma-carboxylation, transport, and amino-terminal cleavage of proteins 2290
25.1.1 Gamma-carboxylation of protein precursors 2291
25.1.1.1 pro-factor IX, uncarboxylated + 12 CO2 + 12 O2 + 12 vitamin K hydroquinone -> pro-factor IX + 12 H2O + 12 vitamin K epoxide
2292
25.1.1.2 pro-factor VII, uncarboxylated + 10 CO2 + 10 O2 + 10 vitamin K hydroquinone -> pro-factor VII + 10 H2O + 10 vitamin K 2292
epoxide
25.1.1.3 pro-factor X, uncarboxylated + 11 CO2 + 11 O2 + 11 vitamin K hydroquinone -> pro-factor X + 11 H2O + 11 vitamin K epoxide2293
25.1.1.4 pro-prothrombin, uncarboxylated + 10 CO2 + 10 O2 + 10 vitamin K hydroquinone -> pro-prothrombin + 10 H2O + 10 vitamin K2294
epoxide
25.1.1.5 pro-protein C, uncarboxylated + 8 CO2 + 8 O2 + 8 vitamin K hydroquinone -> pro-protein C + 8 H2O + 8 vitamin K epoxide 2295
25.1.1.6 pro-protein S, uncarboxylated + 11 CO2 + 11 O2 + 11 vitamin K hydroquinone -> pro-protein S + 11 H2O + 11 vitamin K 2295
epoxide
25.1.1.7 pro-protein Z, uncarboxylated + 13 CO2 + 13 O2 + 13 vitamin K hydroquinone -> pro-protein Z + 13 H2O + 13 vitamin K 2296
epoxide
25.1.1.8 pro-GAS6, uncarboxylated + 11 CO2 + 11 O2 + 11 vitamin K hydroquinone -> pro-GAS6 + 11 H2O + 11 vitamin K epoxide 2297
25.1.2 Transport of gamma-carboxylated protein precursors from the endoplasmic reticulum to the Golgi apparatus 2298
25.1.2.1 Pro-factor IX is transported from the endoplasmic reticulum to the Golgi apparatus 2298
25.1.2.2 Pro-factor VII is transported from the endoplasmic reticulum to the Golgi apparatus 2299
25.1.2.3 Pro-factor X is transported from the endoplasmic reticulum to the Golgi apparatus 2299
25.1.2.4 Pro-prothrombin is transported from the endoplasmic reticulum to the Golgi apparatus 2299
25.1.2.5 Pro-protein C is transported from the endoplasmic reticulum to the Golgi apparatus 2300
25.1.2.6 Pro-protein S is transported from the endoplasmic reticulum to the Golgi apparatus 2300
25.1.2.7 Pro-protein Z is transported from the endoplasmic reticulum to the Golgi apparatus 2301
25.1.2.8 Pro-GAS6 is transported from the endoplasmic reticulum to the Golgi apparatus 2301
25.1.3 Removal of aminoterminal propeptides from gamma-carboxylated proteins 2301
25.1.3.1 pro-factor IX -> factor IX + factor IX propeptide 2302
25.1.3.2 pro-factor VII -> factor VII + factor VII propeptide 2303
25.1.3.3 pro-factor X -> factor X + factor X propeptide 2303
25.1.3.4 pro-prothrombin -> prothrombin + prothrombin propeptide 2304
25.1.3.5 pro-protein C -> protein C + protein C propeptide 2304
25.1.3.6 pro-protein S -> protein S + protein S propeptide 2305
25.1.3.7 pro-protein Z -> protein Z + protein Z propeptide 2305
25.1.3.8 pro-GAS6 -> GAS6 + GAS6 propeptide 2306
25.2 Synthesis of GPI-anchored proteins 2306
25.2.1 Synthesis of dolichol-phosphate mannose 2307
25.2.1.1 dolichol phosphate + GDP-alpha-D-mannose -> dolichyl phosphate D-mannose 2308
25.2.1.2 Reorientation of dolichyl phosphate D-mannose in the endoplasmic reticulum membrane 2308
25.2.2 Synthesis of glycosylphosphatidylinositol (GPI) 2309
25.2.2.1 phosphatidylinositol + UDP-N-acetyl-D-glucosamine -> N-acetylglucosaminyl-PI + UDP 2310
25.2.2.2 N-acetylglucosaminyl-PI + H2O -> glucosaminyl-PI + acetate 2311
25.2.2.3 Reorientation of glucosaminyl-acyl-PI in the endoplasmic reticulum membrane 2312
25.2.2.4 glucosaminyl-PI + fatty acyl-CoA -> glucosaminyl-acyl-PI + CoA-SH 2313
25.2.2.5 glucosaminyl-acyl-PI + dolichol phosphate D-mannose -> mannose(al1-4)glucosaminyl-acyl-PI + dolichol phosphate 2314
25.2.2.6 mannose(a1-4)glucosaminyl-acyl-PI + phosphatidylethanolamine -> (ethanolamineP) mannose(al1-4)glucosaminyl-acyl-PI + 2314
diacylglycerol
25.2.2.7 (ethanolamineP) mannose (a1-4) glucosaminyl-acyl-PI + dolichol phosphate D-mannose -> mannose (a1-6) (ethanolamineP) 2315
mannose (a1-4) glucosaminyl-acyl-PI + dolichol phosphate
25.2.2.8 mannose (a1-6) (ethanolamineP) mannose (a1-4) glucosaminyl-acyl-PI + dolichol phosphate D-mannose -> mannose (a1-2) 2316
mannose (a1-6) (ethanolamineP) mannose (a1-4) glucosaminyl-acyl-PI + dolichol phosphate
25.2.2.9 mannose (a1-2) mannose (a1-6) (ethanolamineP) mannose (a1-4) glucosaminyl-acyl-PI + phosphatidylethanolamine -> 2317
(ethanolamineP) mannose (a1-2) mannose (a1-6) (ethanolamineP) mannose (a1-4) glucosaminyl-acyl-PI (acyl-GPI) + diacylglycerol
25.2.2.10 mannose (a1-2) mannose (a1-6) (ethanolamineP) mannose (a1-4) glucosaminyl-acyl-PI -> mannose (a1) mannose (a1-2) 2318
mannose (a1-6) (ethanolamineP) mannose (a1-4) glucosaminyl-acyl-PI
25.2.2.11 (ethanolamineP) mannose (a1-2) mannose (a1-6) (ethanolamineP) mannose (a1-4) glucosaminyl-acyl-PI -> (ethanolamineP)2319
mannose (a1-2) (ethanolamineP) mannose (a1-6) (ethanolamineP) mannose (a1-4) glucosaminyl-acyl-PI
25.2.3 Attachment of GPI anchor to uPAR 2319
25.2.3.1 uPAR precursor + acyl-GPI -> uPAR-acyl-GPI + uPAR propeptide 2320
25.2.3.2 uPAR-acyl-GPI + H2O -> uPAR + long-chain fatty acid 2321
25.3 Hypusine synthesis from eIF5A-lysine 2322
25.3.1 EIF5A + spermidine <=> EIF5A(Dhp) + 1,3-diaminopropane 2322
25.3.2 EIF5A(Dhp) + O2 => EIF5A(Hyp) 2323
26 Regulation of beta-cell development 2325
26.1 Regulation of gene expression in early pancreatic precursor cells 2326
26.1.1 HNF1B-dependent synthesis of HNF6 protein 2327
26.1.2 HNF1B- and FGF10-dependent synthesis of PTF1A protein 2328
26.1.3 HNF6-dependent synthesis of ONECUT3 protein during early pancreas specification 2329
26.1.4 HNF6- and FGF10-dependent synthesis of PDX1 protein 2330
26.1.5 PDX1-dependent synthesis of NR5A2 protein 2331
26.1.6 PDX1-dependent synthesis of NKX6-1 protein 2332
26.2 Regulation of gene expression in late stage (branching morphogenesis) pancreatic bud precursor cells 2334
26.2.1 HNF6-dependent synthesis of HNF1B protein 2334
26.2.2 HNF6-dependent synthesis of ONECUT3 protein during morphogenesis 2335
26.2.3 HNF6-dependent synthesis of NEUROG3 protein during morphogenesis 2336
26.2.4 RBPJ- and NOTCH1-dependent synthesis of HES1 protein during morphogenesis 2338
26.3 Regulation of gene expression in endocrine-committed (NEUROG3+) progenitor cells 2339
26.3.1 NEUROG3-dependent synthesis of PAX4 protein 2340
26.3.2 NEUROG3-dependent synthesis of NEUROD1 2341
26.3.3 NEUROG3-dependent synthesis of NKX2-2 2342
26.3.4 NEUROG3-dependent synthesis of INSM1 2343
26.4 Regulation of gene expression in beta cells 2344
26.4.1 AKT-mediated inactivation of FOXO1A 2344
26.4.1.1 AKT phosphorylates FOXO1A 2345
26.4.1.2 Phosphorylated FOXO1A is excluded from the nucleus 2346
26.4.2 FOXOA2-, MAFA-, and PAX6-dependent synthesis of PDX1 protein 2347
26.4.3 MAFA-, NKX2-2-, PAX6-, and PDX1-dependent synthesis of insulin precursor protein 2348
26.4.4 PDX1-dependent synthesis of IAPP protein 2349
26.4.5 PDX1-dependent synthesis of NKX6-1 protein 2350
26.4.6 NEUROD1- and PDX1-dependent synthesis of glucokinase (GCK) protein 2351
26.4.7 HNF1A-dependent synthesis of GLUT2 protein 2353
26.4.8 HNF1A-dependent synthesis of the L isoform of PKLR protein 2354
26.4.9 HNF1A-dependent synthesis of HNF4A 2355
26.4.10 HNF1A-dependent synthesis of HNF4G protein 2356
26.4.11 HNF1A-dependent synthesis of FOXA3 2357
27 Regulatory RNA pathways 2359
27.1 MicroRNA biogenesis 2359
27.1.1 Pol II mediated transcription of microRNA genes 2361
27.1.2 Microprocessor complex cleaves pri-miRNA to pre-miRNA 2362
27.1.3 Exportin-5 recognizes 3' overhang of pre-miRNA 2364
27.1.4 Exportin complex translocates pre-miRNA to cytosol 2364
27.1.5 Dicer cleaves pre-miRNA to mature miRNA 2365
28 Signaling by BMP 2367
28.1 The ligand trap binds the ligand BMP2, blocking BMP signalling 2368
28.2 Formation of a heteromeric BMP receptor complex 2369
28.3 BMP2 binds to the receptor complex 2370
28.4 Type II receptor phosphorylates type I receptor 2370
28.5 An anchoring protein, Endofin, recruits R-Smad1/5/8 2371
28.6 Ubiquitin-dependent degradation controls basal levels of R-Smad1/5/8 2372
28.7 I-Smad binds to type I receptor, preventing Smad1/5/8 from being activated 2373
28.8 Activated type I receptor phosphorylates R-Smad1/5/8 directly 2374
28.9 Phospho-R-Smad1/5/8 dissociates from the receptor complex 2375
28.10 I-Smad competes with Co-Smad for R-Smad1/5/8 2376
28.11 Phospho-R-Smad1/5/8 forms a complex with Co-Smad 2376
28.12 The phospho-R-Smad1/5/8:Co-Smad transfers to the nucleus 2377
28.13 Ubiquitin-dependent degradation of the Smad complex terminates BMP2 signalling 2378
28.14 SKI complexes with the Smad complex, suppressing BMP2 signalling 2379
29 Signaling by EGFR 2380
29.1 Pro-EGF is cleaved to form mature EGF 2380
29.2 EGFR binds EGF ligand 2381

29.3 EGFR dimerization 2382
29.4 EGFR autophosphorylation 2383
29.5 Phosphorylation of EGFR by SRC kinase 2384
29.6 EGFR interacts with phospholipase C-gamma 2386
29.6.1 Phospholipase C-gamma1 binds to the activated EGF receptor 2386
29.6.2 EGFR activates PLC-gamma1 by phosphorylation 2387
29.6.3 Active PLC-gamma1 dissociates from EGFR 2388
29.7 Grb2 events in EGFR signaling 2388
29.7.1 Grb2 binds Sos 2389
29.7.2 Sos:Grb2 complex binds to EGF:EGFR complex 2390
29.7.3 Sos-mediated nucleotide exchange of Ras (EGF:EGFR-Sos:Grb2) 2391
29.7.4 Transient dissociation of 14-3-3 upon Ras binding 2393
29.8 Shc events in EGFR signaling 2394
29.8.1 Shc binds to the phospho-receptor:ligand complex 2395
29.8.2 Shc phosphorylation by phospho-EGFR:EGF 2396
29.8.3 Sos:Grb2 binds to Phospho-Shc:EGF:Phospho-EGFR 2397
29.8.4 Sos-mediated nucleotide exchange of Ras (EGF:EGFR-Sos:Grb2:Shc) 2398
29.8.5 Transient dissociation of 14-3-3 upon Ras binding 2400
29.9 Gab1 signalosome 2401
29.9.1 Binding of Grb2 to Gab1 2401
29.9.2 Binding of PI3K regulatory alpha subunit to Gab1:Grb2 2403
29.9.3 Gab1:Grb2:PI3K binds to EGF:Phospho-EGFR 2404
29.9.4 PI3K catalytic subunit binds to Gab1:Grb2:PI3K:EGF:EGFR 2405
29.9.5 PI3K converts Phosphatidylinositol-4,5-bisphosphate to Phosphatidylinositol-3,4,5-trisphosphate 2406
29.9.6 Gab1 binds phosphatidylinositol-3,4,5-trisphosphate 2407
29.9.7 Gab1:Grb2 binds to EGF:Phospho-EGFR 2408
29.9.8 Gab1 phosphorylation by EGFR kinase 2408
29.9.9 Activation of SHP2 through the binding to phospho-Gab1 2409
29.9.10 Dephosphorylation of Gab1 by SHP2 2410
29.9.11 SHP2 dephosphorylates Tyr 992 on EGFR 2411
29.9.12 Dephosphorylation of PAG by SHP2 2412
29.9.13 Sustained activation of SRC kinase by SHP2 2413
29.10 EGFR downregulation 2414
29.10.1 binding of Cbl to EGFR 2414
29.10.2 Phosphorylation of Cbl (EGFR:Cbl) 2415
29.10.3 Cbl binds and ubiquitinates Phospho-Sprouty 2416
29.10.4 Ubiquitination of stimulated EGFR (Cbl) 2417
29.10.5 Cbl binds to Grb2 2417
29.10.6 Localization of Cbl:Grb2 to the membrane 2418
29.10.7 Phosphorylation of Cbl (EGFR:Cbl:Grb2) 2419
29.10.8 Ubiquitination of stimulated EGFR (Cbl:Grb2) 2420
29.10.9 Sprouty lures cytosolic Cbl away from EGFR 2420
29.10.10 Sprouty lures membrane-bound Cbl away from EGFR 2421
29.10.11 Cbl ubiquitinates Sprouty 2422
29.10.12 Cdc42 lures Cbl away from the receptor 2423
29.10.13 betaPIX pushes CIN85 away from Cbl 2423
29.10.14 Cbl escapes Cdc42-mediated inhibition by down-regulating the adaptor molecule betaPix 2424
29.10.15 Assembly of EGFR complex in clathrin-coated vesicles 2425
29.10.16 Assembly in clathrin-coated vesicles (CCVs) 2426
29.10.17 EGFR non-clathrin mediated endocytosis 2426
29.10.18 Cbl-mediated ubiquitination of CIN85 2427
29.10.19 Sprouty sequesters Cbl away from active EGFR 2428
30 Signaling by FGFR 2430
30.1 FGFR ligand binding and activation 2432
30.1.1 FGFR1 ligand binding and activation 2433
30.1.1.1 FGFR1b ligand binding and activation 2433
30.1.1.1.1 FGFR1b binds to FGF 2434
30.1.1.1.2 Autocatalytic phosphorylation of FGFR1b 2435
30.1.1.2 FGFR1c ligand binding and activation 2436
30.1.1.2.1 FGFR1c binds to FGF 2436
30.1.1.2.2 Autocatalytic phosphorylation of FGFR1c 2437
30.1.1.3 FGFR1c and Klotho ligand binding and activation 2438
30.1.1.3.1 FGFR1c binds to Klotho-bound FGF23 2438
30.1.1.3.2 Autocatalytic phosphorylation of Klotho-bound FGFR1c 2439

30.1.4.1 FGFR4 binds to FGF 2450
30.1.4.2 Autocatalytic phosphorylation of FGFR4 2451
31 Signaling in Immune system 2452
31.1 Innate Immunity Signaling 2452
31.1.1 Toll Receptor Cascades 2452
31.1.1.1 Toll Like Receptor 10 (TLR10) Cascade 2453
31.1.1.1.1 Ligand binds to TLR10 2454
31.1.1.1.2 MyD88 cascade 2455
31.1.1.1.2.1 MyD88 and Mal combine with the activated TLR receptor in human 2455
31.1.1.1.2.2 IRAK4 combines with activated TLR receptor complexed with Mal:MyD88 2456
31.1.1.1.2.3 IRAK1 combines with activated TLR complexed with IRAK4 and MyD88 2457
31.1.1.1.2.4 First phosphorylation of IRAK1 by IRAK4 bound to activated TLR 2458
31.1.1.1.2.5 Second phosphorylation of IRAK1 by IRAK4 bound to activated TLR 2459
31.1.1.1.2.6 Multiple IRAK1 autophosphorylation steps 2459
31.1.1.1.2.7 Dissociation of IRAK1-P(n) from TLR 2460
31.1.1.1.2.8 IRAK1-P(n) combines with TRAF6 2461
31.1.1.1.2.9 IRAK1-P(n):TRAF6 binds MEKK1 2462
31.1.1.2.1 Viral dsRNA binds the Toll-Like Receptor 3 (TLR3) 2463
31.1.1.2.2 Viral dsRNA bound TRL3 Recruits TRIF 2464
31.1.1.2.3 Viral dsRNA:TLR3:TRIF Complex Activates RIP1 2464
31.1.1.2.3.1 Viral dsRNA:TLR3:TRIF Complex Recruits RIP1 2465
31.1.1.2.3.2 Viral dsRNA:TLR3:TRIF Complex Releases Activated RIP1 2466
31.1.1.2.3.3 RIP1 phosphorylates IKKs 2466
31.1.1.2.3.4 Phospho-IKK Complex phosphorylates IkB within the IkB:NFkB Complex 2467
31.1.1.2.3.5 Released NFkB complex is transported to the Nucleus 2468
31.1.1.2.4 TRAF6 Mediated Induction of the antiviral cytokine IFN-alpha/beta cascade 2469
31.1.1.2.4.1 TRAF6 is Recruited to the Viral dsRNA:TLR3:TRIF Complex 2469
31.1.1.2.4.2 Formation of the Viral dsRNA:TLR3:TRIF:TRAF6:TAB1:TAB2 Complex 2470
31.1.1.2.4.3 Activation and Release of the TRAF6:Phospho-Tak1:Tab1:Phospho-Tab2 Complex 2470
31.1.1.2.4.4 Phosphorylation of JNK by the Activated TRAF6:Phospho-TAK1:TAB1:Phospho-Tab2 Complex 2471
31.1.1.2.4.5 TRAF6:Phosho-TAK1:Tab1:PhosphoTAB2 mediated phosphorylation of MAPK1/p38 2472
31.1.1.2.4.6 Phosphorylated MAPK1 phosphorylates ATF-2 2473
31.1.1.2.4.7 Dimerisation of Activated Protein 1 (AP-1) heterodimer 2474
31.1.1.2.4.8 TRAF6:Phosho-TAK1:Tab1:PhosphoTAB2 mediated phosphorylation of the IKK Complex 2474
31.1.1.2.4.9 Phospho-IKK Complex phosphorylates IkB within the IkB:NFkB Complex 2475
31.1.1.2.4.10 Released NFkB complex is transported to the Nucleus 2476
31.1.1.2.5 Viral dsRNA:TLR3:TRIF Complex Activates TBK1 2476
31.1.1.2.5.1 TBK1 is Recruited to the Viral dsRNA:TLR3:TRIF Complex 2477
31.1.1.2.5.2 Viral dsRNA:TLR3:TRIF:TBK1 complex recruits IRF3 2477
31.1.1.2.5.3 Phosphorylation and Release of IRF3 2478
31.1.1.2.5.4 Dimerization of Phospho-IRF3 2479
31.1.1.2.5.5 Dimerized Phospho-IRF3 is Transported To The Nucleus 2479
31.1.1.3.1 Flagellin binds to TLR5 2481
31.1.1.3.2 MyD88 cascade 2481
31.1.1.4 Toll Like Receptor 7/8 (TLR7/8) Cascade 2488
31.1.1.4.1 Ligand binds to TLR7 or TLR8 2488

31.1.1.4.2 MyD88 cascade 2489
31.1.1.5.1 Engulfed CpG DNA binds to endosomal TLR9 2497
31.1.1.5.2 Rab5-mediated recruitment of class III PI3K to TLR9 2497
31.1.1.5.3 MyD88 cascade 2498
31.1.1.6.1 Association of LBP with LPS 2505
31.1.1.6.2 LPS transferred from LBP carrier to CD14 2506
31.1.1.6.2.1 GPI-bound CD14 binds LPS 2506
31.1.1.6.2.2 Secreted CD14 binds LPS 2507
31.1.1.6.3 Transfer of LPS onto TLR4 from CD14 2508
31.1.1.6.4 Activated TLR4 signalling 2509
31.1.1.6.4.1 MyD88 cascade 2509
31.1.1.6.4.1.1 MyD88 and Mal combine with the activated TLR receptor in human 2510
31.1.1.6.4.1.2 IRAK4 combines with activated TLR receptor complexed with Mal:MyD88 2511
31.1.1.6.4.1.3 IRAK1 combines with activated TLR complexed with IRAK4 and MyD88 2511
31.1.1.6.4.1.4 First phosphorylation of IRAK1 by IRAK4 bound to activated TLR 2512
31.1.1.6.4.1.5 Second phosphorylation of IRAK1 by IRAK4 bound to activated TLR 2513
31.1.1.6.4.1.6 Multiple IRAK1 autophosphorylation steps 2513
31.1.1.6.4.1.7 Dissociation of IRAK1-P(n) from TLR 2514
31.1.1.6.4.1.8 IRAK1-P(n) combines with TRAF6 2515
31.1.1.6.4.1.9 IRAK1-P(n):TRAF6 binds MEKK1 2515
31.1.1.6.4.2 TRAM Cascade 2516
31.1.1.6.4.2.1 Association of TRAM to activated TLR4 2516
31.1.1.6.4.2.2 Association of TRIF to TRAM complexed to activated TLR4 2517
31.1.1.6.4.2.3 TBK1-mediated phosphorylation of IRF-3 2518
31.1.1.6.4.2.4 Dimerisation of phosphorylated IRF3 2519
31.1.1.6.4.2.5 TRIF:TRAM:TLR4 complex recruits RIP1 2520
31.1.1.6.4.2.6 TRIF:TRAM:TLR4 complex activates RIP1 2520
31.1.1.6.5 Chlamydial HSP60 binding to TLR4 2521
31.1.1.7 Toll Like Receptor 2 Cascade 2522
31.1.1.7.1 Toll Like Receptor TLR1:TLR2 Cascade 2522
31.1.1.7.1.1 Ligand bound to TLR1:TLR2 2523
31.1.1.7.1.2 MyD88 cascade 2524
31.1.1.7.2 Toll Like Receptor TLR6:TLR2 Cascade 2530
31.1.1.7.2.1 Ligand bound to TLR6:TLR2 2531
31.1.1.7.2.2 MyD88 cascade 2532

31.1.1.8 Toll Like Receptor 11 (TLR11) Cascade (Murine) 2538
31.1.1.8.1 Toxomplasma gondii Profilin binds to murine TLR11 2539
31.1.2 Complement cascade 2539
31.1.2.1 Initial triggering of complement 2540
31.1.2.1.1 Creation of C4 and C2 activators 2541
31.1.2.1.1.1 Lectin pathway of complement activation 2541
31.1.2.1.1.1.1 MBL binds to repetitive carbohydrate structures on the surfaces of viruses, bacteria, fungi, and protozoa 2542
31.1.2.1.1.1.2 Activation of MBL 2543
31.1.2.1.1.2 Classical antibody-mediated complement activation 2544
31.1.2.1.1.2.1 Activation of C1R 2545
31.1.2.1.1.2.2 Activation of C1S 2545
31.1.2.1.2 Conversion of C4 into C4a and C4b 2546
31.1.2.1.3 Conversion of C2 into C2a and C2b 2547
31.1.2.1.4 Formation of C3 convertase (C4b:C2a complex) 2548
31.1.2.1.5 Alternative complement activation 2549
31.1.2.1.5.1 Spontaneous hydrolysis of C3 thioester 2549
31.1.2.1.5.2 Factor B binds to Complement factor 3(H2O) (C3(H2O)) 2550
31.1.2.1.5.3 Factor D cleaves C3(H2O)-bound Factor B 2551
31.1.2.1.5.4 Properdin stabilizes C3b:Bb bound to cell surfaces 2552
31.1.2.1.5.5 Formation of alternate C5 convertase 2552
31.1.2.1.5.6 Cleavage of C3 by C3 convertase 2553
31.1.2.1.5.7 C3(H2O):Factor Bb-mediated C3 cleavage leads to C3b deposition on a target cell surface 2554
31.1.2.1.5.8 Factor B binds to surface-associated C3b 2555
31.1.2.1.5.9 Factor D cleaves C3b-bound Factor B 2555
31.1.2.2 Terminal pathway of complement 2556
31.1.2.2.1 Formation of C5b:C6 complex 2556
31.1.2.2.2 Formation of C5b:C6:C7 complex 2557
31.1.2.2.3 C7 allows complex to insert into membrane under attack 2557
31.1.2.2.4 Formation of C5b:C6:C7:C8 complex 2558
31.1.2.2.5 Creation of the Membrane Attack Complex (MAC) 2559
31.1.2.3 Activation of C3 and C5 2559
31.1.2.3.1 Cleavage of C3 by C3 convertase 2559
31.1.2.3.2 Formation of classic C5 convertase 2560
31.1.2.3.3 Activation of C5 2561
31.2 Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell 2562
31.2.1 VCAM-1interacts with VLA-4 2563
31.2.2 TCR complex interacts with peptide antigen-presenting MHC Class I 2564
31.2.3 KIR2DL2/3 interacting with HLA-C group 1 (Cw4) 2565
31.2.4 NKG2D homodimer interacting with ligands 2566
31.2.5 CD96 binds Nectin-like-5 2567
31.2.6 MadCAM interacts with Integrin alpha-4 beta-7 2568
31.2.7 LILRs interact with MHC Class I 2568
31.2.8 Ligands bind L-selectin 2569
31.2.9 ICAMs 1-4 bind to Integrin LFA-1 2570
31.2.10 NKG2A-CD94 heterdimer interacts with HLA-E 2571
31.2.11 Epithelial cadherin binds to KLRG1 2572
31.2.12 CRTAM binds to NECL2 2573
31.2.13 Poliovirus precursor binds CD226 2574
31.2.14 Nectin 2 binds CD226 2575
31.2.15 CD200 binds to CD200R 2576
31.2.16 MHC Class I interacts with CD160 2577
31.2.17 Fc gamma receptors interact with antigen-bound IgG 2577
31.2.18 CD40 interacting with CD40L 2578
31.2.20 C3d-complexed antigen binds to complement receptor 2580
31.2.21 KIR2DL1 interacting with HLA-C group 2 (Cw3) 2581
31.2.22 KIR3DL1 interacting with HLA Bw4 2582
31.2.23 KIR3DL2 interacting with HLA-A3 2583
31.2.24 KIR2DL4 interacting with HLA-G 2584
31.2.25 KIR2DS1 interacting with HLA-C group 2 (Cw3) 2585
31.2.26 KIR2DS2 interacting with HLA-C group 1 (Cw4) 2586
31.3 TCR signaling 2587
31.3.1 Phosphorylation of CD3 and TCR zeta chains 2589

31.3.1.1 Interaction of Csk with PAG 2591
31.3.1.2 Inactivation of Lck by Csk 2592
31.3.1.3 Dephosphorylation of Lck-pY505 by CD45 2593
31.3.1.4 Activation of Lck 2593
31.3.1.5 Phosphorylation of ITAM motifs in CD3 complexes 2594
31.3.2 Translocation of ZAP-70 to Immunological synapse 2595
31.3.2.1 Recruitment of ZAP-70 to phosphorylated ITAMs 2596
31.3.2.2 Phosphorylation of ZAP-70 by Lck 2597
31.3.2.3 Activation of ZAP-70 2598
31.3.3 Generation of second messenger molecules 2599
31.3.3.1 Phosphorylation of TBSMs in LAT 2600
31.3.3.2 Recruitment of Gads to LAT 2601
31.3.3.3 Recruitment of SLP-76 to Gads 2602
31.3.3.4 Phosphorylation of SLP-76 2603
31.3.3.5 Recruitment of ITK to SLP-76 2604
31.3.3.6 Recruitment of PLC-gamma1 to SLP-76 2604
31.3.3.7 Recruitment of PLC-gamma1 to LAT 2605
31.3.3.8 Phosphorylation of PLC-gamma1 2606
31.3.3.9 Disassociation of PLC-gamma1 from LAT 2607
31.3.3.10 Disassociation of PLC-gamma1 from SLP-76 2607
31.3.3.11 Translocation of PLC-gamma1 to PIP2 2608
31.3.3.12 PLC-gamma1 hydrolyses PIP2 2609
31.3.4 Downstream TCR signaling 2609
31.3.4.1 Recruitment of PI3K to plasmamembrane 2611
31.3.4.2 PI3K phosphorylates PIP2 to PIP3 2612
31.3.4.3 Hydrolysis of PIP3 to PI(3,4)P2 2613
31.3.4.4 Hydrolysis of PIP3 to PIP2 2614
31.3.4.5 Translocation of PDK1 to Plasma membrane 2615
31.3.4.6 Translocation of PKB to plasma membrane 2616
31.3.4.7 Phosphorylation of PKB by PDK1 2617
31.3.4.8 Translocation of PKC theta to plasma membrane 2617
31.3.4.9 Change of PKC theta conformation 2618
31.3.4.10 Phosphorylation of PKC theta 2619
31.3.4.11 Translocation of CARMA1 to Plasma membrane 2620
31.3.4.12 Phosphorylation of CARMA1 2621
31.3.4.13 Oligomerization of CARMA1 2622
31.3.4.14 Interaction of Bcl10 to CARMA1 2622
31.3.4.15 Phosphorylation of Bcl10 2623
31.3.4.16 Oligomerization of Bcl10 2624
31.3.4.17 Interaction and oligomerization of MALT1 to Bcl10 2624
31.3.4.18 Translocation of TRAF6 to CBM complex 2625
31.3.4.19 Auto-ubiquitination of TRAF6 2626
31.3.4.20 Activation of TAK1-TAB2 complex 2627
31.3.4.21 Activation of IKK complex 2627
31.3.4.22 Ubiquitination of NEMO by TRAF6 2628
31.3.4.23 Activation of NF-kB complex 2629
31.4 Cell surface interactions at the vascular wall 2630
31.4.1 Binding of GP VI:Fc Epsilon R1 gamma receptor complex with collagen 2631
31.4.2 MAC1 binds JAM-C 2632
31.4.3 JAM-B binds JAM-C 2633
31.4.4 VLA-4 binds JAM-B 2634
31.4.5 JAM-A homodimerises 2635
31.4.6 LFA1 binds JAM-A 2636
31.4.7 Integrin alphaX beta2 binds JAM-C 2637
31.4.8 MERTK receptor binds ligands (Gas6 or Protein S) 2637
31.4.9 CD84 homodimerises 2638
31.4.10 P-selectin binds P-selectin ligand 2639
31.4.11 JAM-B homodimerises 2640
31.4.12 JAM-C homodimerises 2641
31.4.13 CD177 binds PECAM-1 2641
31.4.14 CD47 binds SIRP 2642
31.4.15 CD48 binds CD244 2643
31.4.16 CD58 binds CD2 2644
31.4.17 Integrin alpha 5 beta 1 binds fibronectin 2645
31.4.19 protein C -> activated protein C + protein C heavy chain activation peptide 2646
31.4.20 OLR1 binds to oxidized LDL 2647
31.4.21 Platelet-derived TREM-1 ligand binds to TREM-1 2648

31.4.22 Tie2 Signaling 2648
31.4.22.2 Dimerization of Tie2/Ang1 complex 2650
31.4.22.3 Trans-phosphorylation of Tie2 2651
31.4.22.4 Interaction of Tie2 and p85 of PI3K 2652
31.4.22.5 Interaction of Tie2 and Grb2 2653
31.4.22.6 Interaction of SOS-1 to Tie2 bound Grb2 2654
31.4.22.7 Sos-mediated nucleotide exchange of Ras (Tie2 receptor:Grb2:Sos) 2655
31.4.22.8 Interaction of Tie2 and Shc1 2655
31.4.22.9 Interaction of Tie2 and Dok-2 2656
31.4.22.11 Interaction of Tie2 and Ang4 2658
31.4.23 PECAM1 interactions 2659
31.4.23.1 Trans-homophilic interaction of PECAM-1 2659
31.4.23.2 Heterophilic interaction of PECAM-1 and Integrin alpha-v beta-3 2660
31.4.23.3 Phoshorylation of PECAM-1 by Fyn or Lyn or c-Src 2661
31.4.23.4 Interaction of PECAM-1 and SHIP 2662
31.4.23.6 Interaction of PECAM-1 and PLC gamma1 2663
31.4.24 Basigin interactions 2665
31.4.24.1 CyP60 chaperones Basigin 2666
31.4.24.2 Basigin homodimerises 2666
31.4.24.3 Caveolin-1 binds Basigin 2667
31.4.24.4 Basigin binds CyPA 2668
32 Signaling by Insulin receptor 2670
32.1 Insulin binding 2670
32.2 Autophosphorylation of insulin receptor 2672
32.3 Insulin receptor signalling cascade 2673
32.3.1 SHC-related events 2674
32.3.1.1 SHC activation 2674
32.3.1.1.1 Binding of SHC to insulin receptor 2674
32.3.1.1.2 Phosphorylation of SHC 2675
32.3.1.1.3 Dissociation of SHC-P from insulin receptor 2675
32.3.1.2 SHC-mediated signalling 2676
32.3.1.2.1 GRB2:SOS binds to SHC-P 2676
32.3.1.2.2 SOS mediated nucleotide exchange of RAS (SHC) 2677
32.3.1.2.3 RAF activation 2679
32.3.1.2.3.1 Transient dissociation of 14-3-3 upon Ras binding 2679
32.3.1.2.3.2 Stabilisation of RAS:RAF by 14-3-3 2681
32.3.1.2.3.3 Stabilisation of RAF by further phosphorylation 2682
32.3.1.2.4 MAP kinase cascade 2683
32.3.1.2.4.1 MEK activation 2683
32.3.1.2.4.1.1 Activated RAF complex binds MEK 2684
32.3.1.2.4.1.2 RAF phosphorylates MEK 2686
32.3.1.2.4.1.2.1 RAF phosphorylates MEK1 2686
32.3.1.2.4.2 ERK activation 2689
32.3.1.2.4.2.1 ERK1 activation 2689
32.3.1.2.4.2.1.1 MEK1 binds ERK-1 2690
32.3.1.2.4.2.1.2 MEK1 phosphorylates ERK-1 2691
32.3.1.2.4.2.1.3 Dimerisation of phospho-ERK-1 2692
32.3.1.2.4.2.1.4 Nuclear translocation of phospho-ERK-1 dimer 2693
32.3.1.2.4.2.2 ERK2 activation 2694
32.3.1.2.4.2.2.1 MEK2 binds ERK-2 2694
32.3.2 IRS-related events 2699
32.3.2.1 IRS activation 2699
32.3.2.1.1 Binding of IRS to insulin receptor 2700
32.3.2.1.2 Phosphorylation of IRS 2700
32.3.2.1.3 Dissociation of IRS-P from insulin receptor 2700
32.3.2.2 IRS-mediated signalling 2701
32.3.2.2.1 PI3K Cascade 2701
32.3.2.2.1.1 PI3K activation 2701
32.3.2.2.1.2 PKB-mediated events 2702
32.3.2.2.1.2.1 PDE3B signalling 2702

32.3.2.2.1.2.1.1 Hydrolysis of cAMP to 5' AMP by Phosphorylated PDE3B 2702
32.3.2.2.1.2.1.2 Phosphorylation of PDE3B 2703
32.3.2.2.1.2.2 mTOR signalling 2703
32.3.2.2.1.2.2.1 Inhibition of TSC complex formation by PKB 2703
32.3.2.2.1.2.2.1.1 Phosphorylation of TSC2 by PKB 2704
32.3.2.2.1.2.2.1.2 Phosphorylation of complexed TSC2 by PKB 2704
32.3.2.2.1.2.2.2 GTP loading by Rheb 2705
32.3.2.2.1.2.2.3 Formation of active mTORC1 complex 2705
32.3.2.2.1.2.2.4 mTORC1-mediated signalling 2706
32.3.2.2.1.2.2.4.1 S6K1-mediated signalling 2706
32.3.2.2.1.2.2.4.1.1 Activation of S6K1 2706
32.3.2.2.1.2.2.4.1.2 S6K1 signalling 2707
32.3.2.2.1.2.2.4.1.2.1 Phosphorylation of Ribosomal protein S6 by activated S6K1 2707
32.3.2.2.1.2.2.4.1.2.2 Phosphorylation and inactivation of eEF2K by activated S6K1 2707
32.3.2.2.1.2.2.4.1.2.3 Phosphorylation and activation of eIF4G by activated S6K1 2708
32.3.2.2.1.2.2.4.1.2.4 Phosphorylation and activation of eIF4B by activated S6K1 2709
32.3.2.2.1.2.2.4.2 Release of eIF4E 2709
32.3.2.2.1.2.2.4.2.1 Phosphorylation of 4E-BP1 by activated mTORC1 2709
32.3.2.2.1.2.2.4.2.2 Dissociation of phosphorylated 4EBP1 from eIF4E 2710
32.3.2.2.1.3 Activation of PKB 2711
32.3.2.2.1.3.1 PDK1 attachment to plasma membrane 2711
32.3.2.2.1.3.2 PKB attachment to plasma membrane 2711
32.3.2.2.1.3.3 Phosphorylation of PKB by PDK1 2712
32.3.2.2.2 SOS-mediated signalling 2713
32.3.2.2.2.1 GRB2:SOS binds IRS-P 2713
32.3.2.2.2.2 SOS mediated nucleotide exchange of RAS (IRS) 2714
32.3.2.2.2.3 RAF activation 2715
32.3.2.2.2.3.1 Transient dissociation of 14-3-3 upon Ras binding 2716
32.3.2.2.2.3.2 Stabilisation of RAS:RAF by 14-3-3 2717
32.3.2.2.2.3.3 Stabilisation of RAF by further phosphorylation 2718
32.3.2.2.2.4 MAP kinase cascade 2719
32.3.2.2.2.4.1 MEK activation 2720
32.3.2.2.2.4.1.1 Activated RAF complex binds MEK 2720
32.3.2.2.2.4.1.2 RAF phosphorylates MEK 2722
32.3.2.2.2.4.1.2.1 RAF phosphorylates MEK1 2722
32.3.2.2.2.4.2 ERK activation 2725
32.3.2.2.2.4.2.1 ERK1 activation 2725
32.3.2.2.2.4.2.1.1 MEK1 binds ERK-1 2725
32.3.2.2.2.4.2.1.2 MEK1 phosphorylates ERK-1 2726
32.3.2.2.2.4.2.1.3 Dimerisation of phospho-ERK-1 2727
32.3.2.2.2.4.2.1.4 Nuclear translocation of phospho-ERK-1 dimer 2728
32.3.2.2.2.4.2.2 ERK2 activation 2730
32.3.2.2.2.4.2.2.1 MEK2 binds ERK-2 2730
32.4 Internalisation of the insulin receptor 2734
32.5 Insulin receptor recycling 2736
32.5.1 Endosome acidification 2736
32.5.2 Dissociation of insulin from insulin receptor 2738
32.5.3 Insulin receptor de-phosphorylation 2738
32.5.4 Re-integration of insulin receptor into plasma membrane 2740
33 Signalling by NGF 2742
33.1 NGF processing 2742
33.1.1 The signal peptide is excised from beta-NGF pre-pro-precursor 2743
33.1.2 pro-beta-NGF dimerizes 2744
33.1.3 pro-beta-NGF homodimer transits to the golgi apparatus 2745
33.1.4 Part of pro-beta-NGF is processed to mature beta-NGF 2746
33.1.5 Pro-beta-NGF and mature beta-NGF are secreted 2747
33.2 p75 NTR receptor-mediated signalling 2748
33.2.1 NFG and proNGF binds to p75NTR 2748
33.2.1.1 Binding of pro-NGF to p75NTR:sortilin 2749
33.2.1.2 NGF homodimer binds to p75NTR 2750
33.2.2 Cell death signalling via NRAGE, NRIF and NADE 2751
33.2.2.1 NRAGE signals death through JNK 2752
33.2.2.1.1 A ligand:p75NTR complex binds to NRAGE 2752
33.2.2.1.2 NRAGE sequesters CHE1 in the cytoplasm 2753

33.2.2.1.3 NRAGE activates JNK 2754
33.2.2.1.4 p75NTR indirectly activates RAC and Cdc42 via a guanyl-nucleotide exchange factor 2755
33.2.2.1.5 GTP-bound RAC contributes to JNK activation 2756
33.2.2.1.6 JNK phosphorylates BIM, BAD and other targets 2757
33.2.2.1.7 Active JNK moves to the nucleus and phosphorylates different transcription factors 2758
33.2.2.2 NRIF signals cell death from the nucleus 2759
33.2.2.2.1 NRIF binds to p75NTR 2759
33.2.2.2.2 TRAF6 binds to p75NTR:NRIF 2760
33.2.2.2.3 NRIF and TRAF6 may activate JNK 2761
33.2.2.2.4 gamma-secretase cleaves p75NTR, releasing NRIF and TRAF6 2762
33.2.2.2.5 TRAF6 polyubiquitinates NRIF 2763
33.2.2.2.6 Polyubiquitinated NRIF binds to p62 (Sequestosome) 2764
33.2.2.2.7 Polyubiquitinated NRIF migrates to the nucleus 2765
33.2.2.3 NADE modulates death signalling 2766
33.2.2.3.1 p75NTR binds to NADE 2766
33.2.2.3.2 p75NTR:NADE promotes caspase2/3 activation 2767
33.2.2.3.3 14-3-3epsilon attentuates NADE-related apoptosis 2768
33.2.3 p75NTR negatively regulates cell cycle via SC1 2769
33.2.3.1 PRDM4 (SC1) binds to p75NTR 2769
33.2.3.2 PRDM4 translocates to the nucleus 2770
33.2.3.3 PRDM4 inhibits cyclin E transcription 2771
33.2.4 p75NTR signals via NF-kB 2772
33.2.4.1 p75NTR recruits signalling complexes 2773
33.2.4.1.1 p75NTR interacts with RIP2 2773
33.2.4.1.2 p75NTR interacts with IRAK:MYD88 2774
33.2.4.1.3 IRAK is activated 2775
33.2.4.1.4 IRAK interacts with TRAF6 2776
33.2.4.1.5 MYD88 dissociates 2777
33.2.4.1.6 p62 is recruited and forms a complex with TRAF6 2777
33.2.4.1.7 The TRAF6:p62 complex regulates trafficking of TRKA and other substrates 2778
33.2.4.1.8 TRAF6 is auto-ubiquitinated 2779
33.2.4.1.9 p62 recruits an atypical PKC 2780
33.2.4.1.10 IKK-beta is recruited 2781
33.2.4.2 NF-kB is activated and signals survival 2781
33.2.4.2.1 IKKbeta is activated 2782
33.2.4.2.2 IKKbeta phosphorylates IkB causing NF-kB to dissociate 2783
33.2.4.2.3 IkB is ubiquitinated and degraded 2784
33.2.4.2.4 NF-kB migrates to the nucleus and turns on transcription 2784
33.2.5 Ceramide signalling 2785
33.2.5.1 Sphingomyelinase is activated by the NGF:p75NTR complex 2786
33.2.5.2 Production of ceramide which can activate JNK and other targets 2787
33.2.6 p75NTR regulates axonogenesis 2788
33.2.6.1 Axonal growth stimulation 2789
33.2.6.1.1 p75NTR and RHOA-GDI interact 2789
33.2.6.1.2 NGF binding to p75NTR inactivates RHOA 2790
33.2.6.1.3 p75NTR reduces RHO-GDI activity 2791
33.2.6.1.4 Nucleotide exchange on RHOA 2792
33.2.6.2 Axonal growth inhibition (RHOA activation) 2793
33.2.6.2.1 p75NTR interacts with the NOGO receptor 2793
33.2.6.2.2 p75NTR:NgR complex interacts with the axonal inhibitor LINGO1 2794
33.2.6.2.3 Myelin components can interact with p75NTR:NgR:LINGO1 2795
33.2.6.2.4 The p75NTR:NgR:MDGI complex reduces RHOA-GDI activity, displacing RHOA 2796
33.2.6.2.5 RhoA is activated by nucleotide exchange and inhibits axonal growth 2797
33.2.7 Regulated proteolysis of p75NTR 2797
33.2.7.1 alpha-secretase cleaves the p75NTR extracellular domain 2798
33.2.7.2 The p75NTR C-terminal fragment enters endosomes 2799
33.2.7.3 gamma-secretase cleaves the p75NTR transmembrane domain 2800
33.2.7.4 p75NTR ICD signals to NF-kB 2801
33.2.7.5 Part of the ICD migrates to the nucleus 2802
33.3 Activation of TRKA receptors 2803
33.3.1 TRKA activation by NGF 2804
33.3.1.1 beta-NGF dimer binds to TrkA receptor 2804
33.3.1.2 The bound receptor dimerizes 2805
33.3.1.3 TrkA receptor autophosphorylates 2806
33.3.1.4 Activated TrkA receptor internalizes to endosomes 2807
33.3.2 NGF-independant TRKA activation 2807
33.3.2.1 By adenosine A2a receptor 2808
33.3.2.2 By PACAP type 1 receptor 2809

33.4 TRKA signalling from the plasma membrane 2810
33.4.1 Signalling to ERKs 2810
33.4.1.1 Signalling to RAS 2811
33.4.1.1.1 Shc binds to the activated TrkA receptor 2812
33.4.1.1.2 Shc, complexed with TrkA, is tyrosine-phosphorylated 2812
33.4.1.1.3 Phospho-Shc dissociates from the TrkA receptor 2813
33.4.1.1.4 GRB2:SOS binds to SHC-P 2814
33.4.1.1.5 SOS mediated nucleotide exchange of RAS (SHC) 2815
33.4.1.1.7.2.1 ERK1 activation 2827
33.4.1.1.7.2.1.1 MEK1 binds ERK-1 2828
33.4.1.1.7.2.2 ERK2 activation 2832
33.4.1.1.7.2.2.1 MEK2 binds ERK-2 2832
33.4.1.1.8 p38MAPK events 2837
33.4.1.1.8.1 Ral-GDS binds to Ras-GTP 2838
33.4.1.1.8.2 Guanine nucleotide exchange on Ral 2838
33.4.1.1.8.3 Activation of SRC by Ral-GTP 2839
33.4.1.1.8.4 Binding and activation of MAP Kinase 2840
33.4.1.1.8.5 MAP kinase activates MAPKAPK2, MAPKAPK3 and MSK1 2841
33.4.1.2 Signalling to p38 via RIT and RIN 2842
33.4.1.2.1 TrkA recruits RIT and RIN 2842
33.4.1.2.2 RIT/RIN are activated 2843
33.4.1.2.3 RIT/RIN-GTP binds B-Raf 2844
33.4.1.2.5.2.1 ERK1 activation 2854
33.4.1.2.5.2.1.1 MEK1 binds ERK-1 2854
33.4.1.2.5.2.2 ERK2 activation 2859
33.4.1.2.5.2.2.1 MEK2 binds ERK-2 2859
33.4.1.3 Prolonged ERK activation events 2863
33.4.1.3.1 Frs2-mediated activation 2864
33.4.1.3.1.1 Frs2 binds to active TrkA receptor 2865
33.4.1.3.1.2 Frs2 is phosphorylated by active TrkA receptor 2865
33.4.1.3.1.3 Phospho-Frs2 binds CrkL 2866
33.4.1.3.1.4 Phospho-Frs2:CrkL engages C3G 2867
33.4.1.3.1.5 (Frs2)C3G stimulates nucleotide exchange on Rap1 2868
33.4.1.3.1.6 (Frs2)Rap1-GTP binds to and activates B-Raf 2869

33.4.1.3.1.7.1 MEK activation 2870
33.4.1.3.1.7.2 ERK activation 2875
33.4.1.3.1.7.2.1 ERK1 activation 2875
33.4.1.3.1.7.2.1.1 MEK1 binds ERK-1 2875
33.4.1.3.1.7.2.2 ERK2 activation 2880
33.4.1.3.1.7.2.2.1 MEK2 binds ERK-2 2880
33.4.1.3.2 ARMS-mediated activation 2884
33.4.1.3.2.1 ARMS:Crk complex binds to active TrkA receptor 2885
33.4.1.3.2.2 ARMS is phosphorylated by active TrkA receptor 2886
33.4.1.3.2.3 Crk's SH3 domain engages C3G 2887
33.4.1.3.2.4 C3G stimulates nucleotide exchange on Rap1 2887
33.4.1.3.2.5 (ARMS)Rap1-GTP binds and activates B-Raf 2888
33.4.1.3.2.6.1 MEK activation 2890
33.4.1.3.2.6.2 ERK activation 2895
33.4.1.3.2.6.2.1 ERK1 activation 2895
33.4.1.3.2.6.2.1.1 MEK1 binds ERK-1 2895
33.4.1.3.2.6.2.2 ERK2 activation 2900
33.4.1.3.2.6.2.2.1 MEK2 binds ERK-2 2900
33.4.2 PLC-gamma1 signalling 2904
33.4.2.1 Binding of PLCG1 to active TrkA receptor 2905
33.4.2.2 TrkA phosphorylates PLCG1 2906
33.4.2.3 Active PLCG1 dissociates from TrkA receptor 2906
33.4.2.4 Active PLCG1 hydrolyses PIP2 2907
33.4.2.5 DAG stimulates protein kinase C-delta 2908
33.4.2.6 IP3 binds with the IP3 receptor, opening the Ca2+ channel 2908
33.4.2.7 Release of calcium from intracellular stores by IP3 receptor activation 2909
33.4.3 PI3K/AKT signalling 2910
33.4.3.1 Active TRKA binds IRS1/2 2911
33.4.3.2 TRKA phosphorylates IRS 2911
33.4.3.3 Active IRS recruits PI3K to the plasma membrane and activates it 2912
33.4.3.4 PI3K produces PIP3 and other phosphatidyl inositides 2913
33.4.3.5 PIP3 binds to RhoA and activates it 2913
33.4.3.6 PIP3 recruits PDK1 and AKT to the membrane 2914
33.4.3.7 PDK1 phosphorylates AKT at T308 2915
33.4.3.8 TORC2 (mTOR) phosphorylates AKT at S473 2916
33.4.3.9 AKT phosphorylates targets in the cytosol 2917
33.4.3.9.1 AKT phosphorylates BAD 2917
33.4.3.9.2 AKT phosphorylates GSK3 2918
33.4.3.9.3 AKT phosphorylates caspase-9 2919
33.4.3.9.4 AKT phosphorylates MDM2 2920
33.4.3.9.5 AKT phosphorylates IKKalpha 2921
33.4.3.9.6 AKT phosphorylates p21Cip1 and p27Kip1 2922
33.4.3.9.7 AKT phosphorylates TSC2, inhibiting it 2922
33.4.3.9.8 AKT phosphorylates PRAS40 2924
33.4.3.10 AKT translocates to the nucleus 2925
33.4.3.11 AKT phosphorylates targets in the nucleus 2925

33.4.3.11.1 AKT phosphorylates CREB 2926
33.4.3.11.2 AKT can phosphorylate forkhead box transcription factors 2927
33.4.3.11.2.1 AKT phosphorylates FOXO1A 2927
33.4.3.11.3 AKT can phosphorylate SRK 2928
33.4.3.11.4 AKT can phosphorylate NUR77 2929
33.4.3.12 Negative regulation of the PI3K/AKT network 2930
33.4.3.12.1 PTEN dephosphorylates PIP3 2930
33.4.3.12.2 CTMP and/or TRB3 inhibit AKT phosphorylation 2931
33.4.3.12.3 PHLPP dephosphorylates S473 in AKT 2932
33.4.4 Signalling to STAT3 2933
33.4.4.1 STAT3 activation 2933
33.4.5 Signalling to ERK5 2934
33.4.5.1 ERK5 is activated 2935
33.4.5.2 ERK5 translocates to the nucleus 2936
33.5 Retrograde neurotrophin signalling 2936
33.5.1 Formation of clathrin-coated vesicle 2937
33.5.2 Endocytosis (internalization) of clathrin-coated vesicle 2938
33.5.3 Axonal transport of NGF:Trk complexes 2938
33.6 Nuclear Events (kinase and transcription factor activation) 2939
33.6.1 ERK/MAPK targets 2940
33.6.1.1 ERK1/2 activates ELK1 2940
33.6.1.2 ERK1/2 phosphorylates MSK1 2941
33.6.1.3 p38MAPK phosphorylates MSK1 2942
33.6.1.4 ERK1/2/5 activate RSK1/2/3 2942
33.6.1.5 ERK5 activates the transcription factor MEF2 2943
33.6.1.6 ERKs are inactivated by protein phosphatase 2A 2944
33.6.1.7 ERKs are inactivated 2945
33.6.1.7.1 ERKs are inactivated by protein phosphatase 2A 2946
33.6.1.7.2 ERKs are inactivated by dual-specific phosphatases (DUSPs) 2947
33.6.2 CREB phosphorylation 2948
33.6.2.1 MSK1 activates CREB 2948
33.6.2.2 MSK1 activates ATF1 2949
33.6.2.3 RSK1/2/3 phosphorylates CREB at Serine 133 2950
33.6.2.4 MAPKAPK2 phosphorylates CREB at Serine 133 2950
34 Signaling by Notch 2952
34.1 Transport of Notch receptor precursor to golgi 2953
34.1.1 Notch 1 precursor transport to golgi 2954
34.2 Maturation of Notch precursor via proteolytic cleavage 2956
34.2.1 Notch 1 precursor cleaved to form a heterodimer 2957
34.3 Mature Notch receptor trafficks to plasma membrane 2960
34.3.1 Notch 1 heterodimer trafficks to the plasma membrane 2960
34.4 Notch receptor binds with a ligand 2962
34.4.1 Notch 1 heterodimer binds with a Notch ligand in the extracellular space 2963
34.5 Receptor-ligand binding initiates the second proteolytic cleavage of Notch receptor 2965
34.5.1 Notch 1-ligand complex is cleaved to produce NEXT1 2966
34.6 A third proteolytic cleavage releases NICD 2969
34.6.1 NEXT1 is cleaved to produce NICD1 2969
34.7 NICD trafficks to nucleus 2972
34.7.1 NICD1 trafficks to the nucleus 2972

35 Signaling by Rho GTPases 2976
35.1 Rho GTPase cycle 2977
35.1.1 GEFs activate Rho GTPase:GDP 2978
35.1.2 Rho GTPase:GTP activates downstream effectors 2980
35.1.3 GAPs inactivate Rho GTPase:GTP by hydrolysis 2981
35.1.4 GDIs block activation of Rho GTPase:GDP 2982
35.1.5 Dissociation of Rho GTP:GDP from GDI complex 2983
36 Signaling by TGF beta 2985
36.1 Latent TGF-beta1 is cleaved by furin 2986
36.2 Secretion and activation of the latent large complex of TGF-beta1 2987
36.3 Dimeric TGF-beta1 binds to the receptor 2987
36.4 Type II receptor recruits type I receptor 2988
36.5 Type II receptor phosphorylates type I receptor 2989
36.6 An anchoring protein, SARA, recruits R-SMAD 2990
36.7 Ubiquitin-dependent degradation controls basal levels of R-SMAD 2990
36.8 Activated type I receptor phosphorylates R-SMAD directly 2991
36.9 Phospho-R-SMAD dissociates from the receptor complex 2992
36.10 Phospho-R-SMAD forms a complex with CO-SMAD 2993
36.11 The phospho-R-SMAD:CO-SMAD transfers to the nucleus 2994
36.12 Ubiquitin-dependent degradation of the SMAD complex terminates TGF-beta signaling 2995
36.13 SKI complexes with the SMAD complex, suppressing TGF-beta signaling 2996
36.14 I-SMAD competes with R-SMAD for type I receptor 2996
36.15 I-SMAD recruits type I receptor phosphatase and ubiquitin ligases to mediate receptor downregulation 2997
37 Signaling by VEGF 2998
37.1 VEGF ligand-receptor interactions 3000
37.1.1 Homodimerization of VEGF proteins 3001
37.1.2 VEGF binds to VEGFR leading to receptor dimerization 3002
37.1.2.1 VEGF-A,B,PLGF bind to VEGFR1 leading to receptor dimerization 3003
37.1.2.2 VEGF-A,C,D,E bind to VEGFR2 leading to receptor dimerization 3005
37.1.2.3 VEGF-C,D bind to VEGFR3 leading to receptor dimerization 3006
37.1.3 Neurophilin interactions with VEGF and VEGFR 3008
37.1.3.1 NRP-1 forms a ternary complex with VEGF165 and VEGFR1 3008
37.1.3.2 NRP-2 associates with VEGFR1 forming complexes on cell surface 3010
38 Signaling by Wnt 3011
38.1 Degradation of beta-catenin by the destruction complex 3011
38.1.1 Assembly of the destruction complex 3012
38.1.2 Association of beta-catenin with the destruction complex 3013
38.1.3 Beta-catenin phosphorylation cascade 3014
38.1.3.1 Phosphorylation of beta-catenin at Ser45 by CK1 alpha 3014
38.1.3.2 Phosphorylation of phospho-(Ser45 ) at Thr 41 by GSK-3 3015
38.1.3.3 Phosphoryation of phospho- (Ser45, Thr41) beta-catenin at Ser37 by GSK-3 3016
38.1.3.4 Phosphorylation of phospho-(Ser45,Thr41,Ser37) at Ser33 by GSK-3 3017
38.1.4 Phosphorylation of APC component of the destruction complex 3017
38.1.5 Dissociation of beta-catenin from Axin and association of beta catenin with phospho-(20 aa) APC in the detruction complex 3018
38.1.6 Association of beta-catenin with the SCF-beta-TrCP1 ubiquitin ligase complex 3019
38.1.7 Multi-ubiquitination of phospho-beta-catenin by SCF-beta-TrCP1 3020
38.1.8 Degradation of ubiquitinated -beta catenin by the proteasome 3021
39 Synaptic Transmission 3022
39.1 Transmission across Chemical Synapses 3022
39.1.1 Depolarization of the Presynaptic Terminal Triggers the Opening of Calcium Channels 3023
39.1.1.1 Ca2+ influx through voltage gated Ca2+ channels 3023
39.1.2 Neurotransmitter Release Cycle 3024
39.1.2.1 Glutamate Neurotransmitter Release Cycle 3025
39.1.2.1.1 L-Glutamate [cytosolic] from leucine catabolism 3027
39.1.2.1.2 L-Glutamine transport into neurons 3027
39.1.2.1.3 Transport of L-Glutamine (cytosol) to mitochondrial matrix 3028
39.1.2.1.4 L-Glutamate [mitochondrial] from leucine catabolism 3029
39.1.2.1.5 L-Glutamine conversion to L-Glutamate 3029
39.1.2.1.6 Tranport of L-Glutamate (mitochondrial matrix) to cytosol 3030
39.1.2.1.7 Re-acidification of the synaptic vesicle 3031
39.1.2.1.8 L-Glutamate loading of synaptic vesicle 3031
39.1.2.1.9 Synaptic vesicle docking and priming 3032
39.1.2.1.10 release of L-Glutamate at the synapse 3033
39.1.2.1.11 L-Glutamate uptake by neurons 3034
39.1.3 Neurotransmitter Clearance In The Synaptic Cleft 3035
39.1.4 Neurotransmitter uptake and Metabolism In Glial Cells 3035

39.1.4.1 Astrocytic Glutamate-Glutamine Uptake And Metabolism 3036
39.1.4.1.1 glutamate uptake by astrocytes 3036
39.1.4.1.2 Glutamate conversion to glutamine 3037
39.1.4.1.3 Glutamine transport from astrocytes 3038
40 Telomere Maintenance 3039
40.1 Extension of Telomeres 3041
40.1.1 Telomere Extension By Telomerase 3041
40.1.1.1 Biogenesis And Assembly Of The Telomerase RNP 3042
40.1.1.2 Recruitment of Telomerase RNP to the Telomeric Chromosome End 3045
40.1.1.3 Alignment Of The RNA Template On The Telomeric Chromosome End 3047
40.1.1.4 Elongation Of The Telomeric Chromosome End 3048
40.1.1.5 Translocation Of Telomerase RNP And Alignment Of RNA Template (TERC) To Extended Single Stranded Telomeric 3049
Chromosome-End
40.1.1.6 Elongation of Extended Telomeric Chromosome End 3051
40.1.1.7 Disassociation of Telomerase RNP and the Chromosome End 3052
40.1.2 Telomere C-strand (Lagging Strand) Synthesis 3053
40.1.2.1 Telomere C-strand synthesis initiation 3054
40.1.2.1.1 The primase component of DNA polymerase:primase synthesizes a 6-10 nucleotide RNA primer on the G strand of the 3055
telomere
40.1.2.1.2 The polymerase component of DNA polymerase alpha:primase synthesizes a 20-nucleotide primer on the G strand of the 3055
telomere
40.1.2.2 Polymerase switching on the C-strand of the telomere 3056
40.1.2.2.1 RFC binding displaces Pol Alpha on the C-strand of the telomere 3058
40.1.2.2.2 Loading of PCNA - Sliding Clamp Formation on the C-strand of the telomere 3058
40.1.2.2.3 RFC dissociates after sliding clamp formation on the C-strand of the telomere 3059
40.1.2.2.4 Formation of Processive Complex on the C-strand of the telomere 3060
40.1.2.3 Processive synthesis on the C-strand of the telomere 3061
40.1.2.3.1 Formation of C-strand Okazaki fragments 3062
40.1.2.3.2 Formation of the Flap Intermediate on the C-strand 3063
40.1.2.3.3 Removal of the Flap Intermediate from the C-strand 3064
40.1.2.3.3.1 RPA binds to the Flap on the C-strand 3065
40.1.2.3.3.2 Recruitment of Dna2 endonuclease to the C strand 3065
40.1.2.3.3.3 Removal of RNA primer and dissociation of RPA and Dna2 from the C-strand 3066
40.1.2.3.3.4 Removal of remaining Flap from the C-strand 3067
40.1.2.3.4 Joining of adjacent Okazaki fragments of the C-strand 3068
40.1.2.4 Disassociation of Processive Complex and Completed Telomere End 3069
40.2 Packaging Of Telomere Ends 3070
40.2.1 Incorporation Of Extended And Processed Telomere End Into Associated Protein Structure 3072
40.2.2 Incorporation Of Extended And Processed Telomere End Into Higher Order T-Loop And Associated Protein Structure 3074
41 Transcription 3077
41.1 RNA Polymerase I Transcription 3077
41.1.1 RNA Polymerase I Promoter Clearance 3078
41.1.1.1 RNA Polymerase I Promoter Opening 3078
41.1.1.1.1 UBF-1 Binds rDNA Promoter 3078
41.1.1.1.2 Phosphorylation of UBF-1:rDNA Promoter 3079
41.1.1.2 RNA Polymerase I Transcription Initiation 3080
41.1.1.2.1 Formation of SL1 3081
41.1.1.2.2 Acetylation of SL1 3082
41.1.1.2.3 Recruitment of Acetylated SL1 to phosUBF-1:rDNA Promoter 3083
41.1.1.2.4 Assembly of RNA Polymerase I Holoenzyme (human) 3084
41.1.1.2.5 Binding of Rrn3 to RNA Polymerase I 3085
41.1.1.2.6 Recruitment of Active RNA Polymerase I to SL1:phos.UBF-1:rDNA Promoter 3086
41.1.1.3 RNA Polymerase I Promoter Escape 3087
41.1.1.3.1 Loss of Rrn3 from RNA Polymerase I promoter escape complex 3088
41.1.2 RNA Polymerase I Chain Elongation 3089
41.1.2.1 Elongation of pre-rRNA transcript 3089
41.1.3 RNA Polymerase I Transcription Termination 3089
41.1.3.1 TTF-I binds to the Sal Box 3090
41.1.3.2 Polymerase I Transcription Complex/Nascent Pre rRNA Complex pauses at the TTF-I:Sal Box 3091
41.1.3.3 PTRF Binds the Polymerase I Transcription Complex/Nascent Pre rRNA Complex paused at the TTF-I:Sal Box 3092
41.1.3.4 Dissociation of PTRF:Polymerase I/Nascent Pre rRNA Complex:TTF-I:Sal Box 3093
41.2 RNA Polymerase II Transcription 3094
41.2.1 RNA Polymerase II Transcription Pre-Initiation 3094
41.2.1.1 Recognition and Binding of Core Promoter Elements by TFIID 3095
41.2.1.2 Binding of TFIIA and TFIIB to the pol II promoter:TFIID complex 3095
41.2.1.3 Recruitment of RNA Polymerase II Holoenzyme by TFIIF to the pol II promoter:TFIID:TFIIA:TFIIB complex 3096
41.2.1.4 Binding of TFIIE to the growing preinitiation complex 3097
41.2.1.5 Formation of the closed pre-initiation complex 3098

41.2.2 RNA Polymerase II Promoter Opening: First Transition 3098
41.2.3 RNA Polymerase II Transcription Initiation 3100
41.2.3.1 NTP Binds Active Site of RNA Polymerase II 3100
41.2.3.2 Nucleophillic Attack by 3'-hydroxyl Oxygen of nascent transcript on the Alpha Phosphate of NTP 3101
41.2.3.3 Newly Formed Phosphodiester Bond Stabilized and PPi Released 3101
41.2.4 RNA Polymerase II Promoter Escape 3102
41.2.4.1 Addition of the third nucleotide on the nascent transcript 3102
41.2.4.2 Addition of the fourth nucleotide on the Nascent Transcript: Second Transition 3103
41.2.4.3 Addition of Nucleotides 5 through 9 on the growing Transcript 3104
41.2.4.4 Addition of nucleotides 10 and 11 on the growing transcript: Third Transition 3105
41.2.4.5 Addition of nucleotides between position +11 and +30 3106
41.2.5 RNA Pol II CTD phosphorylation and interaction with CE 3107
41.2.5.1 Extrusion of 5'-end of 30 nt long transcript through the pore in Pol II complex 3107
41.2.5.2 Phosphorylation (Ser5) of RNA pol II CTD 3107
41.2.5.3 RNA Polymerase II CTD (phosphorylated) binds to CE 3108
41.2.5.4 Activation of GT 3108
41.2.5.5 SPT5 subunit of Pol II binds the RNA triphosphatase (RTP) 3109
41.2.6 RNA Polymerase II Transcription Elongation 3109
41.2.6.1 Formation of the Early Elongation Complex 3112
41.2.6.1.1 Hypophosphorylation of RNA Pol II CTD by FCP1P protein 3112
41.2.6.1.2 DSIF complex binds to RNA Pol II (hypophosphorylated) 3113
41.2.6.1.3 Formation of DSIF:NELF:early elongation complex 3113
41.2.6.2 Formation of RNA Pol II elongation complex 3114
41.2.6.2.1 Hyperphosphorylation (Ser2) of RNA Pol II CTD by P-TEFb complex 3115
41.2.6.2.2 Recruitment of elongation factors to form elongation complex 3116
41.2.6.3 Addition of nucleotides leads to transcript elongation 3116
41.2.6.4 Pol II elongation complex moves on the template as transcript elongates 3117
41.2.6.5 Separation of elongating transcript from template 3118
41.2.7 RNA Polymerase II Transcription Termination 3118
41.2.7.1 Cleavage of Growing Transcript in the Termination Region 3119
41.2.7.1.1 Cleavage of mRNA at the 3'-end 3119
41.2.7.1.2 Cleavage of the 3'-end of Replication Dependent Histone Pre-mRNA 3120
41.2.7.1.3 Cleavage of the 3'-end of the Histone Pre-mRNA 3121
41.2.7.1.4 Cleavage of Intronless Pre-mRNA at 3'-end 3122
41.3 RNA Polymerase III Transcription 3123
41.3.1 RNA Polymerase III Transcription Initiation 3124
41.3.1.1 RNA Polymerase III Transcription Initiation From Type 1 Promoter 3125
41.3.1.1.1 Binding of TFIIIA To type 1 Promoter 3126
41.3.1.1.2 Binding of TFIIIC to TFIIIA:Type I Promoter complex 3127
41.3.1.1.3 Binding of TFIIIB to TFIIIC:TFIIIA:Type I Promoter complex 3128
41.3.1.1.4 Recruitment of RNA polymerase III to TFIIIB:TFIIIC:TFIIIA:Type 1 Promoter Complex 3129
41.3.1.1.5 RNA Polymerase III Promoter Opening at Type 1 Promoters 3131
41.3.1.2.1 Binding of TFIIIC to Type 2 promoter 3133
41.3.1.2.2 Binding of TFIIIB to TFIIC: Type 2 Promoter Complex 3134
41.3.1.2.3 Recruitment of RNA Polymerase III to the TFIIIB:TFIIIC: Type 2 Promoter Complex 3135
41.3.1.3.1 Binding of SNAPc, Oct-1, and Staf to Type 3 Promoter 3138
41.3.1.3.2 Binding of TFIIIB to SNAPc:Oct-1:Staf:Type 3 Promoter Complex 3139
41.3.1.3.3 Recruitment of RNA Polymerase III to TFIIIB:SNAPc:Type 3 Promoter Complex 3140
41.3.2 RNA Polymerase III Chain Elongation 3142
41.3.2.1 Initiation of RNA Polymerase III Productive Transcription 3142
41.3.2.2 RNA Polymerase III Productive Transcription 3143
41.3.3 RNA Polymerase III Transcription Termination 3145
41.3.3.1 RNA Polymerase III Transcriptional Pause at Terminator Sequence 3145
41.3.3.2 RNA Polymerase III Termination and release of transcribed mRNA 3146
41.4 Transcription from mitochondrial promoters 3148
41.4.1 Mitochondrial transcription initiation 3150
41.4.1.1 TFAM binds to mitochondrial promoters 3151
41.4.1.2 Association of TFAM:mt promoter complex with POLRMT:TFB2M 3152
41.4.2 POLRMT chain elongation 3152
41.4.3 Mitochondrial transcription termination 3153
41.4.3.1 Association of mTERF with the termination sequence 3154
42 Translation 3156
42.1 Eukaryotic Translation Initiation 3156
42.1.1 Cap-dependent Translation Initiation 3157

42.1.1.1 Formation of a pool of free 40S subunits 3159
42.1.1.1.1 Release of 40S and 60S subunits from the 80S ribosome 3159
42.1.1.1.2 eIF3 and eIF1A bind to the 40S subunit 3159
42.1.1.2 Formation of the ternary complex, and subsequently, the 43S complex 3160
42.1.1.2.1 De novo formation of eIF2:GTP 3160
42.1.1.2.2 Met-tRNAi binds to eIF2:GTP to form the ternary complex 3161
42.1.1.2.3 Formation of the 43S pre-initiation complex 3161
42.1.1.3 Activation of the mRNA upon binding of the cap-binding complex and eIFs, and subsequent binding to 43S 3162
42.1.1.3.1 Release of eIF4E from the inactive eIF4E:4E-BP complex 3163
42.1.1.3.2 Formation of the cap-binding eIF4F complex 3163
42.1.1.3.3 eIF4F binds to mRNP 3164
42.1.1.3.4 Cap-bound mRNA is activated by helicases 3164
42.1.1.3.5 Translation initiation complex formation 3165
42.1.1.3.5.1 Formation of translation initiation complexes containing mRNA that does not circularize 3165
42.1.1.3.5.2 Formation of translation initiation complexes yielding circularized Ceruloplasmin mRNA in a 'closed-loop' conformation3166
42.1.1.4 Ribosomal scanning and start codon recognition 3167
42.1.1.4.1 Ribosomal scanning 3167
42.1.1.4.2 Start codon recognition 3168
42.1.1.5 GTP hydrolysis and joining of the 60S ribosomal subunit 3169
42.1.1.5.1 eIF2:GTP is hydrolyzed, eIFs are released 3169
42.1.1.5.2 eIF5B:GTP is hydrolyzed and released 3171
42.1.1.5.3 The 60S subunit joins the translation initiation complex 3171
42.1.1.6 Recycling of eIF2:GDP 3172
42.1.1.6.1 Formation of eIF2:GDP:eIF2B intermediate 3172
42.1.1.6.2 eIF2 activation 3173
42.1.2 Cap-independent Translation Initiation 3173
42.2 Eukaryotic Translation Elongation 3174
42.2.1 eEF1A complexes with GTP 3175
42.2.2 eEF1A:GTP:aminoacyl tRNA ternary complex formation. 3177
42.2.3 Peptide chain elongation 3178
42.2.3.1 Aminoacyl-tRNA binds to the ribosome at the A-site 3178
42.2.3.2 Hydrolysis of eEF1A:GTP 3180
42.2.3.3 Peptide transfer from P-site tRNA to the A-site tRNA 3181
42.2.3.4 Translocation of ribosome by 3 bases in the 3' direction 3182
42.2.4 Regeneration of eEF1A:GTP by eEF1B activity 3184
42.3 Eukaryotic Translation Termination 3185
42.3.1 GTP bound eRF3:eRF1 complex binds the peptidyl tRNA:mRNA:80S Ribosome complex 3186
42.3.2 GTP Hydrolysis by eRF3 bound to the eRF1:mRNA:polypeptide:80S Ribosome complex 3187
42.3.3 Polypeptide release from the eRF3-GDP:eRF1:mRNA:80S Ribosome complex 3188
43 mRNA Processing 3190
43.1 mRNA Capping 3190
43.1.1 Capping complex formation 3192
43.1.2 Hydrolysis of the 5'-end of the nascent transcript by the capping enzyme 3192
43.1.3 Formation of the CE:GMP intermediate complex 3194
43.1.4 Transfer of GMP from the capping enzyme GT site to 5'-end of mRNA 3194
43.1.5 Dissociation of transcript with 5'-GMP from GT 3195
43.1.6 Methylation of GMP-cap by RNA Methyltransferase 3196
43.1.7 Recognition and binding of the mRNA cap by the cap-binding complex 3197
43.2 Processing of Capped pre-mRNA 3198
43.2.1 Processing of Capped Intron-Containing Pre-mRNA 3198
43.2.1.1 Internal Methylation of mRNA 3198
43.2.1.2 Formation of pre-mRNPs 3199
43.2.1.3 mRNA Splicing 3200
43.2.1.3.1 mRNA Splicing - Major Pathway 3200
43.2.1.3.1.1 Formation of the Spliceosomal E complex 3201
43.2.1.3.1.2 Formation of the Spliceosomal A Complex 3203
43.2.1.3.1.3 Formation of the Spliceosomal B Complex 3203
43.2.1.3.1.4 Formation of an intermediate Spliceosomal C complex 3205
43.2.1.3.1.5 Formation of the active Spliceosomal C complex 3206
43.2.1.3.1.6 Lariat Formation and 5'-Splice Site Cleavage 3206
43.2.1.3.1.7 Formation of Exon Junction Complex 3208
43.2.1.3.1.8 Cleavage at the 3'-Splice Site and Exon Ligation 3208
43.2.1.3.2 mRNA Splicing - Minor Pathway 3209
43.2.1.3.2.1 Formation of AT-AC A complex 3211
43.2.1.3.2.2 Formation of AT-AC B Complex 3213
43.2.1.3.2.3 Formation of AT-AC C complex 3215
43.2.1.3.2.4 ATAC spliceosome mediated Lariat formation,5' splice site cleavage 3217
43.2.1.3.2.5 ATAC spliceosome mediated 3' splice site cleavage, exon ligation 3219
43.2.1.4 mRNA 3'-end processing 3221
43.2.1.4.1 Cleavage of mRNA at the 3'-end 3221
43.2.1.4.2 mRNA polyadenylation 3222
43.2.1.5 Transport of Mature Transcript to Cytoplasm 3223
43.2.1.5.1 Transport of Mature mRNA derived from an Intron-Containing Transcript 3223
43.2.1.5.1.1 Recruitment of TAP to the EJC 3224
43.2.1.5.1.2 Docking of the TAP:EJC Complex with the NPC 3224
43.2.1.5.1.3 Transport of the export-competent complex through the NPC 3225
43.2.1.5.1.4 Release from the NPC and Disassembly of the mRNP 3225
43.2.1.5.2 Transport of Mature mRNAs Derived from Intronless Transcripts 3226
43.2.1.5.2.1 Transport of the SLBP Dependant Mature mRNA 3226
43.2.1.5.2.1.1 Docking of Mature Replication Dependent Histone mRNA with the NPC 3227
43.2.1.5.2.1.2 Transport of the Mature IntronlessTranscript Derived Histone mRNA:SLBP:TAP:Aly/Ref complex through the NPC3228
43.2.1.5.2.1.3 Release of the Mature intronless transcript derived Histone mRNA:SLBP:eIF4E Complex 3228
43.2.1.5.2.2 Transport of the SLBP independent Mature mRNA 3229
43.2.1.5.2.2.1 Docking of Mature Histone mRNA complex:TAP at the NPC 3229
43.2.1.5.2.2.2 Transport of the Mature Intronless Transcript Derived Histone mRNA:TAP:Aly/Ref Complex through the NPC 3230
43.2.1.5.2.2.3 Release of the SLBP independent Histone mRNA from the NPC 3230
43.2.1.5.2.3 Transport of Mature mRNA Derived from an Intronless Transcript 3231
43.2.1.5.2.3.1 Docking of the Mature intronless derived transcript derived mRNA, TAP and Aly/Ref at the NPC 3231
43.2.1.5.2.3.2 Transport of the Mature intronless transcript derived mRNA:TAP:Aly/Ref Complex through the NPC 3232
43.2.1.5.2.3.3 Release of the Mature intronless derived mRNA, TAP, and Aly/Ref from the NPC 3232
43.2.2 Processing of Capped Intronless Pre-mRNA 3233
43.2.2.1 SLBP Dependent Processing of Replication-Dependent Histone Pre-mRNAs 3233
43.2.2.1.1 Binding of SLBP to Replication-Dependent Histone Pre-mRNA 3235
43.2.2.1.2 Recruitment of U7 snRNP:ZFP100 complex to the SLBP Bound Pre-mRNA 3236
43.2.2.1.3 Cleavage of the 3'-end of Replication Dependent Histone Pre-mRNA 3237
43.2.2.2 SLBP independent Processing of Histone Pre-mRNAs 3238
43.2.2.2.1 Recruitment of U7 snRNP:ZFP100 complex to the Histone Pre-mRNA 3238
43.2.2.2.2 Cleavage of the 3'-end of the Histone Pre-mRNA 3239
43.2.2.3 Processing of Intronless Pre-mRNAs 3240
43.2.2.3.1 Recognition of AAUAAA sequence by CPSF 3240
43.2.2.3.2 Recruitment of CstF to the CPSF Bound Pre-mRNA 3241
43.2.2.3.3 Binding of Cleavage factors and Poly(A)Polymerase to the CstF:CPSF:Pre-mRNA Complex 3242
43.2.2.3.4 Cleavage and polyadenylation of Intronless Pre-mRNA 3243
43.3 mRNA Editing 3243
43.3.1 mRNA Editing: C to U Conversion 3245
43.3.1.1 Formation of the Editosome 3245
43.3.1.1.1 Formation of stem-loop structure in ApoB mRNA 3246
43.3.1.1.2 Binding of ACF to stem-looped RNA 3246
43.3.1.1.3 Binding of APOBEC-1 to form editosome 3247
43.3.1.2 C4 deamination of cytidine 3247
43.3.2 mRNA Editing: A to I Conversion 3248
43.3.2.1 Formation of dsRNA structure by looping 3249
43.3.2.2 Formation of editosomes by ADAR proteins 3249
43.3.2.2.1 Binding of ADAR1 homodimer to dsRNA duplex 3249
43.3.2.2.2 Binding of ADAR2 homodimer to dsRNA duplex 3250
43.3.2.3 C6 deamination of adenosine 3250
43.3.2.3.1 Deamination at C6 position of adenosine in Editosome (ADAR1) 3251
43.3.2.3.2 Deamination at C6 position of adenosine in Editosome (ADAR2) 3251
Preface
The Reactome project is a collaboration among Cold Spring Harbor Laboratory, The European Bioinformatics Institute, and The Gene Ontology
Consortium to develop a curated resource of core pathways and reactions in human biology. The information in this database is authored by
biological researchers with expertise in their fields, maintained by the Reactome editorial staff, and cross-referenced with the sequence
databases at NCBI, Ensembl and UniProt, the UCSC Genome Browser, HapMap, KEGG(Gene and Compound ), ChEBI, PubMed and GO. In
addition to curated human events, inferred orthologous events in 22 non-human species including mouse, rat, chicken, puffer fish, worm, fly,
yeast, two plants and E. coli are also available. A description of Reactome has been published in Genome Biology. Reactome is a free on-line
resource, and Reactome software is open-source. However, please take note of our disclaimer.
Acknowledgements
The development of Reactome is supported by an EU STRP, EMI-CD grant from the European Union, the EBI Industry program and grant P41
HG003751 from the National Human Genome Research Institute at the US National Institutes of Health.
We would also like to thank our Scientific Advisory Board: Julie Ahringer </TD><TD>University of Cambridge, Russ Altman </TD><TD>Stanford
University, Richard Belew </TD><TD>University of California, San Diego, Timo Hannay </TD><TD>Nature Publishing Group, Edda Klipp
</TD><TD>Max Planck Institute for Molecular Genetics, Adrian Krainer </TD><TD>Cold Spring Harbor Laboratory, Ed Marcotte
</TD><TD>University of Texas at Austin, Mark McCarthy </TD><TD>Oxford University, Bill Pearson </TD><TD>University of Virginia, Pardis
Sabeti </TD><TD>Broad Institute, David Stewart </TD><TD>Cold Spring Harbor Laboratory.
The Reactome project, and therefore the existence of this book, depends finally on the Reactome team members themselves: Ewan Birney,
Bernard de Bono, Michael Caudy, David Croft, Peter D'Eustachio, Phani Garapati, Marc Gillespie, Gopal Gopinath, Jill Hemish, Bijay Jassal,
Suzanna Lewis, Shahana Mahajan, Lisa Matthews, Bruce May, Esther Schmidt, Lincoln Stein, Imre Vastrik, Guanming Wu.
1 Apoptosis
Authors
Alnemri, E, Hengartner, M, Tschopp, J, Tsujimoto, Y, Hardwick, JM, 2004-01-16.
Editors
Gopinathrao, G, Matthews, L, Gillespie, ME, Joshi-Tope, G, 0000-00-00.
Reviewers
Hengartner, M, Ranganathan, S, Vaux, D, 0000--0-0-.
Description
Apoptosis is a distinct form of cell death that is functionally and morphologically different from necrosis. Nuclear chromatin condensation,
cytoplasmic shrinking, dilated endoplasmic reticulum, and membrane blebbing characterize apoptosis in general. Mitochondria remain
morphologically unchanged. In 1972 Kerr et al introduced the concept of apoptosis as a distinct form of "cell-death", and the mechanisms of
various apoptotic pathways are still being revealed today.
The two principal pathways of apoptosis are (1) the Bcl-2 inhibitable or intrinsic pathway induced by various forms of stress like intracellular
damage, developmental cues, and external stimuli and (2) the caspase 8/10 dependent or extrinsic pathway initiated by the engagement of
death receptors
The caspase 8/10 dependent or extrinsic pathway is a death receptor mediated mechanism that results in the activation of caspase-8 and
caspase-10. Activation of death receptors like Fas/CD95, TNFR1, and the TRAIL receptor is promoted by the TNF family of ligands including
FASL (APO1L OR CD95L), TNF, LT-alpha, LT-beta, CD40L, LIGHT, RANKL, BLYS/BAFF, and APO2L/TRAIL. These ligands are released in
response to microbial infection, or as part of the cellular, humoral immunity responses during the formation of lymphoid organs, activation of
dendritic cells, stimulation or survival of T, B, and natural killer (NK) cells, cytotoxic response to viral infection or oncogenic transformation.
The Bcl-2 inhibitable or intrinsic pathway of apoptosis is a stress-inducible process, and acts through the activation of caspase-9 via Apaf-1 and
cytochrome c. The rupture of the mitochondrial membrane, a rapid process involving some of the Bcl-2 family proteins, releases these molecules
into the cytoplasm. Examples of cellular processes that may induce the intrinsic pathway in response to various damage signals include: auto
reactivity in lymphocytes, cytokine deprivation, calcium flux or cellular damage by cytotoxic drugs like taxol, deprivation of nutrients like glucose
and growth factors like EGF, anoikis, transactivation of target genes by tumor suppressors including p53.
In many non-immune cells, death signals initiated by the extrinsic pathway are amplified by connections to the intrinsic pathway. The connecting
link appears to be the truncated BID (tBID) protein a proteolytic cleavage product mediated by caspase-8 or other enzymes.
References
A Ashkenazi, "Targeting death and decoy receptors of the tumour-necrosis factor superfamily", Nat Rev Cancer, 2, 2002, 420-30.
M MacFarlane, AC Williams, "Apoptosis and disease: a life or death decision", EMBO Rep, 5, 2004, 674-8.
JF Kerr, "History of the events leading to the formulation of the apoptosis concept", Toxicology, 181, 2002, 471-4.
JF Kerr, AH Wyllie, AR Currie, "Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics", Br J Cancer, 26,
1972, 239-57.
S Cory, JM Adams, "The Bcl2 family: regulators of the cellular life-or-death switch", Nat Rev Cancer, 2, 2002, 647-56.
JM Adams, "Ways of dying: multiple pathways to apoptosis.", Genes Dev, 17, 2003, 2481-95.
S Cory, DC Huang, JM Adams, "The Bcl-2 family: roles in cell survival and oncogenesis.", Oncogene, 22, 2003, 8590-607.
1.1 Extrinsic Pathway for Apoptosis
Authors
Gillespie, ME, 2004-08-10.
Editors
Reviewers
Vaux, D, 0000-00-00.
Description
Known as the "death receptor pathway" the extrinsic or caspase 8/10 dependent pathway is activated by ligand binding. The "death receptors"
are specialized cell-surface receptors including Fas/CD95, tumor necrosis factor-alpha (TNF-alpha) receptor 1, and two receptors, DR4 and
DR5, that bind to the TNF-alpha related apoptosis-inducing ligand (TRAIL). The extrinsic and intrinsic pathways unite in the activation of
Caspase-3, though the two pathways communicate through the pro-apoptotic Bcl-2 family member Bid before uniting at the shared activation of
Caspase-3.
References
MO Hengartner, "The biochemistry of apoptosis.", Nature, 407, 2000, 770-6.
1.1.1 Death Receptor Signalling
Authors
Editors

Reviewers
Vaux, D, 0000-00-00.
Description
The death receptors, all cell-surface receptors, begin the process of caspase activation. The common feature of these type 1 transmembrane
proteins is the "death-domain" a conserved cytoplasmic motif found on all of the three receptors (FAS/CD95, TNF-receptor, and TRAIL-receptor)
that binds the Fas-associated protein with death domain (FADD)
1.1.1.1 FasL/ CD95L signaling
Authors
Editors
Reviewers
Vaux, D, 0000-00-00.
Description
The Fas family of cell surface receptors initiate the apototic pathway through interaction with the external ligand, FasL. The cytoplasmic domain
of Fas interacts with a number of molecules in the transduction of the external signal to the cytoplasmic side of the cell membrane. The most
notable cytoplasmic domain is the Death Domain (DD) that is involved in recruiting the FAS-associating death domain-containing protein
(FADD). This interaction drives downstream events.
References
MO Hengartner, "The biochemistry of apoptosis.", Nature, 407, 2000, 770-6.
N Itoh, S Yonehara, A Ishii, M Yonehara, S Mizushima, M Sameshima, A Hase, Y Seto, S Nagata, "The polypeptide encoded by the cDNA for
human cell surface antigen Fas can mediate apoptosis", Cell, 66, 1991, 233-43.
S Yonehara, A Ishii, M Yonehara, "A cell-killing monoclonal antibody (anti-Fas) to a cell surface antigen co-downregulated with the receptor of
tumor necrosis factor", J Exp Med, 169, 1989, 1747-56.
AM Chinnaiyan, K O'Rourke, M Tewari, VM Dixit, "FADD, a novel death domain-containing protein, interacts with the death domain of Fas and
initiates apoptosis", Cell, 81, 1995, 505-12.
1.1.1.1.1 FASL binds FAS Receptor

Description
At the beginning of this reaction, 1 molecule of 'FASL', and 1 molecule of 'FAS Receptor' are present. At the end of this reaction, 1 molecule of
'FASL:FAS Receptor monomer' is present.
This reaction takes place in the 'cell'.
References
T Brunner, RJ Mogil, D LaFace, NJ Yoo, A Mahboubi, F Echeverri, SJ Martin, WR Force, DH Lynch, CF Ware, "Cell-autonomous Fas
(CD95)/Fas-ligand interaction mediates activation-induced apoptosis in T-cell hybridomas", Nature, 373, 1995, 441-4.
Reaction
1.1.1.1.2 Trimerization of the FASL:FAS receptor complex

Description
At the beginning of this reaction, 3 molecules of 'FASL:FAS Receptor monomer' is present. At the end of this reaction, 1 molecule of 'FASL:FAS
Receptor Trimer' is present.
References
FC Kischkel, S Hellbardt, I Behrmann, M Germer, M Pawlita, PH Krammer, ME Peter, "Cytotoxicity-dependent APO-1 (Fas/CD95)-associated
proteins form a death-inducing signaling complex (DISC) with the receptor", EMBO J, 14, 1995, 5579-88.
Reaction
1.1.1.1.3 FasL:Fas binds FADD

Description
At the beginning of this reaction, 1 molecule of 'FADD', and 1 molecule of 'FASL:FAS Receptor Trimer' are present. At the end of this reaction, 1
molecule of 'FASL:FAS Receptor Trimer:FADD complex' is present.
References
MP Boldin, EE Varfolomeev, Z Pancer, IL Mett, JH Camonis, D Wallach, "A novel protein that interacts with the death domain of Fas/APO1
contains a sequence motif related to the death domain", J Biol Chem, 270, 1995, 7795-8.
Reaction
1.1.1.1.4 FASL:FAS Receptor Trimer:FADD complex binds pro-Caspase-8
Description
At the beginning of this reaction, 1 molecule of 'Caspase-8 precursor ', and 1 molecule of 'FASL:FAS Receptor Trimer:FADD complex' are
present. At the end of this reaction, 1 molecule of 'FASL:FAS Receptor Trimer:FADD:pro-Caspase-8 DISC' is present.
References
MP Boldin, TM Goncharov, YV Goltsev, D Wallach, "Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF
receptor-induced cell death", Cell, 85, 1996, 803-15.
Reaction
1.1.1.1.5 FASL:FAS Receptor Trimer:FADD complex binds pro-Caspase-10
Description
At the beginning of this reaction, 1 molecule of 'Caspase-10 precursor ', and 1 molecule of 'FASL:FAS Receptor Trimer:FADD complex' are
present. At the end of this reaction, 1 molecule of 'FASL:FAS Receptor Trimer:FADD:pro-Caspase-10' is present.
References
J Wang, HJ Chun, W Wong, DM Spencer, MJ Lenardo, "Caspase-10 is an initiator caspase in death receptor signaling", Proc Natl Acad Sci U S
A, 98, 2001, 13884-8.
MR Sprick, E Rieser, H Stahl, A Grosse-Wilde, MA Weigand, H Walczak, "Caspase-10 is recruited to and activated at the native TRAIL and
CD95 death-inducing signalling complexes in a FADD-dependent manner but can not functionally substitute caspase-8", EMBO J, 21, 2002,
4520-30.
Reaction
1.1.1.2 TNF signaling
Authors
Editors
Description
The Tumor Necrosis Factor alpha (TNF-alpha) mediated apoptosis pathway has been implicated in the pathogenesis of a number of diseases
including sepsis, diabetes, cancer, osteoporosis, multiple sclerosis, rheumatoid arthritis, and inflammatory bowel diseases. The TNF signaling
network provides extensive cross talk between the apoptotic pathway, and the other NF-B, and JNK pathways that also emanate from TNF-R.
References
G Chen, DV Goeddel, "TNF-R1 signaling: a beautiful pathway", Science, 296, 2002, 1634-5.
1.1.1.2.1 TNF Binds TNF-R1
Description
At the beginning of this reaction, 1 molecule of 'TNF-RI', and 1 molecule of 'TNF-alpha' are present. At the end of this reaction, 1 molecule of
'TNF-alpha:TNF-R1 complex' is present.

References
GB Stauber, RA Aiyer, BB Aggarwal, "Human tumor necrosis factor-alpha receptor. Purification by immunoaffinity chromatography and initial
characterization.", J Biol Chem, 263, 1988, 19098-104.
Reaction
1.1.1.2.2 TNF:TNF-R1 binds TRADD, TRAF2 and RIP Complex
Editors
Reviewers
Vaux, D, 0000-00-00.
Description
Once the TNF-aplha:TNF-R1:TRADD complex is formed the two TNF-alpha mediated pathways are possible. The variable is the recruitment of
FADD to the larger complex formed by the TNF-aplha:TNF-R1 platform via the interaction of the Death Domains. The steps leading to the Jun,
NF kappaB, or apoptotic pathways are rife with modulation.
References
H Hsu, J Xiong, DV Goeddel, "The TNF receptor 1-associated protein TRADD signals cell death and NF-kappa B activation", Cell, 81, 1995,
495-504.
Reaction
1.1.1.2.3 TRADD:TRAF2:RIP1 complex dissociates from the TNF-alpha:TNF-R1 complex.
Editors
Reviewers
Vaux, D, 0000-00-00.
Description
Once formed the TRADD:TRAF2:RIP1 complex may dissociate from the TNF:TNF-R1 platform and become cytosolic. If this complex recruits
FADD then the cell will be pushed along the apoptotic pathway.
References
O Micheau, J Tschopp, "Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes", Cell, 114, 2003, 181-90.
Reaction
1.1.1.2.4 TRADD:TRAF2:RIP1 complex binds FADD
Editors
Reviewers
Vaux, D, 0000-00-00.
Description
Once formed the TRADD:TRAF2:RIP1 complex may dissociate from the TNF:TNF-R1 platform and become cytosolic. If this complex recruits
FADD then the cell will be pushed along the apoptotic pathway.
References
Reaction
1.1.1.2.5 TRADD:TRAF2:RIP1:FADD complex binds Pro-Caspase 8
Description
At the beginning of this reaction, 1 molecule of 'TRAF2:TRADD:RIP1:FADD', and 1 molecule of 'Caspase-8 precursor ' are present. At the end of
this reaction, 1 molecule of 'TRADD:TRAF2:RIP1:FADD:Capase-8 Complex' is present.
References
Reaction
1.1.1.3 TRAIL signaling
Authors
Editors
Description
Tumor necrosis factor-related apoptosis-inducing ligand or Apo 2 ligand (TRAIL/Apo2L) is a member of the tumor necrosis factor (TNF) family.
This group of apoptosis induction pathways all work through protein interactions mediated by the intracellular death domain (DD), encoded within
the cytoplasmic domain of the receptor. TRAIL selectively induces apoptosis through its interaction with the Fas-associated death domain
protein (FADD).
References
S Wang, WS el-Deiry, "TRAIL and apoptosis induction by TNF-family death receptors", Oncogene, 22, 2003, 8628-33.
1.1.1.3.1 TRAIL Binds TRAIL-Receptor2
Description
At the beginning of this reaction, 1 molecule of 'TRAIL receptor-2', and 1 molecule of 'TRAIL' are present. At the end of this reaction, 1 molecule
of 'TRAIL receptor-2:TRAIL complex' is present.
References
SR Wiley, K Schooley, PJ Smolak, WS Din, CP Huang, JK Nicholl, GR Sutherland, TD Smith, C Rauch, CA Smith, "Identification and
characterization of a new member of the TNF family that induces apoptosis", Immunity, 3, 1995, 673-82.
Reaction
1.1.1.3.2 Trimerization of TRAIL: TRAIL receptor-2 complex
Description
At the beginning of this reaction, 3 molecules of 'TRAIL receptor-2:TRAIL complex' is present. At the end of this reaction, 1 molecule of 'TRAIL
receptor-2:TRAIL Trimer' is present.
References
MR Sprick, MA Weigand, E Rieser, CT Rauch, P Juo, J Blenis, PH Krammer, H Walczak, "FADD/MORT1 and caspase-8 are recruited to TRAIL
receptors 1 and 2 and are essential for apoptosis mediated by TRAIL receptor 2", Immunity, 12, 2000, 599-609.
Reaction
1.1.1.3.3 TRAIL:TRAIL receptor-2 Trimer Binds FADD
Description
At the beginning of this reaction, 1 molecule of 'FADD', and 1 molecule of 'TRAIL receptor-2:TRAIL Trimer' are present. At the end of this
reaction, 1 molecule of 'TRAIL:TRAIL receptor-2:FADD complex' is present.
References
Reaction
1.1.1.3.4 TRAIL:TRAIL-Receptor2 Trimer:FADD complex binds Caspase-8
Description
At the beginning of this reaction, 1 molecule of 'Caspase-8 precursor ', and 1 molecule of 'TRAIL:TRAIL receptor-2:FADD complex' are present.
At the end of this reaction, 1 molecule of 'TRAIL:TRAIL receptor-2 Trimer:FADD:Caspase-8 precursor complex' is present.
References
Reaction
1.1.1.3.5 TRAIL:TRAIL-Receptor2 Trimer:FADD complex binds Caspase-10
Description
At the beginning of this reaction, 1 molecule of 'Caspase-10 precursor ', and 1 molecule of 'TRAIL:TRAIL receptor-2:FADD complex' are present.
At the end of this reaction, 1 molecule of 'TRAIL:TRAIL receptor-2 Trimer:FADD:Caspase-10 precursor complex' is present.
References
J Wang, HJ Chun, W Wong, DM Spencer, MJ Lenardo, "Caspase-10 is an initiator caspase in death receptor signaling", Proc Natl Acad Sci U S
A, 98, 2001, 13884-8.
MR Sprick, E Rieser, H Stahl, A Grosse-Wilde, MA Weigand, H Walczak, "Caspase-10 is recruited to and activated at the native TRAIL and
CD95 death-inducing signalling complexes in a FADD-dependent manner but can not functionally substitute caspase-8", EMBO J, 21, 2002,
4520-30.
Reaction
1.1.2 Caspase-8 is formed from procaspase-8
Description
Caspase-8 is formed from the precursor protein pro-Caspase-8 as a cleavage product.
References
KM Boatright, GS Salvesen, "Mechanisms of caspase activation", Curr Opin Cell Biol, 15, 2003, 725-31.
1.1.2.1 Activation of Pro-Caspase 8
Description
Pro-caspase-8 is known to be activated by various factors in response to a variety of external and internal stimuli. These include FAS, TRAIL,
and TNF.
1.1.2.1.1 FAS Mediated Activation of Pro-caspase 8
Description
At the beginning of this reaction, 1 molecule of 'FASL:FAS Receptor Trimer:FADD:pro-Caspase-8 DISC' is present. At the end of this reaction, 1
molecule of 'p10 subunit of Caspase 8', 1 molecule of 'p18 subunit of Caspase 8', and 1 molecule of 'FASL:FAS Receptor Trimer:FADD complex'
are present.

References
A Ashkenazi, VM Dixit, "Death receptors: signaling and modulation", Science, 281, 1998, 1305-8.
Reaction
1.1.2.1.2 TRAIL Mediated Activation of Pro-caspase 8
Description
At the beginning of this reaction, 1 molecule of 'TRAIL:TRAIL receptor-2 Trimer:FADD:Caspase-8 precursor complex' is present. At the end of
this reaction, 1 molecule of 'p10 subunit of Caspase 8', 1 molecule of 'TRAIL:TRAIL receptor-2:FADD complex', and 1 molecule of 'p18 subunit
of Caspase 8' are present.
Reaction
1.1.2.1.3 TNF Mediated Activation of Pro-caspase 8
Description
At the beginning of this reaction, 1 molecule of 'TRADD:TRAF2:RIP1:FADD:Capase-8 Complex' is present. At the end of this reaction, 1
molecule of 'TRAF2:TRADD:RIP1:FADD', 1 molecule of 'p10 subunit of Caspase 8', and 1 molecule of 'p18 subunit of Caspase 8' are present.
Reaction
1.1.2.2 Formation of Caspase-8 dimer
Description
At the beginning of this reaction, 1 molecule of 'p10 subunit of Caspase 8', and 1 molecule of 'p18 subunit of Caspase 8' are present. At the end
of this reaction, 1 molecule of 'Caspase-8 dimer' is present.
This reaction takes place in the 'cytosol'.
Reaction
1.2 Intrinsic Pathway for Apoptosis
Authors
Matthews, L, 2004-08-06.
Description
The intrinsic (Bcl-2 inhibitable or mitochondrial) pathway of apoptosis functions in response to various types of intracellular stress including
growth factor withdrawal, DNA damage, unfolding stresses in the endoplasmic reticulum and death receptor stimulation. Following the reception
of stress signals, proapoptotic BCL-2 family proteins are activated and subsequently interact with and inactivate antiapoptotic BCL-2 proteins.
This interaction leads to the destabilization of the mitochondrial membrane and release of apoptotic factors. These factors induce the caspase
proteolytic cascade, chromatin condensation, and DNA fragmentation, ultimately leading to cell death. The key players in the Intrinsic pathway
are the Bcl-2 family of proteins that are critical death regulators residing immediately upstream of mitochondria. The Bcl-2 family consists of both
anti- and proapoptotic members that possess conserved alpha-helices with sequence conservation clustered in BCL-2 Homology (BH) domains.
Proapoptotic members are organized as follows:
1. "Multidomain" BAX family proteins such as BAX, BAK etc. that display sequence conservation in their BH1-3 regions. These proteins act
downstream in mitochondrial disruption.
2. "BH3-only" proteins such as BID,BAD, NOXA, PUMA,BIM, and BMF have only the short BH3 motif. These act upstream in the pathway,
detecting developmental death cues or intracellular damage. Anti-apoptotic members like Bcl-2, Bcl-XL and their relatives exhibit homology in all
segments BH1-4. One of the critical functions of BCL-2/BCL-XL proteins is to maintain the integrity of the mitochondrial outer membrane.
References
GS Salvesen, CS Duckett, "IAP proteins: blocking the road to death's door", Nat Rev Mol Cell Biol, 3, 2002, 401-10.
JM Adams, "Ways of dying: multiple pathways to apoptosis.", Genes Dev, 17, 2003, 2481-95.
X Wang, "The expanding role of mitochondria in apoptosis.", Genes Dev, 15, 2001, 2922-33.
X Saelens, N Festjens, L Vande Walle, M van Gurp, G van Loo, P Vandenabeele, "Toxic proteins released from mitochondria in cell death",
Oncogene, 23, 2004, 2861-74.
1.2.1 Activation, myristolyation of BID and translocation to mitochondria
Authors
Gopinathrao, G, 2004-08-19.
Reviewers
Vaux, D, 0000-00-00.
Description
BID may promote cell death by activating BAX and BAK while inactivating anti-apoptotic proteins. The engagement of cell surface receptors
activates the caspase-8, a heterodimer, that cleaves BID in its amino terminal region. This particular event may act as a link between Extrinsic
(caspase 8/10 dependent) and Intrinsic (Bcl-2 inhibitable) pathways although some evidences from mouse genetic experiments suggest the
contrary. It has been suggested that the death signals from the extrinsic or death receptor pathway may get amplified by the mechanisms of
intrinsic pathway and that this functional loop may be enabled by the molecules like tBID (truncated BID).
Cleavage of BID to tBID can also be achieved by Granzyme B. The truncated protein is myristoylated and translocates to mitochondria.
References
H Li, H Zhu, CJ Xu, J Yuan, "Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis", Cell, 94,
1998, 491-501.
1.2.1.1 Caspase-8 activates BID by cleavage
Authors
Description
The caspase 8 -mediated cleavage of cytosolic, inactive p22 BID at internal Asp sites yields a major p15 and minor p13 and p11 fragments. After
myristoylation, tBID translocates to mitochondria as an integral membrane protein.
References
A Gross, XM Yin, K Wang, MC Wei, J Jockel, C Milliman, H Erdjument-Bromage, P Tempst, SJ Korsmeyer, "Caspase cleaved BID targets
mitochondria and is required for cytochrome c release, while BCL-XL prevents this release but not tumor necrosis factor-R1/Fas death", J Biol
Chem, 274, 1999, 1156-63.
B Fischer, D Coelho, P Dufour, JP Bergerat, JM Denis, J Gueulette, P Bischoff, "Caspase 8-mediated cleavage of the pro-apoptotic BCL-2
family member BID in p53-dependent apoptosis", Biochem Biophys Res Commun, 306, 2003, 516-22.
Reaction
1.2.1.2 Granzyme-B activates BID by cleavage
Description
At the beginning of this reaction, 1 molecule of 'BID' is present. At the end of this reaction, 1 molecule of 'tBID-p15' is present.
This reaction takes place in the 'cytosol' and is mediated by the 'granzyme B activity' of 'Granzyme B'.
References
JB Alimonti, L Shi, PK Baijal, AH Greenberg, "Granzyme B induces BID-mediated cytochrome c release and mitochondrial permeability
transition", J Biol Chem, 276, 2001, 6974-82.
Reaction
1.2.1.3 Myristoylation of tBID by NMT1
Authors
Description
After proteolytic activation, tBID is myristoylated by NMT-1 at an exposed glycine. N-myristoylation may enable the activated tBID to associate
with the lipid components of the mitochondrial membrane.
References
J Zha, S Weiler, KJ Oh, MC Wei, SJ Korsmeyer, "Posttranslational N-myristoylation of BID as a molecular switch for targeting mitochondria and
apoptosis", Science, 290, 2000, 1761-5.
Reaction
1.2.1.4 Translocation of tBID to mitochondria
Description
In this reaction, 1 molecule of 'tBID' is translocated from cytosol to mitochondrial outer membrane.
Reaction
1.2.2 Activation of BH3-only proteins
Authors
Reviewers
Vaux, D, 0000-00-00.
Description
The BH3-only members act as sentinels that selectively trigger apoptosis in response to developmental cues or stress-signals like DNA
damages. Widely expressed mammalian BH3-only proteins are thought to act by binding to and neutralizing their pro-survival counterparts.
Activation of BH3-only proteins directly or indirectly results in the activation of proapoptotic BAX and BAK to trigger cell death. Anti-apoptotic
BCL-2 or BCL-XL may bind and sequester BH3-only molecules to prevent BAX, BAK activation. The individual BH3-only members are held in
check by various mechanisms with in the cells. They are recruited for death duties in response to death cues by diverse activation
processes.The mechanisms involved in activation and release of BH3-only proteins for apoptosis will be discussed in this section.
The following figure has been reproduced here with the kind permission from the authors.
References
1.2.2.1 Activation of BAD and translocation to mitochondria
Authors
Reviewers
Vaux, D, 0000-00-00.
Description
The switching on/off of its phosphorylation by growth/survival factors regulates BAD activity. BAD remains sequestered by 14-3-3 scaffold
proteins after phosphorylation by Akt1. Calcineurin activates BAD by dephosphorylation.
References
SY Seo, YB Chen, I Ivanovska, AM Ranger, SJ Hong, VL Dawson, SJ Korsmeyer, DS Bellows, Y Fannjiang, JM Hardwick, "BAD Is a
Pro-survival Factor Prior to Activation of Its Pro-apoptotic Function.", J Biol Chem, 279, 2004, 42240-9.
1.2.2.1.1 Akt1 phosphorylates BAD protein
Description
At the beginning of this reaction, 1 molecule of 'BAD protein' is present. At the end of this reaction, 1 molecule of 'Phospho-BAD' is present.
This reaction takes place in the 'cytosol' and is mediated by the 'kinase activity' of 'AKT1'.
References
TO Khor, YA Gul, H Ithnin, HF Seow, "Positive correlation between overexpression of phospho-BAD with phosphorylated Akt at serine
15183529", Cancer Lett, 210, 2004, 139-50.
Reaction
1.2.2.1.2 Sequestration of BAD protein by 14-3-3
Description
At the beginning of this reaction, 1 molecule of '143B protein', and 1 molecule of 'Phospho-BAD' are present. At the end of this reaction, 1
molecule of '143B:phospo-BAD complex' is present.
References
J Won, DY Kim, M La, D Kim, GG Meadows, CO Joe, "Cleavage of 14-3-3 protein by caspase-3 facilitates bad interaction with Bcl-x(L) during
apoptosis", J Biol Chem, 278, 2003, 19347-51.
Reaction
1.2.2.1.3 Activation of BAD by calcineurin
Description
At the beginning of this reaction, 1 molecule of '143B:phospo-BAD complex' is present. At the end of this reaction, 1 molecule of 'BAD protein',
and 1 molecule of '143B protein' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'calcium-dependent protein serine/threonine phosphatase regulator activity' of
'Calcineurin B complex'.
References
HG Wang, N Pathan, IM Ethell, S Krajewski, Y Yamaguchi, F Shibasaki, F McKeon, T Bobo, TF Franke, JC Reed, "Ca2+-induced apoptosis
through calcineurin dephosphorylation of BAD.", Science, 284, 1999, 339-43.
Reaction
1.2.2.1.4 Translocation of activated BAD protein to mitochondria
Description
In this reaction, 1 molecule of 'BAD protein' is translocated from cytosol to mitochondrial outer membrane.
Reaction
1.2.2.1.5 BAD displaces tBID from BCL-2 sequestration
Authors
Reviewers
Vaux, D, 0000-00-00.
Description
Short peptides representing BAD and BIX were found to bind BCL-2 displacing BID-like BH3 domains that initiate mitochondrial dysfunction.
References
A Letai, MC Bassik, LD Walensky, MD Sorcinelli, S Weiler, SJ Korsmeyer, "Distinct BH3 domains either sensitize or activate mitochondrial
apoptosis, serving as prototype cancer therapeutics", Cancer Cell, 2, 2002, 183-92.
Reaction
1.2.2.2 Activation of NOXA and translocation to mitochondria
Description
NOXA is transactivated in a p53-dependent manner and by E2F1. Activated NOXA is translocated to mitochondria.
1.2.2.2.1 Transactivation of NOXA by p53
Description
At the end of this reaction, 1 molecule of 'NOXA protein' is present.
References
CQ Li, AI Robles, CL Hanigan, LJ Hofseth, LJ Trudel, CC Harris, GN Wogan, "Apoptotic signaling pathways induced by nitric oxide in human
lymphoblastoid cells expressing wild-type or mutant p53", Cancer Res, 64, 2004, 3022-9.
Reaction
1.2.2.2.2 Transactivation of NOXA by E2F1
Description
At the end of this reaction, 1 molecule of 'NOXA protein' is present.
References
T Hershko, D Ginsberg, "Up-regulation of Bcl-2 homology 3 (BH3)-only proteins by E2F1 mediates apoptosis", J Biol Chem, 279, 2004, 8627-34.
Reaction
1.2.2.2.3 Translocation of NOXA to mitochondria
Authors
Description
It was observed that cytosolic Noxa underwent BH3 motif-dependent localization to mitochondria and interacted with anti-apoptotic Bcl-2 family
members, resulting in the activation of caspase-9.
References
E Oda, R Ohki, H Murasawa, J Nemoto, T Shibue, T Yamashita, T Tokino, T Taniguchi, N Tanaka, "Noxa, a BH3-only member of the Bcl-2
family and candidate mediator of p53-induced apoptosis", Science, 288, 2000, 1053-8.
Reaction
1.2.2.3 Activation of PUMA and translocation to mitochondria
Description
Puma is transactivated in a p53-dependent manner and by E2F1. Activated Puma is translocated to mitochondria.
1.2.2.3.1 Transactivation of PUMA by p53
Description
At the end of this reaction, 1 molecule of 'PUMA protein' is present.
References
K Nakano, KH Vousden, "PUMA, a novel proapoptotic gene, is induced by p53", Mol Cell, 7, 2001, 683-94.
Reaction
1.2.2.3.2 Transactivation of PUMA by E2F1
Description
At the end of this reaction, 1 molecule of 'PUMA protein' is present.
References
T Hershko, D Ginsberg, "Up-regulation of Bcl-2 homology 3 (BH3)-only proteins by E2F1 mediates apoptosis", J Biol Chem, 279, 2004, 8627-34.
Reaction
1.2.2.3.3 Translocation of PUMA protein to mitochondria
Authors
Reviewers
Vaux, D, 0000-00-00.
Description
It is thought that due to its p53 dependence for expression, PUMA could function as a mediator of p53-induced apoptosis. Newly synthesized
PUMA protein translocates to mitochondria and binds to BCL-2 and Bcl-X(L) through a BH3 domain.
References
J Yu, L Zhang, PM Hwang, KW Kinzler, B Vogelstein, "PUMA induces the rapid apoptosis of colorectal cancer cells", Mol Cell, 7, 2001, 673-82.
Reaction
1.2.2.4 Activation of BIM and translocation to mitochondria
Authors
Reviewers
Vaux, D, 0000-00-00.
Description
BIM acts as a sentinel to check the integrity of the cytoskeleton. It exists as two variant proteins: BIM-EL and BIM-L. In healthy cells, these two
isoforms are sequestered to the dynein motor complex on microtubules via the dynein light chain DLC1. JNK or MAPK8 releases BIM in
response to UV irradiation by phosphorylation.
References
S Mouhamad, L Besnault, MT Auffredou, C Leprince, MF Bourgeade, G Leca, A Vazquez, "B cell receptor-mediated apoptosis of human
lymphocytes is associated with a new regulatory pathway of Bim isoform expression.", J Immunol, 172, 2004, 2084-91.
1.2.2.4.1 Phosphorylation of DLC1 by MAPK 8
Description
At the beginning of this reaction, 1 molecule of 'BIM sequestered to dynein (DLC1)' is present. At the end of this reaction, 1 molecule of 'BIM',
and 1 molecule of 'phospho-dynein(DLC1) on microtubules' are present.
This reaction takes place on the 'plasma membrane' and is mediated by the 'kinase activity' of 'Mitogen-activated protein kinase 8 '.
References
K Lei, RJ Davis, "JNK phosphorylation of Bim-related members of the Bcl2 family induces Bax-dependent apoptosis", Proc Natl Acad Sci U S A,
100, 2003, 2432-7.
Reaction
1.2.2.4.2 Translocation of BIM to mitochondria
Description
In this reaction, 1 molecule of 'BIM' is translocated from cytosol to mitochondrial outer membrane.

Reaction
1.2.2.5 Activation of BMF and translocation to mitochondria
Authors
Reviewers
Vaux, D, 0000-00-00.
Description
In healthy cells, BMF is bound to the myosin V motor complex through its interaction with DLC2. UV irradiation or anoikis induces MAPK8 (JNK)
to phosphorylate Dynein Light Chain 2 (DLC2) to release BMF.
References
1.2.2.5.1 Phosphorylation of DLC2 by MAPK-8
Description
At the beginning of this reaction, 1 molecule of 'BMF sequestered to dynein (DLC2)' is present. At the end of this reaction, 1 molecule of
'phospho-dynein(DLC2) on microtubules', and 1 molecule of 'BMF' are present.
This reaction takes place on the 'plasma membrane' and is mediated by the 'kinase activity' of 'Mitogen-activated protein kinase 8 '.
References
K Lei, RJ Davis, "JNK phosphorylation of Bim-related members of the Bcl2 family induces Bax-dependent apoptosis", Proc Natl Acad Sci U S A,
100, 2003, 2432-7.
Reaction
1.2.2.5.2 Translocation of BMF to mitochondria
Description
In this reaction, 1 molecule of 'BMF' is translocated from cytosol to mitochondrial outer membrane.

Reaction
1.2.3 BH3-only proteins associate with and inactivate anti-apoptotic BCL-2 members
1.2.3.1 Interaction of BAD with BCL2
Description
At the beginning of this reaction, 1 molecule of 'Bcl-2 protein', and 1 molecule of 'BAD protein' are present. At the end of this reaction, 1 molecule
of 'BAD:BCL-2' is present.
This reaction takes place in the 'mitochondrial outer membrane'.
Source reaction
This reaction was inferred from the corresponding reaction "Interaction of BAD with Bcl-2" in species Mus musculus.
The following literature references support the source reaction:
E Yang, J Zha, J Jockel, LH Boise, CB Thompson, SJ Korsmeyer, "Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and
promotes cell death", Cell, 80, 1995, 285-91.
Reaction
1.2.3.2 Interaction of BAD with BCL-xl
Description
At the beginning of this reaction, 1 molecule of 'BAD protein', and 1 molecule of 'Apoptosis regulator Bcl-X' are present. At the end of this
reaction, 1 molecule of 'BAD:BCL-xl' is present.
References
HG Wang, N Pathan, IM Ethell, S Krajewski, Y Yamaguchi, F Shibasaki, F McKeon, T Bobo, TF Franke, JC Reed, "Ca2+-induced apoptosis
through calcineurin dephosphorylation of BAD.", Science, 284, 1999, 339-43.
Reaction
1.2.3.3 Sequestration of tBID by BCL-2
Description
At the beginning of this reaction, 1 molecule of 'Bcl-2 protein', and 1 molecule of 'tBID' are present. At the end of this reaction, 1 molecule of
'tBID:BCL-2' is present.
References
X Yi, XM Yin, Z Dong, "Inhibition of Bid-induced apoptosis by Bcl-2. tBid insertion, Bax translocation, and Bax/Bak oligomerization suppressed.",
J Biol Chem, 278, 2003, 16992-9.
Reaction
1.2.3.4 Interaction of tBID with BCL-xl
Description
At the beginning of this reaction, 1 molecule of 'tBID', and 1 molecule of 'Apoptosis regulator Bcl-X' are present. At the end of this reaction, 1
molecule of 'tBID:BCL-xl' is present.
References
H Li, H Zhu, CJ Xu, J Yuan, "Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis", Cell, 94,
1998, 491-501.
Reaction
1.2.3.5 Interaction of BIM with BCL2
Description
At the beginning of this reaction, 1 molecule of 'BIM', and 1 molecule of 'Bcl-2 protein' are present. At the end of this reaction, 1 molecule of
'BIM:BCL2' is present.
References
H Puthalakath, DC Huang, LA O'Reilly, SM King, A Strasser, "The proapoptotic activity of the Bcl-2 family member Bim is regulated by
interaction with the dynein motor complex", Mol Cell, 3, 1999, 287-96.
Reaction
1.2.3.6 Interaction of BIM with BCL-xl
Description
At the beginning of this reaction, 1 molecule of 'BIM', and 1 molecule of 'Apoptosis regulator Bcl-X' are present. At the end of this reaction, 1
molecule of 'BIM:BCL-xl' is present.
References
H Puthalakath, DC Huang, LA O'Reilly, SM King, A Strasser, "The proapoptotic activity of the Bcl-2 family member Bim is regulated by
interaction with the dynein motor complex", Mol Cell, 3, 1999, 287-96.
Reaction
1.2.3.7 Interaction of NOXA with BCL2
Description
At the beginning of this reaction, 1 molecule of 'Bcl-2 protein', and 1 molecule of 'NOXA protein' are present. At the end of this reaction, 1
molecule of 'NOXA:BCL2' is present.
Source reaction
This reaction was inferred from the corresponding reaction "Interaction of NOXA and Bcl-2" in species Mus musculus.
Reaction
1.2.3.8 Interaction of PUMA and Bcl-2
Description
At the beginning of this reaction, 1 molecule of 'Bcl-2 protein', and 1 molecule of 'PUMA protein' are present. At the end of this reaction, 1
molecule of 'PUMA:Bcl-2 complex' is present.
This reaction takes place in the 'mitochondrial envelope'.
References
Reaction
1.2.3.9 Interaction of PUMA and Bcl-XL
Description
At the beginning of this reaction, 1 molecule of 'PUMA protein', and 1 molecule of 'Apoptosis regulator Bcl-X' are present. At the end of this
reaction, 1 molecule of 'PUMA:Bcl-XL complex' is present.

References
Reaction
1.2.3.10 Interaction of NOXA with BCL-xl
Description
At the beginning of this reaction, 1 molecule of 'NOXA protein', and 1 molecule of 'Apoptosis regulator Bcl-X' are present. At the end of this
reaction, 1 molecule of 'NOXA:BCL-xl' is present.
Source reaction
This reaction was inferred from the corresponding reaction "Interaction of NOXA and Bcl-XL" in species Mus musculus.
Reaction
1.2.4 Activation, translocation and oligomerization of BAX
Description
As a result of binding to Bid, Bax oligomerizes and integrates in the outer mitochondrial membrane that triggers cytochrome c release. Bax
mitochondrial membrane insertion triggered by Bid may represent a key step in pathways leading to apoptosis (Eskes et al., 2000).
References
R Eskes, S Desagher, B Antonsson, JC Martinou, "Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane",
Mol Cell Biol, 20, 2000, 929-35.
B Antonsson, S Montessuit, B Sanchez, JC Martinou, "Bax is present as a high molecular weight oligomer/complex in the mitochondrial
membrane of apoptotic cells.", J Biol Chem, 276, 2001, 11615-23.
X Roucou, S Montessuit, B Antonsson, JC Martinou, "Bax oligomerization in mitochondrial membranes requires tBid (caspase-8-cleaved Bid)
and a mitochondrial protein", Biochem J, 368, 2002, 915-21.
1.2.4.1 tBID activates BAX protein
Authors
Description
During certain types of apoptosis, activated tBID (p15) induces a change in conformation of Bax which leads to the unmasking of its
NH2-terminal domain. This change in confirmation usually results in the release of cytochrome c from mitochondria.
References
S Desagher, A Osen-Sand, A Nichols, R Eskes, S Montessuit, S Lauper, K Maundrell, B Antonsson, JC Martinou, "Bid-induced conformational
change of Bax is responsible for mitochondrial cytochrome c release during apoptosis", J Cell Biol, 144, 1999, 891-901.
R Eskes, S Desagher, B Antonsson, JC Martinou, "Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane",
Mol Cell Biol, 20, 2000, 929-35.
Reaction
1.2.4.2 Translocation of activated BAX to the mitochondria
Description
At the beginning of this reaction, 1 molecule of 'Activated BAX' is present. At the end of this reaction, 1 molecule of 'Activated BAX inserted into
mitochondrial membrane' is present.
References
X Yi, XM Yin, Z Dong, "Inhibition of Bid-induced apoptosis by Bcl-2. tBid insertion, Bax translocation, and Bax/Bak oligomerization suppressed.",
J Biol Chem, 278, 2003, 16992-9.
B Bellosillo, N Villamor, A LÃ³pez-Guillermo, S MarcÃ©, F Bosch, E Campo, E Montserrat, D Colomer, "Spontaneous and drug-induced
apoptosis is mediated by conformational changes of Bax and Bak in B-cell chronic lymphocytic leukemia.", Blood, 100, 2002, 1810-6.
Reaction
1.2.4.3 Oligomerization of BAX at the mitochondrial membrane
Description
At the beginning of this reaction, 2 molecules of 'Activated BAX inserted into mitochondrial membrane' is present. At the end of this reaction, 1
molecule of 'Activated BAX' is present.
References
Reaction
1.2.4.4 tBID binds to inactive BAX protein
Description
At the beginning of this reaction, 1 molecule of 'tBID-p15', and 1 molecule of 'Inactive Bax alpha protein' are present. At the end of this reaction,
1 molecule of 'tBID bound to inactive BAX' is present.
Reaction
1.2.5 Activation and oligomerization of BAK protein
Description
tBID binds to its mitochondrial partner BAK to release cytochrome c. Activated tBID results in an allosteric activation of BAK. This may induce its
intramembranous oligomerization into a pore for cytochrome c efflux.
1.2.5.1 tBID activates BAK protein
Authors
Reviewers
Vaux, D, 0000-00-00.
Description
tBID binds to its mitochondrial partner BAK to release cytochrome c. It has been observed in mouse systems that the activated tBID results in an
allosteric activation of BAK. Activated BAX induces intramembranous oligomerization leading to a pore for cytochrome c efflux.
References
MC Wei, T Lindsten, VK Mootha, S Weiler, A Gross, M Ashiya, CB Thompson, SJ Korsmeyer, "tBID, a membrane-targeted death ligand,
oligomerizes BAK to release cytochrome c", Genes Dev, 14, 2000, 2060-71.
Reaction
1.2.5.2 Oligomerization of BAK at the mitochondrial membrane
Description
At the beginning of this reaction, 2 molecules of 'Activated BAK protein' is present. At the end of this reaction, 1 molecule of 'Active oligomeric
BAK' is present.
References
SC Ruffolo, GC Shore, "BCL-2 selectively interacts with the BID-induced open conformer of BAK, inhibiting BAK auto-oligomerization", J Biol
Chem, 278, 2003, 25039-45.
Reaction
1.2.5.3 tBID binds to inactive BAK protein
Description
tBID binds to its mitochondrial partner BAK to release cytochrome c. It has been observed in mouse systems that the activated tBID results in an
allosteric activation of BAK. Activated BAX induces intramembranous oligomerization leading to a pore for cytochrome c efflux.
References
MC Wei, T Lindsten, VK Mootha, S Weiler, A Gross, M Ashiya, CB Thompson, SJ Korsmeyer, "tBID, a membrane-targeted death ligand,
oligomerizes BAK to release cytochrome c", Genes Dev, 14, 2000, 2060-71.
Reaction
1.2.6 Permeabilization of mitochondria
Authors
Matthews, L, 2004-08-06.
Reviewers
Vaux, D, 0000-00-00.
Description
Activation and oligomerization of Bax and Bak result in the destabilization of the outer mitochondrial membrane. This results in the release of the
apoptotic factors Cytochrome c and SMAC from the intermembrane space.
References
D Arnoult, B Gaume, M Karbowski, JC Sharpe, F Cecconi, RJ Youle, "Mitochondrial release of AIF and EndoG requires caspase activation
downstream of Bax/Bak-mediated permeabilization.", EMBO J, 22, 2003, 4385-99.
1.2.7 Release of apoptotic factors from the mitochondria
Authors
Matthews, L, 2004-08-06.
Reviewers
Vaux, D, 0000-00-00.
Description
Apoptotic factors released from the mitochondria promote apoptosis through several different mechanisms. Cytochrome C participates in
Apoptosome driven effector caspase activation while SMAC relieves IAP mediated caspase inhibition.
References
Z Song, X Yao, M Wu, "Direct interaction between survivin and Smac/DIABLO is essential for the anti-apoptotic activity of survivin during
taxol-induced apoptosis.", J Biol Chem, 278, 2003, 23130-40.
E Daugas, D Nochy, L Ravagnan, M Loeffler, SA Susin, N Zamzami, G Kroemer, "Apoptosis-inducing factor (AIF): a ubiquitous mitochondrial
oxidoreductase involved in apoptosis.", FEBS Lett, 476, 2000, 118-23.
Oncogene, 23, 2004, 2861-74.
1.2.7.1 Release of Cytochrome c from mitochondria
Description
In this reaction, 1 molecule of 'Cytochrome c' is translocated from mitochondrial intermembrane space to cytosol.
References
Reaction
1.2.7.2 Release of SMAC from mitochondria
Description
At the beginning of this reaction, 1 molecule of 'Smac protein, mitochondrial precursor' is present. At the end of this reaction, 1 molecule of
'SMAC' is present.
References
Reaction
1.2.8 Apoptotic factor-mediated response
Description
In response to apoptotic signals, mitochondrial proteins are released into the cytosol and activate both caspase-dependent and -independent cell
death pathways. Cytochrome c induces apoptosome formation, AIF and endonuclease G function in caspase independent apoptotic nuclear
DNA damage. Smac/DIABLO and HtrA2/OMI both promote caspase activation and caspase-independent cytotoxicity (Saelens et al., 2004).
References
Oncogene, 23, 2004, 2861-74.
1.2.8.1 Cytochrome c-mediated apoptotic response
Authors
Matthews, L, 2004-08-06.
Reviewers
Vaux, D, 0000-00-00.
Description
Upon its release from the mitochondrial intermembrane space, Cytochrome c binds to and causes a conformational change in the cytoplasmic
protein Apaf1. This conformational change allows the Cytochrome c:Apaf1 complex to associate with ATP triggering oligomerization of the
Apaf1:Cytochrome c:ATP complex. The apoptosome then associates with Procaspase-9 resulting in the formation of the active caspase-9
holoenzyme which functions in cleaving effector caspases.
References
AG Martin, J Nguyen, JA Wells, HO Fearnhead, "Apo cytochrome c inhibits caspases by preventing apoptosome formation", Biochem Biophys
Res Commun, 319, 2004, 944-50.
1.2.8.1.1 Formation of apoptosome
Description
Cytoplasmic cytochrome c binds to apoptotic protease activating factor-1 (Apaf-1) promoting the formation of an Apaf-1 oligomer (the
apoptosome) which in turn binds and activates caspase-9.
References
H Zou, Y Li, X Liu, X Wang, "An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9.", J Biol
Chem, 274, 1999, 11549-56.
AG Martin, J Nguyen, JA Wells, HO Fearnhead, "Apo cytochrome c inhibits caspases by preventing apoptosome formation", Biochem Biophys
Res Commun, 319, 2004, 944-50.
1.2.8.1.1.1 Cytochrome C Binds to Apaf-1
Description
At the beginning of this reaction, 1 molecule of 'Cytochrome c', and 1 molecule of 'Apaf-1' are present. At the end of this reaction, 1 molecule of
'Apaf-1:Cytochrome C' is present.
References
H Zou, WJ Henzel, X Liu, A Lutschg, X Wang, "Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome
c-dependent activation of caspase-3.", Cell, 90, 1997, 405-13.
Reaction
1.2.8.1.1.2 Cytochrome C:Apaf-1 binds Procaspase-9
Authors
Alnemri, E, 2004-02-17.
Description
Apaf-1 and Caspase-9 form a complex in the presence of dATP and cytochrome c (Li et al.,1997).
References
P Li, D Nijhawan, I Budihardjo, SM Srinivasula, ES Alnemri, X Wang, "Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9
complex initiates an apoptotic protease cascade.", Cell, 91, 1997, 479-89.
Reaction
1.2.8.1.1.3 Cleavage of Procaspase-9 to Caspase-9
Authors
Alnemri, E, 2004-02-17.
Description
Caspase-9 is activated in an ATP-dependent manner following association with Apaf-1 and cytochrome c (Li et al., 1997)
References
Reaction
1.2.8.1.2 Activation of caspases through apoptosome-mediated cleavage
Authors
Alnemri, E, 2004-02-17.
Description
Procaspase-3 and 7 are cleaved by the apoptosome.
References
EA Slee, MT Harte, RM Kluck, BB Wolf, CA Casiano, DD Newmeyer, HG Wang, JC Reed, DW Nicholson, ES Alnemri, DR Green, SJ Martin,
"Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent
manner", J Cell Biol, 144, 1999, 281-92.
1.2.8.1.2.1 Cleavage of Â Procaspase-3 by the apoptosome
Authors
Alnemri, E, 2004-02-17.
Description
Caspases-3 and -7 are directly cleaved downstream of caspase-9 in the cytochrome c/Apaf-1-inducible caspase cascade (Slee et al., 1999).
References
H Zou, Y Li, X Liu, X Wang, "An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9.", J Biol
Chem, 274, 1999, 11549-56.
Reaction
1.2.8.1.2.2 Cleavage of Procaspase-7 by the apoptosome
Authors
Alnemri, E, 2004-02-17.
Description
Caspases-3 and -7 are directly cleaved downstream of caspase-9 in the cytochrome c/Apaf-1-inducible caspase cascade (Slee et al., 1999).
References
Reaction
1.2.8.2 SMAC-mediated apoptotic response
Authors
Matthews, L, 2004-08-06.
Reviewers
Vaux, D, 0000-00-00.
Description
Once released from the mitochondria, SMAC binds to IAP family proteins displacing them from Caspase:IAP complexes liberating the active
caspases.
References
Oncogene, 23, 2004, 2861-74.
1.2.8.2.1 SMAC binds to IAPs
Description
SMAC binds to the XIAP:caspase complexes.
References
1.2.8.2.1.1 SMAC binds XIAP:Caspase-3
Description
At the beginning of this reaction, 1 molecule of 'SMAC', and 1 molecule of 'XIAP:Caspase-3' are present. At the end of this reaction, 1 molecule
of 'SMAC:XIAP:Caspase-3' is present.
References
QH Yang, C Du, "Smac/DIABLO selectively reduces the levels of c-IAP1 and c-IAP2 but not that of XIAP and livin in HeLa cells.", J Biol Chem,
279, 2004, 16963-70.
QL Deveraux, R Takahashi, GS Salvesen, JC Reed, "X-linked IAP is a direct inhibitor of cell-death proteases.", Nature, 388, 1997, 300-4.
Y Huang, YC Park, RL Rich, D Segal, DG Myszka, H Wu, "Structural basis of caspase inhibition by XIAP: differential roles of the linker versus
the BIR domain", Cell, 104, 2001, 781-90.
Source reaction
This reaction was inferred from the corresponding reaction "SMAC binds XIAP:Caspase-7" in species Homo sapiens.
279, 2004, 16963-70.
Reaction
Description
At the beginning of this reaction, 1 molecule of 'XIAP:Caspase-7', and 1 molecule of 'SMAC' are present. At the end of this reaction, 1 molecule
References
279, 2004, 16963-70.
Reaction
Description
At the beginning of this reaction, 1 molecule of 'SMAC', and 1 molecule of 'XIAP:Caspase-9' are present. At the end of this reaction, 1 molecule

References
D Vucic, HR Stennicke, MT Pisabarro, GS Salvesen, VM Dixit, "ML-IAP, a novel inhibitor of apoptosis that is preferentially expressed in human
melanomas.", Curr Biol, 10, 2000, 1359-66.
279, 2004, 16963-70.
Source reaction
This reaction was inferred from the corresponding reaction "SMAC binds XIAP:Caspase-7" in species Homo sapiens.
279, 2004, 16963-70.
Reaction
1.2.8.2.2 SMAC-mediated dissociation of IAP:caspase complexes
Description
Smac/DIABLO regulates IAP function. Residues 56â€"59 of Smac/DIABLO are homologous to the amino-terminal motif that is used by
caspase-9 to bind to the BIR3 domain of XIAP. Binding of Smac/DIABLO to XIAP thus displaces caspase-9 from the XIAP:Caspase complex
(reviewed in Salvesen et al., 2002).
References
1.2.8.2.2.1 Dissociation of Caspase-3 from SMAC:XIAP:Caspase-3
Authors
Alnemri, E, 2004-02-17.
Description
At the beginning of this reaction, 1 molecule of 'SMAC:XIAP:Caspase-3' is present. At the end of this reaction, 1 molecule of 'Active caspase-3',
and 1 molecule of 'SMAC:XIAP' are present.
Source reaction
This reaction was inferred from the corresponding reaction "Dissociation of Caspase-7 from SMAC:XIAP:Caspase-7" in species Homo sapiens.
Reaction
Authors
Alnemri, E, 2004-02-17.
Description
At the beginning of this reaction, 1 molecule of 'SMAC:XIAP:Caspase-7' is present. At the end of this reaction, 1 molecule of 'SMAC:XIAP', and 1
molecule of 'Active Caspase-7' are present.
References
Reaction
Authors
Alnemri, E, 2004-02-17.
Description
At the beginning of this reaction, 1 molecule of 'SMAC:XIAP:Caspase-9' is present. At the end of this reaction, 1 molecule of 'Cleaved
Caspase-9', and 1 molecule of 'SMAC:XIAP' are present.
Source reaction
This reaction was inferred from the corresponding reaction "Dissociation of Caspase-7 from SMAC:XIAP:Caspase-7" in species Homo sapiens.
Reaction
1.3 Activation of Effector Caspases
Authors
Tschopp, J, 2004-01-28.
Description
Effector caspases cleave a number of vital proteins in the cell, including specific cytosolic proteins and nuclear lamins, leading to the controlled
death of the cell.
1.4 Apoptotic execution phase
Authors
Alnemri, E, 2004-02-17.
Editors
Matthews, L, 2008-02-12.
Reviewers
Ranganathan, S, 2007-11-23.
Description
In the execution phase of apoptosis, effector caspases cleave vital cellular proteins leading to the morphological changes that characterize
apoptosis. These changes include destruction of the nucleus and other organelles, DNA fragmentation, chromatin condensation, cell shrinkage
and cell detachment and membrane blebbing (reviewed in Fischer et al., 2003).
References
U Fischer, RU Janicke, K Schulze-Osthoff, "Many cuts to ruin: a comprehensive update of caspase substrates", Cell Death Differ, 10, 2003,
76-100.
1.4.1 Apoptotic cleavage of cellular proteins
Authors
Schulze-Osthoff, K, 2007-09-03.
Editors
Matthews, L, 2008-02-08.
Reviewers
Description
Apoptotic cell death is achieved by the caspase-mediated cleavage of various vital proteins. Among caspase targets are proteins such as
E-cadherin, Beta-catenin, alpha fodrin, GAS2, FADK, alpha adducin, HIP-55, and desmoglein involved in cell adhesion and maintenance of the
cytoskeletal architecture. Cleavage of proteins such as APC and CIAP1 can further stimulate apoptosis by produce proapoptotic proteins
(reviewed in Fischer et al., 2003. See also Wee et al., 2006 and the CASVM Caspase Substrates Database:
http://www.casbase.org/casvm/squery/index.html ).
References
76-100.
LJ Wee, TW Tan, S Ranganathan, "SVM-based prediction of caspase substrate cleavage sites", BMC Bioinformatics, 7, 2006, S14.
LJ Wee, TW Tan, S Ranganathan, "CASVM: web server for SVM-based prediction of caspase substrates cleavage sites", Bioinformatics, 23,
2007, 3241-3.
1.4.1.1 Caspase-mediated cleavage of cytoskeletal proteins
Authors
Editors
Matthews, L, 2008-04-13, Matthews, L, 2008-06-12.

Reviewers
Description
Caspase-mediated cleavage of a number of proteins in the cortical actin network ( ) microfilament system and others involved in maintenance of
the cytoskeletal architecture (vimentin, or Gas2 and plectin) may directly contribute to apoptotic changes in cell shape.
References
76-100.
LJ Wee, TW Tan, S Ranganathan, "SVM-based prediction of caspase substrate cleavage sites", BMC Bioinformatics, 7, 2006, S14.
LJ Wee, TW Tan, S Ranganathan, "CASVM: web server for SVM-based prediction of caspase substrates cleavage sites", Bioinformatics, 23,
2007, 3241-3.
1.4.1.1.1 Caspase-mediated cleavage of alpha adducin
Authors
Editors
Matthews, L, 2007-09-03, Matthews, L, 2007-10-09, Matthews, L, 2007-11-11.
Reviewers
Description
The cortical actin cytoskeletal network is lost during apoptosis. During apoptosis, increased phosphorylation of the actin capping protein
alpha-adducin leads to its dissociation from the cytoskeleton. The caspase-3-mediated cleavage cleavage of alpha adducin at
Asp-Asp-Ser-Asp(633)-Ala prevents its reassociation (van de Water et al, 2000).
References
B van de Water, IB Tijdens, A Verbrugge, M Huigsloot, AA Dihal, JL Stevens, S Jaken, GJ Mulder, "Cleavage of the actin-capping protein alpha
-adducin at Asp-Asp-Ser-Asp633-Ala by caspase-3 is preceded by its phosphorylation on serine 726 in cisplatin-induced apoptosis of renal
epithelial cells", J Biol Chem, 275, 2000, 25805-13.
Reaction
1.4.1.1.2 Caspase mediated cleavage of alpha-II-Fodrin
Authors
Editors
Reviewers
Description
Apoptosis induced caspases cleave cortical actin network components including fodrin and components of the focal adhesion complex
components which links membrane proteins and cortical actin filaments to the extracellular matrix (Janicke et al.,1998). Cleavage of these
proteins results in disruption of the cortical cytoskeleton and may contribute to membrane blebbing (see Fischer et al., 2003). The full length 240
kDa alpha-fodrin protein can be cleaved at several sites within its sequence by activated caspases to yield amino-terminal 150 kDa,
carboxy-terminal 120 kDa and 35 kDa major products. Cleavage of alpha-II fodrin leads to membrane malfunction and cell shrinkage (Janicke et
al., 1998).
References
76-100.
RU Janicke, P Ng, ML Sprengart, AG Porter, "Caspase-3 is required for alpha-fodrin cleavage but dispensable for cleavage of other death
substrates in apoptosis", J Biol Chem, 273, 1998, 15540-5.
Reaction
1.4.1.1.3 Caspase-mediated cleavage of GAS2
Authors
Editors
Reviewers
Description
Cleavage of Gas2 during apoptosis is associated with changes of the microfilament system but does not interfere with its ability to bind F-actin
(Brancolini et al., 1995).
References
A Sgorbissa, R Benetti, S Marzinotto, C Schneider, C Brancolini, "Caspase-3 and caspase-7 but not caspase-6 cleave Gas2 in vitro: implications
for microfilament reorganization during apoptosis", J Cell Sci, 112, 1999, 4475-82.
C Brancolini, M Benedetti, C Schneider, "Microfilament reorganization during apoptosis: the role of Gas2, a possible substrate for ICE-like
proteases", EMBO J, 14, 1995, 5179-90.
Reaction
1.4.1.1.4 Caspase mediated cleavage of HIP-55
Authors
Editors
Matthews, L, 2007-11-11.
Reviewers
Description
HIP-55 is an actin binding SH3 domain protein that is cleaved by caspase-3. Cleavage results in dissociation of the actin-binding domain from
the SH3 domain and may alter cell signaling to and from the actin cytoskeleton. In addition, this cleavage may be involved in the the alteration in
cell morphology that occur during apoptosis (Chen et al., 2001).
References
YR Chen, R Kori, B John, TH Tan, "Caspase-mediated cleavage of actin-binding and SH3-domain-containing proteins cortactin, HS1, and
HIP-55 during apoptosis", Biochem Biophys Res Commun, 288, 2001, 981-9.
Reaction
1.4.1.1.5 Caspase-mediated cleavage of vimentin at DSVD (85)
Authors
Editors
Reviewers
Description
Vimentin is cleaved by several caspases during apoptosis (Morishima et al., 1999, Byun et al., 2001). This cleavage disrupts the cytoplasmic
network of intermediate filaments and coincides temporally with nuclear fragmentation. Caspase-6 recognizes and cleaves C terminal side of
Asp-429. Vimentin is cleaved at Asp85 by caspases-3 and -7 (Byun et al., 2001). This clevage generates a pro-apoptotic amino-terminal
cleavage product (amino acids 1-85) that amplifies the cell death signal (Byun et al., 2001).
References
N Morishima, "Changes in nuclear morphology during apoptosis correlate with vimentin cleavage by different caspases located either upstream
or downstream of Bcl-2 action", Genes Cells, 4, 1999, 401-14.
Y Byun, F Chen, R Chang, M Trivedi, KJ Green, VL Cryns, "Caspase cleavage of vimentin disrupts intermediate filaments and promotes
apoptosis", Cell Death Differ, 8, 2001, 443-50.
Reaction
1.4.1.1.6 Caspase mediated cleavage of vimentin at IDVD (259)
Authors
Editors
Reviewers
Description
Vimentin is cleaved by several caspases during apoptosis (Morishima et al., 1999, Byun et al., 2001). This clevage disrupts the cytoplasmic
network of intermediate filaments and coincides temporally with nuclear fragmentation. Asp259 is recognized and cleaved by caspase-6 (Byun et
al., 2001).
References
Reaction
1.4.1.1.7 Caspase-mediated cleavage of vimentin at TNLD (429)
Authors
Editors
Reviewers
Description
Vimentin is cleaved by several caspases during apoptosis (Morishima et al., 1999, Byun et al., 2001). This clevage disrupts the cytoplasmic
network of intermediate filaments and coincides temporally with nuclear fragmentation. Caspase-6 recognizes and cleaves C terminal side of
Asp-429.
References
Reaction
1.4.1.1.8 Caspase-mediated cleavage of gelsolin
Authors
Editors
Matthews, L, 2007-09-03.
Reviewers
Description
Gelsolin is cleaved by caspase-3 generating a constitutively
active fragment that can depolymerize F-actin contributing to actin cytoskeletal collapse (Kothakota et al., 1997)
References
YJ Geng, T Azuma, JX Tang, JH Hartwig, M Muszynski, Q Wu, P Libby, DJ Kwiatkowski, "Caspase-3-induced gelsolin fragmentation contributes
to actin cytoskeletal collapse, nucleolysis, and apoptosis of vascular smooth muscle cells exposed to proinflammatory cytokines", Eur J Cell Biol,
77, 1998, 294-302.
S Kothakota, T Azuma, C Reinhard, A Klippel, J Tang, K Chu, TJ McGarry, MW Kirschner, K Koths, DJ Kwiatkowski, LT Williams,
"Caspase-3-generated fragment of gelsolin: effector of morphological change in apoptosis", Science, 278, 1997, 294-8.
Reaction
1.4.1.1.9 Caspase-mediated cleavage of plectin-1
Authors
Editors
Reviewers
Description
Plectin is a major cross-linking protein of the three main cytoplasmic filament systems. Caspase-8 mediated cleavage of plectin 1 appears to
contribute to disruption of the microfilament system during the early stages of apoptosis (Stegh et al., 2000).
References
AH Stegh, H Herrmann, S Lampel, D Weisenberger, K AndrÃ¤, M Seper, G Wiche, PH Krammer, ME Peter, "Identification of the cytolinker
plectin as a major early in vivo substrate for caspase 8 during CD95- and tumor necrosis factor receptor-mediated apoptosis", Mol Cell Biol, 20,
2000, 5665-79.
Reaction
1.4.1.1.10 Caspase-mediated cleavage of Tau
Authors
Editors
Reviewers
Description
Caspase-3 cleaves Tau at position 421 in vitro producing an N-terminal fragment that functions as an apoptotic effector (Fasulo et al., 2000).
References
L Fasulo, G Ugolini, M Visintin, A Bradbury, C Brancolini, V Verzillo, M Novak, A Cattaneo, "The neuronal microtubule-associated protein tau is a
substrate for caspase-3 and an effector of apoptosis", J Neurochem, 75, 2000, 624-33.
Reaction
1.4.1.2 Apoptotic cleavage of cell adhesion proteins
Authors
Editors
Matthews, L, 2008-06-12.
Reviewers
Description
Apoptotic cells show dramatic rearrangements of tight junctions, adherens junctions, and desmosomes (Abreu et al., 2000).
Desmosome-specific members of the cadherin superfamily of cell adhesion molecules including desmoglein-3, plakophilin-1 and desmoplakin
are cleaved by caspases after onset of apoptosis (Weiske et al., 2001). Cleavage results in the disruption of the desmosome structure and thus
contributes to cell rounding and disintegration of the intermediate filament system (Weiske et al., 2001).
References
RL Dusek, S Getsios, F Chen, JK Park, EV Amargo, VL Cryns, KJ Green, "The differentiation-dependent desmosomal cadherin desmoglein 1 is
a novel caspase-3 target that regulates apoptosis in keratinocytes", J Biol Chem, 281, 2006, 3614-24.
N Cirillo, M Lanza, A De Rosa, M Cammarota, A La Gatta, F Gombos, A Lanza, "The most widespread desmosomal cadherin, desmoglein 2, is
a novel target of caspase 3-mediated apoptotic machinery", J Cell Biochem, 103, 2008, 598-606.
J Weiske, T Schoneberg, W Schroder, M Hatzfeld, R Tauber, O Huber, "The fate of desmosomal proteins in apoptotic cells", J Biol Chem, 276,
2001, 41175-81.
1.4.1.2.1 Caspase-mediated cleavage of E-Cadherin
Authors
Editors
Reviewers
Description
The cleavage of E-cadherin at both the intracellular and extracellular domains likely contributes to the disruption of cadherin-mediated cell-cell
contacts in apoptotic cells. Loss of cell contact is necessary for cell rounding and exit from the epithelium (Steinhusen et al., 2001).
References
U Steinhusen, J Weiske, V Badock, R Tauber, K Bommert, O Huber, "Cleavage and shedding of E-cadherin after induction of apoptosis", J Biol
Chem, 276, 2001, 4972-80.
Reaction
1.4.1.2.2 Caspase mediated cleavage of beta-catenin

Authors
Editors
Reviewers
Description
Apoptosis-induced cleavage of beta-catenin by caspase 3 results in reduced alpha catenin binding, relocalization to the cytoplasm and a
reduction in cell-cell contact. In addition, the resulting proteolytic fragments have reduced transcription factor activity (Steinhusen et al., 2000 ).
References
U Steinhusen, V Badock, A Bauer, J Behrens, B Wittman-Liebold, B Dorken, K Bommert, "Apoptosis-induced cleavage of beta-catenin by
caspase-3 results in proteolytic fragments with reduced transactivation potential", J Biol Chem, 275, 2000, 16345-53.
Reaction
1.4.1.2.3 Caspase-mediated cleavage of Desmoglein 3
Authors
Editors
Reviewers
Description
In epithelial cells, desmosomes are anchoring junctions that mediate strong cell-cell contacts. Desmosomal proteins are proteolytically targeted
during apoptosis (Weiske et al., 2001). Desmogleins are a major component of the desmosome are specifically cleaved after onset of apoptosis.
Cleavage of desmosomal proteins results in the disruption of the structure of desmosomes and contributes to cell rounding and disassembly of
the intermediate filament network (Weiske et al., 2001). The cytosolic fragment has implications for the autoimmune disease, Pemphigus
vulgaris (Tong et al. 2006).
References
2001, 41175-81.
JC Tong, TW Tan, AA Sinha, S Ranganathan, "Prediction of desmoglein-3 peptides reveals multiple shared T-cell epitopes in HLA DR4- and
DR6-associated pemphigus vulgaris", BMC Bioinformatics, 7, 2006, S7.
Reaction
Authors
Editors
Reviewers
Description
In apoptotic cells, intercellular contacts are disrupted through the activity of caspases. Apoptotic cleavage of Dsg2,the most widespread
desmosomal cadherin, is mediated by caspase 3 in epithelial cells (Cirillo et al., 2008).
References
N Cirillo, M Lanza, A De Rosa, M Cammarota, A La Gatta, F Gombos, A Lanza, "The most widespread desmosomal cadherin, desmoglein 2, is
a novel target of caspase 3-mediated apoptotic machinery", J Cell Biochem, 103, 2008, 598-606.
Reaction
Authors
Editors
Matthews, L, 2007-09-03, Matthews, L, 2007-10-09, Matthews, L, 2007-11-11, Matthews, L, 2008-05-28.

Reviewers
Description
Caspase mediated cleavage of desmoglein 1 leads to decreased expression at the cell surface and re-localization of its C terminus diffusely
throughout the cytoplasm. Cleavage is thought to contribute to the dismantling of desmosomes during keratinocyte apoptosis (Dusek et al.,
2006).
References
RL Dusek, S Getsios, F Chen, JK Park, EV Amargo, VL Cryns, KJ Green, "The differentiation-dependent desmosomal cadherin desmoglein 1 is
a novel caspase-3 target that regulates apoptosis in keratinocytes", J Biol Chem, 281, 2006, 3614-24.
Reaction
1.4.1.2.6 Caspase-mediated cleavage of Desmoplakin
Authors
Editors
Reviewers
Description
Cleavage of desmosomal proteins including desmoplakin contributes to cell rounding and disintegration of the intermediate filament system
(Weiske et al., 2001).
References
2001, 41175-81.
Reaction
1.4.1.2.7 Caspase-mediated cleavage of plakophilin-1
Authors
Editors
Matthews, L, 2008-06-03.
Reviewers
Description
Desmosomes represent one of the anchoring junctions mediating strong cell-cell contacts.Desmosomal plaque proteins including the head
domain of plakophilin provide interaction sites for cytokeratin filaments (see references in Weiske et al.,2001). Proteolytic fragmentation of these
proteins prevents binding of intermediate filaments and in consequence results in remodeling of the intermediate filament cytoskeleton (Weiske
et al., 2001).Cleaved Plakophilin-1 appears to be is impaired in supporting the formation and maintenance of desmosomes during apoptosis
(Weiske et al., 2001).
References
2001, 41175-81.
Reaction
1.4.1.2.8 Caspase-mediated cleavage of Z0-1
Authors
Editors
Reviewers
Description
Cleavage of the C-terminal cytoplasmic domain of occludin during apoptosis generates a fragment that can longer associate with the
cytoplasmic adapter proteins ZO-1, -2 and -3 and, as a consequence, with the actin cytoskeleton (Bojarski et al., 2003) . Cleavage of ZO-1 and
ZO-2 further disrupts tight junction structure and function . Notably, claudins, which are associated with ZO-1, ZO-2 and ZO-3, completely lose
their linkage to the actin cytoskeleton and other ZO-1-, ZO-2-, ZO-3-interacting proteins.(Bojarski et al., 2003) .
References
C Bojarski, J Weiske, T SchÃ¶neberg, W SchrÃ¶der, J Mankertz, JD Schulzke, P Florian, M Fromm, R Tauber, O Huber, "The specific fates of
tight junction proteins in apoptotic epithelial cells", J Cell Sci, 117, 2004, 2097-107.
Reaction
1.4.1.2.9 Caspase-mediated cleavage of Z0-2
Authors
Editors
Matthews, L, 2008-05-20.
Reviewers
Description
Cleavage of the C-terminal cytoplasmic domain of occludin during apoptosis generates a fragment that can longer associate with the
cytoplasmic adapter proteins ZO-1, -2 and -3 and, as a consequence, with the actin cytoskeleton (Bojarski et al., 2003) . Cleavage of ZO-1 and
ZO-2 further disrupts tight junction structure and function . Notably, claudins, which are associated with ZO-1, ZO-2 and ZO-3, completely lose
their linkage to the actin cytoskeleton and other ZO-1-, ZO-2-, ZO-3-interacting proteins.(Bojarski et al., 2003) .
References
Reaction
1.4.1.2.10 Caspase-mediated cleavage of occludin
Authors
Editors
Matthews, L, 2008-06-02.
Reviewers
Description
Following iinduction of apoptosis in epithelial cells, tight junction are disrupted. Tight junction proteins, including the the transmembrane protein
occludin and the cytoplasmic adaptor proteins ZO-1 and ZO-2 are fragmented by caspase cleavage (Bojarski et al., 2004).
References
Reaction
1.4.1.3 Breakdown of the nuclear lamina
Authors
Editors
Matthews, L, 2008-05-20.
Reviewers
Description
Activated caspases cleave nuclear lamins causing the irreversible breakdown of the nuclear lamina.
References
76-100.
1.4.1.3.1 Caspase-mediated cleavage of Lamin A
Authors
Editors
Reviewers
Description
Caspases initiate the destruction of the nucleus cleavage of lamins leads to
disassembly of the nuclear lamina. Lamin A is cleaved by active caspase 6 (Orth et al., 1996).
References
K Orth, AM Chinnaiyan, M Garg, CJ Froelich, VM Dixit, "The CED-3/ICE-like protease Mch2 is activated during apoptosis and cleaves the death
substrate lamin A", J Biol Chem, 271, 1996, 16443-6.
YA Lazebnik, A Takahashi, GG Poirier, SH Kaufmann, WC Earnshaw, "Characterization of the execution phase of apoptosis in vitro using
extracts from condemned-phase cells", J Cell Sci Suppl, 19, 1995, 41-9.
Reaction
1.4.1.3.2 Caspase-mediated cleavage of Lamin B1
Authors
Editors
Reviewers
Description
Caspases initiate the destruction of the nucleus cleavage of lamins leads to Â disassembly of the nuclear lamina. Lamin B is cleaved by active
caspase 6 (Orth et al., 1996) (Rao et al., 1996).
References
K Orth, AM Chinnaiyan, M Garg, CJ Froelich, VM Dixit, "The CED-3/ICE-like protease Mch2 is activated during apoptosis and cleaves the death
substrate lamin A", J Biol Chem, 271, 1996, 16443-6.
L Rao, D Perez, E White, "Lamin proteolysis facilitates nuclear events during apoptosis", J Cell Biol, 135, 1996, 1441-55.
Reaction
1.4.1.4 Caspase mediated cleavage of APC
Authors
Editors
Reviewers
Description
Cleavage of APC by caspase 3 and release of the amino-terminal fragment (1-760) are required for the APC mediated acceleration of
apoptosis-associated caspase activity (Qian et al., 2007).
References
J Qian, K Steigerwald, KA Combs, MC Barton, J Groden, "Caspase cleavage of the APC tumor suppressor and release of an amino-terminal
domain is required for the transcription-independent function of APC in apoptosis", Oncogene, 26, 2007, 4872-6.
SJ Browne, M MacFarlane, GM Cohen, C Paraskeva, "The adenomatous polyposis coli protein and retinoblastoma protein are cleaved early in
apoptosis and are potential substrates for caspases", Cell Death Differ, 5, 1998, 206-13.
SJ Webb, D Nicholson, VJ Bubb, AH Wyllie, "Caspase-mediated cleavage of APC results in an amino-terminal fragment with an intact armadillo
repeat domain", FASEB J, 13, 1999, 339-46.
Reaction
1.4.1.5 Caspase mediated cleavage of C-IAP1
Authors
Editors
Reviewers
Description
c-IAP1 is cleaved by caspase-3 producing a proapoptotic C-terminal fragment.
References
RJ Clem, TT Sheu, BW Richter, WW He, NA Thornberry, CS Duckett, JM Hardwick, "c-IAP1 is cleaved by caspases to produce a proapoptotic
C-terminal fragment", J Biol Chem, 276, 2001, 7602-8.
Reaction
1.4.1.6 Caspase-mediated cleavage of FADK 1
Authors
Editors
Reviewers
Description
FAK is a tyrosine kinase that localizes to focal adhesions and associates temporally and spatially with integrins (see references in Fischer et al.,
2003 ). FAK is cleaved by caspases including caspase-7 (Wen et al., 1997). Caspases also cleave fodrin and components of the focal adhesion
complex which links cortical actin filaments and membrane proteins to the extracellular matrix. Cleavage of these proteins is thought to promote
cell shrinkage and cell detachment and disrupt antiapoptotic integrin signaling (see Fischer et al., 2003).
References
76-100.
LP Wen, JA Fahrni, S Troie, JL Guan, K Orth, GD Rosen, "Cleavage of focal adhesion kinase by caspases during apoptosis", J Biol Chem, 272,
1997, 26056-61.
Reaction
1.4.1.7 Caspase 3-mediated cleavage of PKC delta
Authors
Editors
Matthews, L, 2007-09-03.
Reviewers
Description
Caspase mediated cleavage produces a constitutively active kinase that induces apoptosis (Ghayur et al.,1996).
References
T Ghayur, M Hugunin, RV Talanian, S Ratnofsky, C Quinlan, Y Emoto, P Pandey, R Datta, Y Huang, S Kharbanda, H Allen, R Kamen, W Wong,
D Kufe, "Proteolytic activation of protein kinase C delta by an ICE/CED 3-like protease induces characteristics of apoptosis", J Exp Med, 184,
1996, 2399-404.
Reaction
1.4.1.8 Caspase-mediated cleavage of PKC theta
Authors
Editors
Matthews, L, 2007-09-03.
Reviewers
Description
Cleavage of PKCtheta by Caspase-3 induces nuclear fragmentation and lethality (Datta et al., 1997)
References
K Mizuno, K Noda, T Araki, T Imaoka, Y Kobayashi, Y Akita, M Shimonaka, S Kishi, S Ohno, "The proteolytic cleavage of protein kinase C
isotypes, which generates kinase and regulatory fragments, correlates with Fas-mediated and 12-O-tetradecanoyl-phorbol-13-acetate-induced
apoptosis", Eur J Biochem, 250, 1997, 7-18.
R Datta, H Kojima, K Yoshida, D Kufe, "Caspase-3-mediated cleavage of protein kinase C theta in induction of apoptosis", J Biol Chem, 272,
1997, 20317-20.
Reaction
1.4.1.9 Caspase-mediated cleavage of Acinus
Authors
Editors
Matthews, L, 2007-09-03.
Reviewers
Description
Acinus induces apoptotic chromatin condensation after cleavage and activation by CASP3 (Sahara et al., 1999).
References
S Sahara, M Aoto, Y Eguchi, N Imamoto, Y Yoneda, Y Tsujimoto, "Acinus is a caspase-3-activated protein required for apoptotic chromatin
condensation", Nature, 401, 1999, 168-73.
Reaction
1.4.1.10 Caspase-mediated cleavage of Rock-1
Authors
Editors
Reviewers
Description
Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing (Sebbagh et al., 2001). Cleavage
and activation of ROCK-1 by caspase-3 plays has also been shown to play a crucial role in in cardiac myocyte apoptosis (Chang et al., 2006).
References
J Chang, M Xie, VR Shah, MD Schneider, ML Entman, L Wei, RJ Schwartz, "Activation of Rho-associated coiled-coil protein kinase 1 (ROCK-1)
by caspase-3 cleavage plays an essential role in cardiac myocyte apoptosis", Proc Natl Acad Sci U S A, 103, 2006, 14495-500.
M Sebbagh, C RenvoizÃ©, J Hamelin, N RichÃ©, J Bertoglio, J BrÃ©ard, "Caspase-3-mediated cleavage of ROCK I induces MLC
phosphorylation and apoptotic membrane blebbing", Nat Cell Biol, 3, 2001, 346-52.
Reaction
1.4.1.11 Caspase-mediated cleavage of farnesyltransferase/geranylgeranyltransferase subunit alpha
Authors
Editors
Reviewers
Description
Farnesyltransferase/geranyl-geranyltransferase catalyzes the transfer of a farnesyl or geranyl-geranyl moiety from farnesyl or geranyl-geranyl
pyrophosphate to a cysteine at the fourth position from the C-terminus of proteins having the C-terminal sequence Cys-aliphatic-aliphatic-X. This
enzyme complex consists of a heterodimer of an alpha and a beta subunit. The alpha subunit is thought to function in the formation of a stable
complex with the substrate. This alpha subnit is cleaved by caspase 3. Expression of the cleavage product (60-379) induces cell death (Kim et
al., 2001).
References
KW Kim, HH Chung, CW Chung, IK Kim, M Miura, S Wang, H Zhu, KD Moon, GB Rha, JH Park, DG Jo, HN Woo, YH Song, BJ Kim, J Yuan, YK
Jung, "Inactivation of farnesyltransferase and geranylgeranyltransferase I by caspase-3: cleavage of the common alpha subunit during
apoptosis", Oncogene, 20, 2001, 358-66.
Reaction
1.4.1.12 Caspase mediated cleavage of BAP31
Authors
Editors
Matthews, L, 2008-06-02.
Reviewers
Description
Caspase-8 mediated cleavage of BAP31 at the ER produces a pro-apoptotic p20 fragment that remains at the ER (Breckenridgeet al., 2003).
Cleavage stimulates Ca2+-dependent mitochondrial fission, enhancing the release of cytochrome C (Breckenridgeet al., 2003).
References
DG Breckenridge, M Stojanovic, RC Marcellus, GC Shore, "Caspase cleavage product of BAP31 induces mitochondrial fission through
endoplasmic reticulum calcium signals, enhancing cytochrome c release to the cytosol", J Cell Biol, 160, 2003, 1115-27.
Reaction
1.4.1.13 Caspase-mediated cleavage of Etk
Authors
Editors
Reviewers
Description
Cleavage of Etk by caspase-3 stimulates its kinase activity. Overexpression of the fragment induces apoptosis (Wu et al.,2001).
References
YM Wu, CL Huang, HJ Kung, CY Huang, "Proteolytic activation of ETK/Bmx tyrosine kinase by caspases", J Biol Chem, 276, 2001, 17672-8.
Reaction
1.4.1.14 Caspase-mediated cleavage of MASK
Authors
Editors
Matthews, L, 2008-05-27.
Reviewers
Description
MASK is cleaved in vitro by caspase 3 . C-terminally truncated forms of MASK can both induce apoptosis upon overexpression in mammalian
cells (Dan et al., 2002).
References
I Dan, SE Ong, NM Watanabe, B Blagoev, MM Nielsen, E Kajikawa, TZ Kristiansen, M Mann, A Pandey, "Cloning of MASK, a novel member of
the mammalian germinal center kinase III subfamily, with apoptosis-inducing properties", J Biol Chem, 277, 2002, 5929-39.
Reaction
1.4.1.15 Caspase-mediated cleavage of MST3

Authors
Editors
Matthews, L, 2008-05-27.
Reviewers
Description
Caspase-mediated cleavage of Mst3 activates its intrinsic kinase activity. Proteolytic removal of the COOH-terminal domain promotes nuclear
translocation of its kinase domain. Ectopic expression of COOH-terminal truncated Mst3 results in DNA fragmentation and morphological
changes characteristic of apoptosis (Huang et al., 2002).
References
CY Huang, YM Wu, CY Hsu, WS Lee, MD Lai, TJ Lu, CL Huang, TH Leu, HM Shih, HI Fang, DR Robinson, HJ Kung, CJ Yuan, "Caspase
activation of mammalian sterile 20-like kinase 3 (Mst3). Nuclear translocation and induction of apoptosis", J Biol Chem, 277, 2002, 34367-74.
Reaction
1.4.2 Apoptosis induced DNA fragmentation
Authors
Matthews, L, 2008-04-25.
Editors
Matthews, L, 2008-05-18.
Reviewers
Widlak, P, 2008-04-25.
Description
DNA fragmentation in response to apoptotic signals is achieved through the activity of two apoptotic nucleases, termed DNA fragmentation
factor (DFF) or caspase-activated DNase (CAD) and endonuclease G (Endo G) (reviewed in Widlak and Garrard, 2005). These endonucleases
cleave chromatin producing 3'-hydroxyl groups and 5'-phosphate residues. 50-300 kb cleavage products are produced followed by
internucleosomal DNA fragmentation. Although the activities of DFF and Endo G are similar, they reside in different locations within the cell and
are regulated in different ways.
References
P Widlak, WT Garrard, "Discovery, regulation, and action of the major apoptotic nucleases DFF40/CAD and endonuclease G", J Cell Biochem,
94, 2005, 1078-87.
1.4.2.1 Activation of DNA fragmentation factor
Authors
Matthews, L, 2008-01-29.
Editors
Matthews, L, 2008-05-02.
Reviewers
Widlak, P, 2008-05-07.
Description
DNA fragmentation in response to apoptotic signals is achieved, in part, through the activity of apoptotic nucleases, termed DNA fragmentation
factor (DFF) or caspase-activated DNase (CAD) (reviewed in Widlak and Garrard, 2005). In non-apoptotic cells, DFF is a nuclear heterodimer
consisting of a 45 kD chaperone and inhibitor subunit (DFF45)/inhibitor of CAD (ICAD-L)] and a 40 kD nuclease subunit (DFF40/CAD)( Liu et al.
1997, 1998; Enari et al. 1998). During apoptosis, activated caspase-3 or -7 cleave DFF45/ICAD releasing active DFF40/CAD nuclease. The
activity of DFF is tightly controlled at multiple stages. During translation, DFF45/ICAD, Hsp70, and Hsp40 proteins play a role in insuring the
appropriate folding of DFF40 during translation(Sakahira and Nagata, 2002). The nuclease activity of DFF40 is enhanced by the chromosomal
proteins histone H1, Topoisomerase II and HMGB1/2(Widlak et al., 2000). In addition, the inhibitors (DFF45/35; ICAD-S/L) are produced in
stoichiometric excess (Widlak et al., 2003).
References
P Widlak, P Li, X Wang, WT Garrard, "Cleavage preferences of the apoptotic endonuclease DFF40 (caspase-activated DNase or nuclease) on
naked DNA and chromatin substrates", J Biol Chem, 275, 2000, 8226-32.
P Widlak, WT Garrard, "Discovery, regulation, and action of the major apoptotic nucleases DFF40/CAD and endonuclease G", J Cell Biochem,
94, 2005, 1078-87.
M Enari, H Sakahira, H Yokoyama, K Okawa, A Iwamatsu, S Nagata, "A caspase-activated DNase that degrades DNA during apoptosis, and its
inhibitor ICAD", Nature, 391, 1998, 43-50.
X Liu, H Zou, P Widlak, W Garrard, X Wang, "Activation of the apoptotic endonuclease DFF40 (caspase-activated DNase or nuclease).
Oligomerization and direct interaction with histone H1", J Biol Chem, 274, 1999, 13836-40.
P Widlak, J Lanuszewska, RB Cary, WT Garrard, "Subunit structures and stoichiometries of human DNA fragmentation factor proteins before
and after induction of apoptosis", J Biol Chem, 278, 2003, 26915-22.
1.4.2.1.1 Association of DFF40 with DFF45
Authors
Matthews, L, 2008-01-29.
Editors
Reviewers
Widlak, P, 2008-05-07.
Description
DNA Fragmentation Factor (DFF), is a heterodimer of 40 kDa (DFF40) and 45 kDa (DFF45) subunits (Liu et al., 1997). DFF45 (ICAD) appears to
act as a chaperone for DFF40 (CAD) during its synthesis, remaining complexed with it to inhibit its DNase activity (Enari et al., 1998). The
complex could exist as: a DFF40:DFF45 heterodimer, a (DFF40:DFF45)2 heterotetramer or a (DFF40:DFF40:DFF45:DFF45) heterotetramer
(Lechardeur et al., 2005).
References
X Liu, H Zou, C Slaughter, X Wang, "DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during
apoptosis", Cell, 89, 1997, 175-84.
D Lechardeur, S Dougaparsad, C Nemes, GL Lukacs, "Oligomerization state of the DNA fragmentation factor in normal and apoptotic cells", J
Biol Chem, 280, 2005, 40216-25.
Reaction
1.4.2.1.2 Association of DFF with alpha:beta importin
Authors
Matthews, L, 2008-01-29.
Editors
Matthews, L, 2008-05-02.
Reviewers
Widlak, P, 2008-05-07.
Description
The translocation of the DFF complex from the cytoplasm to the nucleus is mediated by the importin alfa/beta heterodimer. Both DFF40 and
DFF45 possess NLS at their C-termini that interact directly with the importin alfa/beta heterodimer. However, DFF complex binds more tightly
compared with the individual subunits and C-termini of both subunits are required for DFF nuclear import (Neimanis et al., 2007).
References
S Neimanis, W Albig, D Doenecke, J Kahle, "Sequence elements in both subunits of the DNA fragmentation factor are essential for its nuclear
transport", J Biol Chem, 282, 2007, 35821-30.
Reaction
1.4.2.1.3 Translocation of DFF to the nucleus
Authors
Matthews, L, 2008-01-29.
Editors
Matthews, L, 2008-05-08.
Reviewers
Widlak, P, 2008-05-07.
Description
DFF associated with alpha-importin:beta-importin is translocated to the nucleus (Neimanis et al., 2007)
References
S Neimanis, W Albig, D Doenecke, J Kahle, "Sequence elements in both subunits of the DNA fragmentation factor are essential for its nuclear
transport", J Biol Chem, 282, 2007, 35821-30.
Reaction
1.4.2.1.4 Caspase 3-mediated cleavage of DFF45 (117)
Authors
Matthews, L, 2008-01-29.
Editors
Reviewers
Widlak, P, 2008-05-07.
Description
Caspase-3 cleaves the DFF45 subunit of the DFF45:DFF40 complex at two sites to generate an active DNA fragmentation factor. One site of
cleavage is between residues 117,118 (Liu et al., 1997) .
References
apoptosis", Cell, 89, 1997, 175-84.
Reaction
1.4.2.1.5 Cleavage of DFF45 (224) by caspase-3
Authors
Matthews, L, 2008-01-29.
Editors

Reviewers
Widlak, P, 2008-05-07.
Description
Caspase-3 cleaves DFF45 between residues 224,225.
References
apoptosis", Cell, 89, 1997, 175-84.
Reaction
1.4.2.1.6 Cleaved fragments of DFF45 dissociate from DFF40
Authors
Matthews, L, 2008-01-29.
Editors
Matthews, L, 2008-05-02.
Reviewers
Widlak, P, 2008-05-07.
Description
Following caspase-3 cleavage, the fragments of DFF45 dissociate from DFF40, the active component of DFF (Liu et al. 1998).
References
apoptosis", Cell, 89, 1997, 175-84.
Reaction
1.4.2.1.7 Homodimerization of DFF40
Authors
Matthews, L, 2008-01-29.
Editors
Matthews, L, 2008-05-02.
Reviewers
Widlak, P, 2008-05-07.
Description
Following its release from DFF45, DFF40 forms homodimers, which are the basic structures of the enzymatically active nuclease (Woo et al.,
2004). Following dimerization, DFF40 can further oligomerize forming units containing at least 4 monomers (Liu et al., 1999; Widlak et al., 2003).
References
P Widlak, J Lanuszewska, RB Cary, WT Garrard, "Subunit structures and stoichiometries of human DNA fragmentation factor proteins before
and after induction of apoptosis", J Biol Chem, 278, 2003, 26915-22.
EJ Woo, YG Kim, MS Kim, WD Han, S Shin, H Robinson, SY Park, BH Oh, "Structural mechanism for inactivation and activation of CAD/DFF40
in the apoptotic pathway", Mol Cell, 14, 2004, 531-9.
Reaction
1.4.2.1.8 Association of DFF40 with chromatin
Authors
Matthews, L, 2008-01-29.
Editors
Reviewers
Widlak, P, 2008-05-07.
Description
Direct interactions between the histone H1 C-terminal domain and DFF40/CAD possibly target the nuclease to chromatin linker DNA promoting
the linker DNA cleavage during the terminal stages of apoptosis (Widlak et al., 2005). Noteworthy, it has been reported that DFF40/DFF45
complexes could also associate with chromatin and be activated with caspase-3 in DNA-bound state (Korn et al., 2005).
References
C Korn, SR Scholz, O Gimadutdinow, R Lurz, A Pingoud, G Meiss, "Interaction of DNA fragmentation factor (DFF) with DNA reveals an
unprecedented mechanism for nuclease inhibition and suggests that DFF can be activated in a DNA-bound state", J Biol Chem, 280, 2005,
6005-15.
P Widlak, M Kalinowska, MH Parseghian, X Lu, JC Hansen, WT Garrard, "The histone H1 C-terminal domain binds to the apoptotic nuclease,
DNA fragmentation factor (DFF40/CAD) and stimulates DNA cleavage", Biochemistry, 44, 2005, 7871-8.
Reaction
1.4.2.1.9 Cleavage of DNA by DFF40
Authors
Matthews, L, 2008-01-29.
Editors
Reviewers
Widlak, P, 2008-05-07.
Description
The DFF40 cleaves DNA substrates to generate fragments possessing ends with 5â€™-phosphate and 3â€™-hydroxyl groups, and generates
exclusively double strand breaks (primarily blunt ends). It has some sequence preferences on naked DNA substrates and prefers
purine/pyrimidine blocks with rotational symmetry (Widlak et al., 2000). DFF is both a deoxyribonucleotide-specific and a double-strand-specific
endonuclease (Hanus et al., 2008).
References
P Widlak, P Li, X Wang, WT Garrard, "Cleavage preferences of the apoptotic endonuclease DFF40 (caspase-activated DNase or nuclease) on
naked DNA and chromatin substrates", J Biol Chem, 275, 2000, 8226-32.
J Hanus, M Kalinowska-Herok, P Widlak, "The major apoptotic endonuclease DFF40/CAD is a deoxyribose-specific and double-strand-specific
enzyme", Apoptosis, 13, 2008, 377-82.
Reaction
1.4.3 Stimulation of the cell death response by PAK-2p34
Authors
Jakobi, R, 2008-02-05.
Editors
Reviewers
Chang, E, 2008-05-21.
Description
In response to stress signals, the p21-activated protein kinase PAK-2 stimulates a cell death response characterized by increased cell rounding
and apoptotic chromatin condensation (see Jakobi et al., 2003). PAK-2 is proteolytically cleaved by caspase-3 producing a constitutively active
fragment, PAK-2p34. Following cleavage, PAK-2p34 is autophosphorylated at Thr 402 and transported to the nucleus where it accumulates due
to the loss of its nuclear export signal motif (Jakobi et al., 2003). The activity of PAK-2p34 appears to be regulated both by proteosomal
degradation (Jakobi et al., 2003) and by association with the GTPase-activating protein PS-GAP/ RHG-10. This interaction inhibits the kinase
activity of PAK-2p34 and changes the localization of PAK-2p34 from the nucleus to the perinuclear region (Koeppel et al., 2004). PAK-2p34 may
function in the down-regulation of translation initiation in apoptosis through phosphorylation of Mnk1 (Orton et al.,2004).
References
BN Walter, Z Huang, R Jakobi, PT Tuazon, ES Alnemri, G Litwack, JA Traugh, "Cleavage and activation of p21-activated protein kinase
gamma-PAK by CPP32 (caspase 3). Effects of autophosphorylation on activity", J Biol Chem, 273, 1998, 28733-9.
KC Orton, J Ling, AJ Waskiewicz, JA Cooper, WC Merrick, NL Korneeva, RE Rhoads, N Sonenberg, JA Traugh, "Phosphorylation of Mnk1 by
caspase-activated Pak2/gamma-PAK inhibits phosphorylation and interaction of eIF4G with Mnk", J Biol Chem, 279, 2004, 38649-57.
R Jakobi, CC McCarthy, MA Koeppel, DK Stringer, "Caspase-activated PAK-2 is regulated by subcellular targeting and proteasomal
degradation", J Biol Chem, 278, 2003, 38675-85.
MA Koeppel, CC McCarthy, E Moertl, R Jakobi, "Identification and characterization of PS-GAP as a novel regulator of caspase-activated
PAK-2", J Biol Chem, 279, 2004, 53653-64.
1.4.3.1 Cleavage of PAK-2 at 212
Authors
Jakobi, R, 2008-02-05.
Editors
Reviewers
Chang, E, 2008-05-21.
Description
p21-activated protein kinase (PAK-2), also known as gamma-PAK, is cleaved by caspase-3 during apoptosis and plays a role in regulating cell
death. Cleavage produces two peptides; 1-212 containing most of the regulatory domain and 213-524 containing 34 amino acids of the
regulatory domain as well as the catalytic domain (Walter et al., 1998). Proteolytic cleavage of PAK by caspase-3 creates the constitutively
active PAK-2p34 fragment (Jakobi et al., 2003). Evidence for this reaction comes from experiments using both human and rabbit proteins.
References
Source reaction
This reaction was inferred from the corresponding reaction "Cleavage of PAK-2 at 212" in species Oryctolagus cuniculus.
Reaction
1.4.3.2 Autophosphorylation of PAK-2p34 in the activation loop
Authors
Jakobi, R, 2008-02-05.
Editors
Reviewers
Chang, E, 2008-05-21.
Description
Activation of PAK-2p34 coincides with autophosphorylation of Thr 402 in the the catalytic domain (Walter et al., 1998).
References
Source reaction
This reaction was inferred from the corresponding reaction "Autophosphorylation of PAK-2p34" in species Oryctolagus cuniculus.
Reaction
1.4.3.3 Proteolytic PAK-2p34 fragment translocates to the nucleus
Authors
Jakobi, R, 2008-02-05.
Editors
Reviewers
Chang, E, 2008-05-21.
Description
The subcellular localization of PAK-2 is controlled by nuclear localization and nuclear export signal motifs (Jakobi et al.,2003). The regulatory
domain contains a nuclear export signal motif that prevents the nuclear accumulation of full-length PAK-2. The activating proteolytic cleavage
disrupts the nuclear export signal in PAK-2 and removes most its regulatory domain. The resulting activated PAK-2p34 fragment contains a
nuclear localization signal and translocates to and is retained in the nucleus (Jakobi et al.,2003).
References
Source reaction
This reaction was inferred from the corresponding reaction "Proteolytic PAK-2p34 fragment translocates to the nucleus" in species Oryctolagus
cuniculus.
Reaction
1.5 Regulation of Apoptosis
Authors
Jakobi, R, 2008-02-05.
Editors
Reviewers
Chang, E, 2008-05-21.
Description
A regulated balance between cell survival and apoptosis is essential for normal development and homeostasis of multicellular organisms (see
Matsuzawa, 2001). Defects in control of this balance may contribute to autoimmune disease, neurodegeneration and cancer. Protein
ubiquitination and degradation is one of the major mechanisms that regulate apoptotic cell death (reviewed in Yang and Yu 2003).
References
Y Yang, X Yu, "Regulation of apoptosis: the ubiquitous way", FASEB J, 17, 2003, 790-9.
A Matsuzawa, H Ichijo, "Molecular mechanisms of the decision between life and death: regulation of apoptosis by apoptosis signal-regulating
kinase 1", J Biochem, 130, 2001, 1-8.
1.5.1 Regulation of activated PAK-2p34 by proteasome mediated degradation
Authors
Jakobi, R, 2008-02-05.
Editors

Reviewers
Chang, E, 2008-05-21.
Description
Stimulation of cell death by PAK-2 requires the generation and stabilization of the caspase-activated form, PAK-2p34 (Walter et al., 1998;Jakobi
et al., 2003). Levels of proteolytically activated PAK-2p34 protein are controlled by ubiquitin-mediated proteolysis. PAK-2p34 but not full-length
PAK-2 is degraded by the 26 S proteasome (Jakobi et al., 2003). It is not known whether ubiquitination and degradation of PAK-2p34 occurs in
the cytoplasm or in the nucleus.
References
Source pathway
This pathway was inferred from the corresponding pathway "Regulation of activated PAK-2p34 by proteasome mediated degradation" in species
Homo sapiens.
The following literature references support the source pathway:
1.5.1.1 Ubiquitination of PAK-2p34
Authors
Jakobi, R, 2008-02-05.
Editors

Reviewers
Chang, E, 2008-05-21.
Description
PAK-2p34 is ubiquitinated prior to degradation (Jakobi et al., 2003). Here, ubiquitination of PAK-2p34 is described as occurring in the cytosol.
However, to date it is not known whether this occurs in the nucleus or in the cytoplasm. Evidence for this reaction comes from experiments using
both human and rabbit proteins.
References
Source reaction
This reaction was inferred from the corresponding reaction "Ubiquitination of PAK-2p34" in species Oryctolagus cuniculus.
Reaction
1.5.1.2 Proteasome mediated degradation of PAK-2p34
Authors
Jakobi, R, 2008-02-05.
Editors
Matthews, L, 2008-02-03.
Reviewers
Chang, E, 2008-05-21.
Description
Proteolytically activated PAK-2p34, but not full-length PAK-2, is degraded rapidly by the proteasome (Jakobi et al., 2003). Here, degradation of
PAK-2p34 is described as occurring in the cytosol. However, to date it is not known whether this occurs in the nucleus or in the cytoplasm.
References
Source reaction
This reaction was inferred from the corresponding reaction "Proteasome mediated degradation of PAK-2p34" in species Homo sapiens.
Reaction
1.5.2 Regulation of PAK-2p34 activity by PS-GAP/RHG10
Authors
Jakobi, R, 2008-02-05.
Editors
Reviewers
Chang, E, 2008-05-21.
Description
PS-GAP (RGH10) interacts specifically with caspase-activated PAK-2p34 reducing the ability of PAK-2p34 to induce cell death. This interaction
Â inhibits the kinase activity of Â PAK-2p34 and changes the localization of PAK-2p34 from the nucleus to the perinuclear region (Koeppel et al.,
2004). Â
References
PAK-2", J Biol Chem, 279, 2004, 53653-64.
Source pathway
This pathway was inferred from the corresponding pathway "Regulation of PAK-2p34 activity by PS-GAP" in species Mus musculus.
PAK-2", J Biol Chem, 279, 2004, 53653-64.
1.5.2.1 RHG10 interacts with caspase-activated PAK-2p34
Authors
Jakobi, R, 2008-02-05.
Editors
Reviewers
Chang, E, 2008-05-21.
Description
Murine PS-GAP interacts specifically with caspase-activated PAK-2p34, but not active or inactive full-length PAK-2, through a region between
the GAP and SH3 domains (Koeppel et al.,2004). Evidence for this reaction comes from experiments using both mouse and rabbit proteins.
References
PAK-2", J Biol Chem, 279, 2004, 53653-64.
Source reaction
This reaction was inferred from the corresponding reaction "PS-GAP interacts Â with caspase-activated PAK-2p34" in species Mus musculus.
PAK-2", J Biol Chem, 279, 2004, 53653-64.
Reaction
1.5.2.2 Interaction of PAK-2p34 with RGH10/ PS-GAP results in accumulation of PAK-2p34 in the perinuclear region
Authors
Jakobi, R, 2008-02-05.
Editors
Reviewers
Chang, E, 2008-05-21.
Description
Following caspase mediated cleavage, PAK-2p34 translocates to the nucleus (Jakobi et al., 2003). The interaction with PS-GAP changes the
localization of PAK-2p34 from the nucleus to the perinuclear region (Koeppel et al.,2004).
References
PAK-2", J Biol Chem, 279, 2004, 53653-64.
Source reaction
This reaction was inferred from the corresponding reaction "Interaction of PAK-2p34 with PS-GAP results in accumulation of PAK-2p34 in the
perinuclear region" in species Mus musculus.
PAK-2", J Biol Chem, 279, 2004, 53653-64.
Reaction
2 Biological oxidations
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
D'Eustachio, P, 2008-05-28.
Description
All organisms are constantly exposed to foreign chemicals every day. These can be man-made (drugs, industrial chemicals) or natural
(alkaloids, toxins from plants and animals). Uptake is usually via ingestion but inhalation and transdermal routes are also common.
The very nature of many chemicals that make them suitable for uptake by these routes, in other words their lipophilicty (favours fat solubility) is
also the main reason organisms have developed mechanisms that convert them to hydrophilic (favours water solubility) compounds which are
readily excreted via bile and urine. Otherwise, lipophilic chemicals would accumulate in the body and overwhelm defense mechanisms. This
process is called biotransformation and is catalyzed by enzymes mainly in the liver of higher organisms but a number of other organs have
considerable ability to process xenobiotica such as kidneys, gut and lungs. As well as xenobiotics, many endogenous compounds are commonly
eliminated by this process.
This mechanism is of ancient origin and a major factor for its development in animals is plants. Most animals are plant eaters and thus are
subject to a huge variety of chemical compounds which plants produce to stop themselves being eaten. This complex set of enzymes have
several features which make them ideal for biotransformation;
(1) metabolites of the parent chemical are usually made more water soluble so it favours rapid excretion via bile and urine
(2) the enzymes possess broad and overlapping specificity to be able to deal with newly exposed chemicals
(3) metabolites of the parent generally don't have adverse biological effects.
In the real world however, all these criteria have exceptions. Many chemicals are transformed into reactive metabolites. In pharmacology, the
metabolites of some parent drugs exert the desired pharmacological effect but in the case of polycyclic aromatic hydrocarbons (PAHs), which
can undergo epoxidation, it results in the formation of an electrophile which can attack proteins and DNA.
Metabolism of xenobiotica occurs in several steps called Phase 1 (functionalization) and Phase 2 (conjugation). To improve water solubility, a
functional group is added to or exposed on the chemical in one or more steps (Phase 1) to which hydrophilic conjugating species can be added
(Phase 2). Functional groups can either be electrophilic (epoxides, carbonyl groups) or nucleophilic (hydroxyls, amino and sulfhydryl groups,
carboxylic groups) (see picture).
Once chemicals undergo functionalization, the electrophilic or nucleophilic species can be detrimental to biological systems. Electrophiles can
react with electron-rich macromolecules such as proteins, DNA and RNA by covalent interaction whilst nucleophiles have the potential to interact
with biological receptors. That's why conjugation is so important as it mops up these potentially reactive species.
Many chemicals, when exposed to certain metabolizing enzymes can induce those enzymes, a process called enzyme induction. The effect of
this is that these chemicals accelerate their own biotransformation and excretion. The reverse is also true where some chemicals cause enzyme
inhibition. Some other factors that alter enzyme levels are sex, age and genetic predisposition. Between species, there can be considerable
differences in biotransformation ability which is a problem faced by drug researchers interpreting toxicological results to humans.
References
F Oesch, M Arand, "Toxicology", 1999, 83-107.
2.1 Phase 1 - Functionalization of compounds
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Phase 1 of metabolism is concerned with functionalization, that is the introduction or exposure of functional groups on the chemical structure of a
compound. This provides a 'handle' for phase 2 conjugating species with which to react with. Many xenobiotics are lipophilic and almost
chemically inert (e.g. PAHs) so would not necessarily undergo a phase 2 reaction. Making them more chemically reactive would facilitate their
excretion but also increases their chance of reacting with cellular macromolecules (e.g. proteins, DNA). There is a fine balance between
producing a more reactive metabolite and conjugation reactions.
There are two groups of enzymes in phase 1 - oxidoreductases and hydrolases. Oxidoreductases introduce an oxygen atom into or remove
electrons from their substrates. The major oxidoreductase enzyme system is called the P450 monooxygenases. Other systems include
flavin-containing monooxygenases (FMO), cyclooxygenases (COX) and monoamine oxidases (MAO). Hydrolases hydrolyse esters, amides,
epoxides and glucuronides.
References
F Oesch, M Arand, "Toxicology", 1999, 83-107.
FP Guengerich, "Cytochrome P450s and other enzymes in drug metabolism and toxicity", AAPS J, 8, 2006, E101-11.
PB Danielson, "The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism in humans", Curr Drug Metab, 3, 2002, 561-97.
M Strolin Benedetti, R Whomsley, E Baltes, "Involvement of enzymes other than CYPs in the oxidative metabolism of xenobiotics", Expert Opin
Drug Metab Toxicol, 2, 2006, 895-921.
2.1.1 Cytochrome P450 - arranged by substrate type
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
The P450 isozyme system is the major phase 1 biotransforming system in man, accounting for more than 90% of drug biotransformations. This
system has huge catalytic versatility and a broad substrate specificity, acting upon xenobiotica and endogenous compounds. It is also called the
mixed-function oxidase system, the P450 monooxygenases and the heme-thiolate protein system. All P450 enzymes are a group of
heme-containing isozymes which are located on the membrane of the smooth endoplasmic reticulum. They can be found in all tissues of the
human body but are most concentrated in the liver. The name "cytochrome P450" (CYP) is derived from the spectral absorbance maximum at
450nm when carbon monoxide binds to CYP in its reduced (ferrous, Fe2+) state. The basic reaction catalyzed by CYP is mono-oxygenation,
that is the transfer of one oxygen atom from molecular oxygen to a substrate. The other oxygen atom is reduced to water during the reaction with
the equivalents coming from the cofactor NADPH. The basic reaction is;
RH (substrate) + O2 + NADPH + H+ = ROH (product) + H2O + NADP+
The end results of this reaction can be (N-)hydroxylation, epoxidation, heteroatom (N-, S-) oxygenation, heteroatom (N-, S-, O-) dealkylation,
ester cleavage, isomerization, dehydrogenation, replacement by oxygen or even reduction under anaerobic conditions.
The metabolites produced from these reactions can either be mere intermediates which have relatively little reactivity towards cellular sysytems
and are readily conjugated, or, they can be disruptive to cellular systems. Indeed, inert compounds need to be prepared for conjugation and thus
the formation of potentially reactive metabolites is in most cases unavoidable.
There are 57 human CYPs (in 18 families and 42 subfamilies), mostly concentrated in the endoplasmic reticulum of liver cells although many
tissues have them to some extent (Nelson DR et al, 2004). CYPs are grouped into 14 families according to their sequence similarity. Generally,
enzymes in the same family share 40% sequence similarity and 55% within a subfamily. Nomenclature of P450s is as follows. CYP (to represent
cytochrome P450 superfamily), followed by an arabic number for the family, a capital letter for the subfamily and finally a second arabic number
to denote the isoenzyme. An example is CYP1A1 which is the first enzyme in subfamily A of cytochrome P450 family 1.
The majority of the enzymes are present in the CYP1-4 families. CYP1-3 are primarily concerned with xenobiotic biotransformation whereas the
other CYPs deal primarily with endogenous compounds. The CYP section is structured by the typical substrate they act upon. Of the 57 human
CYPs, 7 encode mitochondrial enzymes, all involved in sterol biosynthesis. Of the remaining 50 microsomal enzymes, 20 act upon endogenous
compounds, 15 on xenobiotics and 15 are the so-called "orphan enzymes" with no substrate identified.
The P450 catalytic cycle (picture) shows the steps involved when a substrate binds to the enzyme.
(1) The normal state of a P450 with the iron in its ferric [Fe3+] state.
(2) The substrate binds to the enzyme.
(3) The enzyme is reduced to the ferrous [Fe2+] state by the addition of an electron from NADPH cytochrome P450 reductase. The bound
substrate facilitates this process.
(4,5) Molecular oxygen binds and forms an Fe2+OOH complex with the addition of a proton and a second donation of an electron from either
NADPH cytochrome P450 reductase or cytochrome b5. A second proton cleaves the Fe2+OOH complex to form water.
(6) An unstable [FeO]3+ complex donates its oxygen to the substrate (7). The oxidised substrate is released and the enzyme returns to its initial
state (1).
References
DR Nelson, DC Zeldin, SM Hoffman, LJ Maltais, HM Wain, DW Nebert, "Comparison of cytochrome P450 (CYP) genes from the mouse and
human genomes, including nomenclature recommendations for genes, pseudogenes and alternative-splice variants", Pharmacogenetics, 14,
2004, 1-18.
DF Lewis, "57 varieties: the human cytochromes P450", Pharmacogenomics, 5, 2004, 305-18.
2.1.1.1 Endogenous sterols
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
A number of CYPs take part in cholesterol biosynthesis and elimination, thus playing an important role in maintaining cholesterol homeostasis.
Under normal physiological conditions, cholesterol intake (diet or synthesized de novo from acetyl CoA) equals cholesterol elimination (degraded
to bile salts, secreted in bile and used in steroid hormone synthesis). These processes are under tight regulatory control and any disruption
leads to increased cholesterol levels resulting in cardiovacular disease. The CYPs involved in cholesterol homeostasis could serve as potential
targets for cholesterol-lowering drugs.
References
IA Pikuleva, "Cytochrome P450s and cholesterol homeostasis", Pharmacol Ther, 112, 2006, 761-73.
2.1.1.1.1 CYP1B1 4-hydroxylates estradiol-17beta
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Cytochrome P450 1B1 (CYP1B1) can oxidize a variety of structurally unrelated compounds, including steroids, fatty acids, and xenobiotics.
CYP1B1 can also activate a range of procarcinogens. A specific substrate is the female sex hormone estradiol-17beta which is hydroxylated at
position 4.
References
AF Badawi, EL Cavalieri, EG Rogan, "Role of human cytochrome P450 1A1, 1A2, 1B1, and 3A4 in the 2-, 4-, and 16alpha-hydroxylation of
17beta-estradiol", Metabolism, 50, 2001, 1001-3.
Reaction
2.1.1.1.2 Cholesterol is hydroxylated to 7alpha-hydroxycholesterol by CYP7A1
Authors
Jassal, B, 2008-05-19.
Editors
Reviewers
Description
Cholesterol, NADPH + H+, and O2 form 7alpha-cholesterol (5-cholesten-3beta, 7alpha-diol), NADP+,and H2O, in a reaction catalyzed by
CYP7A1 (cholesterol 7alpha-hydroylase) in the endoplasmic reticulum membrane. In the body, this enzyme is expressed only in liver, and its
expression is tightly regulated at the level of transcription to determine the overall rate of bile acid and bile salt production (Russell 2003;
Pullinger et al. 2002).
References
CR Pullinger, C Eng, G Salen, S Shefer, AK Batta, SK Erickson, A Verhagen, CR Rivera, SJ Mulvihill, MJ Malloy, JP Kane, "Human cholesterol
7alpha-hydroxylase (CYP7A1) deficiency has a hypercholesterolemic phenotype", J Clin Invest, 110, 2002, 109-17.
DW Russell, "The enzymes, regulation, and genetics of bile acid synthesis", Annu Rev Biochem, 72, 2003, 137-74.
M Noshiro, K Okuda, "Molecular cloning and sequence analysis of cDNA encoding human cholesterol 7 alpha-hydroxylase", FEBS Lett, 268,
1990, 137-40.
Reaction
2.1.1.1.3 25-hydroxycholesterol is 7alpha-hydroxylated by CYP7B1
Authors
Jassal, B, 2008-05-19.
Editors
Reviewers
Description
25-hydroxycholesterol is 7alpha-hydroxylated to cholest-5-ene-3beta,7alpha,25-triol by CYP7B1.

References
Z Wu, KO Martin, NB Javitt, JY Chiang, "Structure and functions of human oxysterol 7alpha-hydroxylase cDNAs and gene CYP7B1", J Lipid
Res, 40, 1999, 2195-203.
Reaction
2.1.1.1.4 Sterols are 12-hydroxylated by CYP8B1
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Cytochrome P450 8B1 (CYP8B1, sterol 12-alpha- hydroxylase) has a broad substrate specificity including a number of 7-alpha- hydroxylated
C27 steroids. It is also involved in bile acid synthesis and is responsible for the balance between the formation of cholic acid and
chenodeoxycholic acid.
References
M Gafvels, M Olin, BP Chowdhary, T Raudsepp, U Andersson, B Persson, M Jansson, I Bjorkhem, G Eggertsen, "Structure and chromosomal
assignment of the sterol 12alpha-hydroxylase gene (CYP8B1) in human and mouse: eukaryotic cytochrome P-450 gene devoid of introns",
Genomics, 56, 1999, 184-96.
2.1.1.1.4.1 4-Cholesten-7alpha,24(S)-diol-3-one is 12alpha-hydroxylated to 4-cholesten-7-alpha,12-alpha,24(S)-triol-3-one
Authors
Jassal, B, 2008-05-19.
Editors
Reviewers
Description
4-Cholesten-7alpha,24(S)-diol-3-one, NADPH + H+, and O2 form 4-cholesten-7-alpha,12-alpha,24(S)-triol-3-one + NADP+ + H2O. This reaction
is catalyzed by sterol 12alpha hydroxylase (CYP8B1), an enzyme associated with the endoplasmic reticulum membrane. While the human gene
has been cloned (Gafvels et al. 1999), its protein product has not been characterized, and the enzymatic properties of human CYP8B1 protein
are inferred from those of its well-characterized rabbit homolog (Ishida et al. 1992).
References
H Ishida, M Noshiro, K Okuda, MJ Coon, "Purification and characterization of 7 alpha-hydroxy-4-cholesten-3-one 12 alpha-hydroxylase", J Biol
Chem, 267, 1992, 21319-23.
Genomics, 56, 1999, 184-96.
Reaction
2.1.1.1.4.2 4-Cholesten-7alpha,27-diol-3-one is 12alpha-hydroxylated to 4-Cholesten-7alpha,12alpha,27-triol-3-one
Authors
Jassal, B, 2008-05-19.
Editors
Reviewers
Description
4-Cholesten-7alpha,27-diol-3-one, NADPH + H+, and O2 form 4-Cholesten-7alpha,12alpha,27-triol-3-one + NADP+ + H2O. This reaction is
catalyzed by sterol 12alpha hydroxylase (CYP8B1), an enzyme associated with the endoplasmic reticulum membrane. While the human gene
References
Chem, 267, 1992, 21319-23.
Genomics, 56, 1999, 184-96.
Reaction
2.1.1.1.4.3 4-Cholesten-7alpha-ol-3-one is 12alpha-hydroxylated to 7-alpha,12-alpha-dihydroxycholest-4-en-3-one
Authors
Jassal, B, 2008-05-19.
Editors
Reviewers
Description
7-Alpha-hydroxycholest-4-en-3-one, NADPH + H+, and O2 form 7-alpha,12-alpha-dihydroxycholest-4-en-3-one + NADP+ + H2O. This reaction
References
Chem, 267, 1992, 21319-23.
Genomics, 56, 1999, 184-96.
Reaction
2.1.1.1.5 20alpha,22beta-hydroxycholesterol is cleaved by CYP11A1 to yield pregnenolone and isocaproaldehyde
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2007-04-20.
Reviewers
D'Eustachio, P, 2008-05-28, D'Eustachio, P, 2007-04-20.
Description
20alpha,22beta-hydroxycholesterol, NADPH + H+, and O2 react to form pregnenolone, isocaproaldehyde, NADP+ and H2O. This cleavage
reaction is catalyzed by CYP11A (P450scc) associated with the inner mitochondrial membrane. Pregnenolone is substantially more hydrophilic
than cholesterol and hydroxycholesterol and is released into the mitochondrial matrix.
References
ST Woods, J Sadleir, T Downs, T Triantopoulos, MJ Headlam, RC Tuckey, "Expression of catalytically active human cytochrome p450scc in
Escherichia coli and mutagenesis of isoleucine-462", Arch Biochem Biophys, 353, 1998, 109-15.
BC Chung, KJ Matteson, R Voutilainen, TK Mohandas, WL Miller, "Human cholesterol side-chain cleavage enzyme, P450scc: cDNA cloning,
assignment of the gene to chromosome 15, and expression in the placenta", Proc Natl Acad Sci U S A, 83, 1986, 8962-6.
Reaction
2.1.1.1.6 11-deoxycortisol is oxidised to cortisol by CYP11B1

Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
11-Deoxycortisol, NADPH + H+, and O2 react to form cortisol, NADP+, and H2O. This reaction is catalyzed by CYP11B1 associated with the
inner mitochondrial membrane.
References
E Mornet, J Dupont, A Vitek, PC White, "Characterization of two genes encoding human steroid 11 beta-hydroxylase (P-450(11) beta)", J Biol
Chem, 264, 1989, 20961-7.
T Kawamoto, Y Mitsuuchi, K Toda, Y Yokoyama, K Miyahara, S Miura, T Ohnishi, Y Ichikawa, K Nakao, H Imura, S Ulick, Y Shizuta, "Role of
steroid 11 beta-hydroxylase and steroid 18-hydroxylase in the biosynthesis of glucocorticoids and mineralocorticoids in humans", Proc Natl Acad
Sci U S A, 89, 1992, 1458-62.
Reaction
2.1.1.1.7 11-deoxycorticosterone is oxidised to corticosterone by CYP11B2
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
P450 11B2 (steroid 11 beta-hydroxylase) is a mitochondrial cytochrome P-450 enzyme necessary for cortisol biosynthesis.The synthesis of the
most important glucocorticoid and mineralocorticoid hormones in humans (cortisol and aldosterone, respectively), take place in the adrenal
gland. The 11?- and 18-hydroxylation of the substrate 11-deoxycorticosterone (DOC) leads to corticosterone (B)band 18-hydroxycorticosterone
(18-OH-B), whose 18-oxidation yields aldosterone. CYP11B1 is also able to produce corticosterone from 11-deoxycorticosterone but it cannot
convert corticosterone into aldosterone.
References
Chem, 264, 1989, 20961-7.
S Bechtel, N Belkina, R Bernhardt, "The effect of amino-acid substitutions I112P, D147E and K152N in CYP11B2 on the catalytic activities of the
enzyme", Eur J Biochem, 269, 2002, 1118-27.
Reaction
2.1.1.1.8 Hydroxylation of pregnenolone to form 17alpha-hydroxypregnenolone
Authors
Jassal, B, 2007-02-13.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
Pregnenolone and NADPH + H+ react to form 17alpha-hydroxypregnenolone, NADP+, and H2O. CYP17A1 (steroid 17alpha-monooxygenase)
associated with the endoplasmic reticulum membrane catalyzes this reaction.
References
RJ Auchus, WL Miller, "Molecular modeling of human P450c17 (17alpha-hydroxylase/17,20-lyase): insights into reaction mechanisms and
effects of mutations", Mol Endocrinol, 13, 1999, 1169-82.
Reaction
2.1.1.1.9 Adrostenedione is converted to estrone by Aromatase (CYP19A1)
Authors
Jassal, B, 2007-02-13.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
The conversion of androstenedione to estrone is catalyzed by aromatase (CYP19A1) associated with the endoplasmic reticulum membrane.
References
K Toda, M Terashima, T Kawamoto, H Sumimoto, Y Yokoyama, I Kuribayashi, Y Mitsuuchi, T Maeda, Y Yamamoto, Y Sagara, "Structural and
functional characterization of human aromatase P-450 gene", Eur J Biochem, 193, 1990, 559-65.
ER Simpson, MS Mahendroo, GD Means, MW Kilgore, MM Hinshelwood, S Graham-Lorence, B Amarneh, Y Ito, CR Fisher, MD Michael, CR
Mendelson, SE Bulun, "Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis", Endocr Rev, 15, 1994, 342-55.
Reaction
2.1.1.1.10 21-hydroxylation of progesterone to form 11-deoxycorticosterone
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
Progesterone, NAPDH + H+, and O2 react to form 11-deoxycorticosterone, NADP+ and H2O. This reaction is catalyzed by CYP21A2 associated
with the endoplasmic reticulum membrane.
References
NR Rodrigues, I Dunham, CY Yu, MC Carroll, RR Porter, RD Campbell, "Molecular characterization of the HLA-linked steroid 21-hydroxylase B
gene from an individual with congenital adrenal hyperplasia", EMBO J, 6, 1987, 1653-61.
PC White, MI New, B Dupont, "Structure of human steroid 21-hydroxylase genes", Proc Natl Acad Sci U S A, 83, 1986, 5111-5.
Reaction
2.1.1.1.11 Cholesterol is hydroxylated to 27-hydroxycholesterol by CYP27
Authors
Jassal, B, 2008-05-19.
Editors
Reviewers
Description
Cholesterol, NADPH + H+, and O2 react to form 27-hydroxycholesterol, H2O, and NADP+, in the mitochondrial matrix, catalyzed by CYP27A1.
27-Hydroxycholesterol is the most abundant oxysterol in the plasma in humans; its formation is thought to play a central role in the mobilization
of cholesterol from non-hepatic tissues.
References
A Babiker, O Andersson, D Lindblom, J van der Linden, B Wiklund, D Lutjohann, U Diczfalusy, I Bjorkhem, "Elimination of cholesterol as
cholestenoic acid in human lung by sterol 27-hydroxylase: evidence that most of this steroid in the circulation is of pulmonary origin", J Lipid Res,
40, 1999, 1417-25.
JJ Cali, DW Russell, "Characterization of human sterol 27-hydroxylase. A mitochondrial cytochrome P-450 that catalyzes multiple oxidation
reaction in bile acid biosynthesis.", J Biol Chem, 266, 1991, 7774-8.
Reaction
2.1.1.1.12 24-hydroxycholesterol is 7alpha-hydroxylated to yield cholest-5-ene-3beta,7alpha,24-triol
Authors
Jassal, B, 2008-05-19.
Editors
Reviewers
Description
24-hydroxycholesterol, NADPH + H+, and O2 react to form cholest-5-ene-3beta,7alpha,24-triol, NADP+, and H2O. This hydroxylation reaction is
catalyzed by CYP39A1 in the membrane of the endoplasmic reticulum. In the body, expression of CYP39A1 is restricted to the liver.
References
J Li-Hawkins, EG Lund, AD Bronson, DW Russell, "Expression cloning of an oxysterol 7alpha-hydroxylase selective for 24-hydroxycholesterol",
J Biol Chem, 275, 2000, 16543-9.
Reaction
2.1.1.1.13 Cholesterol is hydroxylated to 24-hydroxycholesterol by CYP46A1
Authors
Jassal, B, 2008-05-19.
Editors
Reviewers
Description
Cholesterol, NADPH + H+, and O2 react to form 24-hydroxycholesterol , NADP+, and H2O. This reaction is catalyzed by CYP46A1 in the
endoplasmic reticulum membrane. In the body, this enzyme is expressed predominantly in the brain and is thought to play a major role in
cholesterol turnover there (Javitt 2002).
References
EG Lund, JM Guileyardo, DW Russell, "cDNA cloning of cholesterol 24-hydroxylase, a mediator of cholesterol homeostasis in the brain", Proc
Natl Acad Sci U S A, 96, 1999, 7238-43.
NB Javitt, "Cholesterol, hydroxycholesterols, and bile acids", Biochem Biophys Res Commun, 292, 2002, 1147-53.
N Mast, R Norcross, U Andersson, M Shou, K Nakayama, I Bjorkhem, IA Pikuleva, "Broad substrate specificity of human cytochrome P450 46A1
which initiates cholesterol degradation in the brain", Biochemistry, 42, 2003, 14284-92.
Reaction
2.1.1.1.14 Lanosterol is oxidatively demethylated to 4,4-dimethylcholesta-8(9),14,24-trien-3beta-ol
Authors
Jassal, B, 2008-05-19.
Editors
Reviewers
Description
Lanosterol, NADPH + H+, and O2 react to form 4,4-dimethylcholesta-8(9),14,24-trien-3beta-ol, NADP+, H2O and formate. This oxidative
demethylation reaction, in the endoplasmic reticulum, is catalyzed by lanosterol 14alpha-demethylase (CYP51A1). Although the reaction is
annotated here as a single concerted event, studies with purified rat enzyme indicate that the methyl group is converted successively to an
alcohol and an aldehyde before being released as formate (Trzaskos et al. 1986; Fischer et al. 1991; Gaylor 2002).
References
M Stromstedt, D Rozman, MR Waterman, "The ubiquitously expressed human CYP51 encodes lanosterol 14 alpha-demethylase, a cytochrome
P450 whose expression is regulated by oxysterols", Arch Biochem Biophys, 329, 1996, 73-81.
RT Fischer, JM Trzaskos, RL Magolda, SS Ko, CS Brosz, B Larsen, "Lanosterol 14 alpha-methyl demethylase. Isolation and characterization of
the third metabolically generated oxidative demethylation intermediate.", J Biol Chem, 266, 1991, 6124-32.
JM Trzaskos, S Kawata, JL Gaylor, "Microsomal enzymes of cholesterol biosynthesis. Purification of lanosterol 14 alpha-methyl demethylase
cytochrome P-450 from hepatic microsomes.", J Biol Chem, 261, 1986, 14651-7.
JL Gaylor, "Membrane-bound enzymes of cholesterol synthesis from lanosterol", Biochem Biophys Res Commun, 292, 2002, 1139-46.
Reaction
2.1.1.2 Xenobiotics
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Of the 50 microsomal CYPs, 15 act on xenobiotics. They all possess wide substrate specificity to cater for most foreign compounds that find their
way into the body.
References
2.1.1.2.1 Ethylene is oxidized to Ethylene oxide by CYP1A1
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
A simple example of epoxidation is the oxidation of an alkene (olefin) to the epoxide (oxirane), catalysed by CYP1A1. Even the simplest of
epoxides (ethylene oxide) can react with DNA and amino groups in a protein.
References
de Montell Ortiz, HS Beilan, KL Kunze, BA Mico, "Destruction of cytochrome P-450 by ethylene. Structure of the resulting prosthetic heme
adduct.", J Biol Chem, 256, 1981, 4395-9.
FP Guengerich, "Cytochrome P450 oxidations in the generation of reactive electrophiles: epoxidation and related reactions", Arch Biochem
Biophys, 409, 2003, 59-71.
Reaction
2.1.1.2.2 Aromatic amines can be N-hydroxylated or N-dealkylated by CYP1A2
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
CYP1A2 oxidizes a variety of structurally unrelated compounds, including steroids, fatty acids, and xenobiotics. It is most active in catalyzing
N-hydroxylation or N-dealkylation reactions.
2.1.1.2.2.1 N-atom dealkylation of caffeine
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Caffeine is one of the world's most frequently consumed xenobiotic. The major source of caffeine comes from tea and coffee. Caffeine is
extensively metabolized in humans with at least 17 metabolites formed in its biotransformation. CYP1A2 is a prominent enzyme in the formation
of an important metabolite of caffeine (paraxanthine) by N3-demethylation.
References
JC Bloomer, SE Clarke, RJ Chenery, "Determination of P4501A2 activity in human liver microsomes using [3-14C-methyl]caffeine", Xenobiotica,
25, 1995, 917-27.
Reaction
2.1.1.2.2.2 N-hydroxylation of 4-aminobiphenyl
Authors
Jassal, B, 2008-05-19.
Reviewers
Description
Heterocyclic and aromatic amines require metabolic activation to convert them to genotoxic metabolites.The initial step is N-hydroxylation and
cytochrome P450 1A2 can carry out the catalysis.
References
RJ Turesky, NP Lang, MA Butler, CH Teitel, FF Kadlubar, "Metabolic activation of carcinogenic heterocyclic aromatic amines by human liver and
colon", Carcinogenesis, 12, 1991, 1839-45.
GJ Hammons, D Milton, K Stepps, FP Guengerich, RH Tukey, FF Kadlubar, "Metabolism of carcinogenic heterocyclic and aromatic amines by
recombinant human cytochrome P450 enzymes", Carcinogenesis, 18, 1997, 851-4.
Reaction
2.1.1.2.3 Coumarin is 7-hydroxylated by CYP2A6
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
The 7-hydroxylation of coumarin is used as an assay of P450 activity in animal and human liver microsomes. CYP2A6 is the major coumarin
7-hydroxylase in human liver.
References
CH Yun, KH Kim, MW Calcutt, FP Guengerich, "Kinetic analysis of oxidation of coumarins by human cytochrome P450 2A6", J Biol Chem, 280,
2005, 12279-91.
Reaction
2.1.1.2.4 Coumarin is 7-hydroxylated by CYP2A13
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
CYP2A13 can also 7-hydroxylate coumarin. It shares a 93.5% identity with CYP2A6 in the amino acid sequence but it is only about one-tenth as
active as CYP2A6 in catalyzing coumarin 7-hydroxylation.
References
XY He, J Shen, WY Hu, X Ding, AY Lu, JY Hong, "Identification of Val117 and Arg372 as critical amino acid residues for the activity difference
between human CYP2A6 and CYP2A13 in coumarin 7-hydroxylation", Arch Biochem Biophys, 427, 2004, 143-53.
Reaction
2.1.1.2.5 Cyclophosphamide is 4-hydroxylated by CYP2B6
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Cyclophosphamide (CPA) is an alkylating agent used in cancer chemotherapy and an immunosuppressant. CYP2B6 converts CPA to the active
metabolite 4-hydroxy-CPA.
References
HJ Xie, U Yasar, S Lundgren, L Griskevicius, Y Terelius, M Hassan, A Rane, "Role of polymorphic human CYP2B6 in cyclophosphamide
bioactivation", Pharmacogenomics J, 3, 2003, 53-61.
Reaction
2.1.1.2.6 CYP2C8 inactivates paclitaxel by 6alpha-hydroxylation
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Paclitaxel (Taxol) is a naturally occurring member of the taxane family of antitumor drugs. It acts by stabilizing microtubules. Paclitaxel is
inactivated in human liver by CYP2C8, which catalyzes 6alpha-hydroxylation of paclitaxel.
References
PB Desai, JZ Duan, YW Zhu, S Kouzi, "Human liver microsomal metabolism of paclitaxel and drug interactions", Eur J Drug Metab
Pharmacokinet, 23, 1998, 417-24.
Reaction
2.1.1.2.7 CYP2C9 inactivates tolbutamide by 4methyl-hydroxylation
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Tolbutamide is an oral hypoglycemic agent whose action is terminated by hydroxylation of the tolylsulfonyl methyl moiety. The reaction is
catalyzed by CYP2C9.
References
ME Veronese, PI Mackenzie, CJ Doecke, ME McManus, JO Miners, DJ Birkett, "Tolbutamide and phenytoin hydroxylations by cDNA-expressed
human liver cytochrome P4502C9", Biochem Biophys Res Commun, 175, 1991, 1112-8.
Reaction
2.1.1.2.8 CYP2C18 initiates bioactivation of phenytoin by 4-hydroxylation
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Phenytoin is a widely used anti-epileptic drug which can be hydroxylated by several P450s including CYP2C18 to its major metabolite,
(5-(4-hydroxyphenyl)-phenylhydantoin (HPPH).
References
RT Kinobe, OT Parkinson, DJ Mitchell, EM Gillam, "P450 2C18 catalyzes the metabolic bioactivation of phenytoin", Chem Res Toxicol, 18, 2005,
1868-75.
Reaction
2.1.1.2.9 CYP2C19 5-hydroxylates omeprazole
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Omeprazole is a potent long-acting inhibitor of gastric acid secretion by irreversible binding to the proton pump (H+,K+) ATPase in the gastric
parietal cell. CYP2C19 is the major P450 which involved in 5-hydroxylation of omeprazole.
References
GC Ibeanu, BI Ghanayem, P Linko, L Li, LG Pederson, JA Goldstein, "Identification of residues 99, 220, and 221 of human cytochrome P450
2C19 as key determinants of omeprazole activity", J Biol Chem, 271, 1996, 12496-501.
Reaction
2.1.1.2.10 CYP2E1 reactions
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
CYP2E1 can metabolize and activate a large number of solvents and industrial monomers as well as drugs. This quality of CYP2E1 may make it
an important determinant of human susceptibility to the toxic effects of industrial and environmental chemicals. Typical CYP2E1 substrates
include acetaminophen, benzene, CCl4, halothane, ethanol and vinyl chloride. CYP2E1 contributes to oxidative stress by producing oxidising
species called reactive oxygen species (ROS) which can lead to damage to mitochondria, DNA and initiate lipid peroxidation or even cell death.
References
CS Lieber, "Microsomal ethanol-oxidizing system (MEOS): the first 30 years (1968-1998)--a review", Alcohol Clin Exp Res, 23, 1999, 991-1007.
AA Caro, AI Cederbaum, "Oxidative stress, toxicology, and pharmacology of CYP2E1", Annu Rev Pharmacol Toxicol, 44, 2004, 27-42.
FJ Gonzalez, "Role of cytochromes P450 in chemical toxicity and oxidative stress: studies with CYP2E1", Mutat Res, 569, 2005, 101-10.
2.1.1.2.10.1 Acetaminophen oxidised to N-acetylbenzoquinoneimine (NAPQI)
Authors
Jassal, B, 2008-05-19.
Reviewers
Description
N-acetyl-p-benzoquinone imine (NAPQI) is the reactive intermediate of the analgesic and antipyretic, acetaminophen (INN, paracetamol). At
usual doses, NAPQI is quickly detoxified by conjugation but in overdose situations, NAPQI is extremely toxic to liver tissue.
References
PT Manyike, ED Kharasch, TF Kalhorn, JT Slattery, "Contribution of CYP2E1 and CYP3A to acetaminophen reactive metabolite formation", Clin
Pharmacol Ther, 67, 2000, 275-82.
Reaction
2.1.1.2.10.2 Benzene is hydroxylated to phenol
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Benzene is an occupational and environmental toxicant and is implicated in myelogenous leukemia. For toxicity to occur, benzene is oxidised to
phenol and subsequently to catechol and hydroquinone. CYP2E1 is the enzyme responsible for oxidation of benzene to phenol.
References
MW Powley, GP Carlson, "Cytochrome P450 isozymes involved in the metabolism of phenol, a benzene metabolite", Toxicol Lett, 125, 2001,
117-23.
Reaction
2.1.1.2.10.3 Dehalogenation of carbon tetrachloride to form a free radical
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Carbon tetrachloride (CCl4) has been widely used as a dry-cleaning agent, in fire extinguishers and in the manufacture of other halogenated
hydrocarbons. At toxic doses, CCl4 exposure can damage the liver and kidneys. This toxicity results from CYP2E1-dependant reduction of CCl4
to the reactive trichloromethyl radical (CCl3.).
References
RC Zangar, JM Benson, VL Burnett, DL Springer, "Cytochrome P450 2E1 is the primary enzyme responsible for low-dose carbon tetrachloride
metabolism in human liver microsomes", Chem Biol Interact, 125, 2000, 233-43.
Reaction
2.1.1.2.10.4 Dehalogenation of the poly-halogenated hydrocarbon Halothane to form the acylhalide Trifluoroacetlychloride and
hydrogen bromide
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
The volatile anesthetic halothane can undergo CYP2E1-catalyzed oxidation to form a reactive intermediate which can acetylate liver proteins.
These proteins can then stimulate an immune reaction that mediates severe hepatic necrosis ("halothane hepatitis").
References
ED Kharasch, D Hankins, D Mautz, KE Thummel, "Identification of the enzyme responsible for oxidative halothane metabolism: implications for
prevention of halothane hepatitis", Lancet, 347, 1996, 1367-71.
DK Spracklin, DC Hankins, JM Fisher, KE Thummel, ED Kharasch, "Cytochrome P450 2E1 is the principal catalyst of human oxidative
halothane metabolism in vitro", J Pharmacol Exp Ther, 281, 1997, 400-11.
Reaction
2.1.1.2.10.5 MEOS oxidizes ethanol to acetaldehyde
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
The MEOS (microsomal ethanol oxidizing system) is an accessory pathway in the liver which increases in activity on chronic alcohol induction.
The MEOS utilizes a cytochrome P450 which has since been deciphered to be CYP2E1, an ethanol-inducible form of P450. CYP2E1 also
increases acetaldehyde formation and free radicals which can initiate lipid peroxidation. CYP2E1 can also activate many over-the-counter
medicines and solvents to toxic metabolites and deplete retinoids resulting in their depletion and deletrious effects. This is because, being a
cytochrome P450 and using NADPH and oxygen, it has the ability to biotransform drugs when it has been induced by ethanol.
References
JM Lasker, J Raucy, S Kubota, BP Bloswick, M Black, CS Lieber, "Purification and characterization of human liver cytochrome P-450-ALC",
Biochem Biophys Res Commun, 148, 1987, 232-8.
K Ohnishi, CS Lieber, "Reconstitution of the microsomal ethanol-oxidizing system. Qualitative and quantitative changes of cytochrome P-450
after chronic ethanol consumption.", J Biol Chem, 252, 1977, 7124-31.
KS Salmela, IG Kessova, IB Tsyrlov, CS Lieber, "Respective roles of human cytochrome P-4502E1, 1A2, and 3A4 in the hepatic microsomal
ethanol oxidizing system", Alcohol Clin Exp Res, 22, 1998, 2125-32.
H Asai, S Imaoka, T Kuroki, T Monna, Y Funae, "Microsomal ethanol oxidizing system activity by human hepatic cytochrome P450s", J
Pharmacol Exp Ther, 277, 1996, 1004-9.
CS Lieber, "Ethanol metabolism, cirrhosis and alcoholism", Clin Chim Acta, 257, 1997, 59-84.
Reaction
2.1.1.2.10.6 Vinyl chloride is oxidized to 2-Chloroethylene oxide
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
CYP2E1 can catalyze the oxidation of the vinyl halide vinyl chloride to the epoxide 2-chloroethylene oxide. The epoxide is very unstable and
rearranges quickly to 2-chloroacetaldehyde. Both these products can interact with DNA and proteins.
References
FP Guengerich, WM Jr Crawford, PG Watanabe, "Activation of vinyl chloride to covalently bound metabolites: roles of 2-chloroethylene oxide
and 2-chloroacetaldehyde", Biochemistry, 18, 1979, 5177-82.
FP Guengerich, DH Kim, M Iwasaki, "Role of human cytochrome P-450 IIE1 in the oxidation of many low molecular weight cancer suspects",
Chem Res Toxicol, 4, 1991, 168-79.
Reaction
2.1.1.2.11 CYP2D6 4-hydroxylates debrisoquine
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
CYP2D6 (debrisoquine 4-hydroxylase) has a wide substrate specificity and is an important cytochrme P450 in drug metabolism. It has extensive
genetic polymorphism (called the debrisoquine/sparteine oxidation polymorphism) that influences its expression and function.The polymorphism
is responsible for populations being poor metabolizers (PM) or extensive metabolizers (EM, normal). Approximately 10% of Caucasians and less
than 1% of Asians lack the CYP2D6 protein because of two null alleles which do not encode the functional product. Further polymorphisms
discovered recently have identified ultrarapid metabolizers (PM) (alleles with multiple gene copies) and intermediate metabolizers (IM)
(deficiency in their metabolism capacity) (Zanger UM et al, 2004).
References
UM Zanger, S Raimundo, M Eichelbaum, "Cytochrome P450 2D6: overview and update on pharmacology, genetics, biochemistry", Naunyn
Schmiedebergs Arch Pharmacol, 369, 2004, 23-37.
AR Boobis, S Murray, GC Kahn, GM Robertz, DS Davies, "Substrate specificity of the form of cytochrome P-450 catalyzing the 4-hydroxylation
of debrisoquine in man", Mol Pharmacol, 23, 1983, 474-81.
I Johansson, E Lundqvist, L Bertilsson, ML Dahl, F SjÃ¶qvist, M Ingelman-Sundberg, "Inherited amplification of an active gene in the cytochrome
P450 CYP2D locus as a cause of ultrarapid metabolism of debrisoquine", Proc Natl Acad Sci U S A, 90, 1993, 11825-9.
Reaction
2.1.1.2.12 CYP2F1 dehydrogenates 3-methylindole
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
3-methylindole (3MI) is a fermentation product of tryptophan. It is usually formed in the rumen of goats and cattle and in the large intestine of
humans. CYP2F1 shows the highest activity towards the dehydrogenation of 3MI to form a methylene imine-reactive intermediate.
References
DL Lanza, E Code, CL Crespi, FJ Gonzalez, GS Yost, "Specific dehydrogenation of 3-methylindole and epoxidation of naphthalene by
recombinant human CYP2F1 expressed in lymphoblastoid cells", Drug Metab Dispos, 27, 1999, 798-803.
Reaction
2.1.1.2.13 CYP3A4 can N-demethylate loperaminde

Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
The CYP3A family are the most abundantly expressed P450s in human liver, accounting for around 50% of xenobiotic drug metabolism.
CYP3A4 is the most abundant member of the family and possesses broad specificity to a range of xenobiotics. Loperamide (LOP), an
antidiarrheal, is mainly metabolized to desmethylloperamide (DLOP) through the N-demethylation pathway. This initial N-demethylation is carried
out by CYP3A4.
References
KA Kim, J Chung, DH Jung, JY Park, "Identification of cytochrome P450 isoforms involved in the metabolism of loperamide in human liver
microsomes", Eur J Clin Pharmacol, 60, 2004, 575-81.
Reaction
2.1.1.2.14 CYP3A5 oxidises aflatoxin B1 to aflatoxin-8,9-oxide

Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Aflatoxins are produced by the fungal molds Aspergillus flavus and Aspergillus parasiticus. Dietary contamination accounts for adverse health
problems including liver cancer therby classifying aflatoxins as a Group 1 carcinogen in humans.Aflatoxin B1 (AFB1) is especially carcinogenic in
a number of species including humans.
AFB1 requires microsomal oxidation to produce epoxides which are the cause of their toxic and carcinogenic effects. In humans, both CYP3A4
and CYP3A5 are able to produce epoxide stereoisomers of AFB1, the most potent being aflatoxin exo-8,9-oxide (AFBO).
References
L Wojnowski, PC Turner, B Pedersen, E Hustert, J BrockmÃ¶ller, M Mendy, HC Whittle, G Kirk, CP Wild, "Increased levels of aflatoxin-albumin
adducts are associated with CYP3A5 polymorphisms in The Gambia, West Africa", Pharmacogenetics, 14, 2004, 691-700.
Reaction
2.1.1.2.15 CYP3A7 can 6beta-hydroxylate testosterone
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
CYP3A7 is only expressed in fetal liver and not in adults. It has lower biotransformation capability than other members of the CYP3A family such
as 3A4 or 3A5 but possesses a similar broad specificity. CYP3A7 plays a major role in fetal steroid hydroxylation, an example being the
6beta-hydroxylation of testosterone.
References
S Ohmori, N Fujiki, H Nakasa, H Nakamura, I Ishii, K Itahashi, M Kitada, "Steroid hydroxylation by human fetal CYP3A7 and human
NADPH-cytochrome P450 reductase coexpressed in insect cells using baculovirus", Res Commun Mol Pathol Pharmacol, 100, 1998, 15-28.
Reaction
2.1.1.3 Fatty acids

Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
The CYP4 family are the main CYPs involved in the metabolism of long-chain fatty acids.
References
RH Elbekai, AO El-Kadi, "Cytochrome P450 enzymes: central players in cardiovascular health and disease", Pharmacol Ther, 112, 2006,
564-87.
M Medhora, A Dhanasekaran, SK Gruenloh, LK Dunn, M Gabrilovich, JR Falck, DR Harder, ER Jacobs, PF Pratt, "Emerging mechanisms for
growth and protection of the vasculature by cytochrome P450-derived products of arachidonic acid and other eicosanoids", Prostaglandins Other
Lipid Mediat, 82, 2007, 19-29.
2.1.1.3.1 CYP2J2 epoxygenates arachidonic acid
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Activation of phospholipases releases free arachidonic acid (AA) from phospholipid bilayers which can then be metabolized to biologically active
eicosanoids (signaling molecules which exert effects in inflammation and immunity). The cytochrome P450 enzyme CYP2J2 (arachidonic acid
epoxygenase) is mainly expressed in human heart and can metabolize AA to epoxyeicosatrienoic acid (EET). Four cis-EETs can be produced:
5,6-, 8,9-, 11,12- and 14,15-EET. Each of these can be formed as the R,S or the S,R enantiomer (Zeldin DC, 2001). The most abundant
regioisomer in human heart is 14,15-EET although 11,12-EET possesses the most potent anti-inflammatory effect (Wu et al, 1996).
References
S Wu, CR Moomaw, KB Tomer, JR Falck, DC Zeldin, "Molecular cloning and expression of CYP2J2, a human cytochrome P450 arachidonic
acid epoxygenase highly expressed in heart", J Biol Chem, 271, 1996, 3460-8.
DC Zeldin, "Epoxygenase pathways of arachidonic acid metabolism", J Biol Chem, 276, 2001, 36059-62.
DC Zeldin, J Foley, J Ma, JE Boyle, JM Pascual, CR Moomaw, KB Tomer, C Steenbergen, S Wu, "CYP2J subfamily P450s in the lung:
expression, localization, and potential functional significance", Mol Pharmacol, 50, 1996, 1111-7.
Reaction
2.1.1.3.2 CYP4A11 omega-hydroxylates laurate
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Dodecanoic acid (Lauric acid) is a medium-chain fatty acid which serves as a model substrate for studying the CYP4A gene subfamily of
cytochrome P450s. CYP4A11 and CYP2E1 are the principal isozymes involved in omega-hydroxylation and omega-1 hydroxylation respectively
of dodecanoic acid.
References
PK Powell, I Wolf, JM Lasker, "Identification of CYP4A11 as the major lauric acid omega-hydroxylase in human liver microsomes", Arch
Biochem Biophys, 335, 1996, 219-26.
Reaction
2.1.1.3.3 CYP4B1 can 12-hydroxylate arachidonic acid
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Injury to the eye's surface provokes an inflammatory response, mediated, in part, by 12-hydroxyeicosanoids. CYP4B1 catalyzes the
12-hydroxylation of arachidonic acid to 12-HETE and 12-HETrE (12-hydroxy-5,8,11,14-eicosatetraenoic acid and
12-hydroxy-5,8,14-eicosatrienoic acid respectively). Both these metabolites possess potent inflammatory and angiogenic properties. The
example of 12-HETE formation only is shown here.
References
S Ashkar, A Mesentsev, WX Zhang, V Mastyugin, MW Dunn, M Laniado-Schwartzman, "Retinoic acid induces corneal epithelial CYP4B1 gene
expression and stimulates the synthesis of inflammatory 12-hydroxyeicosanoids", J Ocul Pharmacol Ther, 20, 2004, 65-74.
Reaction
2.1.1.3.4 CYP4F12 hydroxylates arachidonic acid
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Human CYP4F12 is involved in metabolism of endogenous compounds such as inflammatory mediators (arachidonic acid and prostaglandin H2)
as well as xenobiotics like terfenadine (an antihistaminic drug). The omega-hydroxylation of arachidonic acid is shown here as an example.
References
J Bylund, M Bylund, EH Oliw, "cDna cloning and expression of CYP4F12, a novel human cytochrome P450", Biochem Biophys Res Commun,
280, 2001, 892-7.
Reaction
2.1.1.4 Eicosanoids
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Arachidonic acid is metabolized via three major enzymatic pathways: cyclooxygenase, lipoxygenase, and cytochrome P450. The cytochrome
P450 pathway metabolites are oxygenated metabolites of arachidonic acid.
References
JA Cook, J Geisel, PV Halushka, HD Reines, "Prostaglandins, thromboxanes, leukotrienes, and cytochrome P-450 metabolites of arachidonic
acid", New Horiz, 1, 1993, 60-9.
2.1.1.4.1 CYP4F2 omega-hydroxylate leukotriene B4, thus inactivating it
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Leukotriene B4 (LTB4) is formed from arachidonic acid and is a potent inflammatory mediator. LTB4's activity is terminated by formation of its
omega hydroxylated metabolite, 20-hydroxyleukotriene B4 (20-OH LTB4), catalyzed by CYP4F2 primarily in human liver.
References
R Jin, DR Koop, JL Raucy, JM Lasker, "Role of human CYP4F2 in hepatic catabolism of the proinflammatory agent leukotriene B4", Arch
Reaction
2.1.1.4.2 CYP4F3 omega-hydroxylate leukotriene B4, thus inactivating it
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Leukotriene B4 (LTB4) is formed from arachidonic acid and is a potent inflammatory mediator. LTB4's activity is terminated by formation of its
omega hydroxylated metabolite, 20-hydroxyleukotriene B4 (20-OH LTB4). This deactivation can be carried out by CYP4F3 in addition to
CYP4F2.
References
Y Kikuta, M Kato, Y Yamashita, Y Miyauchi, K Tanaka, N Kamada, M Kusunose, "Human leukotriene B4 omega-hydroxylase (CYP4F3) gene:
molecular cloning and chromosomal localization", DNA Cell Biol, 17, 1998, 221-30.
Reaction
2.1.1.4.3 CYP4F8 hydroxylates prostaglandin H2
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
19-hydroxyprostaglandins E1 and E2 (19-OH PGE1/2) are major components of human seminal fluid. The initial step in their formation is the
19-hydroxylation of prostaglandin H1 and H2 (PGH1/2). CYP4F8 performs this initial conversion. The example of PGH2 is used here.
References
J Bylund, M Hidestrand, M Ingelman-Sundberg, EH Oliw, "Identification of CYP4F8 in human seminal vesicles as a prominent 19-hydroxylase of
prostaglandin endoperoxides", J Biol Chem, 275, 2000, 21844-9.
J Bylund, N FinnstrÃ¶m, EH Oliw, "Gene expression of a novel cytochrome P450 of the CYP4F subfamily in human seminal vesicles", Biochem
Biophys Res Commun, 261, 1999, 169-74.
Reaction
2.1.1.4.4 Thromboxane synthase (CYP5A1) mediates the isomerization of prostaglandin H2 to thromboxane A2
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
This reaction is not coupled with any P450 reductase proteins nor consumes NADPH. An electron transfer occurs from P450-Fe(III) to the
peroxidic species on PGH2
References
D Chevalier, JM Lo-Guidice, E Sergent, D Allorge, H DebuysÃ¨re, N Ferrari, C Libersa, M Lhermitte, F Broly, "Identification of genetic variants in
the human thromboxane synthase gene (CYP5A1)", Mutat Res, 432, 2001, 61-7.
A Miyata, C Yokoyama, H Ihara, S Bandoh, O Takeda, E Takahashi, T Tanabe, "Characterization of the human gene (TBXAS1) encoding
thromboxane synthase", Eur J Biochem, 224, 1994, 273-9.
Reaction
2.1.1.4.5 Prostacyclin synthase (CYP8A1) mediates the isomerization of prostaglandin H2 to prostaglandin I2
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
This reaction is not coupled with any P450 reductase proteins nor consumes NADPH. An electron transfer occurs from P450-Fe(III) to the
peroxidic species on PGH2
References
M Wada, C Yokoyama, T Hatae, M Shimonishi, M Nakamura, Y Imai, V Ullrich, T Tanabe, "Purification and characterization of recombinant
human prostacyclin synthase", J Biochem, 135, 2004, 455-63.
Reaction
2.1.1.5 Vitamins
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
A number of CYPs can act upon vitamins.
References
2.1.1.5.1 25-Hydroxylation of vitamin D3 in liver
Authors
Jassal, B, 2008-01-07.
Editors
Jassal, B, 2008-05-28, Jassal, B, 2008-05-19.
Reviewers
Description
To be functionally active, vitamin D is required to be dihydroxylated. The first hydroxylation at position 25 is carried out by vitamin D
25-hydroxylase (CYP2R1) in the liver, forming calcidiol.
References
R Shinkyo, T Sakaki, M Kamakura, M Ohta, K Inouye, "Metabolism of vitamin D by human microsomal CYP2R1", Biochem Biophys Res
Commun, 324, 2004, 451-7.
Reaction
2.1.1.5.2 CYP24A1 catalyzes tjhe initial step in the deactivation of the hormonally active form of vitamin D3
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Catabolic inactivation of the active, hormonal form of vitamin D3 (1,25-dihydroxyvitamin D3) is initially carried out by 24-hydroxylation, mediated
by CYP24A1 (1,25-dihydroxyvitamin D3 24-hydroxylase). The product formed is eventually transformed to calcitroic acid, the major
water-soluble metabolite that can be excreted in bile.
References
MJ Beckman, P Tadikonda, E Werner, J Prahl, S Yamada, HF DeLuca, "Human 25-hydroxyvitamin D3-24-hydroxylase, a multicatalytic enzyme",
Biochemistry, 35, 1996, 8465-72.
T Sakaki, N Sawada, K Komai, S Shiozawa, S Yamada, K Yamamoto, Y Ohyama, K Inouye, "Dual metabolic pathway of 25-hydroxyvitamin D3
catalyzed by human CYP24", Eur J Biochem, 267, 2000, 6158-65.
DE Prosser, M Kaufmann, B O'Leary, V Byford, G Jones, "Single A326G mutation converts human CYP24A1 from 25-OH-D3-24-hydroxylase
into -23-hydroxylase, generating 1alpha,25-(OH)2D3-26,23-lactone", Proc Natl Acad Sci U S A, 104, 2007, 12673-8.
Reaction
2.1.1.5.3 CYP26A1 breaks down all-trans-retinoic acid by 4-hydroxylation
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Retinoic acid (RA) is a biologically active analogue of vitamin A (retinol). RA plays an important role in regulating cell growth and
differentiation.CYP26A1 is involved in the metabolic breakdown of RA by 4-hydroxylation. CYP26A1-mediated 4-hydroxylation is specific for
all-trans-RA but not for the isomers 13-cis-RA and 9-cis-RA.
References
E Sonneveld, CE van den Brink, BM van der Leede, RK Schulkes, M Petkovich, B van der Burg, PT van der Saag, "Human retinoic acid (RA)
4-hydroxylase (CYP26) is highly specific for all-trans-RA and can be induced through RA receptors in human breast and colon carcinoma cells",
Cell Growth Differ, 9, 1998, 629-37.
Reaction
2.1.1.5.4 CYP26B1 also deactivates all-trans-retinoic acid by 4-hydroxylation
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
CYP26B1 can also deactivate all-trans-retinoic acid by 4-hydroxylation. High expression levels in the cerebellum and pons of human brain
suggests a protective role of specific tissues against retinoid damage.
References
JA White, H Ramshaw, M Taimi, W Stangle, A Zhang, S Everingham, S Creighton, SP Tam, G Jones, M Petkovich, "Identification of the human
cytochrome P450, P450RAI-2, which is predominantly expressed in the adult cerebellum and is responsible for all-trans-retinoic acid
metabolism", Proc Natl Acad Sci U S A, 97, 2000, 6403-8.
Reaction
2.1.1.5.5 CYP26C1 deactivates 9-cis-retinoic acid by 4-hydroxylation
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Endogenous retinoic acids (RA) which play a role in gene regulation exist as either cis or trans isomers. While CYP26C1 can also hydroxylate
the trans form, it is unique in hydroxylating the 9-cis isomer of RA.
References
M Taimi, C Helvig, J Wisniewski, H Ramshaw, J White, M Amad, B Korczak, M Petkovich, "A novel human cytochrome P450, CYP26C1,
involved in metabolism of 9-cis and all-trans isomers of retinoic acid", J Biol Chem, 279, 2004, 77-85.
Reaction
2.1.1.5.6 Further hydroxylation of calcidiol in kidney to form calcitriol
Authors
Jassal, B, 2008-01-07.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
The second step in vitamin D3 activation requires further hydroxylation of 25-hydroxyvitamin D3 (calcidiol) to 1alpha-25-dihydroxyvitamin D3
(calcitriol). This conversion is mediated by 25-hydroxyvitamin D-1alpha hydroxylase (CYP27B1).
References
D Zehnder, R Bland, RS Chana, DC Wheeler, AJ Howie, MC Williams, PM Stewart, M Hewison, "Synthesis of 1,25-dihydroxyvitamin D(3) by
human endothelial cells is regulated by inflammatory cytokines: a novel autocrine determinant of vascular cell adhesion", J Am Soc Nephrol, 13,
2002, 621-9.
J Fritsche, K Mondal, A Ehrnsperger, R Andreesen, M Kreutz, "Regulation of 25-hydroxyvitamin D3-1 alpha-hydroxylase and production of 1
alpha,25-dihydroxyvitamin D3 by human dendritic cells", Blood, 102, 2003, 3314-6.
Reaction
2.1.1.6 Unknown
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Approximately a quarter of the 57 human CYPs still remain "orphans" in the sense that their function, expression sites, and regulation are largely
not elucidated. While there is enough experimental evidence to know that all these proteins get made and can catalyze CYP-like reactions in
vitro, evidence of in vivo function and substrate specificity is insufficient to allow them to be placed in any of the classes in the functional scheme.
References
K Stark, FP Guengerich, "Characterization of orphan human cytochromes P450", Drug Metab Rev, 39, 2007, 627-37.
FP Guengerich, ZL Wu, CJ Bartleson, "Function of human cytochrome P450s: characterization of the orphans", Biochem Biophys Res Commun,
338, 2005, 465-9.
2.1.1.6.1 CYP2S1 can deactivate all-trans-retinoic acid
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
CYP2S1 is a recently discovered cytochrome P450 enzyme on the basis of homology searches of databases and was found to be homologous
to the CYP2 family of enzymes that are known to metabolize xenobiotics (Rylander T et al, 2001). CYP2S1 is expressed in skin cells and is
inducible by UV radiation, coal tar and all-trans retinoate, the latter also serving as a substrate for the enzyme. Expression of CYP2S1 was
significantly higher in psoriatic plaque than in normal skin. Psoriasis is a chronic hyperproliferative and inflammatory disorder.
References
G Smith, CR Wolf, YY Deeni, RS Dawe, AT Evans, MM Comrie, J Ferguson, SH Ibbotson, "Cutaneous expression of cytochrome P450
CYP2S1: individuality in regulation by therapeutic agents for psoriasis and other skin diseases", Lancet, 361, 2003, 1336-43.
T Rylander, EP Neve, M Ingelman-Sundberg, M Oscarson, "Identification and tissue distribution of the novel human cytochrome P450 2S1
(CYP2S1)", Biochem Biophys Res Commun, 281, 2001, 529-35.
Reaction
2.1.1.6.2 CYP2U1 can omega-hydroxylate arachidonate
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
A novel cytochrome P450, CYP2U1, may play an important role in modulating the arachidonic acid signaling pathway. It was discovered by
searching the human EST database for homology to existing CYPs and subsequent cloning and expression to obtain the enzyme. CYP2U1 was
found to be highly expressed in the thymus and the brain (cerebellum) and found to metabolize arachidonic acid to 19- and 20-hydroxy-modified
arachidonic acids. It is thought that CYP2U1 plays an important physiological role in fatty acid signaling processes in both the cerebellum and
thymus. The omega-hydroxylation (19) example is provided here.
References
SS Chuang, C Helvig, M Taimi, HA Ramshaw, AH Collop, M Amad, JA White, M Petkovich, G Jones, B Korczak, "CYP2U1, a novel human
thymus- and brain-specific cytochrome P450, catalyzes omega- and (omega-1)-hydroxylation of fatty acids", J Biol Chem, 279, 2004, 6305-14.
Reaction
2.1.1.6.3 CYP2W1 can oxidize indole
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
CYP2W1 is a so-called "ophan P450", a P450 which has no defined function or endogenous/xenobiotic substrates. CYP2W1 has recently been
shown to be selectively expressed in some forms of cancers and, with the low expression in normal tissues, could be rendered as a possible
drug target during cancer therapy (Karlgren M, Ingelman-Sundberg M, 2007). CYP2W1 can bioactivate several procarcinogens but at lower
levels than other P450s (Wu, ZL et al, 2006). CYP2W1 also shows monooxygenase activity towards the pigment indole, an ingredient of
perfumes and coal tar.
References
H Yoshioka, N Kasai, S Ikushiro, R Shinkyo, M Kamakura, M Ohta, K Inouye, T Sakaki, "Enzymatic properties of human CYP2W1 expressed in
Escherichia coli", Biochem Biophys Res Commun, 345, 2006, 169-74.
M Karlgren, M Ingelman-Sundberg, "Tumour-specific expression of CYP2W1: its potential as a drug target in cancer therapy", Expert Opin Ther
Targets, 11, 2007, 61-7.
ZL Wu, CD Sohl, T Shimada, FP Guengerich, "Recombinant enzymes overexpressed in bacteria show broad catalytic specificity of human
cytochrome P450 2W1 and limited activity of human cytochrome P450 2S1", Mol Pharmacol, 69, 2006, 2007-14.
Reaction
2.1.1.6.4 CYP3A43 catalyzes the 6beta-hydroxylation of testosterone
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
CYP3A43 belongs to the CYP3A family, of which CYP3A4 is the most active member in the biotransformation of xenobiotics. Testosterone
metabolites are a major determinant of prostate growth and differentiation. CYP3A43, which is expressed in the prostate, exhibits minor
6-beta-hydroxylation activity towards testosterone. Thus, CYP3A43 may be involved in the etiology of prostate cancer.
References
C Zeigler-Johnson, T Friebel, AH Walker, Y Wang, E Spangler, S Panossian, M Patacsil, R Aplenc, AJ Wein, SB Malkowicz, TR Rebbeck,
"CYP3A4, CYP3A5, and CYP3A43 genotypes and haplotypes in the etiology and severity of prostate cancer", Cancer Res, 64, 2004, 8461-7.
TL Domanski, C Finta, JR Halpert, PG Zaphiropoulos, "cDNA cloning and initial characterization of CYP3A43, a novel human cytochrome P450",
Mol Pharmacol, 59, 2001, 386-92.
Reaction
2.1.1.6.5 CYP4F11 omega-hydroxylates 3-hydroxypalmitate
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Long-chain 3-hydroxy fatty acids (3-OHFAs) are omega-hydroxylated to form 3-hydroxydicarboxylic acid (3-OHDCAs) precursors in human liver.
These products may be implicated in pathological states where fatty acid mobilzation or impairment of mitochondrial fatty acid beta-oxidation
increases 3-OHFA levels. CYP4A11, an orphan P450, has been shown to omega-hydroxylate 3-hydroxystearate and 3-hydroxypalmitate; the
latter is shown here.
References
M Dhar, DW Sepkovic, V Hirani, RP Magnusson, JM Lasker, "Omega oxidation of 3-hydroxy fatty acids by the human CYP4F gene subfamily
enzyme CYP4F11", J Lipid Res, 49, 2008, 612-24.
Reaction
2.1.2 FMO oxidizes nucleophiles
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Flavin-containing monooxygenases (FMOs) are the second family of microsomal oxidative enzymes with broad and overlapping specificity. The
major reactions FMOs catalyze are nucleophilic hetero-atom compounds such as nitrogen, sulfur or phosphorus as the hetero-atom to form
N-oxides, S-oxides or P-oxides respectively. Despite the functional overlap with cytochrome P450s, the mechanism of action differs. FMOs bind
and activate molecular oxygen before the substrate binds to the enzyme (picture). They also require flavin adenosine dinucleotide (FAD) as a
cofactor. Unlike cytochrome P450 enzymes, FMOs are heat-labile, a useful way to distinguish which enzyme system is at work for researchers
studying metabolism. Also, FMOs are not inducible by substrates, unlike the P450 enzymes.\n(1) NADPH binds to the enzyme and reduces the
prosthetic group FAD to FADH2. NADP+ remains bound to the enzyme.\n(2) Incorporation of molecular oxygen to form a hydroperoxide.\n(3) A
peroxide oxygen is transferred to the substrate.\n(4) Water is released.\n(5) NADP+ dissociates returning the enzyme to its initial state.\n\nTo
date, there are 6 isozymes of FMO (FMO1-6) in humans, the most prominent and active one being FMO3. The FMO6 gene does not encode for
a functional enzyme although it has the greatest sequence similarity with FMO3 (71%), whilst the others range from 50-58% sequence similarity
with FMO3. FMO1-3 are the ones that exhibit activity towards nucleophiles, the others are insignificant in this respect.
References
SK Krueger, DE Williams, "Mammalian flavin-containing monooxygenases: structure/function, genetic polymorphisms and role in drug
metabolism", Pharmacol Ther, 106, 2005, 357-87.
JR Cashman, "The role of flavin-containing monooxygenases in drug metabolism and development", Curr Opin Drug Discov Devel, 6, 2003,
486-93.
2.1.2.1 FMO1 N-oxidizes the anti-cancer drug tamoxifen
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Tamoxifen (TAM) is an antiestrogen and currently used extensively for breast cancer therapy. FMOs, especially FMO1 can N-oxidze TAM to
tamoxifen N-oxide (TNO). TNO can be reduced back to TAM by the P450 system. TNO appears to be just as potent as TAM but with fewer
side-effects so this metabolic cycling could play a part in the use of TNO in the treatment of breast cancer.
References
P Parte, D Kupfer, "Oxidation of tamoxifen by human flavin-containing monooxygenase (FMO) 1 and FMO3 to tamoxifen-N-oxide and its novel
reduction back to tamoxifen by human cytochromes P450 and hemoglobin", Drug Metab Dispos, 33, 2005, 1446-52.
Reaction
2.1.2.2 FMO2 S-oxidizes the antithyroid drug methimazole
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Methimazole is a drug used to treat hyperthyroidism, a condition arising when the thyroid gland is producing too much thyroid hormone. FMO2 is
able to the S-oxidized form of methimazole.
References
SK Krueger, SR Martin, MF Yueh, CB Pereira, DE Williams, "Identification of active flavin-containing monooxygenase isoform 2 in human lung
and characterization of expressed protein", Drug Metab Dispos, 30, 2002, 34-41.
Reaction
2.1.2.3 FMO3 N-oxidizes the tertiary amine trimethylamine

Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-14.
Reviewers
Description
Trimethylamine (TMA) is present in the diet (in fish) but primarily formed in vivo from the breakdown of choline. It is N-oxidized by FMO3 in the
liver, the major isoform active towards TMA Trimethylaminuria ("fish-odour syndrome") is a human genetic disorder characterized by an impaired
ability to convert the malodourous TMA to its odourless N-oxide.
References
JA Humbert, KB Hammond, WE Hathaway, "Trimethylaminuria: the fish-odour syndrome", Lancet, 2, 1970, 770-1.
T Higgins, S Chaykin, KB Hammond, JR Humbert, "Trimethylamine N-oxide synthesis: a human variant", Biochem Med, 6, 1972, 392-6.
Reaction
2.1.3 COX reactions

Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Arachidonic acid (AA) is a 20 carbon unsaturated fatty acid which is present in the lipid bilayer of all mammalian cells. AA is released from the
membrane by phospholipases, thus making it available for conversion to bioactive lipids. The cyclooxygenase pathway is one of three pathways
(the others being lipoxygenase and P450 monooxygenase pathways) that perform this conversion.\n\nThe enzyme that acts in the
cyclooxygenase pathway is called cyclooxygenase (COX) or prostaglandin H synthase (PGHS). PGHS exhibits a dual catalytic activity, a
cyclooxygenase and a peroxidase. The cyclooxygenase catalyzes the initial conversion of AA to an intermediate, prostaglandin G2 (PGG2)
whilst the peroxidase converts PGG2 to prostaglandin H2 (PGH2) via a two-electron reduction. PGH2 is the intermediate for products that play
critical roles in immune function regulation, kidney development and mucosal integrity of the GI tract.\n\nPGHS exists in two isoforms, 1 and 2
and both forms can perform the above reactions. Form 1 is constitutively expressed in most tissues and is involved in performing normal
physiological functions. Form 2, in contrast, is inducible and is involved in critical steps of rheumatic disease, inflammation and tumorigenesis.
References
WL Smith, DL DeWitt, RM Garavito, "Cyclooxygenases: structural, cellular, and molecular biology", Annu Rev Biochem, 69, 2000, 145-82.
2.1.3.1 Arachidonic acid oxidised to PGG2
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-28, Jassal, B, 2008-05-19.
Reviewers
Description
PGHS exhibits a dual catalytic activity, a cyclooxygenase and a peroxidase. The cyclooxygenase function catalyzes the initial conversion of AA
to an intermediate, prostaglandin G2 (PGG2)
References
DH Nugteren, E Hazelhof, "Isolation and properties of intermediates in prostaglandin biosynthesis", Biochim Biophys Acta, 326, 1973, 448-61.
M Hamberg, J Svensson, T Wakabayashi, B Samuelsson, "Isolation and structure of two prostaglandin endoperoxides that cause platelet
aggregation", Proc Natl Acad Sci U S A, 71, 1974, 345-9.
Reaction
2.1.3.2 Peroxidative reduction of PGG2 to PGH2
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
PGHS exhibits a dual catalytic activity, a cyclooxygenase and a peroxidase. The peroxidase function of PGHS converts PGG2 to prostaglandin
H2 (PGH2) via a two-electron reduction.
References
M Hamberg, B Samuelsson, "Detection and isolation of an endoperoxide intermediate in prostaglandin biosynthesis", Proc Natl Acad Sci U S A,
70, 1973, 899-903.
Reaction
2.1.4 Amine Oxidase reactions
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Human amine oxidases (AO) catalyze the oxidative deamination of biogenic amines (neurotransmitters such as serotonin, noradrenaline, the
hormone adrenaline and polyamines such as the spermines) and xenobiotic amines (exogenous dietary tyramine and phenylethylamine). The
basic reaction is the oxidative cleavage of the alpha-H to form an imine product with the concomitant reduction of a FAD cofactor. The imine
product then hydrolyses to an aldehyde and ammonia (or amine for secondary and tertiary amine substrates). Reduced FAD is reoxidized to
form hydrogen peroxide to complete the catalytic cycle.
The reaction can be summarized as
RCH2NH2 + H2O + O2 = RCHO + NH3 + H2O2
The resultant hydrogen peroxide is the source of the most toxic free radical, the hydroxyl radical (.OH). This free radical is produced in the
Fenton reaction with the use of ferrous (Fe2+) iron.
References
C Beedham, "The role of non-P450 enzymes in drug oxidation", Pharm World Sci, 19, 1997, 255-63.
MS Benedetti, "Biotransformation of xenobiotics by amine oxidases", Fundam Clin Pharmacol, 15, 2001, 75-84.
CW Tabor, H Tabor, "Polyamines", Annu Rev Biochem, 53, 1984, 749-90.
2.1.4.1 Monoamines are oxidized to aldehydes by MAOA and MAOB, producing NH3 and H2O2
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Human monoamine oxidases (MAOs) are flavin-containing enzymes that are present on the outer mitochondrial membrane and act on primary,
secondary and tertiary amines. In contrast to the P450s which have a large number of isozymes, MAOs number only two isozymes, MAO-A and
MAO-B. These gene products share over 70% sequence identity, are approximately 59KDa in size and have overlapping substrates (for
example dopamine, tryamine and tryptamine) but each form also has distinct substrate specificities. MAO-A (primary type in fibroblasts)
preferentially oxidises serotonin (5-Hydroxytryptamine) whereas MAO-B (primary type in platelets) prefers phenylethylamine. MAOs are of
particular clinical interest because of the use of MAO inhibitors (MAOI) as antidepressants or in the treatment of neurodegenerative diseases.
References
C Beedham, "The role of non-P450 enzymes in drug oxidation", Pharm World Sci, 19, 1997, 255-63.
2.1.4.1.1 Dietary tyramine is oxidatively deaminated to an aldehyde by MAOB
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Monoamine oxidases (MAO-A/B), present in the outer mitochondrial membrane, catalyze the oxidation of biogenic amines, releasing hydrogen
peroxide (H2O2). H2O2 produced during the oxidative deamination of these amines appears to be involved in the progress of neurodegenerative
disorders such as Parkinson disease, presumably via oxidative damage to the mitochondrial membrane.
References
LB Pearce, JA Roth, "Human brain monoamine oxidase type B: mechanism of deamination as probed by steady-state methods", Biochemistry,
24, 1985, 1821-6.
Reaction
2.1.4.1.2 Oxidative deamination of 5-Hydroxytryptamine by MAOA
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
At the beginning of this reaction, 1 molecule of 'Oxygen', 1 molecule of 'H2O', and 1 molecule of '5-Hydroxytryptamine' are present. At the end of
this reaction, 1 molecule of 'NH3', 1 molecule of '5-Hydroxyindoleacetaldehyde', and 1 molecule of 'H2O2' are present.
This reaction takes place in the 'mitochondrial outer membrane' and is mediated by the 'amine oxidase activity' of 'MAOA-FAD complex'.
References
AM O'Carroll, CJ Fowler, JP Phillips, I Tobbia, KF Tipton, "The deamination of dopamine by human brain monoamine oxidase. Specificity for the
two enzyme forms in seven brain regions", Naunyn Schmiedebergs Arch Pharmacol, 322, 1983, 198-202.
Reaction
2.1.4.1.3 Oxidative deamination of Phenyethylamine by MAOB
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
At the beginning of this reaction, 1 molecule of 'Oxygen', 1 molecule of 'H2O', and 1 molecule of '2-Phenylethylamine' are present. At the end of
this reaction, 1 molecule of 'NH3', 1 molecule of 'H2O2', and 1 molecule of 'Phenylacetaldehyde' are present.
This reaction takes place in the 'mitochondrial outer membrane' and is mediated by the 'amine oxidase activity' of 'MAOB-FAD complex'.
References
K Oguchi, S Kobayashi, T Uesato, K Kamijo, "Studies on beta-phenylethylamine deamination by human placental monoamine oxidase", Jpn J
Pharmacol, 31, 1981, 7-14.
Reaction
2.1.4.2 Polyamines are oxidized to amines, aldehydes and H2O2 by PAOs
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Polyamine oxidases (PAOs), like MAOs, are also FAD-dependant and form aldehydes and hydrogen peroxide. PAOs are approximately 60KDa
in size, are monomers and are located in peroxisomes. They act on endogenous polyamines as well as some xenobiotics. The polyamines of the
spermine and spermidine families are important physiologically as they mediate cell function and growth and also play a role in programmed cell
death. The balance between biosynthesis, degradation and uptake of these polyamines is strictly controlled in cells and the PAO system is one
of a set of enzymes that maintains this regulation.
References
CW Tabor, H Tabor, "Polyamines", Annu Rev Biochem, 53, 1984, 749-90.
2.1.4.2.1 Spermine is oxidized to spermidine
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Spermine oxidase (SMOX/PAOh1/SMO) is a polyamine oxidase flavoenzyme which catalyzes the oxidation of spermine to spermidine. It plays
an important role in the regulation of endogenous polyamine intracellular concentration. Five different isozymes are produced by alternative
splicing with isozyme 3 being the major isoform and possessing the highest affinity for spermine. It is highly inducible by specific antitumor
polyamine analogues (Wang et al., 2001).
References
Y Wang, W Devereux, PM Woster, TM Stewart, A Hacker, Jr Casero RA, "Cloning and characterization of a human polyamine oxidase that is
inducible by polyamine analogue exposure", Cancer Res, 61, 2001, 5370-3.
Y Wang, T Murray-Stewart, W Devereux, A Hacker, B Frydman, PM Woster, Jr Casero RA, "Properties of purified recombinant human
polyamine oxidase, PAOh1/SMO", Biochem Biophys Res Commun, 304, 2003, 605-11.
S Vujcic, P Diegelman, CJ Bacchi, DL Kramer, CW Porter, "Identification and characterization of a novel flavin-containing spermine oxidase of
mammalian cell origin", Biochem J, 367, 2002, 665-75.
Reaction
2.1.4.2.2 N-acetylspermidine is oxidized to putrescine
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Acetylated spermidine is oxidized by the flavoenzyme polyamine oxidase (PAO) to produce putrescine. PAO is involved in the back-conversion
of polyamines and thus the regulation of their intracellular concentrations.
References
S Vujcic, P Liang, P Diegelman, DL Kramer, CW Porter, "Genomic identification and biochemical characterization of the mammalian polyamine
oxidase involved in polyamine back-conversion", Biochem J, 370, 2003, 19-28.
Reaction
2.1.4.2.3 N-acetylspermine is oxidised to spermidine
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Acetylated spermine is oxidized by the flavoenzyme polyamine oxidase (PAO) to produce spermidine. PAO is involved in the back-conversion of
polyamines and thus the regulation of their intracellular concentrations.
References
S Vujcic, P Liang, P Diegelman, DL Kramer, CW Porter, "Genomic identification and biochemical characterization of the mammalian polyamine
oxidase involved in polyamine back-conversion", Biochem J, 370, 2003, 19-28.
Reaction
2.1.5 Ethanol catabolism
Authors
Jassal, B, 2008-05-19.
Editors
Description
Ethanol can be ingested as part of the diet, and is formed by microorganisms in the intestinal tract. The catabolism of ethanol occurs primarily in
liver cells, and has three steps. First, in the cytosol, ethanol is oxidized to acetaldehyde, with the formation of NADH. Second, in the
mitochondrion, acetaldehyde is oxidized to acetate with the formation of NADH. Finally, acetate can be condensed with coenzyme A to form
acetyl-CoA. Polymorphisms in the enzymes catalyzing the first two steps are associated with variation in the efficiency of alcohol catabolism in
human populations.
References
LG Lange, AJ Sytkowski, BL Vallee, "Human liver alcohol dehydrogenase: purification, composition, and catalytic features.", Biochemistry, 15,
1976, 4687-93.
2.1.5.1 Alcohol Dehydrogenase
Authors
Jassal, B, 2004-10-11.
Editors
Jassal, B, 2008-05-19.
Description
Alcohol dehydrogenase (ADH) is a zinc-containing, cytosolic, dimeric enzyme present in the liver, kidney, lung and gastric mucosa. NAD+ is the
prefered cofactor. ADH consists of two 40KDa subunits which can combine as either homo- or hetero-dimers. The subunits are encoded by at
least 7 gene loci (ADH1-7). Two of the subunits have more than one allelic variant (3 beta and 2 gamma encoded by ADH2 and ADH3
respectively) so there are many combinations for the formation of the dimer.
ADH falls into 5 main classes. Subunits combine within but not between classes. Class I ADHs are made up from alpha, beta and gamma
subunits. They can oxidise ethanol and other small, aliphatic alcohols. The homodimer beta2beta2 and heterodimers containing at least one
beta2 are the most active towards ethanol and are known as atypical ADH. This atypical form is present in high frequencies in Far East
populations (approx 85%) whereas Caucasian populations have low frequencies (5-20%).
References
A Parkinson, "Casarett and Doull's Toxicology 5th Edn", 1995, 113-186.
CS Lieber, "Ethanol metabolism, cirrhosis and alcoholism", Clin Chim Acta, 257, 1997, 59-84.
2.1.5.1.1 Ethanol is oxidized by NAD+ to form acetaldehyde, NADH, and H+
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Alcohol dehydrogenase enzymes are encoded by a family of seven genes, four of which, ADH1A, ADH1B, ADH1C, and ADH4, encode proteins
with substantial roles in human ethanol catabolism. The first three genes encode the so-called class I alpha, beta, and gamma proteins, and the
fourth encodes class IV pi protein. The active form of alcohol dehydrogenase is a dimer, and seven functional dimers have been identified in
human liver. The class I monomers associate to form both homodimers (alpha/alpha, beta/beta, gamma/gamma) and heterodimers (alpha/beta,
alpha/gamma, beta/gamma), while the class IV monomers associate to form homodimers (pi/pi) only. Expression of class I proteins is also
developmentally regulated: alpha monomer is abundant in the fetus, but expressed only at low levels in adulthood, when beta and gamma
proteins are abundant. This diversity is functionally important because the various dimers differ substantially in the efficiency with which they
oxidize ethanol. In addition, common polymorphic variants of beta and gamma proteins have been described that likewise differ substantially in
this respect.
References
RF Murray, PH Price, "Ontogenetic, polymorphic, and interethnic variation in the isoenzymes of human alcohol dehydrogenase.", Ann N Y Acad
Sci, 197, 1972, 68-72.
SJ Yin, WF Bosron, LJ Magnes, TK Li, "Human liver alcohol dehydrogenase: purification and kinetic characterization of the beta 2 beta 2, beta 2
beta 1, alpha beta 2, and beta 2 gamma 1 "Oriental" isoenzymes.", Biochemistry, 23, 1985, 5847-53.
HJ Edenberg, "Regulation of the mammalian alcohol dehydrogenase genes.", Prog Nucleic Acid Res Mol Biol, 64, 2000, 295-341.
1976, 4687-93.
CC Chen, RB Lu, YC Chen, MF Wang, YC Chang, TK Li, SJ Yin, "Interaction between the functional polymorphisms of the alcohol-metabolism
genes in protection against alcoholism.", Am J Hum Genet, 65, 1999, 795-807.
M Smith, DA Hopkinson, H Harris, "Studies on the subunit structure and molecular size of the human alcohol dehydrogenase isozymes
determined by the different loci, ADH1, ADH2, and ADH3.", Ann Hum Genet, 36, 1974, 401-14.
TK Li, WF Bosron, WP Dafeldecker, LG Lange, BL Vallee, "Isolation of pi-alcohol dehydrogenase of human liver: is it a determinant of
alcoholism?", Proc Natl Acad Sci U S A, 74, 1978, 4378-81.
2.1.5.1.1.1 Ethanol + NAD+ <=> Acetaldehyde + NADH + H+ [class I, alpha/alpha]
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
At the beginning of this reaction, 1 molecule of 'Ethanol', and 1 molecule of 'NAD+' are present. At the end of this reaction, 1 molecule of 'H+', 1
molecule of 'NADH', and 1 molecule of 'Acetaldehyde' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'alcohol dehydrogenase activity' of 'alcohol dehydrogenase 1 (class I),
alpha/alpha dimer'.
References
1976, 4687-93.
Reaction
2.1.5.1.1.2 Ethanol + NAD+ <=> Acetaldehyde + NADH + H+ [class I, alpha/beta]
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
This reaction takes place in the 'cytosol' and is mediated by the 'alcohol dehydrogenase activity' of 'alcohol dehydrogenase 1 (class I), alpha/beta
dimer'.
References
1976, 4687-93.
Reaction
2.1.5.1.1.3 Ethanol + NAD+ <=> Acetaldehyde + NADH + H+ [class I, alpha/gamma]
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
alpha/gamma dimer'.
References
1976, 4687-93.
Reaction
2.1.5.1.1.4 Ethanol + NAD+ <=> Acetaldehyde + NADH + H+ [class I, beta/beta]
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
This reaction takes place in the 'cytosol' and is mediated by the 'alcohol dehydrogenase activity' of 'alcohol dehydrogenase 1 (class I), beta/beta
dimer'.
References
1976, 4687-93.
Reaction
2.1.5.1.1.5 Ethanol + NAD+ <=> Acetaldehyde + NADH + H+ [class I, beta/gamma]
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
beta/gamma dimer'.
References
1976, 4687-93.
Reaction
2.1.5.1.1.6 Ethanol + NAD+ <=> Acetaldehyde + NADH + H+ [class I, gamma/gamma]
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
gamma/gamma dimer'.
References
1976, 4687-93.
Reaction
2.1.5.1.1.7 Ethanol + NAD+ <=> Acetaldehyde + NADH + H+ [class II, pi/pi]
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
This reaction takes place in the 'cytosol' and is mediated by the 'alcohol dehydrogenase activity' of 'alcohol dehydrogenase 4 (class II), pi dimer'.
References
TK Li, WF Bosron, WP Dafeldecker, LG Lange, BL Vallee, "Isolation of pi-alcohol dehydrogenase of human liver: is it a determinant of
alcoholism?", Proc Natl Acad Sci U S A, 74, 1978, 4378-81.
WF Bosron, TK Li, WP Dafeldecker, BL Vallee, "Human liver pi-alcohol dehydrogenase: kinetic and molecular properties.", Biochemistry, 18,
1979, 1101-5.
Reaction
2.1.5.1.1.8 Ethanol + NAD+ <=> Acetaldehyde + NADH + H+ [class IV, mu or sigma]
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
This reaction takes place in the 'cytosol' and is mediated by the 'alcohol dehydrogenase activity' of 'alcohol dehydrogenase 7 (class IV), mu or
sigma dimer'.
References
A Moreno, X ParÃ©s, "Purification and characterization of a new alcohol dehydrogenase from human stomach.", J Biol Chem, 266, 1991,
1128-33.
H Yokoyama, E Baraona, CS Lieber, "Molecular cloning of human class IV alcohol dehydrogenase cDNA.", Biochem Biophys Res Commun,
203, 1994, 219-24.
Reaction
2.1.5.1.1.9 Ethanol + NAD+ <=> Acetaldehyde + NADH + H+ [class V]
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
This reaction takes place in the 'cytosol' and is mediated by the 'alcohol dehydrogenase activity' of 'alcohol dehydrogenase 6 (class V) complex'.
References
CS Chen, A Yoshida, "Enzymatic properties of the protein encoded by newly cloned human alcohol dehydrogenase ADH6 gene", Biochem
Reaction
2.1.5.2 Aldehyde Dehydrogenase
Authors
Jassal, B, 2004-10-22.
Editors
Jassal, B, 2008-05-19.
Description
Aldehyde Dehydrogenases (ALDHs) are able to oxidize a wide range of endogenous and exogenous aldehydes. Endogenous aldehydes are
formed during the metabolism of amino acids, carbohydrates, lipids, biogenic amines, vitamins, and steroids. A large number of xenobiotics can
generate aldehydes during their biotransformation. Aldehydes are highly reactive electrophilic compounds, which interact with thiol and amino
groups and the aldehyde-mediated effects vary from physiologic and therapeutic to cytotoxic, genotoxic, and mutagenic or carcinogenic.
ALDH oxidizes aldehydes to carboxylic acids. Two acetaldehyde dehydrogenase isozymes, one cytosolic (ALDH1) and one mitochondrial
(ALDH2), catalyze this reaction. There is a third isozyme, ALDH3, present in the stomach but acetaldehyde is not a substrate for this isozyme.
ALDH2 isozymes are responsible for oxidizing simple aldehydes due to their low Km (high affinity) for them. Individuals who have the
reduced-activity allele typically show elevated blood levels of acetaldehyde and facial flushing following ethanol ingestion.
\n
References
H Jornvall, "Alcohol dehydrogenases, aldehyde dehydrogenases, and related enzymes", Alcohol, 2, 1985, 61-6.
2.1.5.2.1 Acetaldehyde is oxidized by NAD+ to form acetate, NADH, and H+
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
The acetaldehyde generated by the liver alcohol dehydrogenases is further oxidized in the liver to form acetate. Two acetaldehyde
dehydrogenase isozymes, one mitochondrial and one cytosolic, catalyze this reaction. An allele of the mitochondrial enzyme with reduced
enzymatic activity is common in human populations. Individuals who have the reduced-activity allele typically show elevated blood levels of
acetaldehyde and facial flushing following ethanol ingestion.
References
HW Goedde, DP Agarwal, S Harada, D Meier-Tackmann, D Ruofu, U Bienzle, A Kroeger, L Hussein, "Population genetic studies on aldehyde
dehydrogenase isozyme deficiency and alcohol sensitivity.", Am J Hum Genet, 35, 1983, 769-72.
J Hempel, R Kaiser, H JÃƒÂ¶rnvall, "Mitochondrial aldehyde dehydrogenase from human liver. Primary structure, differences in relation to the
cytosolic enzyme, and functional correlations.", Eur J Biochem, 153, 1985, 13-28.
K Inoue, H Nishimukai, K Yamasawa, "Purification and partial characterization of aldehyde dehydrogenase from human erythrocytes.", Biochim
Biophys Acta, 569, 1979, 117-23.
2.1.5.2.1.1 Acetaldehyde + NAD+ <=> Acetate + NADH + H+ [cytosolic]
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
At the beginning of this reaction, 1 molecule of 'NAD+', and 1 molecule of 'Acetaldehyde' are present. At the end of this reaction, 1 molecule of
'H+', 1 molecule of 'Acetate', and 1 molecule of 'NADH' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'aldehyde dehydrogenase (NAD) activity' of 'aldehyde dehydrogenase 1A1
tetramer'.
References
K Inoue, H Nishimukai, K Yamasawa, "Purification and partial characterization of aldehyde dehydrogenase from human erythrocytes.", Biochim
Biophys Acta, 569, 1979, 117-23.
Reaction
2.1.5.2.1.2 Acetaldehyde + NAD+ <=> Acetate + NADH + H+ [mitochondrial]
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
At the beginning of this reaction, 1 molecule of 'NAD+', and 1 molecule of 'Acetaldehyde' are present. At the end of this reaction, 1 molecule of
'H+', 1 molecule of 'Acetate', and 1 molecule of 'NADH' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'aldehyde dehydrogenase (NAD) activity' of 'aldehyde
dehydrogenase, mitochondrial, tetramer'.
References
NJ Greenfield, R Pietruszko, "Two aldehyde dehydrogenases from human liver. Isolation via affinity chromatography and characterization of the
isozymes.", Biochim Biophys Acta, 483, 1977, 35-45.
J Hempel, R Kaiser, H JÃƒÂ¶rnvall, "Mitochondrial aldehyde dehydrogenase from human liver. Primary structure, differences in relation to the
cytosolic enzyme, and functional correlations.", Eur J Biochem, 153, 1985, 13-28.
Reaction
2.1.5.3 Acetate, ATP, and coenzyme A react to form acetyl CoA, AMP, and pyrophosphate
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
Formation of acetyl CoA.
2.1.5.3.1 ATP + Acetate + CoA <=> AMP + Pyrophosphate + Acetyl-CoA
Authors
Jassal, B, 2008-05-19.
Description
Synthesis of acetyl-CoA.
References
A Luong, VC Hannah, JL Goldstein, "Molecular characterization of human acetyl-CoA synthetase, an enzyme regulated by sterol regulatory
element-binding proteins.", J Biol Chem, 275, 2000, 26458-66.
Reaction
2.2 Phase II conjugation
Authors
Jassal, B, 2004-11-29.
Editors
Jassal, B, 2008-05-19.
Description
Phase II of biotransformation is concerned with conjugation, that is using groups from cofactors to react with functional groups present or
introduced from phase I on the compound. The enzymes involved are a set of transferases which perform the transfer of the cofactor group to
the substrate. The resultant conjugation results in greatly increasing the excretory potential of compounds. Although most conjugations result in
pharmacological inactivation or detoxification, some can result in bioactivation. Most of the phase II enzymes are located in the cytosol except
UDP-glucuronosyltransferases (UGT), which are microsomal. Phase II reactions are typically much faster than phase I reactions therefore the
rate-limiting step for biotransformation of a compound is usually the phase I reaction.
Phase II metabolism can deal with all the products of phase I metabolism, be they reactive (Type I substrate) or unreactive/poorly active (Type II
substrate) compounds. With the exception of glutathione, the conjugating species needs to be made chemically reactive after synthesis. The
availability of the cofactor in the synthesis may be a rate-limiting factor in some phase II pathways as it may prevent the formation of enough
conjugating species to deal with the substrate or it's metabolite. As many substrates and/or their metabolites are chemically reactive, their
continued presence may lead to toxicity.
References
DG McCarver, RN Hines, "The ontogeny of human drug-metabolizing enzymes: phase II conjugation enzymes and regulatory mechanisms", J
Pharmacol Exp Ther, 300, 2002, 361-6.
2.2.1 Glucuronidation
Authors
Jassal, B, 2004-11-29.
Editors
Jassal, B, 2008-05-19.
Description
Glucuronidation conjugation utilizes UDP-glucuronosyltransferases (UGTs; EC 2.4.1.17) to catalyze a wide range of diverse endogenous and
xenobiotic compounds. Glucuronidation is the major pathway in phase II metabolism and accounts for approximately 35% of drug conjugation.
UGTs are microsomal membrane-bound and catalyze the transfer of a glucuronate group of uridine diphosphoglucuronate (UDPGA, a
co-substrate) to the functional group of specific substrates. UDPGA is synthesized from glucose-1-phosphate (G1P). G1P is required for
glycolysis and is present in high concentrations in the cell, making it is unlikely to be a limiting factor in UDPGA synthesis. UDP is added to G1P
to form UDP-glucose which is then dehydrogenated to form UDPGA. The basic reaction is
UDP-Glucuronate + acceptor -> UDP + acceptor-beta-D-glucuronide
The effect of this conjugation is to confer polarity to the substrate which can then be easily excreted in urine or bile. Functional groups acted on
include hydroxyl, carboxylate, amino and sulfate groups. There are 2 families of UGTs, UGT1 and UGT2 which are further sub-divided into 3
subfamilies, UGT1A, UGT2A and UGT2B. There are more than 26 different isozymes in humans, of which 18 are functional proteins. They are
composed of 527-530 residues and have a molecular weight of 50-57KDa.
The UGT1 family comprises of 9 proteins (UGT1A1, 1A3-1A10) but only 5 have been isolated in humans. Example substrates which are
glucuronidated are acetaminophen by UGT1A6 and bilirubin by UGT1A1. Members of the UGT2 subfamily are each encoded by their own
genes, in contrast to UGT1As which are encoded at the UGT1 locus. Example substrates are morphine conjugation by UGT2B7 and androgenic
steroid conjugation by UGT2B17.
Xenobiotics conjugated with glucuronic acid can be substrates for beta-glucuronidase, an enzyme common in gut microflora. This enzyme can
release the parent or phase I metabolite which can be reabsorbed. It can then either re-exert it's original effects or be conjugated by glucuronic
acid again. This cycle is called enterohepatic circulation and can delay the elimination of the xenobiotic.
References
RH Tukey, CP Strassburg, "Human UDP-glucuronosyltransferases: metabolism, expression, and disease", Annu Rev Pharmacol Toxicol, 40,
2000, 581-616.
PG Wells, PI Mackenzie, JR Chowdhury, C Guillemette, PA Gregory, Y Ishii, AJ Hansen, FK Kessler, PM Kim, NR Chowdhury, JK Ritter,
"Glucuronidation and the UDP-glucuronosyltransferases in health and disease", Drug Metab Dispos, 32, 2004, 281-90.
2.2.1.1 Formation of the active cofactor, UDP-glucuronate
Authors
Jassal, B, 2004-11-30.
Editors
Jassal, B, 2008-05-19.
Description
Glucose 1-phosphate and UTP are the precursors to UDP-glucuronate formation. After oxidation of the resultant complex, UDP-glucuronate is
transported to the ER lumen.
References
T Iyanagi, "Molecular mechanism of phase I and phase II drug-metabolizing enzymes: implications for detoxification", Int Rev Cytol, 260, 2007,
35-112.
2.2.1.1.1 UTP + D-glucose 1-phosphate <=> pyrophosphate + UDP-glucose [liver]
Reviewers
Description
UDPglucose pyrophosphorylase (UDPGPase) catalyses the transfer of a glucose moiety from glucose-1-phosphate to UTP, forming
UDP-Glucose and PPi. Liver requires UDP-glucose for the formation of UDPglucuronate, which acts as a conjugating source for the formation of
soluble glucuronides of xenobiotic and endobiotic metabolites.
References
HL Peng, HY Chang, "Cloning of a human liver UDP-glucose pyrophosphorylase cDNA by complementation of the bacterial galU mutation.",
FEBS Lett, 329, 1993, 153-8.
Reaction
2.2.1.1.2 UDP-glucose is oxidised to UDP-glucuronate
Reviewers
Description
UDP-glucose dehydrogenase (UGDH) catalyzes the conversion of UDP-glucose to UDP-glucuronic acid. The cytosolic enzyme is active as a
hexamer and performs two successive oxidations to convert the 6'-hydroxyl of UDP-glucose to a carboxylate with concurrent reduction of 2 mol
of NAD+ to NADH.
References
BJ Sommer, JJ Barycki, MA Simpson, "Characterization of human UDP-glucose dehydrogenase. CYS-276 is required for the second of two
successive oxidations", J Biol Chem, 279, 2004, 23590-6.
Reaction
2.2.1.1.3 UDP-glucuronate transport from the cytosol to ER lumen
Reviewers
Description
The UDP-glucuronic acid transporter transports both UDP-glucuronic acid (UDP-GlcA) and UDP-N-acetylgalactosamine (UDP-GalNAc) from the
cytoplasm to into the endoplasmic reticulum lumen.
References
M Muraoka, M Kawakita, N Ishida, "Molecular characterization of human UDP-glucuronic acid/UDP-N-acetylgalactosamine transporter, a novel
nucleotide sugar transporter with dual substrate specificity", FEBS Lett, 495, 2001, 87-93.
Reaction
2.2.1.2 Formation of O-glucuronides
Reviewers
Description
Typical O-centred substrates were chosen as examples for these isozymes.
References
SR Babu, VM Lakshmi, GP Huang, TV Zenser, BB Davis, "Glucuronide conjugates of 4-aminobiphenyl and its N-hydroxy metabolites. pH
stability and synthesis by human and dog liver.", Biochem Pharmacol, 51, 1996, 1679-85.
Reaction
2.2.1.3 Formation of N-glucuronides
Reviewers
Description
Typical N-centred substrates were chosen as examples for these isozymes.

References
T Kaku, K Ogura, T Nishiyama, T Ohnuma, K Muro, A Hiratsuka, "Quaternary ammonium-linked glucuronidation of tamoxifen by human liver
microsomes and UDP-glucuronosyltransferase 1A4", Biochem Pharmacol, 67, 2004, 2093-102.
M Nakajima, E Tanaka, JT Kwon, T Yokoi, "Characterization of nicotine and cotinine N-glucuronidations in human liver microsomes", Drug
Metab Dispos, 30, 2002, 1484-90.
GE Kuehl, SE Murphy, "N-glucuronidation of nicotine and cotinine by human liver microsomes and heterologously expressed
UDP-glucuronosyltransferases", Drug Metab Dispos, 31, 2003, 1361-8.
Reaction
2.2.2 Cytosolic sulfonation of small molecules
Authors
Jassal, B, 2004-11-29.
Editors
Jassal, B, 2008-05-19.
Description
Two groups of sulfotransferease (SULT) enzymes catalyze the transfer of a sulfate group from 3-phosphoadenosine 5-phosphosulfate (PAPS) to
a hydroxyl group on an acceptor molecule, yielding a sulfonated acceptor and 3-phosphoadenosine 5-phosphate (PAP). One is localized to the
Golgi apparatus and mediates the sulfonation of proteoglycans. The second, annotated here, is cytosolic and mediates the sulfonation of a
diverse array of small molecules, increasing their solubilities in water and modifying their physiological functions. There are probably thirteen or
more human cytosolic SULT enzymes; eleven of these have been purified and characterized enzymatically, and are annotated here (Blanchard
et al. 2004; Gamage et al. 2005). These enzymes appear to be active as dimers. Their substrate specificities are typically broad, and not related
in an obvious way to their structures; indeed, apparently orthologous human and rodent SULT enzymes can have different substrate specificities
(Glatt 2000), and none has been exhaustively characterized. The substrates listed in the table and annotated here are a sample of the known
ones, chosen to indicate the range of activity of these enzymes and to capture some of their known physiologically important targets. Absence of
a small molecule - enzyme pair from the table, however, may only mean that it has not yet been studied.
References
Y Yamazoe, K Nagata, K Yoshinari, K Fujita, T Shiraga, K Iwasaki, "Sulfotransferase catalyzing sulfation of heterocyclic amines", Cancer Lett,
143, 1999, 103-7.
N Gamage, A Barnett, N Hempel, RG Duggleby, KF Windmill, JL Martin, ME McManus, "Human Sulfotransferases and Their Role in Chemical
Metabolism", Toxicol Sci, 90, 2006, 5-22.
RL Blanchard, RR Freimuth, J Buck, RM Weinshilboum, MW Coughtrie, "A proposed nomenclature system for the cytosolic sulfotransferase
(SULT) superfamily", Pharmacogenetics, 14, 2004, 199-211.
RM Weinshilboum, DM Otterness, IA Aksoy, TC Wood, C Her, RB Raftogianis, "Sulfation and sulfotransferases 1: Sulfotransferase molecular
biology: cDNAs and genes", FASEB J, 11, 1997, 3-14.
H Glatt, "Sulfotransferases in the bioactivation of xenobiotics", Chem Biol Interact, 129, 2000, 141-70.
CA Strott, "Sulfonation and molecular action", Endocr Rev, 23, 2002, 703-32.
2.2.2.1 Acetaminophen can form an O- sulfate conjugate
Reviewers
Description
At the beginning of this reaction, 1 molecule of 'Acetaminophen (TN TYLENOL)', and 1 molecule of 'PAPS' are present. At the end of this
reaction, 1 molecule of 'PAP', and 1 molecule of 'Acetaminophen O-sulfate conjugate' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'aryl sulfotransferase activity' of 'SULT1A1 homodimer'.
References
143, 1999, 103-7.
Reaction
2.2.2.2 N-hydroxy-4-aminobiphenyl can form a sulfate conjugate
Reviewers
Description
At the beginning of this reaction, 1 molecule of 'PAPS', and 1 molecule of 'N-Hydroxy-4-aminobiphenyl' are present. At the end of this reaction, 1
molecule of 'N-hydroxy-4-aminobiphenyl O-sulfate', and 1 molecule of 'PAP' are present.
References
143, 1999, 103-7.
Reaction
2.2.2.3 Phenol can form a sulfate conjugate
Reviewers
Description
At the beginning of this reaction, 1 molecule of 'PAPS', and 1 molecule of 'Phenol' are present. At the end of this reaction, 1 molecule of 'Phenyl
sulfate', and 1 molecule of 'PAP' are present.
References
Reaction
2.2.2.4 Dopamine can form an O-sulfate conjugate
Authors
Jassal, B, 2005-03-03.
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
The catecholamine neurotransmitter dopamine (DA) is predominantly (>95%) conjugated with sulfate in human blood circulation. Human
SULT1A3 is the major sulfotransferase that sulfonates DA.
References
C Reiter, G Mwaluko, J Dunnette, J Van Loon, RM Weinshilboum, "Thermolabile and thermostable human platelet phenol sulfotransferase.
Substrate specificity and physical separation.", Naunyn Schmiedebergs Arch Pharmacol, 324, 1983, 140-7.
GA Johnson, CA Baker, RT Smith, "Radioenzymatic assay of sulfate conjugates of catecholamines and DOPA in plasma", Life Sci, 26, 1980,
1591-8.
Reaction
2.2.2.5 3,3'-diiodothyronine + PAPS => 3,3'-diiodothyronine 4-sulfate + PAP
Editors
Reviewers
Description
Six SULT enzymes, SULT1A1 (Li et al. 2001), 1A3 (Kester, Kaptein et al. 1999), 1B1 (Wang et al. 1998), 1C1 (Li et al. 2000), 1E1 (Li and
Anderson 1999; Kester, van Dijk et al. 1999), and 2A1 (Li and Anderson 1999), can catalyze the sulfonation of 3,3'-diiodothyronine (T2) in vitro
or in tissue culture model systems. These six enzymes also catalyze the sulfonation of 3,5,3'-triiodothyronine (T3).
References
X Li, RJ Anderson, "Sulfation of iodothyronines by recombinant human liver steroid sulfotransferases", Biochem Biophys Res Commun, 263,
1999, 632-9.
J Wang, JL Falany, CN Falany, "Expression and characterization of a novel thyroid hormone-sulfating form of cytosolic sulfotransferase from
human liver", Mol Pharmacol, 53, 1998, 274-82.
X Li, DL Clemens, RJ Anderson, "Sulfation of iodothyronines by human sulfotransferase 1C1 (SULT1C1)*", Biochem Pharmacol, 60, 2000,
1713-6.
MH Kester, E Kaptein, TJ Roest, CH van Dijk, D Tibboel, W Meinl, H Glatt, MW Coughtrie, TJ Visser, "Characterization of human iodothyronine
sulfotransferases", J Clin Endocrinol Metab, 84, 1999, 1357-64.
X Li, DL Clemens, JR Cole, RJ Anderson, "Characterization of human liver thermostable phenol sulfotransferase (SULT1A1) allozymes with
3,3',5-triiodothyronine as the substrate", J Endocrinol, 171, 2001, 525-32.
Reaction
2.2.2.6 3,5,3'-triiodothyronine + PAPS => 3,5,3'-triiodothyronine 4-sulfate + PAP
Editors
Reviewers
Description
3,5,3'-Triiodothyronine (T3) 4-sulfate is a major metabolite of T3 in humans (LoPresti and Nicoloff 1994), and seven SULT enzymes, SULT1A1
(Li et al. 2001), 1A3 (Kester, Kaptein et al. 1999), 1B1 (Wang et al. 1998), 1C1 (Li et al. 2000), 1E1 (Li and Anderson 1999; Kester, van Dijk et
al. 1999), 2A1 (Li and Anderson 1999), and 4A1 (Sakakibara et al. 2002) can catalyze this reaction in vitro or in tissue culture model systems. All
of these enzymes except SULT4A1 also catalyze the sulfonation of 3,3'-diiodothyronine (T2).
References
X Li, RJ Anderson, "Sulfation of iodothyronines by recombinant human liver steroid sulfotransferases", Biochem Biophys Res Commun, 263,
1999, 632-9.
J Wang, JL Falany, CN Falany, "Expression and characterization of a novel thyroid hormone-sulfating form of cytosolic sulfotransferase from
human liver", Mol Pharmacol, 53, 1998, 274-82.
JS LoPresti, JT Nicoloff, "3,5,3'-Triiodothyronine (T3) sulfate: a major metabolite in T3 metabolism in man", J Clin Endocrinol Metab, 78, 1994,
688-92.
Y Sakakibara, M Suiko, TG Pai, T Nakayama, Y Takami, J Katafuchi, MC Liu, "Highly conserved mouse and human brain sulfotransferases:
molecular cloning, expression, and functional characterization", Gene, 285, 2002, 39-47.
X Li, DL Clemens, RJ Anderson, "Sulfation of iodothyronines by human sulfotransferase 1C1 (SULT1C1)*", Biochem Pharmacol, 60, 2000,
1713-6.
MH Kester, E Kaptein, TJ Roest, CH van Dijk, D Tibboel, W Meinl, H Glatt, MW Coughtrie, TJ Visser, "Characterization of human iodothyronine
sulfotransferases", J Clin Endocrinol Metab, 84, 1999, 1357-64.
X Li, DL Clemens, JR Cole, RJ Anderson, "Characterization of human liver thermostable phenol sulfotransferase (SULT1A1) allozymes with
3,3',5-triiodothyronine as the substrate", J Endocrinol, 171, 2001, 525-32.
Reaction
2.2.2.7 p-nitrophenol + PAPS => p-nitrophenol sulfate + PAP
Editors
Reviewers
Description
The sulfonation of the xenobiotic p-nitrophenol (4-nitrophenol) can be catalyzed by five well-characterized SULT enzymes, 1A1 (Brix et al. 1998),
1A2 (Brix et al. 1998; Zhu et al. 1996), 1C2 (Sakakibara et al. 1998), and 4A1 (Sakakibara et al. 2002).
References
Y Sakakibara, K Yanagisawa, J Katafuchi, DP Ringer, Y Takami, T Nakayama, M Suiko, MC Liu, "Molecular cloning, expression, and
characterization of novel human SULT1C sulfotransferases that catalyze the sulfonation of N-hydroxy-2-acetylaminofluorene", J Biol Chem, 273,
1998, 33929-35.
LA Brix, R Nicoll, X Zhu, ME McManus, "Structural and functional characterisation of human sulfotransferases", Chem Biol Interact, 109, 1998,
123-7.
Y Sakakibara, M Suiko, TG Pai, T Nakayama, Y Takami, J Katafuchi, MC Liu, "Highly conserved mouse and human brain sulfotransferases:
molecular cloning, expression, and functional characterization", Gene, 285, 2002, 39-47.
X Zhu, ME Veronese, P Iocco, ME McManus, "cDNA cloning and expression of a new form of human aryl sulfotransferase", Int J Biochem Cell
Biol, 28, 1996, 565-71.
Reaction
2.2.2.8 N-hydroxy-2-acetylaminofluorene + PAPS => 2-acetylaminofluorene-N-sulfate + PAP
Editors
Reviewers
Description
Sulfonation of the xenobiotic N-hydroxy-2-acetylaminofluorene converts it to a potent carcinogen. SULT1A2 (Glatt 2000) and SULT1C1 and 1C2
(Sakakibara et al. 1998) catalyze this reaction.
References
Y Sakakibara, K Yanagisawa, J Katafuchi, DP Ringer, Y Takami, T Nakayama, M Suiko, MC Liu, "Molecular cloning, expression, and
characterization of novel human SULT1C sulfotransferases that catalyze the sulfonation of N-hydroxy-2-acetylaminofluorene", J Biol Chem, 273,
1998, 33929-35.
H Glatt, "Sulfotransferases in the bioactivation of xenobiotics", Chem Biol Interact, 129, 2000, 141-70.
Reaction
2.2.2.9 cholesterol + PAPS => cholesterol sulfate + PAP
Editors
Reviewers
Description
The 3-hydroxyl groups of a number of sterols can undergo sulfonation. Cholesterol sulfate is particularly abundant in the body, and may have
both regulatory and biosynthetic functions (Strott and Higashi 2003). Its synthesis is catalyzed by the b isoform of SULT2B1 (Fuda et al. 2002;
Javitt et al. 2001).
References
CA Strott, Y Higashi, "Cholesterol sulfate in human physiology: what's it all about?", J Lipid Res, 44, 2003, 1268-78.
NB Javitt, YC Lee, C Shimizu, H Fuda, CA Strott, "Cholesterol and hydroxycholesterol sulfotransferases: identification, distinction from
dehydroepiandrosterone sulfotransferase, and differential tissue expression", Endocrinology, 142, 2001, 2978-84.
H Fuda, YC Lee, C Shimizu, NB Javitt, CA Strott, "Mutational analysis of human hydroxysteroid sulfotransferase SULT2B1 isoforms reveals that
exon 1B of the SULT2B1 gene produces cholesterol sulfotransferase, whereas exon 1A yields pregnenolone sulfotransferase", J Biol Chem,
277, 2002, 36161-6.
Reaction
2.2.2.10 27-hydroxycholesterol + PAPS => 27-hydroxycholesterol 3-sulfate + PAP
Editors
Reviewers
Description
The sulfonation of 27-hydroxycholesterol is catalyzed by both the a and b isoforms of SULT2B1 (Javitt et al. 2001).
References
NB Javitt, YC Lee, C Shimizu, H Fuda, CA Strott, "Cholesterol and hydroxycholesterol sulfotransferases: identification, distinction from
dehydroepiandrosterone sulfotransferase, and differential tissue expression", Endocrinology, 142, 2001, 2978-84.
Reaction
2.2.2.11 pregnenolone + PAPS => pregnenolone sulfate + PAP
Editors
Reviewers
Description
The sulfonation of pregnenolone is catalyzed by both the a and b isoforms of SULT2B1, although the a isoform is more active in assays in vitro
(Fuda et al. 2002; Meloche and Falany 2001).
References
CA Meloche, CN Falany, "Expression and characterization of the human 3 beta-hydroxysteroid sulfotransferases (SULT2B1a and SULT2B1b)",
J Steroid Biochem Mol Biol, 77, 2001, 261-9.
H Fuda, YC Lee, C Shimizu, NB Javitt, CA Strott, "Mutational analysis of human hydroxysteroid sulfotransferase SULT2B1 isoforms reveals that
exon 1B of the SULT2B1 gene produces cholesterol sulfotransferase, whereas exon 1A yields pregnenolone sulfotransferase", J Biol Chem,
277, 2002, 36161-6.
Reaction
2.2.2.12 estrone + PAPS => estrone 3-sulfate + PAP
Editors
Reviewers
Description
Sulfonation of estrone is catalyzed by SULT1E1 (Aksoy et al. 1994; Falany et al. 1995), and also by SULT2A1 (Comer et al. 1993), although the
efficiency of SULT2A1 catalysis is unknown.
References
CN Falany, V Krasnykh, JL Falany, "Bacterial expression and characterization of a cDNA for human liver estrogen sulfotransferase", J Steroid
Biochem Mol Biol, 52, 1995, 529-39.
IA Aksoy, RM Weinshilboum, TC Wood, "Human liver estrogen sulfotransferase: identification by cDNA cloning and expression", Biochem
KA Comer, JL Falany, CN Falany, "Cloning and expression of human liver dehydroepiandrosterone sulphotransferase", Biochem J, 289, 1993,
233-40.
Reaction
2.2.2.13 dehydroepiandrosterone (DHEA) + PAPS => DHEA sulfate + PAP
Editors
Reviewers
Description
Sulfonation of dehydroepiandrosterone (DHEA) is catalyzed by SULT1E1 (Aksoy et al. 1994), SULT2A1 (Radominska et al. 1990) and the a and
b isoforms of SULT2B1 (Meloche and Falany 2001).
References
IA Aksoy, RM Weinshilboum, TC Wood, "Human liver estrogen sulfotransferase: identification by cDNA cloning and expression", Biochem
CA Meloche, CN Falany, "Expression and characterization of the human 3 beta-hydroxysteroid sulfotransferases (SULT2B1a and SULT2B1b)",
J Steroid Biochem Mol Biol, 77, 2001, 261-9.
A Radominska, KA Comer, P Zimniak, JL Falany, M Iscan, CN Falany, "Human liver steroid sulphotransferase sulphates bile acids", Biochem J,
272, 1990, 597-604.
Reaction
2.2.2.14 beta-estradiol + PAPS => beta-estradiol 3-sulfate + PAP
Editors
Reviewers
Description
Sulfonation of beta-estradiol is catalyzed by SULT1E1 (Falany et al. 1995).
References
CN Falany, V Krasnykh, JL Falany, "Bacterial expression and characterization of a cDNA for human liver estrogen sulfotransferase", J Steroid
Biochem Mol Biol, 52, 1995, 529-39.
Reaction
2.2.2.15 lithocholate + PAPS => lithocholate sulfate + PAP
Editors
Reviewers
Description
Sulfonation of lithocholate is catalyzed by SULT2A1 (Radominska et al. 1990).
References
272, 1990, 597-604.
Reaction
2.2.2.16 taurolithocholate + PAPS => taurolithocholate sulfate + PAP
Editors
Reviewers
Description
Sulfonation of taurolithocholate is catalyzed by SULT2A1 (Radominska et al. 1990).
References
272, 1990, 597-604.
Reaction
2.2.3 Acetylation
Authors
Jassal, B, 2004-11-30.
Editors
Jassal, B, 2008-05-19.
Description
N-acetyltransferases (NATs; EC 2.3.1.5) utilize acetyl Co-A in acetylation conjugation reactions. This is the preferred route of conjugating
aromatic amines (R-NH2, converted to aromatic amides R-NH-COCH3) and hydrazines (R-NH-NH2, converted to R-NH-NH-COCH3). Aliphatic
amines are not substrates for NAT. The basic reaction is
Acetyl-CoA + an arylamine = CoA + an N- acetylarylamine
NATs are cytosolic and in humans, 2 isoforms are expressed, NAT1 and NAT2. A third isoform, NATP, is a pseudogene and is not expressed.
The NAT2 gene contains mutations that decrease NAT2 activity. This mutations was first seen as slow acetylation compared to the normal, fast
acetylation of the antituberculosis drug isoniazid. Incidence of the slow acetylator phenotype is high in Middle Eastern populations (70%),
average (50%) in Europeans, Americans and Australians and low in Asians (<25% in Chinese, Japanese and Koreans). N-acetylation and
methylation pathways differ from other conjugation pathways in that they mask an amine with a nonionizable group so that the conjugates are
less water soluble than the parent compound. However, certain N-acetlylations facilitate urinary excretion.
N-acetylation occurs in two sequential steps via a ping-pong Bi-Bi mechanism. In the first step, the acetyl group from acetyl-CoA is transferred to
a cysteine residue in NAT, with consequent release of coenzyme-A. In the second step, the acetyl group is released from the acetylated NAT to
the substrate, subsequently regenerating the enzyme.
References
JM Dupret, DM Grant, "Site-directed mutagenesis of recombinant human arylamine N-acetyltransferase expressed in Escherichia coli. Evidence
for direct involvement of Cys68 in the catalytic mechanism of polymorphic human NAT2.", J Biol Chem, 267, 1992, 7381-5.
M Blum, DM Grant, W McBride, M Heim, UA Meyer, "Human arylamine N-acetyltransferase genes: isolation, chromosomal localization, and
functional expression", DNA Cell Biol, 9, 1990, 193-203.
P Meisel, "Arylamine N-acetyltransferases and drug response", Pharmacogenomics, 3, 2002, 349-66.

2.2.3.1 The acetyl group from acetyl-CoA is transferred to the NAT1
Authors
Jassal, B, 2005-02-09.
Reviewers
Description
a conserved cysteine residue (position 68) in the active site of NAT, with consequent release of coenzyme-A. In the second step, the acetyl
group is transferred to the acceptor substrate and the enzyme returns to its initial state.
References
Reaction
2.2.3.2 The acetyl group from acetyl-CoA is transferred to the NAT2
Authors
Jassal, B, 2005-02-09.
Reviewers
Description
a conserved cysteine residue (position 68) in the active site of NAT, with consequent release of coenzyme-A. In the second step, the acetyl
group is transferred to the acceptor substrate and the enzyme returns to its initial state.
References
Reaction
2.2.3.3 NAT1 acetylation
Reviewers
Description
Typical NAT 1 substrates were chosen as examples. They are sulfanilamide, 4-aminosalicylate, 4-aminobenzoate and N-hydroxy
4-aminobiphenyl.
Reaction
2.2.3.4 NAT2 acetylation

Reviewers
Description
Typical NAT 2 substrates were chosen as examples. They are paraxanthine, isoniazid and N-hydroxy 4-aminobiphenyl.
Reaction
2.2.4 Methylation
Authors
Jassal, B, 2005-02-03.
Editors
Jassal, B, 2008-05-19.
Description
Methylation is a common but minor pathway of Phase II conjugation compared to glucuronidation or sulfonation. The cofactor used in
methylation conjugation is S-adenosylmethionine (SAM). SAM is the second most widely used enzyme substrate after ATP and is involved in a
wide range of important biological processes. SAM is sythesized from methionine's reaction with ATP, catalyzed by methionine
adenosyltransferase (MAT). There are two genes, MAT1A and MAT2A, which encode for two homologous MAT catalytic subunits, 1 and 2.
During conjugation with nucleophilic substrates, the methyl group attached to the sulfonium ion of SAM is transferred to the substrate forming the
conjugate. SAM, having lost the methyl moiety, is converted to S-adenosylhomocysteine (SAH). SAH can be hydrolyzed to form adenosine and
homocysteine. Homocysteine can either be converted to glutathione or methylated to form methionine, thus forming the starting point for SAM
synthesis and completing the cycle.
Fuctional groups attacked are phenols, catechols, aliphatic and aromatic amines and sulfhydryl-containing groups. The enzymes that catalyze
the transfer of the methyl group to these functional groups are the methyltransferases (MT). MTs are small, cytosolic, monomeric enzymes that
utilize SAM as a methyl donor. There are many MTs but the best studied ones are named on the basis of their prototypical substrates: COMT
(catechol O-methyltransferase), TPMT (thiopurine methyltransferase), TMT (thiol methyltransferase), HNMT (histamine N-methyltransferase)
and NNMT (nicotinamide N-methyltransferase). An example of each enzyme mentioned is given. In each case, a typical substrate for the
enzyme is shown.
References
SC Lu, ZZ Huang, H Yang, JM Mato, MA Avila, H Tsukamoto, "Changes in methionine adenosyltransferase and S-adenosylmethionine
homeostasis in alcoholic rat liver", Am J Physiol Gastrointest Liver Physiol, 279, 2000, G178-85.
M Fontecave, M Atta, E Mulliez, "S-adenosylmethionine: nothing goes to waste", Trends Biochem Sci, 29, 2004, 243-9.
RM Weinshilboum, DM Otterness, CL Szumlanski, "Methylation pharmacogenetics: catechol O-methyltransferase, thiopurine methyltransferase,

and histamine N-methyltransferase", Annu Rev Pharmacol Toxicol, 39, 1999, 19-52.
2.2.4.1 SAM is sythesized from methionine's reaction with ATP
Reviewers
Description
S-adenosylmethionine (SAM) is an essential metabolite in all cells. SAM is a precursor in the synthesis of polyamines. Methionine
adenosyltransferase (MAT; EC 2.5.1.6) catalyzes the only known SAM biosynthetic reaction from methionine and ATP. In mammalian tissues,
three different forms of MAT (MAT I, MAT III and MAT II) have been identified that are the product of two different genes (MAT1A and MAT2A).
A third gene MAT2B has been identified and is known to be a part of MAT II heterotetramer complex.
References
FJ Corrales, I PÃ©rez-Mato, MM SÃ¡nchez Del Pino, F Ruiz, C Castro, ER GarcÃ-a-Trevijano, U Latasa, ML MartÃ-nez-Chantar, A
MartÃ-nez-Cruz, MA Avila, JM Mato, "Regulation of mammalian liver methionine adenosyltransferase", J Nutr, 132, 2002, 2377S-2381S.
JM Mato, L Alvarez, P Ortiz, MA Pajares, "S-adenosylmethionine synthesis: molecular mechanisms and clinical implications", Pharmacol Ther,
73, 1997, 265-80.
Reaction
2.2.4.2 6-mercaptopurine can be S-methylated
Reviewers
Description
Methylation is the major biotransformation route of thiopurine drugs such as 6-mercaptopurine (6MP), used in the treatment of inflammatory
diseases such as rheumatoid arthritis and childhood acute lymphoblastic leukemia. 6MP and it's thioguanine nucleotide metabolites are
principally inactivated by thiopurine methyltransferase (TPMT)-catalyzed S-methylation.
TPMT exhibits an autosomal co-dominant trait: About one in 300 Caucasian, African, African-American, and Asian populations are TPMT
deficient. Approximately 6-10% of these populations inherit intermediate TPMT activity and are heterozygous at the TPMT locus. The rest are
homozygous for the wild-type allele and have high levels of TPMT activity. Low or undetectable levels of TPMT results in patients having this
trait being at risk to thiopurine drug-induced toxicity such as myelosuppression.
6-MP is a typical substrate for TPMT as shown in this example.
References
CN REMY, "Metabolism of thiopyrimidines and thiopurines. S-Methylation with S-adenosylmethionine transmethylase and catabolism in
mammalian tissues.", J Biol Chem, 238, 1963, 1078-84.
AF Al Hadithy, NK de Boer, LJ Derijks, JC Escher, CJ Mulder, JR Brouwers, "Thiopurines in inflammatory bowel disease: pharmacogenetics,
therapeutic drug monitoring and clinical recommendations", Dig Liver Dis, 37, 2005, 282-97.
S Coulthard, L Hogarth, "The thiopurines: an update", Invest New Drugs, 23, 2005, 523-32.
RM Weinshilboum, DM Otterness, CL Szumlanski, "Methylation pharmacogenetics: catechol O-methyltransferase, thiopurine methyltransferase,

and histamine N-methyltransferase", Annu Rev Pharmacol Toxicol, 39, 1999, 19-52.
Reaction
2.2.4.3 2-mercaptoethanol can be S-methylated
Editors
Jassal, B, 2008-05-19.
Reviewers
Description
2-mercaptoethanol is a typical substrate for Thiol S-methyltransferase.
Reaction
2.2.4.4 Pyridine can be N-methylated
Reviewers
Description
Pyridine is a typical substrate for Nicotinamide N-methyltransferase
Reaction
2.2.4.5 3,4-dihydroxybenzoate can be O-methylated
Reviewers
Description
Dihydroxybenzoate is a typical substrate for Catechol O-methyltransferase
References
D Leclerc, E Campeau, P Goyette, CE Adjalla, B Christensen, M Ross, P Eydoux, DS Rosenblatt, R Rozen, RA Gravel, "Human methionine
synthase: cDNA cloning and identification of mutations in patients of the cblG complementation group of folate/cobalamin disorders", Hum Mol
Genet, 5, 1996, 1867-74.
Reaction
2.2.4.6 S-adenoylhomocysteine is hydrolyzed
Reviewers
Description
At the beginning of this reaction, 1 molecule of 'S-adenosylhomocysteine', and 1 molecule of 'H2O' are present. At the end of this reaction, 1
molecule of 'adenosine', and 1 molecule of 'Homocysteine' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'adenosylhomocysteinase activity' of 'SAH hydrolase homotetramer'.
Reaction
2.2.4.7 Methionine is reformed
Reviewers
Description
Cytosolic methionine synthase catalyzes the reaction of 5-methyltetrahydrofolate polyglutamate and homocysteine to form L-methionine and
tetrahydrofolate polyglutamate (Leclerc et al. 1996).
References
D Leclerc, E Campeau, P Goyette, CE Adjalla, B Christensen, M Ross, P Eydoux, DS Rosenblatt, R Rozen, RA Gravel, "Human methionine
synthase: cDNA cloning and identification of mutations in patients of the cblG complementation group of folate/cobalamin disorders", Hum Mol
Genet, 5, 1996, 1867-74.
Reaction
2.2.5 Glutathione conjugation
Authors
Jassal, B, 2004-11-30.
Editors
Jassal, B, 2008-05-19.
Description
Glutathione S-Transferases (GSTs; EC 2.5.1.18) are another major set of phase II conjugation enzymes. They can be found in the cytosol as
well as being microsomal membrane-bound. Cytosolic GSTs are encoded by at least 5 gene families (alpha, mu, pi, theta and zeta GST)
whereas membrane-bound enzymes are encoded by single genes. Soluble GSTs are homo- or hetero-dimeric enzymes (approximately 25KDa
subunits) which can act on a wide range of endogenous and exogenous electrophiles. GSTs mediate conjugation using glutathione (GSH), a
tripeptide synthesized from its precursor amino acids gamma-glutamate, cysteine and glycine. A generalized reaction is
RX + GSH -> HX + GSR
Glutathione conjugates are excreted in bile and converted to cysteine and mercapturic acid conjugates in the intestine and kidneys. GSH is the
major, low molecular weight, non-protein thiol synthesized de novo in mammalian cells. As well as taking part in conjugation reactions, GSH also
has antioxidant ability and can metabolize endogenous and exogenous compounds. The nucleophilic GSH attacks the electrophilic substrate
forming a thioether bond between the cysteine residue of GSH and the electrophile. The result is generally a less reactive and more
water-soluble conjugate that can be easily excreted. In some cases, GSTs can activate compounds to reactive species such as certain
haloalkanes and haloalkenes. Substrates for GSTs include epoxides, alkenes and compounds with electrophilic carbon, sulfur or nitrogen
centres. There are two types of conjugation reaction with glutathione: displacement reactions where glutathione displaces an
electron-withdrawing group and addition reactions where glutathione is added to activated double bond structures or strained ring systems.
References
2.2.5.1 Glutathione synthesis
Authors
Jassal, B, 2004-11-30.
Editors
Jassal, B, 2008-05-19.
Description
The combination of glutamate, cysteine and ATP is required to form glutathione.
2.2.5.1.1 Glutamate and cysteine combine
Reviewers
Description
At the beginning of this reaction, 1 molecule of 'ATP', 1 molecule of 'L-cysteine', and 1 molecule of 'L-Glutamate' are present. At the end of this
reaction, 1 molecule of 'gamma-glutamyl-L-cysteine', 1 molecule of 'ADP', and 1 molecule of 'Orthophosphate' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'glutamate-cysteine ligase activity' of 'Glutamate-cysteine ligase'.
Reaction
2.2.5.1.2 gamma-glutamylcysteine combines with glycine to form glutathione
Reviewers
Description
At the beginning of this reaction, 1 molecule of 'gamma-glutamyl-L-cysteine', and 1 molecule of 'ATP' are present. At the end of this reaction, 1
molecule of 'Glutathione', 1 molecule of 'ADP', and 1 molecule of 'Orthophosphate' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'glutathione synthase activity' of 'Glutathione synthase homodimer'.
Reaction
2.2.5.2 Glutathione conjugation of luminal substrates
Reviewers
Description
Typical substrates are chosen as examples for which the majority of the GST isozymes act on.
Reaction
2.2.5.3 Glutathione conjugation of cytosolic substrates
Reviewers
Description
Typical substrates are chosen as examples for which the majority of the GST isozymes act on.
Reaction
2.2.6 Amino Acid conjugation
Authors
Jassal, B, 2005-03-10.
Editors
Jassal, B, 2008-05-19.
Description
Xenobiotics that contain either a carboxylic group or an aromatic hydroxylamine group are possible substrates for amino acid conjugation.
Xenobiotics with a carboxylic group conjugate with an amino group of amino acids such as glycine, taurine and glutamine. The hydroxylamine
group conjugates with the carboxylic group of amino acids such as proline and serine. The amino acid is first activated by an
aminoacyl-tRNA-synthetase which then reacts with the hydroxylamine group to form a reactive N-ester. N-esters can degrade to form
electrophilic nitrenium (R-N+-R') and carbonium (R-C+H2) ions. The pyrolysis product of tryptophan, an N-hydroxy intermediate, can potentially
form these reactive electrophilic ions.
References
2.2.6.1 Conjugation of carboxylic acids
Authors
Jassal, B, 2005-03-10.
Editors
Jassal, B, 2008-05-19.
Description
Xenobiotics and endogenous compounds containing carboxylate groups can be activated with coenzyme A to produce acyl-CoA thioesters and
then conjugated with the amino groups of glycine or glutamine to form amide-linked conjugates. Clinically important substrates include benzoic
acid, phenylacetic acid, and salicylic acid.
2.2.6.1.1 Conjugation of benzoate with glycine
Authors
Jassal, B, 2004-11-30.
Editors
Description
Benzoic acid, widely used as a food preservative, is converted to hippuric acid by activation and conjugation with glycine. This was one of the
first detoxification pathways discovered, and was formerly exploited clinically as an alternative means of nitrogen excretion in patients with urea
cycle defects (Brusilow and Horwich 2001).
References
SW Brusilow, AL Horwich, "Urea cycle enzymes", The Metabolic and Molecular Bases of Inherited Disease, 8th ed (Scriver CR, et al., editors),
2, 2001, 1909-1963.
2.2.6.1.1.1 benzoate + Coenzyme A + ATP => benzoyl-CoA + AMP + pyrophosphate
Authors
Jassal, B, 2005-03-10.
Reviewers
Description
Benzoate and ATP react with coenzyme A to form benzoyl CoA, AMP, and pyrophosphate (Vessey et al. 1999, 2003). Two human CoA ligases
have been characterized that catalyze this reaction efficiently in vitro: acyl-CoA synthetase medium-chain family member 1 (BUCS1) (Fujino et
al. 2001) and xenobiotic/medium-chain fatty acid:CoA ligase (Vessey et al. 2003). Their relative contributions to benzoate metabolism in vivo are
unknown.
References
DA Vessey, E Lau, M Kelley, RS Warren, "Isolation, sequencing, and expression of a cDNA for the HXM-A form of xenobiotic/medium-chain fatty
acid:CoA ligase from human liver mitochondria", J Biochem Mol Toxicol, 17, 2003, 1-6.
T Fujino, YA Takei, H Sone, RX Ioka, A Kamataki, K Magoori, S Takahashi, J Sakai, TT Yamamoto, "Molecular identification and
characterization of two medium-chain acyl-CoA synthetases, MACS1 and the Sa gene product", J Biol Chem, 276, 2001, 35961-6.
DA Vessey, M Kelley, RS Warren, "Characterization of the CoA ligases of human liver mitochondria catalyzing the activation of short- and
medium-chain fatty acids and xenobiotic carboxylic acids", Biochim Biophys Acta, 1428, 1999, 455-62.
Reaction
2.2.6.1.1.2 benzoyl-CoA + glycine => benzoyl glycine (hippuric acid) + Coenzyme A
Reviewers
Description
Benzoyl CoA and glycine react to form benzoyl glycine (hippuric acid) and Coenzyme A (Mawal and Qureshi 1994).
References
YR Mawal, IA Qureshi, "Purification to homogeneity of mitochondrial acyl coa:glycine n-acyltransferase from human liver", Biochem Biophys Res
Commun, 205, 1994, 1373-9.
LT Webster, UA Siddiqui, SV Lucas, JM Strong, JJ Mieyal, "Identification of separate acyl- CoA:glycine and acyl-CoA:L-glutamine
N-acyltransferase activities in mitochondrial fractions from liver of rhesus monkey and man", J Biol Chem, 251, 1976, 3352-8.
Reaction
2.2.6.1.2 Conjugation of phenylacetate with glutamine
Authors
Jassal, B, 2004-11-30.
Editors
Description
Phenylacetate metabolism is of clinical importance because its conjugation with glutamine to form phenylacetylglutamine, which can be excreted
in the urine, provides an alternative pathway for nitrogen excretion in patients with urea cycle defects (James et al. 1972; Batshaw et al. 2001;
Brusilow and Horwich 2001). This conjugation proceeds in two steps. First, phenylacetate and ATP react with coenzyme A to form phenylacetyl
CoA, AMP, and pyrophosphate (Vessey et al. 1999). Two human CoA ligases have been characterized that catalyze this reaction efficiently in
vitro: acyl-CoA synthetase medium-chain family member 1 (BUCS1) (Fujino et al. 2001) and xenobiotic/medium-chain fatty acid:CoA ligase
(Vessey et al. 2003). Their relative contributions to phenylacetate metabolism in vivo are unknown. Second, phenylacetyl CoA and glutamine
react to form phenyacetyl glutamine and Coenzyme A. The enzyme that catalyzes this reaction has been purified from human liver mitochondria
and shown to be a distinct polypeptide species from glycine-N-acyltransferase (Webster et al. 1976). This human glutamine-N-acyltransferase
activity has not been characterized by sequence analysis at the protein or DNA level, however, and thus cannot be associated with a known
human protein in the annotation of phenylacetate conjugation.
References
MO James, RL Smith, RT Williams, M Reidenberg, "The conjugation of phenylacetic acid in man, sub-human primates and some non-primate
species", Proc R Soc Lond B Biol Sci, 182, 1972, 25-35.
2, 2001, 1909-1963.
ML Batshaw, RB MacArthur, M Tuchman, "Alternative pathway therapy for urea cycle disorders: twenty years later", J Pediatr, 138, 2001,
S46-54; discussion S.
2.2.6.1.2.1 phenylacetate + Coenzyme A + ATP => phenylacetyl-CoA + AMP + pyrophosphate

Editors
Reviewers
Description
Phenylacetate and ATP react with coenzyme A to form phenylacetyl CoA, AMP, and pyrophosphate (Vessey et al. 1999). Two human CoA
ligases have been characterized that catalyze this reaction efficiently in vitro: acyl-CoA synthetase medium-chain family member 1 (BUCS1)
(Fujino et al. 2001) and xenobiotic/medium-chain fatty acid:CoA ligase (Vessey et al. 2003). Their relative contributions to phenylacetate
metabolism in vivo are unknown.
References
Reaction
2.2.6.1.2.2 phenylacetyl-CoA + glutamine => phenylacetyl glutamine + Coenzyme A
Editors
Reviewers
Description
Phenylacetyl CoA and glutamine react to form phenylacetyl glutamine and Coenzyme A. The enzyme that catalyzes this reaction has been
purified from human liver mitochondria and shown to be a distinct polypeptide species from glycine-N-acyltransferase (Webster et al. 1976). This
human glutamine-N-acyltransferase activity has not been characterized by sequence analysis at the protein or DNA level, however, and thus
cannot be associated with a known human protein in the annotation of phenylacetate conjugation.
References
Reaction
2.2.6.1.3 Conjugation of salicylate with glycine
Authors
Jassal, B, 2004-11-30.
Editors
Description
In the body, aspirin (acetylsalicylic acid) is hydrolyzed to salicylic acid. Salicylic acid (2-hydroxybenzoic acid) can then be hydroxylated to yield
gentisic acid, conjugated with glucuronate, or conjugated with glycine to yield molecules that are excreted by the kidneys. The third of these
conjugation processes is annotated here, and proceeds in two steps. First, salicylate and ATP react with coenzyme A to form salicylate CoA,
AMP, and pyrophosphate in a reaction catalyzed by xenobiotic/medium-chain fatty acid:CoA ligase (Vessey et al. 2003). Second, salicylate CoA
and glycine react to form salicyluric acid and Coenzyme A (Mawal and Qureshi 1994).
References
Commun, 205, 1994, 1373-9.
2.2.6.1.3.1 salicylic acid + Coenzyme A + ATP => salicylate-CoA + AMP + pyrophosphate
Authors
Jassal, B, 2005-03-11.
Reviewers
Description
Salicylate and ATP react with coenzyme A to form salicylate CoA, AMP, and pyrophosphate in a reaction catalyzed by xenobiotic/medium-chain
fatty acid:CoA ligase (Vessey et al. 2003).
References
G Levy, "Pharmacokinetics of salicylate elimination in man", J Pharm Sci, 54, 1965, 959-67.
Reaction
2.2.6.1.3.2 salicylate-CoA + glycine => salicyluric acid + Coenzyme A
Reviewers
Description
Salicylate CoA and glycine react to form salicyluric acid and Coenzyme A (Mawal and Qureshi 1994).
References
Commun, 205, 1994, 1373-9.
Reaction
3 Botulinum neurotoxicity
Authors
Krupa, S, Gopinathrao, G, 2006-06-15.
Editors
Reviewers
Ichtchenko, K, 2007-08-03.
Description
Botulism, caused by botulinum neurotoxin (BoNT), is characterized by descending flaccid paralysis as a result of inhibition of neurotransmitter
release at the neuromuscular junction - NMJ (Turton et al., 2002). According to their antigenic properties, BoNTs are classified into seven
different toxin types (A, B, C1, D, E, F and G) although more than 50 sequences encoding 18 subtypes are known (Smith et al., 2005). The toxin
is released as a 900 kDa complex containing some accessory proteins of unknown functions (Chen et al., 1998). The toxin types A, B and E are
mainly involved in human botulism whereas C and D predominantly cause animal botulism (Poulain et al, 2006). The toxin is absorbed from the
gut or other epithelium and reaches neuromuscular junctions by transcytosis (Park and Simpson, 2003). The binding sites for the toxins are
distributed across the apical surface of the epithelium (Ahsan et al., 2005). It has been observed that the neurotoxin alone is capable of
transcytosis across epithelial cells (Maksymowych and Simpson, 2004). Once internalized, the neurotoxin is dissociated from the non-toxic
components of the progenitor toxin in endosome (Uotsu et al., 2006).
The neurological inhibition is caused by the specific cleavage of a group of proteins integral to NMJ exocytosis, SNARE proteins (soluble
NSF-attachment protein receptors). One or more SNARE proteins are cleaved by BoNT, blocking the release of synaptic vesicular contents like
acetylcholine as in the case of motor neurons.
BoNTs are synthesized as polypeptides of 150 kDa that are cleaved into heavy and light chains linked by a single disulfide bond. Cleavage takes
place within a surface-exposed loop at the N-terminal of the Heavy chain subunit. Both bacterial and host endopeptidases can catalyze BoNT
cleavage into heavy and light chains, but bacterial enzymes are thought to carry out this function in vivo.The Heavy Chain (HC) has two 50 kDa
functional domains: the N-terminal translocation domain is capable of forming channels in lipid bilayers; the C-terminal ganglioside-binding
domain is important for membrane binding and subsequent internalization of toxins by host neurons. The 50 kDa Light chain (LC) is a
zinc-dependent endopeptidase specific for core components of neurotransmitter release complexes.
BoNT action proceeds in the following steps: binding of cleaved toxin to the target cell membrane; transcytosis from epithelial membrane to
target neuromuscular junction cells; release of BoNT Light chain into the target cell cytosol; and proteolytic cleavage of target cell proteins
catalyzed by the BoNT Light chain.
References
CR Ahsan, G Hajnoczky, AB Maksymowych, LL Simpson, "Visualization of binding and transcytosis of botulinum toxin by human intestinal
epithelial cells", J Pharmacol Exp Ther, 315, 2005, 1028-35.
AB Maksymowych, LL Simpson, "Structural features of the botulinum neurotoxin molecule that govern binding and transcytosis across polarized
human intestinal epithelial cells", J Pharmacol Exp Ther, 310, 2004, 633-41.
F Chen, GM Kuziemko, RC Stevens, "Biophysical characterization of the stability of the 150-kilodalton botulinum toxin, the nontoxic component,
and the 900-kilodalton botulinum toxin complex species", Infect Immun, 66, 1998, 2420-5.
TJ Smith, J Lou, IN Geren, CM Forsyth, R Tsai, SL Laporte, WH Tepp, M Bradshaw, EA Johnson, LA Smith, JD Marks, "Sequence variation
within botulinum neurotoxin serotypes impacts antibody binding and neutralization", Infect Immun, 73, 2005, 5450-7.
JB Park, LL Simpson, "Inhalational poisoning by botulinum toxin and inhalation vaccination with its heavy-chain component", Infect Immun, 71,
2003, 1147-54.
K Turton, JA Chaddock, KR Acharya, "Botulinum and tetanus neurotoxins: structure, function and therapeutic utility", Trends Biochem Sci, 27,
2002, 552-8.
B Poulain, BG Stiles, MR Popoff, J Molgo, "Attack of the nervous system by clostridial toxins...", Bacterial protein toxins, third edition; edited by
Alouf and Popoff., 19, 2006, 348.
N Uotsu, A Nishikawa, T Watanabe, T Ohyama, T Tonozuka, Y Sakano, K Oguma, "Cell internalization and traffic pathway of Clostridium
botulinum type C neurotoxin in HT-29 cells", Biochim Biophys Acta, 1763, 2006, 120-8.
C Montecucco, G Schiavo, "Mechanism of action of tetanus and botulinum neurotoxins", Mol Microbiol, 13, 1994, 1-8.
3.1 Binding of BoNT toxins to gut epithelial membrane
Authors
Editors
Reviewers
Description
Botulinum neurotoxins (BoNTs) bind to polysialogangliosides, including GT1b, GD1b and GQ1b and synaptotagmin polypeptides on the
neuronal plasma membrane (Verderio et al., 2006). In the body, this dual binding may have the effect of targeting BoNTs to specific regions of
the neuromuscular junction for endocytosis. Different serotypes are known to bind to different receptors: Bont/A to SV2, Bont/B and G to Syt1
and Syt2 with different affinities.
References
2002, 552-8.
M Dong, DA Richards, MC Goodnough, WH Tepp, EA Johnson, ER Chapman, "Synaptotagmins I and II mediate entry of botulinum neurotoxin B
into cells", J Cell Biol, 162, 2003, 1293-303.
S Swaminathan, S Eswaramoorthy, "Structural analysis of the catalytic and binding sites of Clostridium botulinum neurotoxin B", Nat Struct Biol,
7, 2000, 693-9.
C Verderio, O Rossetto, C Grumelli, C Frassoni, C Montecucco, M Matteoli, "Entering neurons: botulinum toxins and synaptic vesicle recycling",
EMBO Rep, 7, 2006, 995-9.
L Li, BR Singh, "Isolation of synaptotagmin as a receptor for types A and E botulinum neurotoxin and analysis of their comparative binding using
a new microtiter plate assay", J Nat Toxins, 7, 1998, 215-26.
Reaction
3.2 Transcytosis and internalization of BoNT
Authors
Editors
Reviewers
Description
Once BoNT molecules are bound to the host cell surface via their HC domains, they undergo transcytosis which include sorting and endocytosis
into an acidic vesicular compartment within the cytosol. As a result of endocytosis, the toxin becomes resistant to neutralization by antisera.
Endocytosis is temperature and energy-dependent. In the body, endocytosed BoNT molecules remain associated with the neuromuscular
junction which they finally reach by transcytosis.
References
E Habermann, "125I-labeled neurotoxin from Clostridium botulinum A: preparation, binding to synaptosomes and ascent to the spinal cord",
Naunyn Schmiedebergs Arch Pharmacol, 281, 1974, 47-56.
2002, 552-8.
ME Schwab, K Suda, H Thoenen, "Selective retrograde transsynaptic transfer of a protein, tetanus toxin, subsequent to its retrograde axonal
transport", J Cell Biol, 82, 1979, 798-810.
R Pellizzari, O Rossetto, G Schiavo, C Montecucco, "Tetanus and botulinum neurotoxins: mechanism of action and therapeutic uses", Philos
Trans R Soc Lond B Biol Sci, 354, 1999, 259-68.
Reaction
3.3 Translocation of BoNT Light chain
Authors
Editors
Reviewers
Description
Translocation of the BoNT Light chain (LC) from an endocytic vesicle into the cytosol is essential for neurotoxicity. It has been proposed that
acidic pH within the endosome triggers a conformational change in the Heavy chain (HC) N-terminal domain enabling its insertion into the
endosomal lipid bilayer membrane to form a channel large enough to accommodate the unfolded L chain. The L chain moiety of the BoNT
protein then traverses this channel towards the cytosol, the disulfide bond holding the H and L chains together is broken, and the L chain refolds
and is released into the cytosol. Koriazova and Montal (2003) proposed that the "LC refolds at the interface and dissociates from the HC in the
reducing cytosolic milieu where it cleaves its substrate SNAP-25".The molecular details of this process are not yet well understood.
References
G Lalli, S Bohnert, K Deinhardt, C Verastegui, G Schiavo, "The journey of tetanus and botulinum neurotoxins in neurons", Trends Microbiol, 11,
2003, 431-7.
LK Koriazova, M Montal, "Translocation of botulinum neurotoxin light chain protease through the heavy chain channel", Nat Struct Biol, 10, 2003,
13-8.
2002, 552-8.
C Montecucco, E Papini, G Schiavo, "Bacterial protein toxins penetrate cells via a four-step mechanism", FEBS Lett, 346, 1994, 92-8.
3.3.1 Conformational change in BoNT induced by pH
Authors

Editors
Reviewers
Description
The N-terminal half of the BoNT Heavy Chain undergoes conformational changes effected by endosomal pH resulting in ion channel formation
(Blaustein et al., 1987). This process has been demonstrated experimentally for BoNT serotypes A and B, but all serotypes are thought to have
this property (Pellizzari et al. 1999).
References
RO Blaustein, WJ Germann, A Finkelstein, BR DasGupta, "The N-terminal half of the heavy chain of botulinum type A neurotoxin forms channels
in planar phospholipid bilayers", FEBS Lett, 226, 1987, 115-20.
DB Lacy, RC Stevens, "Sequence homology and structural analysis of the clostridial neurotoxins", J Mol Biol, 291, 1999, 1091-104.
JJ Donovan, JL Middlebrook, "Ion-conducting channels produced by botulinum toxin in planar lipid membranes", Biochemistry, 25, 1986, 2872-6.
Reaction
3.3.2 Pore or channel formation in endosomal membrane
Authors

Editors
Reviewers
Description
Acidic pH triggers a conformation change in the Heavy chain N-terminal domain leading to its insertion into the lipid bilayer and formation of a
trans-membrane channel large enough to accommodate the unfolded Light chain. It has been observed that in the closely related Diptheria toxin,
a 10-aa motif is critical for pore formation. Ratts et al. identified this motif in some of the virulent BoNTs (Ratts et al., 2005).
References
R Ratts, C Trujillo, A Bharti, J vanderSpek, R Harrison, JR Murphy, "A conserved motif in transmembrane helix 1 of diphtheria toxin mediates
catalytic domain delivery to the cytosol", Proc Natl Acad Sci U S A, 102, 2005, 15635-40.
RO Blaustein, WJ Germann, A Finkelstein, BR DasGupta, "The N-terminal half of the heavy chain of botulinum type A neurotoxin forms channels
in planar phospholipid bilayers", FEBS Lett, 226, 1987, 115-20.
C Montecucco, E Papini, G Schiavo, "Bacterial protein toxins penetrate cells via a four-step mechanism", FEBS Lett, 346, 1994, 92-8.
Reaction
3.3.3 Transportation of BoNT Light chain to cytosol
Authors
Editors
Reviewers
Description
The BoNT L chain traverses the H chain channel into the cytosol, refolds, and is released into the cytosol. The complete molecular details of
cleavage of the L- H disulfide bond and L chain refolding are not yet known (Pellizzari et al.,1999). The cleavage of host proteins may require the
toxins binding to specific recogntion sites as well as cleavage sites (Rossetto et al., 1994).
References
O Rossetto, G Schiavo, C Montecucco, B Poulain, F Deloye, L Lozzi, CC Shone, "SNARE motif and neurotoxins", Nature, 372, 1994, 415-6.
H Kurazono, S Mochida, T Binz, U Eisel, M Quanz, O Grebenstein, K Wernars, B Poulain, L Tauc, H Niemann, "Minimal essential domains
specifying toxicity of the light chains of tetanus toxin and botulinum neurotoxin type A", J Biol Chem, 267, 1992, 14721-9.
Reaction
3.4 Proteolytic cleavage of SNARE complex proteins
Authors
Editors
Reviewers
Description
VAMP/synaptobrevin, SNAP-25 and syntaxin are important for synaptic vesicle fusion at the nerve terminal. These proteins constitute the
synaptic members of SNARE family (soluble N-ethylmaleimide-sensitive fusion protein) attachment protein receptor, which is central to all
membrane fusion events (Umland et al. 1997). These proteins are involved in docking and/or fusion of synaptic vesicles with the presynaptic
membrane. BoNTs achieve total blockage of neurotransmitter release by selectively inactivating the synaptic SNAREs by proteolysis.
The L chains of BoNTs of different serotypes specifically cleave distinct members of the SNARE family: serotypes B, D, F and G act on
VAMP/synaptobrevin localized on synaptic vesicles; BoNT-A and E cleave SNAP-25; and BoNT-C cleaves both syntaxin 1 and SNAP-25, two
proteins of the pre-synaptic plasma membrane.
Sudhof et al. (2001) and Liu et al. (2005) had observed that alpha-laterotoxins from black widow spider target identical neurmuscular junctions
by opposite mechanism resulting in massive vesicle exocytosis. The exact molecular details of the action of these toxins may reveal the
underlying processes of synaptic vesicle exocytosis/inbition and their regulation.
References
J Liu, Q Wan, X Lin, H Zhu, K Volynski, Y Ushkaryov, T Xu, "Alpha-latrotoxin modulates the secretory machinery via receptor-mediated
activation of protein kinase C", Traffic, 6, 2005, 756-65.
TC Umland, LM Wingert, S Swaminathan, WF Furey, JJ Schmidt, M Sax, "Structure of the receptor binding fragment HC of tetanus neurotoxin",
Nat Struct Biol, 4, 1997, 788-92.
2002, 552-8.
TC Sudhof, "alpha-Latrotoxin and its receptors: neurexins and CIRL/latrophilins", Annu Rev Neurosci, 24, 2001, 933-62.
Y Humeau, F Doussau, NJ Grant, B Poulain, "How botulinum and tetanus neurotoxins block neurotransmitter release", Biochimie, 82, 2000,
427-46.
3.4.1 BoNT Light Chain Types B, D, and F cleave VAMP/Synaptobrevin
Authors
Editors
Reviewers
Description
VAMP1 is located on the synaptic vesicle membranes and interacts with other VAMP family members. This protein can be cleaved by BoNTs of
types B, D and G.
References
JW Arndt, W Yu, F Bi, RC Stevens, "Crystal structure of botulinum neurotoxin type G light chain: serotype divergence in substrate recognition",
Biochemistry, 44, 2005, 9574-80.
P Foran, CC Shone, JO Dolly, "Differences in the protease activities of tetanus and botulinum B toxins revealed by the cleavage of
vesicle-associated membrane protein and various sized fragments", Biochemistry, 33, 1994, 15365-74.
JW Arndt, Q Chai, T Christian, RC Stevens, "Structure of botulinum neurotoxin type D light chain at 1.65 A resolution: repercussions for VAMP-2
substrate specificity.", Biochemistry, 45, 2006, 3255-62.
S Yamasaki, A Baumeister, T Binz, J Blasi, E Link, F Cornille, B Roques, EM Fykse, TC Sudhof, R Jahn, "Cleavage of members of the
synaptobrevin/VAMP family by types D and F botulinal neurotoxins and tetanus toxin", J Biol Chem, 269, 1994, 12764-72.
3.4.1.1 BoNT Light Chain Type B cleaves VAMP-2

Authors
Editors
Reviewers
Description
BoNT Light Chain type B protein cleaves Vamp-2 protein, a member of SNARE complex.
References
Biochemistry, 44, 2005, 9574-80.
Reaction
3.4.1.2 BoNT Light Chain Type D cleaves VAMP
Authors
Editors
Reviewers
Description
BoNT Light Chain type D protein cleaves VAMP proteins of human SNARE complex.
References
Biochemistry, 44, 2005, 9574-80.
Reaction
3.4.1.3 BoNT Light Chain Type F cleaves VAMP
Authors
Editors
Reviewers
Description
BoNT Light Chain type F protein cleaves VAMP proteins of human SNARE complex.
References
Biochemistry, 44, 2005, 9574-80.
Reaction
3.4.2 BoNT Light Chain Types A, C1, E cleave SNAP-25
Authors
Editors
Reviewers
Description
BoNTs of serotypes A, C1 and E cleave SNAP-25 (synaptosomal-associated protein, 25kDa), a presynaptic plasma membrane protein involved
in the regulation of neurotransmitter release. BoNT A removes nine amino-acid residues from the carboxyl terminus, whereas BoNT E removes
26 C-terminal amino-acid residues (Bajohrs et al.,2004).
References
M Bajohrs, C Rickman, T Binz, B Davletov, "A molecular basis underlying differences in the toxicity of botulinum serotypes A and E", EMBO
Rep, 5, 2004, 1090-5.
T Binz, J Blasi, S Yamasaki, A Baumeister, E Link, TC Sudhof, R Jahn, H Niemann, "Proteolysis of SNAP-25 by types E and A botulinal
neurotoxins", J Biol Chem, 269, 1994, 1617-20.
G Schiavo, A Santucci, BR DasGupta, PP Mehta, J Jontes, F Benfenati, MC Wilson, C Montecucco, "Botulinum neurotoxins serotypes A and E
cleave SNAP-25 at distinct COOH-terminal peptide bonds", FEBS Lett, 335, 1993, 99-103.
VV Vaidyanathan, K Yoshino, M Jahnz, C Dorries, S Bade, S Nauenburg, H Niemann, T Binz, "Proteolysis of SNAP-25 isoforms by botulinum
neurotoxin types A, C, and E: domains and amino acid residues controlling the formation of enzyme-substrate complexes and cleavage", J
Neurochem, 72, 1999, 327-37.
P Foran, GW Lawrence, CC Shone, KA Foster, JO Dolly, "Botulinum neurotoxin C1 cleaves both syntaxin and SNAP-25 in intact and
permeabilized chromaffin cells: correlation with its blockade of catecholamine release", Biochemistry, 35, 1996, 2630-6.
3.4.2.1 BoNT Light Chain Type A cleaves SNAP-25
Authors

Editors
Reviewers
Description
BoNT Light Chain type A protein cleaves SNAP-25 protein of human SNARE complex.
References
Neurochem, 72, 1999, 327-37.
Reaction
3.4.2.2 BoNT Light Chain Type C1 cleaves SNAP-25

Authors
Editors
Reviewers
Description
BoNT Light Chain type C1 protein cleaves SNAP-25 protein of human SNARE complex.
References
Neurochem, 72, 1999, 327-37.
Reaction
3.4.2.3 BoNT Light Chain Type E cleaves SNAP-25
Authors
Editors
Reviewers
Description
BoNT Light Chain type E protein cleaves SNAP-25 protein of human SNARE complex.
References
Neurochem, 72, 1999, 327-37.
Reaction
3.4.3 BoNT Light Chain Type C1 cleaves Syntaxin
Authors
Editors
Reviewers
Description
Syntaxins are involved in the localization (docking) of both synaptic vesicles and calcium channels to the presynaptic active zone. Syntaxin 1A
interacts with SNAP-25 in forming t-SNARE part of SNARE complex. BoNT Type C specifically cleaves Syntaxin 1A although a broader target
spectrum is suspected.
References
Reaction
4 Cell Cycle Checkpoints
Authors
Hoffmann, I, Khanna, K, O'Connell, M, Walworth, N, Yen, TJ, 2005-01-01.
Editors
Matthews, L, 0000-00-00.
Reviewers
Sanchez, Y, Knudsen, E, Hardwick, KG, 0000-00-00.
Description
A hallmark of the human cell cycle in normal somatic cells is its precision. This remarkable fidelity is achieved by a number of signal transduction
pathways, known as checkpoints, which monitor cell cycle progression ensuring an interdependency of S-phase and mitosis, the integrity of the
genome and the fidelity of chromosome segregation.
Checkpoints are layers of control that act to delay CDK activation when defects in the division program occur. As the CDKs functioning at
different points in the cell cycle are regulated by different means, the various checkpoints differ in the biochemical mechanisms by which they
elicit their effect. However, all checkpoints share a common hierarchy of a sensor, signal transducers, and effectors that interact with the CDKs.
The stability of the genome in somatic cells contrasts to the almost universal genomic instability of tumor cells. There are a number of
documented genetic lesions in checkpoint genes, or in cell cycle genes themselves, which result either directly in cancer or in a predisposition to
certain cancer types. Indeed, restraint over cell cycle progression and failure to monitor genome integrity are likely prerequisites for the
molecular evolution required for the development of a tumor. Perhaps most notable amongst these is the p53 tumor suppressor gene, which is
mutated in >50% of human tumors. Thus, the importance of the checkpoint pathways to human biology is clear.
4.1 G1/S DNA Damage Checkpoints
Authors
Hoffmann, I, Khanna, K, 2003-06-05.
Description
In the G1 phase there are two types of DNA damage responses, the p53-dependent and the p53-independent pathways. The p53-dependent
responses inhibit CDKs through the up-regulation of genes encoding CKIs mediated by the p53 protein, whereas the p53-independent
mechanisms inhibit CDKs through the inhibitory T14Y15 phosphorylation of Cdk2. Failure of DNA damage checkpoints in G1 leads to mutagenic
replication of damaged templates and other replication defects.
4.1.1 p53-Dependent G1/S DNA damage checkpoint
Authors
Khanna, K, 2003-06-05.
Editors
Joshi-Tope, G, 0000-00-00.
Description
The arrest at G1/S checkpoint is mediated by the action of a widely known tumor suppressor protein, p53. Loss of p53 functions, as a result of
mutations in cancer prevent the G1/S checkpoint (Kuerbitz et al, 1992). P53 is rapidly induced in response to damaged DNA. A number of
kinases, phosphatases, histone acetylases and ubiquitin-conjugating enzymes regulate the stability as well as transcriptional activity of p53 after
DNA damage.
References
SJ Kuerbitz, BS Plunkett, WV Walsh, MB Kastan, "Wild-type p53 is a cell cycle checkpoint determinant following irradiation.", Proc Natl Acad Sci
U S A, 89, 1992, 7491-5.
4.1.1.1 Detection of DNA Damage
Description
Following the detection of DNA damage, the appropriate cell cycle checkpoint mechanisms kick in to stall the cell in the appropriate phase of the
cell cycle. If the DNA damage can be repaired in a timely fashion, the cell can then step through the cell cycle. However, if the DNA damage
cannot be repaired, the cell may remain arrested in the G1 or G2 phases, and even undergo apoptosis under severe conditions. Disruption of
the cell cycle checkpoints can lead to cancer.
The surveillance and detection mechanisms of DNA damage will be addressed in a later version of Reactome.
4.1.1.2 p53-Dependent G1 DNA Damage Response
Reviewers
Manfredi, J, 0000-00-00.
Description
Most of the damage-induced modifications of p53 are dependent on the ATM kinase. The first link between ATM and p53 was predicted based
on the earlier studies that showed that AT cells exhibit a reduced and delayed induction of p53 following exposure to IR (Kastan et al, 1992 and
Khanna and Lavin, 1993).
Under normal conditions, p53 is a short-lived protein. The MDM2 protein, usually interacts with p53 (Haupt et al, 1997 and Kubbutat et al, 1997),
and by virtue of its E3 ubiquitin ligase activity, shuttles p53 to the cytoplasm and mediates its degradation by the ubiquitin-proteasome
machinery. Upon detection of DNA damage, the ATM kinase mediates the phosphorylation of the Mdm2 protein to block its interaction with p53.
Also, phosphorylation of p53 at multiple loci, by the ATM kinase and by other kinases activated by the ATM kinase, stabilizes and activates the
p53 protein.
The p53 protein activates the transcription of cyclin-dependent kinase inhibitor, p21. p21 inactivates the CyclinE:Cdk2 complexes, and prevent
entry of the cell into S phase, leading to G1 arrest. Under severe conditions, the cell may undergo apoptosis.
References
KK Khanna, MF Lavin, "Ionizing radiation and UV induction of p53 protein by different pathways in ataxia-telangiectasia cells.", Oncogene, 8,
1993, 3307-12.
Y Haupt, R Maya, A Kazaz, M Oren, "Mdm2 promotes the rapid degradation of p53.", Nature, 387, 1997, 296-9.
MH Kubbutat, SN Jones, KH Vousden, "Regulation of p53 stability by Mdm2.", Nature, 387, 1997, 299-303.
MB Kastan, Q Zhan, WS el-Deiry, F Carrier, T Jacks, WV Walsh, BS Plunkett, B Vogelstein, AJ Fornace, "A mammalian cell cycle checkpoint
pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia.", Cell, 71, 1992, 587-97.
4.1.1.2.1 Stabilization of p53
Editors
Matthews, L, 2008-05-12.
Reviewers
Sanchez, Y, 2008-05-07.
Description
Later studies pin-pointed that a single serine (Ser-15) was phosphorylated by ATM and phosphorylation of Ser-15 was rapidly-induced in
IR-treated cells and this response was ATM-dependent (Canman et al, 1998; Banin et al, 1998 and Khanna et al, 1998). ATM also regulates the
phosphorylation of p53 at other sites, especially Ser-20, by activating other serine/threonine kinases in response to IR (Chehab et al, 2000;
Shieh et al, 2000; Hirao et al 2000). Phosphorylation of p53 at Ser-20 interferes with p53-MDM2 interaction. MDM2 is transcriptionally activated
by p53 and is a negative regulator of p53 that targets it for degradation (Haupt et al, 1997; Kubbutat et al, 1997). In addition modification of
MDM2 by ATM also affects p53 stabilization (Maya et al, 2001).
References
A Hirao, YY Kong, S Matsuoka, A Wakeham, J Ruland, H Yoshida, D Liu, SJ Elledge, TW Mak, "DNA damage-induced activation of p53 by the
checkpoint kinase Chk2.", Science, 287, 2000, 1824-7.
SY Shieh, J Ahn, K Tamai, Y Taya, C Prives, "The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple
DNA damage-inducible sites.", Genes Dev, 14, 2000, 289-300.
R Maya, M Balass, ST Kim, D Shkedy, JF Leal, O Shifman, M Moas, T Buschmann, Z Ronai, Y Shiloh, MB Kastan, E Katzir, M Oren,
"ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage.", Genes Dev, 15, 2001, 1067-77.
NH Chehab, A Malikzay, M Appel, TD Halazonetis, "Chk2/hCds1 functions as a DNA damage checkpoint in G(1) by stabilizing p53.", Genes
Dev, 14, 2000, 278-88.
4.1.1.2.1.1 Phosphorylation of p53 at ser-15 by ATM kinase
Reviewers
Sanchez, Y, 2008-05-07.
Description
In response to DNA damage due to ionizing radiation, the serine at position 15 of the p53 tumor suppressor protein is rapidly phosphorylated by
the ATM kinase. This serves to stabilize the p53 protein. A rise in the levels of the p53 protein induce the expression of the p21 cyclin-dependent
kinase inhibitor. This prevents the normal progression from G1 to S phase, thus providing a check on replication of damaged DNA.
References
CE Canman, DS Lim, Y Taya, K Tamai, K Sakaguchi, E Appella, MB Kastan, JD Siliciano, "Activation of the ATM kinase by ionizing radiation
and phosphorylation of p53.", Science, 281, 1998, 1677-9.
KK Khanna, KE Keating, S Kozlov, S Scott, M Gatei, K Hobson, Y Taya, B Gabrielli, D Chan, SP Lees-Miller, MF Lavin, "ATM associates with
and phosphorylates p53: mapping the region of interaction.", Nat Genet, 20, 1998, 398-400.
S Banin, L Moyal, S Shieh, Y Taya, CW Anderson, L Chessa, NI Smorodinsky, C Prives, Y Reiss, Y Shiloh, Y Ziv, "Enhanced phosphorylation of
p53 by ATM in response to DNA damage.", Science, 281, 1998, 1674-7.
Reaction
4.1.1.2.1.2 Phosphorylation of MDM2 at serine-395 by ATM kinase

Reviewers
Sanchez, Y, 2008-05-07.
Description
At the beginning of this reaction, 1 molecule of 'Mdm2' is present. At the end of this reaction, 1 molecule of 'phospho-MDM2' is present.
This reaction takes place in the 'nucleoplasm' and is mediated by the 'kinase activity' of 'phospho-ATM (Ser 1981)'.
References
R Maya, M Balass, ST Kim, D Shkedy, JF Leal, O Shifman, M Moas, T Buschmann, Z Ronai, Y Shiloh, MB Kastan, E Katzir, M Oren,
"ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage.", Genes Dev, 15, 2001, 1067-77.
Reaction
4.1.1.2.2 Transcriptional activation of p53 responsive genes
Description
p53 causes G1 arrest by inducing the expression of a cell cycle inhibitor, p21 (El-Deiry et al, 1993; Harper et al, 1993; Xiong et al, 1993). P21
binds and inactivates Cyclin-Cdk complexes that mediate G1/S progression, resulting in lack of phosphorylation of Rb, E2F sequestration and
cell cycle arrest at the G1/S transition. Mice with a homozygous deletion of p21 gene are deficient in their ability to undergo a G1/S arrest in
response to DNA damage (Deng et al, 1995).
References
Y Xiong, GJ Hannon, H Zhang, D Casso, R Kobayashi, D Beach, "p21 is a universal inhibitor of cyclin kinases.", Nature, 366, 1994, 701-4.
JW Harper, GR Adami, N Wei, K Keyomarsi, SJ Elledge, "The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent
kinases.", Cell, 75, 1993, 805-16.
C Deng, P Zhang, JW Harper, SJ Elledge, P Leder, "Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G1
checkpoint control.", Cell, 82, 1995, 675-84.
WS el-Deiry, T Tokino, VE Velculescu, DB Levy, R Parsons, JM Trent, D Lin, WE Mercer, KW Kinzler, B Vogelstein, "WAF1, a potential mediator
of p53 tumor suppression.", Cell, 75, 1993, 817-25.
4.1.1.2.2.1 Transcriptional activation of cell cycle inhibitor p21
Description
Both p53-independent and p53-dependent mechanisms of induction of p21 mRNA have been demonstrated. p21 is transcriptionally activated by
p53 after DNA damage (el-Deiry et al., 1993).
References
4.1.1.2.2.1.1 Transcriptional activation of p21 by p53 after DNA damage
Authors
Matthews, L, 2006-09-29.
Editors
Matthews, L, 2006-10-10.
Reviewers
Coqueret, O, 2006-10-06.
Description
p21 is transcriptionally activated by p53 after DNA damage.
References
Reaction
4.1.1.2.3 Inactivation of Cyclin E:Cdk2 complexes by p27/p21
Editors
Matthews, L, 2006-10-02.
Description
During G1, the activity of cyclin-dependent kinases (CDKs) is kept in check by the CDK inhibitors (CKIs) p27 and p21, thereby preventing
premature entry into S phase (see Guardavaccaro and Pagano, 2006). The efficient recognition and ubiquitination of p27 by the SCF(Skp2)
complex requires the formation of a trimeric complex containing p27 and cyclin E/A:Cdk2.
References
W Wang, L Nacusi, RJ Sheaff, X Liu, "Ubiquitination of p21Cip1/WAF1 by SCFSkp2: substrate requirement and ubiquitination site selection",
Biochemistry, 44, 2005, 14553-64.
D Guardavaccaro, M Pagano, "Stabilizers and destabilizers controlling cell cycle oscillators", Mol Cell, 22, 2006, 1-4.
A Montagnoli, F Fiore, E Eytan, AC Carrano, GF Draetta, A Hershko, M Pagano, "Ubiquitination of p27 is regulated by Cdk-dependent
phosphorylation and trimeric complex formation", Genes Dev, 13, 1999, 1181-9.
Reaction
4.1.1.2.3.1 Inactivation of Cyclin E1:Cdk2 complex by p27/p21
Description
At the beginning of this reaction, 1 molecule of 'Cyclin E1:Cdk2 complex', and 1 molecule of 'CKI' are present. At the end of this reaction, 1
molecule of 'Cyclin E1:Cdk2:CKI' is present.
This reaction takes place in the 'nucleoplasm'.
Reaction
4.1.1.2.3.2 Inactivation of Cyclin E2:Cdk2 complex by p27/p21
Description
References
M Zariwala, J Liu, Y Xiong, "Cyclin E2, a novel human G1 cyclin and activating partner of CDK2 and CDK3, is induced by viral oncoproteins",
Oncogene, 17, 1998, 2787-98.
Reaction
4.1.2 p53-Independent G1/S DNA damage checkpoint
Description
The G1 arrest induced by DNA damage has been ascribed to the transcription factor and tumor suppressor protein p53. To be effective within
minutes after DNA damage, induction of the G1 block should exploit transcription and protein synthesis independent mechanisms.
Upon exposure to ultraviolet light (UV) or ionizing radiation (IR), the abundance and activity of a protein, Cdc25A, rapidly decreases; this DNA
damage response is not dependent on p53. The rapid destruction of Cdc25A phosphatase prevents entry of a cell into S-phase, by maintaining
the CyclinE:Cdk2 complexes in their T14Y15 phosphorylated form.
References
N Mailand, C Lukas, RG SyljuÃ¢sen, M Welcker, J Lukas, "Rapid destruction of human Cdc25A in response to DNA damage.", Science, 288,
2000, 1425-9.
Description
4.1.2.2 p53-Independent DNA Damage Response
Description
In response to DNA damage due to exposure to ultraviolet light or to ionizing radiation, Cdc25A is phosphorylated by Chk1 or Chk2. The
phosphorylation of Cdc25A at ser-123, in response to DNA damage from ionizing radiation is a signal for ubiquitination and subsequent
degradation of Cdc25A. The destruction of Cdc25A prevents the normal G1/S transition. Cdc25A is required for the activation of the Cyclin
E:Cdk2 complexes via dephosphorylation.
Chk1 is activated in response to DNA damage due to uv light. However, the phosphorylation occurs at a different site.
References
N Mailand, RG SyljuÃ¥sen, J Lukas, "The ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA synthesis.", Nature, 410,
2001, 842-7.
4.1.2.2.1 Phosphorylation of Cdc25A at Ser-123 in response to DNA damage
Authors
O'Connell, M, Walworth, N, 2003-06-05.
Editors
Matthews, L, 2003-09-10.
Reviewers
Manfredi, J, 0000-00-00.
Description
Chk1 directly phosphorylates Cdc25A at Ser-123. Chk1 phosphorylation is required for cells to delay cell cycle progression in response to
double-strand DNA breaks (Zhao et al., 2002).
References
H Zhao, JL Watkins, H Piwnica-Worms, "Disruption of the checkpoint kinase 1/cell division cycle 25A pathway abrogates ionizing
radiation-induced S and G2 checkpoints.", Proc Natl Acad Sci U S A, 99, 2002, 14795-800.
Reaction
4.1.2.2.2 Ubiquitin Mediated Degradation of Phosphorylated Cdc25A
Description
cdc25A protein is degraded by the ubiquitin-proteasome machinery in both terminally differentiating and cycling cells (Bernardi et al. 2000).
References
R Bernardi, DA Liebermann, B Hoffman, "Cdc25A stability is controlled by the ubiquitin-proteasome pathway during cell cycle progression and
terminal differentiation", Oncogene, 19, 2000, 2447-54.
4.1.2.2.2.1 Ubiquitination of phosphorylated Cdc25A
Description
At the beginning of this reaction, 1 molecule of 'ubiquitin', and 1 molecule of 'phospho-Cdc25A' are present. At the end of this reaction, 1
molecule of 'Ubiquitinated Phospho-Cdc25A' is present.
This reaction takes place in the 'cytosol' and is mediated by the 'ubiquitin-protein ligase activity' of 'Ubiquitin ligase'.
Reaction
4.1.2.2.2.2 Proteolytic degradation of ubiquitinated-Cdc25A
Description
At the beginning of this reaction, 1 molecule of 'Ubiquitinated Phospho-Cdc25A' is present. At the end of this reaction, 1 molecule of 'Amino Acid'
is present.
This reaction takes place in the 'cytosol' and is mediated by the 'endopeptidase activity' of '26S proteasome'.
Reaction
4.2 G2/M Checkpoints
Description
G2/M checkpoints include the checks for damaged DNA, unreplicated DNA, and checks that ensure that the genome is replicated once and only
once per cell cycle. If cells pass these checkpoints, they follow normal transition to the M phase. However, if any of these checkpoints fail, mitotic
entry is prevented by specific G2/M checkpoint events.
The G2/M checkpoints can fail due to the presence of unreplicated DNA or damaged DNA. In such instances, the cyclin-dependent kinase,
Cdc2(Cdk1), is maintained in its inactive, phosphorylated state, and mitotic entry is prevented. Events that ensure that origins of DNA replication
fire once and only once per cell cycle are also an example of a G2/M checkpoint.
In the event of high levels of DNA damage, the cells may also be directed to undergo apopotosis (not covered).
4.2.1 G2/M DNA damage checkpoint

Description
Throughout the cell cycle, the genome is constantly monitored for damage, resulting either from errors of replication, by-products of metabolism
or through extrinsic sources such as ultra-violet or ionizing radiation. The different DNA damage checkpoints act to inhibit or maintain the
inhibition of the relevant CDK that will control the next cell cycle transition. The G2 DNA damage checkpoint prevents mitotic entry solely through
T14Y15 phosphorylation of Cdc2 (Cdk1). Failure of the G2 DNA damage checkpoint leads to catastrophic attempts to segregate unrepaired
chromosomes.
Description
4.2.1.2 Recruitment and activation of Chk1
Authors
Borowiec, JA, 2006-02-25.
Editors
Description
Chk1 is a checkpoint kinase activated during genotoxic stress. Like ATR, Chk1 is essential for viability in mammals. Targeted gene disruption in
mice shows that loss of Chk1 causes peri-implantation embryonic lethality. Even though ATR-ATRIP not bound to ssDNA can phosphorylate
Chk1, Chk1 activation is greatly enhanced when recruited to stalled replication forks by physical interaction with a modified form of claspin and
the Rad9-Hus1-Rad1 sliding clamp. Activation of Chk1 occurs following phosphorylation of two sites (serine 317 and serine 345). Mutational
analysis indicates that modification of both sites is essential for maximal kinase activity, while phosphorylation of only a single site causes only
weak activation of Chk1. Following phosphorylation, Chk1 can diffuse away from the complex to further amplify the checkpoint signal. ATR
appears to be the primary kinase activating Chk1 as conditions that activate ATR (ultraviolet irradiation or treatment with hydroxyurea) also
activate Chk1. Stresses that activate ATM, e.g., ionizing irradiation, do not cause significant Chk1 activation. While the ATR and ATM pathways
are distinct, there is interplay between the two. For example, double-strand DNA breaks can be processed in an ATM-dependent manner to
generate structures that can cause ATR and hence Chk1 activation. The ATR and ATM pathways also have mechanistic similarities. Analogous
to the Chk1 kinase existing downstream of ATR, the Chk2 checkpoint kinase is modified and activated by ATM. Although having distinct
structures, Chk1 and Chk2 also have overlapping targets with some substrate sites phosphorylatable by both kinases (e.g., serine 20 of p53).
References
A Kumagai, WG Dunphy, "Claspin, a novel protein required for the activation of Chk1 during a DNA replication checkpoint response in Xenopus
egg extracts", Mol Cell, 6, 2000, 839-49.
VA Smits, PM Reaper, SP Jackson, "Rapid PIKK-Dependent Release of Chk1 from Chromatin Promotes the DNA-Damage Checkpoint
Response", Curr Biol, 16, 2006, 150-9.
CC Chini, J Chen, "Human claspin is required for replication checkpoint control", J Biol Chem, 278, 2003, 30057-62.
Q Liu, S Guntuku, XS Cui, S Matsuoka, D Cortez, K Tamai, G Luo, S Carattini-Rivera, F DeMayo, A Bradley, LA Donehower, SJ Elledge, "Chk1
is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint.", Genes Dev, 14, 2000, 1448-59.
Y Chen, Y Sanchez, "Chk1 in the DNA damage response: conserved roles from yeasts to mammals", DNA Repair (Amst), 3, 2004, 1025-32.
K Uto, D Inoue, K Shimuta, N Nakajo, N Sagata, "Chk1, but not Chk2, inhibits Cdc25 phosphatases by a novel common mechanism", EMBO J,
23, 2004, 3386-96.
CS Sorensen, RG Syljuasen, J Lukas, J Bartek, "ATR, Claspin and the Rad9-Rad1-Hus1 complex regulate Chk1 and Cdc25A in the absence of
DNA damage", Cell Cycle, 3, 2004, 941-5.
H Zhao, H Piwnica-Worms, "ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1", Mol Cell Biol, 21,
2001, 4129-39.
H Takai, K Tominaga, N Motoyama, YA Minamishima, H Nagahama, T Tsukiyama, K Ikeda, K Nakayama, M Nakanishi, K Nakayama, "Aberrant
cell cycle checkpoint function and early embryonic death in Chk1(-/-) mice", Genes Dev, 14, 2000, 1439-47.
J Bartek, J Lukas, "Chk1 and Chk2 kinases in checkpoint control and cancer", Cancer Cell, 3, 2003, 421-9.
JS Myers, D Cortez, "Rapid activation of ATR by ionizing radiation requires ATM and Mre11", J Biol Chem, 2006.
Reaction
4.2.1.3 Phosphorylation and activation of CHK2 by ATM
Description
At the beginning of this reaction, 1 molecule of 'ATP', and 1 molecule of 'Chk2' are present. At the end of this reaction, 1 molecule of
'phospho-Chk2', and 1 molecule of 'ADP' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'kinase activity' of 'phospho-ATM (Ser 1981)'.
References
S Matsuoka, G Rotman, A Ogawa, Y Shiloh, K Tamai, SJ Elledge, "Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro", Proc
Natl Acad Sci U S A, 97, 2000, 10389-94.
N Foray, D Marot, A Gabriel, V Randrianarison, M Perricaudet, A Ashworth, P Jeggo, "A subset of ATM- and ATR-dependent phosphorylation
events requires the BRCA1 protein.", EMBO J, 22, 2003, 2860-71.
Reaction
4.2.1.4 Chk1/Chk2(Cds1) mediated inactivation of Cyclin B:Cdk1 complex

Authors
Matthews, L, 2003-08-05.
Description
DNA damage induced activation of the checkpoint kinases Chk1/Chk2(Cds1) results in the conversion and/or maintenance of CyclinB:Cdc2
complex in its Tyrosine 15 phosphorylated (inactive) state. Cdc2 activity is regulated by a balance between the phosphorylation and
dephosphorylation by the Wee1/Myt1 kinase and Cdc25 phosphatase. Inactivation of the Cyclin B:Cdc2 complex likely involves both inactivation
of Cdc25 and/or stimulation of Wee1/Myt1 kinase activity.
4.2.1.4.1 Phosphorylation of Cdc25C at Ser216
Authors
Sanchez, Y, 2004-02-10.
Editors
Matthews, L, 2004-03-22.
Reviewers
Manfredi, J, 0000-00-00.
Description
Cdc25C is negatively regulated by phosphorylation on Ser 216, the 14-3-3-binding site. This is an important regulatory mechanism used by cells
to block mitotic entry under normal conditions and after DNA damage (Bulavin et al., 2003).
References
DV Bulavin, Y Higashimoto, ZN Demidenko, S Meek, P Graves, C Phillips, H Zhao, SA Moody, E Appella, H Piwnica-Worms, Jr Fornace AJ,
"Dual phosphorylation controls Cdc25 phosphatases and mitotic entry", Nat Cell Biol, 5, 2003, 545-51.
Reaction
4.2.1.4.2 Association of phospho-Cdc25C(Ser 216) with 14-3-3 proteins
Authors
Matthews, L, 2003-08-05.
Description
Association of Cdc25C with 14-3-3 proteins with phospho-Cdc25C (Ser216) is believed to result in retention of this complex within the cytoplasm
( Dalal et al., 1999)
References
PR Graves, CM Lovly, GL Uy, H Piwnica-Worms, "Localization of human Cdc25C is regulated both by nuclear export and 14-3-3 protein
binding.", Oncogene, 20, 2001, 1839-51.
SN Dalal, CM Schweitzer, J Gan, JA DeCaprio, "Cytoplasmic localization of human cdc25C during interphase requires an intact 14-3-3 binding
site.", Mol Cell Biol, 19, 1999, 4465-79.
Reaction
4.2.1.4.3 Retention of phospho-Cdc25C:14-3-3 complexes within the cytoplasm

Authors
Matthews, L, 2003-08-05.
Description
Cdc25C is phosphorylated by Chk1 at ser-216 (Blasina et al.,1999 ) resulting in both inhibition of the Cdc25 phosphatase activity and creation of
a 14-3-3 docking site (Peng et al., 1997). Association of 14-3-3 protein is believed to exclude Cdc25C from the nucleus via cytoplasmic retention
of the Cdc25C:14-3-3 complex.
References
CY Peng, PR Graves, RS Thoma, Z Wu, AS Shaw, H Piwnica-Worms, "Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by
phosphorylation of Cdc25C on serine-216.", Science, 277, 1997, 1501-5.
A Blasina, IV de Weyer, MC Laus, WH Luyten, AE Parker, CH McGowan, "A human homologue of the checkpoint kinase Cds1 directly inhibits
Cdc25 phosphatase.", Curr Biol, 9, 1999, 1-10.
SN Dalal, CM Schweitzer, J Gan, JA DeCaprio, "Cytoplasmic localization of human cdc25C during interphase requires an intact 14-3-3 binding
site.", Mol Cell Biol, 19, 1999, 4465-79.
Reaction
4.2.1.4.4 Phosphorylation of Wee1 kinase by Chk1
Authors
Matthews, L, 2003-08-08.
Description
Phosphorylation of Wee1 by Chk1 stimulates Wee1 kinase activity.
Source reaction
This reaction was inferred from the corresponding reaction "Phosphorylation of Wee1 kinase by Chk1" in species Schizosaccharomyces pombe.
MJ O'Connell, JM Raleigh, HM Verkade, P Nurse, "Chk1 is a wee1 kinase in the G2 DNA damage checkpoint inhibiting cdc2 by Y15
phosphorylation.", EMBO J, 16, 1997, 545-54.
Reaction
4.2.1.4.5 Wee1- mediated phosphorylation of Cyclin B1:phospho-Cdc2 complexes
Description
Wee1, a nuclear kinase, phosphorylates cyclin B1:Cdc2 on tyrosine 15 inactivating the complex.
References
LL Parker, H Piwnica-Worms, "Inactivation of the p34cdc2-cyclin B complex by the human WEE1 tyrosine kinase", Science, 257, 1992, 1955-7.
Reaction
4.2.2 G2/M DNA replication checkpoint
Description
The G2/M DNA replication checkpoint ensures that mitosis is not initiated until DNA replication is complete. If replication is blocked, the DNA
replication checkpoint signals to maintain Cyclin B - Cdc2 complexes in their T14Y15 phosphorylated and inactive state. This prevents the
phosphorylation of proteins involved in G2/M transition, and prevents mitotic entry.
Failure of these checkpoints results in changes of ploidy: in the case of mitosis without completion of DNA replication, aneuploidy of <2C will
result, and the opposite is true if DNA replication is completed more than once in a single cell cycle with an overall increase in ploidy. The
mechanism by which unreplicated DNA is first detected by the cell is unknown.
4.2.2.1 Myt-1 mediated phosphorylation of Cyclin B:Cdc2 complexes
Description
Myt1, which localizes preferentially to the endoplasmic reticulum and Golgi complex, phosphorylates Cdc2 on threonine 14 ( Liu et al., 1997).
References
F Liu, JJ Stanton, Z Wu, H Piwnica-Worms, "The human Myt1 kinase preferentially phosphorylates Cdc2 on threonine 14 and localizes to the
endoplasmic reticulum and Golgi complex.", Mol Cell Biol, 17, 1997, 571-83.
RN Booher, PS Holman, A Fattaey, "Human Myt1 is a cell cycle-regulated kinase that inhibits Cdc2 but not Cdk2 activity", J Biol Chem, 272,
1997, 22300-6.
Reaction
4.2.2.2 Wee1- mediated phosphorylation of Cyclin B1:phospho-Cdc2 complexes
Description
References
Reaction
4.2.3 Activation of ATR in response to replication stress
Authors
Editors
Description
Genotoxic stress caused by DNA damage or stalled replication forks can lead to genomic instability. To guard against such instability,
genotoxically-stressed cells activate checkpoint factors that halt or slow cell cycle progression. Among the pathways affected are DNA
replication by reduction of replication origin firing, and mitosis by inhibiting activation of cyclin-dependent kinases (Cdks). A key factor involved in
the response to stalled replication forks is the ATM- and rad3-related (ATR) kinase, a member of the phosphoinositide-3-kinase-related kinase
(PIKK) family. Rather than responding to particular lesions in DNA, ATR and its binding partner ATRIP (ATR-interacting protein) sense
replication fork stalling indirectly by associating with persistent ssDNA bound by RPA. These structures would be formed, for example, by
dissociation of the replicative helicase from the leading or lagging strand DNA polymerase when the polymerase encounters a DNA lesion that
blocks DNA synthesis. Along with phosphorylating the downstream transducer kinase Chk1 and the tumor suppressor p53, activated ATR
modifies numerous factors that regulate cell cycle progression or the repair of DNA damage. The persistent ssDNA also stimulates recruitment
of the RFC-like Rad17Ã¢â‚¬"Rfc2-5 alternative clamp-loading complex, which subsequently loads the Rad9-Hus1-Rad1 complex onto the DNA.
The latter '9-1-1' complex serves to facilitate Chk1 binding to the stalled replication fork, where Chk1 is phosphorylated by ATR and thereby
activated. Upon activation, Chk1 can phosphorylate additional substrates including the Cdc25 family of phosphatases (Cdc25A, Cdc25B, and
Cdc25C). These enzymes catalyze the removal of inhibitory phosphate residues from cyclin-dependent kinases (Cdks), allowing their activation.
In particular, Cdc25A primarily functions at the G1/S transition to dephosphorylate Cdk2 at Thr 14 and Tyr 15, thus positively regulating the
Cdk2-cyclin E complex for S-phase entry. Cdc25A also has mitotic functions. Phosphorylation of Cdc25A at Ser125 by Chk1 leads to Cdc25A
ubiquitination and degradation, thus inhibiting DNA replication origin firing. In contrast, Cdc25B and Cdc25C regulate the onset of mitosis
through dephosphorylation and activation of Cdk1-cyclin B complexes. In response to replication stress, Chk1 phosphorylates Cdc25B and
Cdc25C leading to Cdc25B/C complex formation with 14-3-3 proteins. As these complexes are sequestered in the cytoplasm, they are unable to
activate the nuclear Cdk1-cyclin B complex for mitotic entry.
These events are outlined in the figure. Persistent single-stranded DNA associated with RPA binds claspin (A) and ATR:ATRIP (B), leading to
claspin phosphorylation (C). In parallel, the same single-stranded DNA:RPA complex binds RAD17:RFC (D), enabling the loading of
RAD9:HUS1:RAD1 (9-1-1) complex onto the DNA (E). The resulting complex of proteins can then repeatedly bind (F) and phosphorylate (G)
CHK1, activating multiple copies of CHK1.
References
H Niida, M Nakanishi, "DNA damage checkpoints in mammals", Mutagenesis, 2005.
L Zou, SJ Elledge, "Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes", Science, 300, 2003, 1542-8.
CH McGowan, P Russell, "The DNA damage response: sensing and signaling", Curr Opin Cell Biol, 16, 2004, 629-33.
4.2.3.1 Stalling of DNA replication fork and RPA binding
Authors
Editors
Description
When a DNA replication fork encounters DNA lesions (e.g., cyclobutane pyrimidine dimers or alkylated bases) stalling of the replicative DNA
polymerase may occur. This can lead to dissociation or 'uncoupling' of the DNA polymerase from the DNA helicase and generation of long
regions of persistent ssDNA. Uncoupling can also occur in response to other genotoxic stresses such as reduced dNTP pools caused by
hydroxyurea treatment which inhibits cellular ribonucleotide diphosphate reductase. The exposed ssDNA is bound by the single-stranded DNA
binding protein RPA. The persistent nature of this RPA-ssDNA complex (as opposed to a more-transient complex found at an active replication
fork) allows it to serve as a signal for replication stress that can be recognized by the ATR-ATRIP and Rad17-Rfc2-5 complexes.
RPA associates with ssDNA in distinct complexes that can be distinguished by the length of ssDNA occluded by each RPA molecule. These
complexes reflect the progressive association of distinct DNA-binding domains present in the RPA heterotrimeric structure. Binding is coupled to
significant conformational changes within RPA that are observable at the microscopic level. Presumably, the different conformations of free and
ssDNA-bound RPA allow the protein to selectively interact with factors such as ATR-ATRIP when bound to DNA.
References
MS Wold, "Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism.", Annu Rev
Biochem, 66, 1997, 61-92.
TS Byun, M Pacek, MC Yee, JC Walter, KA Cimprich, "Functional uncoupling of MCM helicase and DNA polymerase activities activates the
ATR-dependent checkpoint", Genes Dev, 19, 2005, 1040-52.
LJ Blackwell, JA Borowiec, "Human replication protein A binds single-stranded DNA in two distinct complexes", Mol Cell Biol, 14, 1994,
3993-4001.
JM Sogo, M Lopes, M Foiani, "Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects", Science, 297,
2002, 599-602.
M Cordeiro-Stone, AM Makhov, LS Zaritskaya, JD Griffith, "Analysis of DNA replication forks encountering a pyrimidine dimer in the template to
the leading strand", J Mol Biol, 289, 1999, 1207-18.
C Iftode, Y Daniely, JA Borowiec, "Replication protein A (RPA): the eukaryotic SSB.", Crit Rev Biochem Mol Biol, 34, 1999, 141-80.
E Raderschall, EI Golub, T Haaf, "Nuclear foci of mammalian recombination proteins are located at single-stranded DNA regions formed after
DNA damage", Proc Natl Acad Sci U S A, 96, 1999, 1921-6.
M Lopes, M Foiani, JM Sogo, "Multiple mechanisms control chromosome integrity after replication fork uncoupling and restart at irreparable UV
lesions", Mol Cell, 21, 2006, 15-27.
Reaction
4.2.3.2 Binding of ATR-ATRIP to the RPA-ssDNA complex
Authors
Editors
Description
ATR kinase activity is stimulated upon binding of the ATR-ATRIP complex to an RPA-ssDNA complex. ATR can subsequently phosphorylate
and activate the checkpoint kinase Chk1, allowing further amplification of the checkpoint signal. The ATR and Chk1 kinases then modify a
variety of factors that can lead to stabilization of stalled DNA replication forks, inhibition of origin firing, inhibition of cell cycle progression,
mobilization of DNA repair factors, and induction of apoptosis. This checkpoint signaling mechanism is highly conserved in eukaryotes, and
homologues of ATR and ATRIP are found in such organisms as S. cerevisiae (Mec1 and Ddc2, respectively), S. pombe (rad3 and rad26,
respectively), and X. laevis (Xatr and Xatrip, respectively).
The ATR (ATM- and rad3-related) kinase is an essential checkpoint factor in human cells. In response to replication stress (i.e., stresses that
cause replication fork stalling) or ultraviolet radiation, ATR becomes active and phosphorylates numerous factors involved in the checkpoint
response including the checkpoint kinase Chk1. ATR is invariably associated with ATRIP (ATR-interacting protein) in human cells. Depletion of
ATRIP by siRNA causes a loss of ATR protein without affecting ATR mRNA levels indicating that complex formation stabilizes the ATR protein.
ATRIP is also a substrate for the ATR kinase, but modification of ATRIP does not significantly regulate the recruitment of ATR-ATRIP to sites of
damage, the activation of Chk1, or the modification of p53.
While the ATR-ATRIP complex binds only poorly to RPA complexed with ssDNA lengths of 30 or 50 nt, binding is significantly enhanced in the
presence of a 75 nt ssDNA molecule. Complex formation is primarily mediated by physical interaction between ATRIP and RPA. Multiple
elements within the ATRIP molecule can bind to the RPA-ssDNA complex, including residues 1-107 (highest affinity), 218-390, and 390-791
(lowest affinity). Although the full-length ATRIP is unable to bind ssDNA, an internal region (108-390) can weakly bind ssDNA when present in
rabbit reticulocyte lysates. ATR can bind to the ssDNA directly independent of RPA, but this binding is inhibited by ATRIP. Upon binding, the
ATR kinase becomes activated and can directly phosphorylate substrates such as Rad17.
References
Z You, L Kong, J Newport, "The role of single-stranded DNA and polymerase alpha in establishing the ATR, Hus1 DNA replication checkpoint", J
Biol Chem, 277, 2002, 27088-93.
E Itakura, K Umeda, E Sekoguchi, H Takata, M Ohsumi, A Matsuura, "ATR-dependent phosphorylation of ATRIP in response to genotoxic
stress", Biochem Biophys Res Commun, 323, 2004, 1197-202.
F al-Khodairy, E Fotou, KS Sheldrick, DJ Griffiths, AR Lehmann, AM Carr, "Identification and characterization of new elements involved in
checkpoint and feedback controls in fission yeast", Mol Biol Cell, 5, 1994, 147-60.
V Paciotti, M Clerici, G Lucchini, MP Longhese, "The checkpoint protein Ddc2, functionally related to S. pombe Rad26, interacts with Mec1 and
is regulated by Mec1-dependent phosphorylation in budding yeast.", Genes Dev, 14, 2000, 2046-59.
HL Ball, JS Myers, D Cortez, "ATRIP binding to replication protein A-single-stranded DNA promotes ATR-ATRIP localization but is dispensable
for Chk1 phosphorylation", Mol Biol Cell, 16, 2005, 2372-81.
A Kumagai, SM Kim, WG Dunphy, "Claspin and the activated form of ATR-ATRIP collaborate in the activation of Chk1", J Biol Chem, 279, 2004,
49599-608.
Y Namiki, L Zou, "ATRIP associates with replication protein A-coated ssDNA through multiple interactions", Proc Natl Acad Sci U S A, 103,
2006, 580-5.
RJ Edwards, NJ Bentley, AM Carr, "A Rad3-Rad26 complex responds to DNA damage independently of other checkpoint proteins", Nat Cell
Biol, 1, 1999, 393-8.
A Jazayeri, J Falck, C Lukas, J Bartek, GC Smith, J Lukas, SP Jackson, "ATM- and cell cycle-dependent regulation of ATR in response to DNA
double-strand breaks", Nat Cell Biol, 8, 2006, 37-45.
Reaction
4.2.3.3 Recruitment of Rad17-RFC complex to DNA
Authors
Editors
Description
The Rad17-RFC complex is involved in an early stage of the genotoxic stress response. The major function of the protein complex is to load the
Rad9-Hus1-Rad1 (9-1-1) complex onto DNA at sites of damage and/or stalled replication forks. This reaction is conceptually similar to the
loading of the PCNA sliding clamp onto DNA by RFC. The association of the Rad17-RFC complex with ssDNA or gapped or primed DNA is
significantly stimulated by RPA, but not by the heterologous E. coli SSB. Loading of the human 9-1-1 complex onto such DNA templates is also
strongly stimulated by cognate RPA, but not yeast RPA. Although Rad17 and Rad9 are substrates of the ATR kinase activity, loading of the
Rad17 and 9-1-1 complexes onto DNA occurs independent of ATR.
The Rad17-RFC complex is a heteropentamer structurally similar to RFC. The complex contains the four smaller RFC subunits (Rfc2 [p37], Rfc3
[p36], Rfc4 [p40], and Rfc5 [p38]) and the 75 kDa Rad17 subunit in place of the Rfc1 [p140] subunit. The Rad17 complex contains a weak
ATPase that is slightly stimulated by primed DNA. Along with binding the 9-1-1 complex and RPA, the Rad17-RFC complex interacts with human
MCM7 protein. Each of these interactions is critical for Chk1 activation.
The Rad17 subunit is conserved evolutionarily with the protein showing 49% identity at the amino acid level with the S. pombe rad17 protein.
Targeted deletion of the N-terminal region of mouse Rad17 leads to embryonic lethality, strongly suggesting that human Rad17 is also essential
for long-term viability.
Rad17-RFC complex associates with DNA substrates containing ssDNA regions including gapped or primed DNA in an ATP-independent
reaction. Loading of the Rad9-Hus1-Rad1 (9-1-1) complex occurs preferentially on DNA substrates containing a 5' recessed end. This contrasts
with the loading of PCNA by RFC which preferentially occurs on DNA with 3' recessed ends.
References
ER Parrilla-Castellar, SJ Arlander, L Karnitz, "Dial 9-1-1 for DNA damage: the Rad9-Hus1-Rad1 (9-1-1) clamp complex", DNA Repair (Amst), 3,
2004, 1009-14.
V Ellison, B Stillman, "Biochemical characterization of DNA damage checkpoint complexes: clamp loader and clamp complexes with specificity
for 5' recessed DNA", PLoS Biol, 1, 2003, E33.
VP Bermudez, LA Lindsey-Boltz, AJ Cesare, Y Maniwa, JD Griffith, J Hurwitz, A Sancar, "Loading of the human 9-1-1 checkpoint complex onto
DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro", Proc Natl Acad Sci U S A, 100, 2003, 1633-8.
L Zou, D Liu, SJ Elledge, "Replication protein A-mediated recruitment and activation of Rad17 complexes", Proc Natl Acad Sci U S A, 100, 2003,
13827-32.
Reaction
4.2.3.4 Recruitment of the Rad9-Hus1-Rad1 complex to DNA
Authors
Editors
Description
The 9-1-1 complex is a heterotrimeric ring-shaped structure that is loaded onto DNA by the Rad17-RFC complex. In vitro studies indicate that
loading is preferred onto DNA substrates containing ssDNA gaps that presumably resemble structures found upon replication fork stalling and
DNA polymerase/helicase uncoupling. The Rad17-RFC and 9-1-1 complexes are structurally similar to the RFC (replication factor C) clamp
loader and PCNA sliding clamp, respectively, and similar mechanisms are thought to be used during loading of the 9-1-1 complex and PCNA.
Upon loading, the 9-1-1 complex can recruit Chk1 onto sites of replication fork uncoupling or DNA damage.
The purified Rad17 and Rad9-Hus1-Rad1 (9-1-1) complexes can form a stable co-complex in the presence of ATP, using Rad17-Rad9
interactions. From computer modeling studies, the Rad17 subunit of the complex is also proposed to interact with the C-terminus of Rad1, p36
with the C-terminus of Hus1, and p38 with the C-terminus of Rad9. A major known function of the 9-1-1 complex is to recruit Chk1 to stalled
replication forks for activation by ATR. However, the presence of the 9-1-1 complex also alters the ability of Rad17 to become phosphorylated,
perhaps suggesting that 9-1-1 may regulate the recruiment of additional ATR substrates. The 9-1-1 complex has also been found to interact with
base excision repair factors human DNA polymerase beta, flap endonuclease FEN1, and the S. pombe MutY homolog (SpMYH), indicating that
9-1-1 also plays a direct role in DNA repair.
References
KM Hopkins, X Wang, A Berlin, H Hang, HM Thaker, HB Lieberman, "Expression of mammalian paralogues of HRAD9 and Mrad9 checkpoint
control genes in normal and cancerous testicular tissue", Cancer Res, 63, 2003, 5291-8.
J Majka, PM Burgers, "The PCNA-RFC families of DNA clamps and clamp loaders", Prog Nucleic Acid Res Mol Biol, 78, 2004, 227-60.
V Ellison, B Stillman, "Biochemical characterization of DNA damage checkpoint complexes: clamp loader and clamp complexes with specificity
for 5' recessed DNA", PLoS Biol, 1, 2003, E33.
C Venclovas, ME Colvin, MP Thelen, "Molecular modeling-based analysis of interactions in the RFC-dependent clamp-loading process", Protein
Sci, 11, 2002, 2403-16.
HB Lieberman, KM Hopkins, M Nass, D Demetrick, S Davey, "A human homolog of the Schizosaccharomyces pombe rad9+ checkpoint control
gene", Proc Natl Acad Sci U S A, 93, 1996, 13890-5.
H Hang, HB Lieberman, "Physical interactions among human checkpoint control proteins HUS1p, RAD1p, and RAD9p, and implications for the
regulation of cell cycle progression", Genomics, 65, 2000, 24-33.
VP Bermudez, LA Lindsey-Boltz, AJ Cesare, Y Maniwa, JD Griffith, J Hurwitz, A Sancar, "Loading of the human 9-1-1 checkpoint complex onto
DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro", Proc Natl Acad Sci U S A, 100, 2003, 1633-8.
L Zou, D Liu, SJ Elledge, "Replication protein A-mediated recruitment and activation of Rad17 complexes", Proc Natl Acad Sci U S A, 100, 2003,
13827-32.
Reaction
4.2.3.5 Loading of claspin onto DNA during replication origin firing
Authors
Editors
Description
Claspin is loaded onto DNA replication origins during replication initiation. Studies in Xenopus egg extracts indicate claspin loading requires the
presence of Cdc45, a factor that promotes the initial unwinding of the origin DNA in the presence of Cdk2. This step is followed by RPA binding
which is a prerequisite for recruitment of PCNA and DNA polymerases alpha and delta. As RPA is not required for claspin binding, it is
postulated that claspin binds at the time of initial origin unwinding but prior to the initiation of DNA synthesis. Claspin would then continue to
associate with replication fork machinery where it can serve as a checkpoint sensor protein. Even though associated with the replication fork,
claspin is not an essential DNA replication factor.
Studies of Xenopus claspin indicate that it can physically associate with cognate Cdc45, DNA polymerase epsilon, RPA, RFC, and Rad17-RFC
on chromatin. Studies of purified human claspin indicate that it binds with high affinity to branched (or forked) DNA structures that resemble
stalled replication forks. Electron microscopy of these complexes indicates that claspin binds as a ring-like structure near the branch. The protein
is hypothesized to encircle the DNA at these sites.
References
J Lee, A Kumagai, WG Dunphy, "Claspin, a Chk1-regulatory protein, monitors DNA replication on chromatin independently of RPA, ATR, and
Rad17", Mol Cell, 11, 2003, 329-40.
A Kumagai, WG Dunphy, "Repeated phosphopeptide motifs in Claspin mediate the regulated binding of Chk1", Nat Cell Biol, 5, 2003, 161-5.
F Sar, LA Lindsey-Boltz, D Subramanian, DL Croteau, SQ Hutsell, JD Griffith, A Sancar, "Human claspin is a ring-shaped DNA-binding protein
with high affinity to branched DNA structures", J Biol Chem, 279, 2004, 39289-95.
CC Chini, J Chen, "Claspin, a regulator of Chk1 in DNA replication stress pathway", DNA Repair (Amst), 3, 2004, 1033-7.
J Lee, DA Gold, A Shevchenko, A Shevchenko, WG Dunphy, "Roles of replication fork-interacting and Chk1-activating domains from Claspin in
a DNA replication checkpoint response", Mol Biol Cell, 16, 2005, 5269-82.
Reaction
4.2.3.6 Activation of claspin
Authors
Editors
Description
Claspin is a replication fork-associated protein important for Chk1 activation. Claspin loads onto the fork during replication origin firing and
travels with the fork during DNA synthesis. Upon fork uncoupling and ATR-ATRIP binding to persistent ssDNA, the activated ATR kinase
phosphorylates claspin at two primary sites. Modification increases the affinity of claspin for Chk1. Studies of human or Xenopus claspin indicate
that phosphorylation of both sites is essential for significant claspin-Chk1 association. Following claspin modification by ATR, Chk1 can be
transiently recruited to the stalled replication fork for subsequent phosphorylation and activation by ATR. Activation of Chk1 allows modification
of additional downstream targets, thus amplifying the checkpoint signal. While much of the mechanistic information concerning claspin action
has been obtained using Xenopus laevis egg extracts and Xenopus claspin, factors with similar activity have been found in various eukaryotic
species including S. cerevisiae (MRC1), S. pombe (mrc1), and humans.
Activated ATR phosphorylates human claspin on two sites, threonine 916 and serine 945.
References
J Lee, A Kumagai, WG Dunphy, "Claspin, a Chk1-regulatory protein, monitors DNA replication on chromatin independently of RPA, ATR, and
Rad17", Mol Cell, 11, 2003, 329-40.
A Kumagai, WG Dunphy, "Repeated phosphopeptide motifs in Claspin mediate the regulated binding of Chk1", Nat Cell Biol, 5, 2003, 161-5.
CC Chini, J Chen, "Claspin, a regulator of Chk1 in DNA replication stress pathway", DNA Repair (Amst), 3, 2004, 1033-7.
CA Clarke, PR Clarke, "DNA-dependent phosphorylation of Chk1 and Claspin in a human cell-free system", Biochem J, 388, 2005, 705-12.
Reaction
4.2.3.7 Recruitment and activation of Chk1
Authors
Editors
Description
Chk1 is a checkpoint kinase activated during genotoxic stress. Like ATR, Chk1 is essential for viability in mammals. Targeted gene disruption in
mice shows that loss of Chk1 causes peri-implantation embryonic lethality. Even though ATR-ATRIP not bound to ssDNA can phosphorylate
Chk1, Chk1 activation is greatly enhanced when recruited to stalled replication forks by physical interaction with a modified form of claspin and
the Rad9-Hus1-Rad1 sliding clamp. Activation of Chk1 occurs following phosphorylation of two sites (serine 317 and serine 345). Mutational
analysis indicates that modification of both sites is essential for maximal kinase activity, while phosphorylation of only a single site causes only
weak activation of Chk1. Following phosphorylation, Chk1 can diffuse away from the complex to further amplify the checkpoint signal. ATR
appears to be the primary kinase activating Chk1 as conditions that activate ATR (ultraviolet irradiation or treatment with hydroxyurea) also
activate Chk1. Stresses that activate ATM, e.g., ionizing irradiation, do not cause significant Chk1 activation. While the ATR and ATM pathways
are distinct, there is interplay between the two. For example, double-strand DNA breaks can be processed in an ATM-dependent manner to
generate structures that can cause ATR and hence Chk1 activation. The ATR and ATM pathways also have mechanistic similarities. Analogous
to the Chk1 kinase existing downstream of ATR, the Chk2 checkpoint kinase is modified and activated by ATM. Although having distinct
structures, Chk1 and Chk2 also have overlapping targets with some substrate sites phosphorylatable by both kinases (e.g., serine 20 of p53).
References
VA Smits, PM Reaper, SP Jackson, "Rapid PIKK-Dependent Release of Chk1 from Chromatin Promotes the DNA-Damage Checkpoint
Response", Curr Biol, 16, 2006, 150-9.
CC Chini, J Chen, "Human claspin is required for replication checkpoint control", J Biol Chem, 278, 2003, 30057-62.
Q Liu, S Guntuku, XS Cui, S Matsuoka, D Cortez, K Tamai, G Luo, S Carattini-Rivera, F DeMayo, A Bradley, LA Donehower, SJ Elledge, "Chk1
is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint.", Genes Dev, 14, 2000, 1448-59.
Y Chen, Y Sanchez, "Chk1 in the DNA damage response: conserved roles from yeasts to mammals", DNA Repair (Amst), 3, 2004, 1025-32.
K Uto, D Inoue, K Shimuta, N Nakajo, N Sagata, "Chk1, but not Chk2, inhibits Cdc25 phosphatases by a novel common mechanism", EMBO J,
23, 2004, 3386-96.
CS Sorensen, RG Syljuasen, J Lukas, J Bartek, "ATR, Claspin and the Rad9-Rad1-Hus1 complex regulate Chk1 and Cdc25A in the absence of
DNA damage", Cell Cycle, 3, 2004, 941-5.
H Zhao, H Piwnica-Worms, "ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1", Mol Cell Biol, 21,
2001, 4129-39.
H Takai, K Tominaga, N Motoyama, YA Minamishima, H Nagahama, T Tsukiyama, K Ikeda, K Nakayama, M Nakanishi, K Nakayama, "Aberrant
cell cycle checkpoint function and early embryonic death in Chk1(-/-) mice", Genes Dev, 14, 2000, 1439-47.
JS Myers, D Cortez, "Rapid activation of ATR by ionizing radiation requires ATM and Mre11", J Biol Chem, 2006.
Reaction
4.2.3.8 Phosphorylation of Cdc25A at Ser-123 by Chk1
Description
Detection of DNA damage caused by ionizing radiation results in the phosphorylation of Cdc25A at Ser-123 by Chk1, inhibiting Cdc25A.
References
H Zhao, JL Watkins, H Piwnica-Worms, "Disruption of the checkpoint kinase 1/cell division cycle 25A pathway abrogates ionizing
radiation-induced S and G2 checkpoints.", Proc Natl Acad Sci U S A, 99, 2002, 14795-800.
Reaction
4.2.3.9 Phosphorylation of Cdc25C at Ser 216 by Chk1
Authors
Matthews, L, 2003-08-05.
Editors
Matthews, L, 2006-07-10.
Description
Phosphorylation of Cdc25C at Ser 216 results in both the inhibition of Cdc25C phosphatase activity and the creation of a 14-3-3 docking site
(Peng et al. 1997).
References
CY Peng, PR Graves, RS Thoma, Z Wu, AS Shaw, H Piwnica-Worms, "Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by
phosphorylation of Cdc25C on serine-216.", Science, 277, 1997, 1501-5.
Reaction
4.3 Mitotic Spindle Checkpoint
Authors
Yen, T, 2004-05-05.
Description
The mitotic checkpoint or spindle assembly checkpoint is an evolutionarily conserved mechanism that ensures that cells with misaligned
chromosomes do not exit mitosis and divide to form aneuploid cells. As chromosome attachment to the spindle microtubules is a stochastic
process, not all chromosomes achieve alignment at the spindle equator at the same time. It is therefore essential that even a single unaligned
chromosome can prevent the onset of anaphase. The ability of the checkpoint to monitor the status of chromosome alignment is achieved by
assigning checkpoint proteins to the kinetochore, a macromolecular complex that resides at centromeres of chromosomes that establishes
connections with spindle microtubules.
The checkpoint proteins monitor, in an unknown way, the mechanical activities between kinetochore-associated proteins and microtubules.
Defects in mechanical activities at kinetochores activate the resident checkpoint proteins to initiate a signal that is amplified throughout the cell
that ultimately prevents the activation of the proteolytic process that is required for sister chromatid separation and the onset of anaphase.
References
A Musacchio, KG Hardwick, "The spindle checkpoint: structural insights into dynamic signalling", Nat Rev Mol Cell Biol, 3, 2002, 731-41.
GK Chan, TJ Yen, "The mitotic checkpoint: a signaling pathway that allows a single unattached kinetochore to inhibit mitotic exit", Prog Cell
Cycle Res, 5, 2003, 431-9.
4.3.1 Initiation of checkpoint signal from defective kinetochores
Authors
Yen, T, 2004-05-05.
Description
Kinetochores of unaligned chromosomes differ from those of aligned chromosomes in two ways. Kinetochores of aligned chromosomes are
saturated with between 20 to 30 microtubules. In addition, poleward directed forces exerted at each sister kinetochore generates tension
between them. Unaligned kinetochores on the other hand, are not saturated with microtubules and are not under tension. The mitotic checkpoint
detects the presence of unattached kinetochores rather than monitoring for the presence of attached kinetochores. Consequently, unattached
kinetochores emit an inhibitory signal that inhibits the biochemical events that are required to initiate the onset of anaphase. The mechanism by
which this inhibitory signal is generated at unattached kinetochores has not precisely been determined but the signal is generated as a result of
the lack of microtubule occupancy and kinetochore tension.
References
4.3.1.1 Detection of kinetochores lacking bipolar connections to spindle microtubules
Authors
Yen, T, 2004-05-05.
Description
Checkpoint proteins are thought to detect kinetochores that have not established proper bipolar attachments by monitoring microtubule
occupancy and tension at kinetochores. Although microtubule attachment is intimately linked to kinetochore tension, these two parameters are
separately monitored by the checkpoint because kinetochores can establish aberrant interactions despite being fully saturated with microtubules.
Syntelic attachments occur where sister kinetochores are attached to a single pole. Merotelic attachments arise when one of the sister
kinetochores of a bipolar attached chromosome becomes attached to microtubules from opposite poles. In both cases, the kinetochores are fully
saturated with microtubules. If microtubule occupancy is the only parameter that is monitored by the checkpoint, syntelic and merotelic
attachments would escape detection by the checkpoint. These aberrant attachments would result in chromosome non-disjunction and lagging
chromosomes that become trapped between the dividing cells. Syntelic and merotelic attachments are detected by the cell because these
kinetochores lack the proper level of tension despite being saturated with microtubules. Thus, a tension-sensitive monitoring system prevents the
accumulation of merotelic and syntelic attachments.
There is a semantics argument as to whether tension-sensing proteins should be considered as checkpoint proteins. There is evidence that
these proteins respond to inappropriate levels of tension by releasing microtubule attachments. The loss of microtubule attachments is then
detected by checkpoint proteins which then arrest cells in mitosis.
References
4.3.1.1.1 Detection of insufficient microtubule occupancy at kinetochore
Description
Entry of a cell into anaphase requires the attachment of all chromosomes in a bipolar manner. Cells monitor both the attachment of microtubules
to kinetochores and the tension that is exerted at kinetochores upon bipolar attachment (Musacchio and Hardwick 2002).
References
4.3.1.1.2 Detection of insufficient tension at kinetochore
Authors
Yen, T, 2004-05-05.
Description
Although the mechanism by which insufficient tension at kinetochores is detected has not been determined, a tension-sensitive monitoring
system appears to prevent the accumulation of merotelic and syntelic attachments. There is evidence that these proteins respond to
inappropriate levels of tension by releasing microtubule attachments. The loss of microtubule attachments is then detected by checkpoint
proteins which then arrest cells in mitosis.
References
4.3.2 Amplification of signal from the kinetochores
Authors
Yen, T, 2004-05-05.
Description
A single unattached kinetochore is capable of preventing cells from exiting mitosis. The mitotic checkpoint provides a way for a localized defect
to affect the global biochemical status of the cell. In principle, the signal that is generated at an unattached kinetochore diffuses throughout the
cell to affect its target. There are currently two models for how this is achieved. One model is based on the observation that the Mad2 checkpoint
protein binds and is rapidly released from unattached kinetochores. The kinetochore is believed to act as a catalyst that converts Mad2 into an
inhibitory state that diffuses throughout the cell upon its release from the kinetochore. A second model proposes that the signal is amplified by a
kinase cascade much like a conventional signal transduction pathway. This kinase cascade is believed to be comprised of the checkpoint
kinases, hBUBR1, hBUB1, hMPS1.
References
Cycle Res, 5, 2003, 431-9.
4.3.2.1 Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal
Authors
Yen, T, 2004-05-05.
Description
The signal from unattached kinetochores is amplified through a Mad2 inhibitory signal that is propagated by the binding of Mad1 to the
kinetochore, the association of Mad2 with Mad1, the conversion of Mad2 conformation to an inhibitory form through its association with Mad1
and finally the release of the inhibitory form of Mad2 from the kinetochore.
References
Cycle Res, 5, 2003, 431-9.
4.3.2.1.1 Mad1 binds kinetochore
Authors
Yen, T, 2004-05-05.
Description
The association of Mad1 with the kinetochore is the first step in the process of Mad2 mediated amplification of the signal from defective
kinetochores.
References
MS Campbell, GK Chan, TJ Yen, "Mitotic checkpoint proteins HsMAD1 and HsMAD2 are associated with nuclear pore complexes in interphase",
J Cell Sci, 114, 2001, 953-63.
Reaction
4.3.2.1.2 MAD2 associates with the Mad1 kinetochore complex
Authors
Yen, T, 2004-05-05.
Description
Mad2 is recruited to the kinetochore through an interaction with Mad1.
References
MS Campbell, GK Chan, TJ Yen, "Mitotic checkpoint proteins HsMAD1 and HsMAD2 are associated with nuclear pore complexes in interphase",
J Cell Sci, 114, 2001, 953-63.
Reaction
4.3.2.1.3 MAD2 converted to an inhibitory state via interaction with Mad1
Authors
Yen, T, 2004-05-05.
Description
In vitro structural studies have shown that Mad2 undergoes a major conformational change upon binding to Mad1. This conformational change is
postulated to activate Mad2 into a high affinity state which can bind and sequester Cdc20 from the APC.
References
X Luo, Z Tang, J Rizo, H Yu, "The Mad2 spindle checkpoint protein undergoes similar major conformational changes upon binding to either
Mad1 or Cdc20", Mol Cell, 9, 2002, 59-71.
Reaction
4.3.2.1.4 Release of activated MAD2 from kinetochores
Authors
Yen, T, 2004-05-05.
Description
The mechanism by which the conformationally altered inhibitory form of Mad2 is released from its association with Mad1 at the kinetochore is not
known. Mad1 and Cdc20 have a common 10 residue Mad2 binding motif. Therefore, one possibility is that Mad2 is transferred competitively
from Mad1 to Cdc20 (Luo et al., 2002; Sironi et al., 2002).
References
L Sironi, M Mapelli, S Knapp, A De Antoni, KT Jeang, A Musacchio, "Crystal structure of the tetrameric Mad1-Mad2 core complex: implications
of a 'safety belt' binding mechanism for the spindle checkpoint", EMBO J, 21, 2002, 2496-506.
Reaction
4.3.2.2 Amplification of signal from unattached kinetochore via a checkpoint kinase cascade
Authors
Yen, T, 2004-05-05.
Description
An alternate model for the amplification of signal from unattached kinetochores proposes that the signal is amplified by a kinase cascade much
like a conventional signal transduction pathway. This kinase cascade is believed to be comprised of the checkpoint kinases, hBUBR1, hBUB1,
hMPS1.
References
Cycle Res, 5, 2003, 431-9.
4.3.3 Inhibition of the proteolytic activity of APC/C required for the onset of anaphase by mitotic spindle
checkpoint components
Authors
Yen, T, 2004-05-05.
Reviewers
Peters, JM, 2006-03-27.
Description
The target of the mitotic checkpoint is the Anaphase Promoting Complex/Cyclosome (APC/C) an E3 ubiquitin ligase that targets proteins whose
destruction is essential for mitotic exit. Currently, there are two proposed mechanism by which inhibition of the APC/C is achieved. These
mechanisms differ depending on the mechanism of signal transduction. The APC/C may be inhibited directly by association with the Mitotic
Checkpoint Complex (MCC) or through the sequestration of its activator, Cdc20.
References
Cycle Res, 5, 2003, 431-9.
4.3.3.1 Inactivation of APC/C via CDC20 sequestration
Authors
Yen, T, 2004-05-05.
Editors
Matthews, L, 2006-02-17.
Reviewers
Peters, JM, 2006-03-27.
Description
In the sequestration model, the Mad2 molecules that dissociate from unattached kinetochores are perceived to bind to Cdc20, a protein that
recruits specific substrates to the APC/C. Consequently, Mad2 indirectly inhibits the APC/C by sequestering its activator, Cdc20. This requires
interaction between Mad1 and Mad2. Cdc20 and Mad1 bind to the same site on Mad2.
Reaction
4.3.3.2 Inactivation of APC/C via direct inhibition of the APC/C complex
Authors
Yen, T, 2004-05-05.
Reviewers
Peters, JM, 2006-03-27.
Description
In the direct inhibition model, the cytosolic Mitotic Checkpoint Complex, consisting of hBUBR1, hBUB3, Cdc20 and Mad2, directly inhibits APC/C
by binding to it.
References
V Sudakin, GK Chan, TJ Yen, "Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and
MAD2", J Cell Biol, 154, 2001, 925-36.
Cycle Res, 5, 2003, 431-9.
G Fang, H Yu, MW Kirschner, "The checkpoint protein MAD2 and the mitotic regulator CDC20 form a ternary complex with the
anaphase-promoting complex to control anaphase initiation", Genes Dev, 12, 1998, 1871-83.
4.3.3.2.1 Formation of the MCC complex
Authors
Yen, T, 2004-05-05.
Reviewers
Peters, JM, 2006-03-27.
Description
Upon release from the kinetochore, Mad2 associates with Cdc20, hBUBR1, and hBUB3 to form the Mitotic Checkpoint Complex (MCC).
Assembly of this complex does not depend on kinetochores but this complex can only inhibit APC/C that has undergone mitotic modifications.
References
MAD2", J Cell Biol, 154, 2001, 925-36.
Reaction
4.3.3.2.2 Binding of the MCC complex to the APC/C complex
Authors
Yen, T, 2004-05-05.
Editors
Matthews, L, 2006-03-07.
Reviewers
Peters, JM, 2006-03-27.
Description
In the direct inhibition model, association of the MCC with APCC results in the inactivation of APC/C. However, the affinity between MCC and
APC/C is not high, so that the inhibition is readily reversible. The role of unattached kinetochores is to sensitize the APC/C to prolonged
inhibition by the MCC.
References
MAD2", J Cell Biol, 154, 2001, 925-36.
Reaction
5 Cell Cycle, Mitotic
Authors
O'Connell, M, Walworth, N, Bosco, G, 2005-01-01.
Editors
Matthews, L, Gopinathrao, G, 0000-00-00.
Reviewers
Manfredi, J, 0000-00-00.
Description
The replication of the genome and the subsequent segregation of chromosomes into daughter cells are controlled by a series of events
collectively known as the cell cycle. DNA replication is carried out during a discrete temporal period known as the S (synthesis)-phase, and
chromosome segregation occurs during a massive reorganization to cellular architecture at mitosis. Two gap-phases separate these major cell
cycle events: G1 between mitosis and S-phase, and G2 between S-phase and mitosis. In the development of the human body, cells can exit the
cell cycle for a period and enter a quiescent state known as G0, or terminally differentiate into cells that will not divide again, but undergo
morphological development to carry out the wide variety of specialized functions of individual tissues.
A family of protein serine/threonine kinases known as the cyclin-dependent kinases (CDKs) controls progression through the cell cycle. As the
name suggests, the activity of the catalytic subunit is dependent on binding to a cyclin partner. The human genome encodes several cyclins and
several CDKs, with their names largely derived from the order in which they were identified. The oscillation of cyclin abundance is one important
mechanism by which these enzymes phosphorylate key substrates to promote events at the relevant time and place. Additional regulatory
proteins and post-translational modifications ensure that CDK activity is precisely regulated, frequently confined to a narrow window of activity.
5.1 G1 Phase
Description
Early cell cycle progression in G1 is under the control of the D-type cyclins together with Cdk4 and Cdk6. An important target for these CDKs is
the Retinoblastoma (Rb) protein, which when phosphorylated promotes cell cycle progression by releasing E2F transcription factors that
transactivate several important genes for later cell cycle events. The formation of Cyclin D - Cdk4/6 complexes is promoted by two proteins,
p21Cip1/Waf1 and p27kip1, and their activity can be inhibited by the binding of several small CDK-inhibitory proteins (CKIs): p15INK4B,
p16INK4A, p18INK4C and p19INK4D.
5.1.1 Cyclin D associated events in G1
Description
Three D-type cyclins are essential for progression from G1 to S-phase. These D cyclins bind to and activate both CDK4 and CDK6. The
formation of all possible complexes between the D-type cyclins and CDK4/6 is promoted by the proteins, p21(CIP1/WAF1) and p27(KIP1). The
cyclin-dependent kinases are then activated due to phosphorylation by CAK. The cyclin-dependent kinases phosphorylate the Rb protein leading
to release of the E2F transcription factors, which then results in proper G1/S transition. The binding and sequestration of p27Kip may also
contribute to the activation of CDK2-cyclin E/CDK2-cyclin A complexes at the G1/S transition (Yew et al., 2001).
5.1.1.1 Formation of Cyclin D:Cdk4/6 complexes
Editors
Matthews, L, 2006-07-03.
Description
The formation of CyclinD:CDK4/6 complexes is promoted by two proteins, p21Cip1/Waf1 and p27kip1. Activity of the cyclin-dependent kinases 4
and 6 can be inhibited by the binding of several small CDK-inhibitory proteins (CKIs).
References
J LaBaer, MD Garrett, LF Stevenson, JM Slingerland, C Sandhu, HS Chou, A Fattaey, E Harlow, "New functional activities for the p21 family of
CDK inhibitors.", Genes Dev, 11, 1997, 847-62.
Reaction
5.1.1.2 Translocation of Cyclin D:Cdk4/6 complexes from the cytoplasm to the nucleus
Editors
Matthews, L, 2006-07-10.
Reaction
5.1.1.3 Phosphorylation of Cyclin D:Cdk4/6 complexes
Authors
O'Connell, M, Walworth, N, 2003-06-05.
Editors
Matthews, L, 2005-10-07.
Reviewers
Manfredi, J, 0000-00-00.
Description
Phosphorylation of CDK4 on Thr172 depends on prior D-type cyclin binding (Bockstaele et al., 2006).
References
L Bockstaele, H Kooken, F Libert, S Paternot, JE Dumont, Y de Launoit, PP Roger, K Coulonval, "Regulated activating Thr172 phosphorylation
of cyclin-dependent kinase 4(CDK4): its relationship with cyclins and CDK "inhibitors"", Mol Cell Biol, 26, 2006, 5070-85.
Reaction
5.1.1.4 Cyclin D:Cdk4/6 mediated phosphorylation of Rb and dissociation of Rb from the Rb:E2F:DP-1 complexes
Description
Cyclin D:Cdk4 mediated phosphorylation of RB releases RB from the transcriptional regulator E2F and activates E2F function.
References
L Connell-Crowley, JW Harper, DW Goodrich, "Cyclin D1/Cdk4 regulates retinoblastoma protein-mediated cell cycle arrest by site-specific
phosphorylation", Mol Biol Cell, 8, 1997, 287-301.
Reaction
5.1.2 G1-Specific Transcription
Description
G1 phase- specific transcription will be described in a future release.

5.2 G1/S Transition
Description
Cyclin E - Cdk2 complexes control the transition from G1 into S-phase. In this case, the binding of p21Cip1/Waf1 or p27kip1 is inhibitory.
Important substrates for Cyclin E - Cdk2 complexes include proteins involved in the initiation of DNA replication. The two Cyclin E proteins are
subjected to ubiquitin-dependent proteolysis, under the control of an E3 ubiquitin ligase known as the SCF. Cyclin A - Cdk2 complexes, which
are also regulated by p21Cip1/Waf1 and p27kip1, are likely to be important for continued DNA synthesis, and progression into G2. An additional
level of control of Cdk2 is reversible phosphorylation of Threonine-14 (T14) and Tyrosine-15 (Y15), catalyzed by the Wee1 and Myt1 kinases,
and dephosphorylated by the three Cdc25 phosphatases, Cdc25A, B and C.
5.2.1 Cyclin E associated events during G1/S transition
Description
The transition from the G1 to S phase is controlled by the Cyclin E:Cdk2 complexes. As the Cyclin E:Cdk2 complexes are formed, the Cdk2 is
phosphorylated by the Wee1 and Myt1 kinases. This phosphorylation keeps the Cdk2 inactive. In yeast this control is called the cell size
checkpoint control. The dephosphorylation of the Cdk2 by Cdc25A activates the Cdk2, and is coordinated with the cells reaching the proper size,
and with the DNA synthesis machinery being ready. The Cdk2 then phosphorylates G1/S specific proteins, including proteins required for DNA
replication initiation. The beginning of S-phase is marked by the first nucleotide being laid down on the primer during DNA replication at the
early-firing origins
5.2.1.1 Formation of Cyclin E:Cdk2 complexes
Description
The E-type cyclins and Cyclin Dependent Kinase 2 control the transition from G1 to S phase. Cdk2 is competent to carry out the necessary
reactions only when complexed with Cyclin E.
References
D Desai, HC Wessling, RP Fisher, DO Morgan, "Effects of phosphorylation by CAK on cyclin binding by CDC2 and CDK2.", Mol Cell Biol, 15,
1995, 345-50.
Reaction
5.2.1.1.1 Formation of Cyclin E1:Cdk2 complexes

Description
At the beginning of this reaction, 1 molecule of 'G1/S-specific cyclin E1', and 1 molecule of 'Cdk2' are present. At the end of this reaction, 1
molecule of 'Cyclin E1:Cdk2 complex' is present.
Reaction
5.2.1.1.2 Formation of Cyclin E2:Cdk2 complexes
Description
At the beginning of this reaction, 1 molecule of 'G1/S-Specific cyclin E2', and 1 molecule of 'Cdk2' are present. At the end of this reaction, 1
molecule of 'Cyclin E2:Cdk2 complex' is present.
References
Oncogene, 17, 1998, 2787-98.
Reaction
5.2.1.2 Translocation of Cyclin E:Cdk2 complex to the nucleus
Description
Follow their formation, the Cyclin E:Cdk2 complexes are translocated to the nucleus.
References
M Jackman, Y Kubota, N Den Elzen, A Hagting, J Pines, "Cyclin A- and cyclin E-Cdk complexes shuttle between the nucleus and the
cytoplasm", Mol Biol Cell, 13, 2002, 1030-45.
Reaction
5.2.1.2.1 Translocation of cyclin E1:Cdk2 complexes to the nucleus
Description
In this reaction, 1 molecule of 'Cyclin E1:Cdk2 complex' is translocated from cytosol to nucleoplasm.
This reaction takes place in the 'nuclear envelope'.
Reaction
5.2.1.2.2 Translocation of cyclin E2:Cdk2 complexes to the nucleus
Description
In this reaction, 1 molecule of 'Cyclin E2:Cdk2 complex' is translocated from cytosol to nucleoplasm.
Reaction
5.2.1.3 Inactivation of Cyclin E:Cdk2 complexes by p27/p21
Editors
Matthews, L, 2006-10-02.
Description
premature entry into S phase (see Guardavaccaro and Pagano, 2006). The efficient recognition and ubiquitination of p27 by the SCF(Skp2)
complex requires the formation of a trimeric complex containing p27 and cyclin E/A:Cdk2.
References
Biochemistry, 44, 2005, 14553-64.
Reaction
5.2.1.3.1 Inactivation of Cyclin E1:Cdk2 complex by p27/p21
Description
Reaction
5.2.1.3.2 Inactivation of Cyclin E2:Cdk2 complex by p27/p21
Description
References
Oncogene, 17, 1998, 2787-98.
Reaction
5.2.1.4 SCF(Skp2)-mediated degradation of p27/p21
Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-09-19.
Reviewers
Coqueret, O, 2006-10-06.
Description
premature entry into S phase (see Guardavaccaro and Pagano, 2006). These two CKIs are degraded in late G1 phase by the ubiquitin pathway
(Pagano et al., 1995; Bloom et al., 2003) involving the ubiquitin ligase SCF(Skp2) (Tsvetkov et al., 1999; Carrano et al., 1999; Sutterluty et al.,
1999, Bornstein et al., 2003) and the cell-cycle regulatory protein Cks1 (Ganoth et al., 2001; Spruck et al 2001; Bornstein et al., 2003).
Recognition of p27 by SCF(Skp2) and its subsequent ubiquitination is dependent upon Cyclin E/A:Cdk2- mediated phosphorylated at Thr 187 of
p27 (Montagnoli et al., 1999). There is evidence that Cyclin A/B:Cdk1 complexes can also bind and phosphorylate p27 on Th187 (Nakayama et
al., 2004). Degradation of polyubiquitinated p27 by the 26S proteasome promotes the activity of CDKs in driving cells into S phase. (Montagnoli
et al., 1999; Tsvetkov et al., 1999, Carrano et al 1999). The mechanism of SCF(Skp2)-mediated degradation of p21 is similar to that of p27 in
terms of its requirements for the presence of Cks1 and of Cdk2/cyclin E/A (Bornstein et al.,2003; Wang et al., 2005). In addition, as observed for
p27, p21 phosphorylation at a specific site (Ser130) stimulates its ubiquitination. In contrast to p27, however, ubiquitination of p21 can take place
in the absence of phosphorylation, although with less efficiency (Bornstein et al.,2003). SCF(Skp2)-mediated degradation of p27/p21 continues
from late G1 through M-phase. During G0 and from early G1 to G1/S, Skp2 is degraded by the anaphase promoting complex/Cyclosome and its
activator Cdh1 [APC/C(Cdh1)] (Bashir et al, 2004; Wei et al, 2004). The tight regulation of APC/C(Cdh1) activity ensures the timely elimination
Skp2 and, thus, plays a critical role in controlling the G1/S transition. APC/C(Cdh1) becomes active in late M-phase by the association of
unphosphorylated Cdh1 with the APC/C. APC/C(Cdh1) remains active until the G1/S phase at which time it interacts with the inhibitory protein,
Emi1 (Hsu et al., 2002). Inhibition of APC/C(Cdh1) activity results in an accumulation of cyclins, which leads to the phosphorylation and
consequently to a further inactivation of Cdh1 at G1/S (Lukas et al., 1999). Finally, to make the inactivation of APC/C(Cdh1) permanent, Cdh1
and its E2, namely Ubc10, are eliminated in an auto-ubiquitination event (Listovsky et al., 2004; Rape and Kirschner, 2004). At G1/S, Skp2
reaccumulates as Cdh1 is inactivated, thus allowing the ubiquitination of p21 and p27 and resulting in a further increase in CDK activity.
References
T Listovsky, YS Oren, Y Yudkovsky, HM Mahbubani, AM Weiss, M Lebendiker, M Brandeis, "Mammalian Cdh1/Fzr mediates its own
degradation", EMBO J, 23, 2004, 1619-26.
T Bashir, NV Dorrello, V Amador, D Guardavaccaro, M Pagano, "Control of the SCF(Skp2-Cks1) ubiquitin ligase by the APC/C(Cdh1) ubiquitin
ligase", Nature, 428, 2004, 190-3.
W Wei, NG Ayad, Y Wan, GJ Zhang, MW Kirschner, Jr Kaelin WG, "Degradation of the SCF component Skp2 in cell-cycle phase G1 by the
anaphase-promoting complex", Nature, 428, 2004, 194-8.
D Ganoth, G Bornstein, TK Ko, B Larsen, M Tyers, M Pagano, A Hershko, "The cell-cycle regulatory protein Cks1 is required for
SCF(Skp2)-mediated ubiquitinylation of p27", Nat Cell Biol, 3, 2001, 321-4.
G Bornstein, J Bloom, D Sitry-Shevah, K Nakayama, M Pagano, A Hershko, "Role of the SCFSkp2 ubiquitin ligase in the degradation of p21Cip1
in S phase", J Biol Chem, 278, 2003, 25752-7.
K Nakayama, H Nagahama, YA Minamishima, S Miyake, N Ishida, S Hatakeyama, M Kitagawa, S Iemura, T Natsume, KI Nakayama,
"Skp2-mediated degradation of p27 regulates progression into mitosis", Dev Cell, 6, 2004, 661-72.
LM Tsvetkov, KH Yeh, SJ Lee, H Sun, H Zhang, "p27(Kip1) ubiquitination and degradation is regulated by the SCF(Skp2) complex through
phosphorylated Thr187 in p27", Curr Biol, 9, 1999, 661-4.
AC Carrano, E Eytan, A Hershko, M Pagano, "SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27", Nat Cell Biol, 1,
1999, 193-9.
Biochemistry, 44, 2005, 14553-64.
M Pagano, SW Tam, AM Theodoras, P Beer-Romero, G Del Sal, V Chau, PR Yew, GF Draetta, M Rolfe, "Role of the ubiquitin-proteasome
pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27", Science, 269, 1995, 682-5.
JY Hsu, JD Reimann, CS Sorensen, J Lukas, PK Jackson, "E2F-dependent accumulation of hEmi1 regulates S phase entry by inhibiting
APC(Cdh1)", Nat Cell Biol, 4, 2002, 358-66.
C Lukas, CS Sorensen, E Kramer, E Santoni-Rugiu, C Lindeneg, JM Peters, J Lukas, "Accumulation of cyclin B1 requires E2F and
cyclin-A-dependent rearrangement of the anaphase-promoting complex", Nature, 401, 1999, 815-8.
J Bloom, V Amador, F Bartolini, G DeMartino, M Pagano, "Proteasome-mediated degradation of p21 via N-terminal ubiquitinylation", Cell, 115,
2003, 71-82.
H Sutterluty, E Chatelain, A Marti, C Wirbelauer, M Senften, U Muller, W Krek, "p45SKP2 promotes p27Kip1 degradation and induces S phase
in quiescent cells", Nat Cell Biol, 1, 1999, 207-14.
M Rape, MW Kirschner, "Autonomous regulation of the anaphase-promoting complex couples mitosis to S-phase entry", Nature, 432, 2004,
588-95.
C Spruck, H Strohmaier, M Watson, AP Smith, A Ryan, TW Krek, SI Reed, "A CDK-independent function of mammalian Cks1: targeting of
SCF(Skp2) to the CDK inhibitor p27Kip1", Mol Cell, 7, 2001, 639-50.
5.2.1.4.1 Cyclin E/A:Cdk2-mediated phosphorylation of p27/p21
Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-09-19.
Reviewers
Coqueret, O, 2006-10-06.
Description
The interaction between the Skp2 subunit of the SCF(Skp2) complex and p27 is dependent upon Cdk2:Cyclin A/E mediated phosphorylation of
p27 at Thr 187 (Carrano et al, 1999; Tsvetkov et al, 1999). There is evidence that Cyclin A/B:Cdk1 can also bind and phosphorylate p27 on Thr
187 (Nakayama et al., 2004). This phosphorylation is also essential for the subsequent ubiquitination of p27.
References
1999, 193-9.
Reaction
5.2.1.4.1.1 Cyclin A:Cdk2 mediated phosphorylation of p27/p21
Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-09-28.
Reviewers
Coqueret, O, 2006-10-06.
Description
Recognition of p27 by SCF(Skp2) and the subsequent ubiquitination of p27 is dependent upon Cyclin E/A:Cdk2- mediated phosphorylated of
p27 at Thr 187 (Montagnoli et al., 1999). p21 is also phosphorylation at a specific site (Ser130) by Cyclin E/A:Cdk2 stimulating its ubiquitination.
Unlike p27, however, p21 ubiquitination can take place in the absence of phosphorylation, although with less efficiency (Bornstein et al.,2003).
References
Reaction
5.2.1.4.1.2 Cyclin E:Cdk2- mediated phosphorylation of p27/p21
Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-09-29.
Reviewers
Coqueret, O, 2006-10-06.
Description
References
1999, 193-9.
Reaction
5.2.1.4.2 Association of Cks1 with SCF(Skp2) complex
Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-09-19.
Reviewers
Coqueret, O, 2006-10-06.
Description
The accessory protein, Cks1 promotes efficient interaction between phosphorylated p27 and the SCF (Skp2) complex (Ganoth et al., 2001;
Spruck et al., 2001). Cks1 binds to Skp2 in the leucine-rich repeat (LRR) domain and C-terminal tail (Hao et al., 2005). The phosphorylated
Thr187 side chain of p27 associates with a phosphate binding site on Cks1, and the side chain containing Glu185 is positioned in the interface
between Skp2 and Cks1 where it interacts with both (Hao et al., 2005).
References
Reaction
5.2.1.4.3 Binding of phospho-p27/p21:Cdk2:Cyclin E/A to the SCF(Skp2):Cks1 complex

Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-09-19.
Reviewers
Coqueret, O, 2006-10-06.
Description
The association of Cks1 with both Skp2 and phosphorylated p27 promotes a tight interaction between p27 and the SCF complex (Hao et al.,
2005).
References
B Hao, N Zheng, BA Schulman, G Wu, JJ Miller, M Pagano, NP Pavletich, "Structural basis of the Cks1-dependent recognition of p27(Kip1) by
the SCF(Skp2) ubiquitin ligase", Mol Cell, 20, 2005, 9-19.
XH Zhu, H Nguyen, HD Halicka, F Traganos, A Koff, "Noncatalytic requirement for cyclin A-cdk2 in p27 turnover", Mol Cell Biol, 24, 2004,
6058-66.
Reaction
5.2.1.4.4 Ubiquitination of phospho-p27/p21

Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-09-19.
Reviewers
Coqueret, O, 2006-10-06.
Description
Once in tight contact with the SCF (Skp2):Cks1 complex, phosphorylated p27/p21 is ubiquitinated.
References
Reaction
5.2.1.4.5 Degradation of ubiquitinated p27/p21 by the 26S proteasome
Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-09-19.
Reviewers
Coqueret, O, 2006-10-06.
Description
Following ubiquitination by the SCF(Skp2):Cks1 complex, phospho-p27/p21 is degraded by the 26S proteasome.
References
1999, 193-9.
Reaction
5.2.1.5 Phosphorylation of Cyclin E:Cdk2 complexes
Description
Wee1 phosphorylates Cdk2, inhibiting entry into S-phase (Watanabe et al., 1995; Wu et al., 2001).
References
CL Wu, SD Kirley, H Xiao, Y Chuang, DC Chung, LR Zukerberg, "Cables enhances cdk2 tyrosine 15 phosphorylation by Wee1, inhibits cell
growth, and is lost in many human colon and squamous cancers", Cancer Res, 61, 2001, 7325-32.
N Watanabe, M Broome, T Hunter, "Regulation of the human WEE1Hu CDK tyrosine 15-kinase during the cell cycle", EMBO J, 14, 1995,
1878-91.
Source reaction
This reaction was inferred from the corresponding reaction "Phosphorylation of Cyclin E2:Cdk2 complexes by Myt1" in species Homo sapiens.
Reaction
5.2.1.5.1 Phosphorylation of Cyclin E1:Cdk2 complexes by Wee1
Description
At the beginning of this reaction, 1 molecule of 'ATP', and 1 molecule of 'Cyclin E1:Cdk2 complex' are present. At the end of this reaction, 1
molecule of 'Cyclin E1:phospho-Cdk2 complex', and 1 molecule of 'ADP' are present.
This reaction takes place in the 'nucleoplasm' and is mediated by the 'kinase activity' of 'Wee1'.
Reaction
5.2.1.5.2 Phosphorylation of Cyclin E2:Cdk2 complexes by Wee1
Description
At the beginning of this reaction, 1 molecule of 'Cyclin E2:Cdk2 complex', and 1 molecule of 'ATP' are present. At the end of this reaction, 1
molecule of 'Cyclin E2:phospho-Cdk2 complex', and 1 molecule of 'ADP' are present.
This reaction takes place in the 'nucleoplasm' and is mediated by the 'kinase activity' of 'Wee1'.
Reaction
5.2.1.6 Dephosphorylation of Cyclin E:Cdk2 complexes by Cdc25A

Authors
Joshi-Tope, G, 2003-06-12.
Editors
Matthews, L, 2003-09-10.
Reviewers
Manfredi, J, 0000-00-00.
Description
Cdc25A dephosphorylates Cdk2 and activates cyclin E-Cdk2 and cyclin A-Cdk2 kinases (Blomberg and Hoffmann, 1999).
References
I Blomberg, I Hoffmann, "Ectopic expression of Cdc25A accelerates the G(1)/S transition and leads to premature activation of cyclin E- and
cyclin A-dependent kinases", Mol Cell Biol, 19, 1999, 6183-94.
Reaction
5.2.1.6.1 Dephosphorylation of Cyclin E1:Cdk2 complexes by Cdc25A
Authors
Joshi-Tope, G, 2003-06-12.
Editors
Matthews, L, 2003-09-10.
Reviewers
Manfredi, J, 0000-00-00.
Description
At the beginning of this reaction, 1 molecule of 'Cyclin E1:phospho-Cdk2 complex', and 1 molecule of 'H2O' are present. At the end of this
reaction, 1 molecule of 'Orthophosphate', and 1 molecule of 'Cyclin E1:Cdk2 complex' are present.
This reaction takes place in the 'nucleoplasm' and is mediated by the 'phosphoprotein phosphatase activity' of 'Cdc25A'.
Reaction
5.2.1.6.2 Dephosphorylation of Cyclin E2:Cdk2 complexes by Cdc25A
Authors
Joshi-Tope, G, 2003-06-12.
Editors
Matthews, L, 2003-09-10.
Reviewers
Manfredi, J, 0000-00-00.
Description
At the beginning of this reaction, 1 molecule of 'H2O', and 1 molecule of 'Cyclin E2:phospho-Cdk2 complex' are present. At the end of this
reaction, 1 molecule of 'Cyclin E2:Cdk2 complex', and 1 molecule of 'Orthophosphate' are present.
This reaction takes place in the 'nucleoplasm' and is mediated by the 'phosphoprotein phosphatase activity' of 'Cdc25A'.
Reaction
5.2.1.7 CAK-mediated phosphorylation of Cyclin E:Cdk2
Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-09-28.
Reviewers
Coqueret, O, 2006-10-06.
Description
Phosphorylation of cyclin-dependent kinases (CDKs) by the CDK-activating kinase (CAK) is required for the activation of the CDK kinase activity.
The association of p21/p27 with the Cyclin A/E:Cdk2 complex prevents CAK mediated phosphorylation of Cdk2 (Aprelikova et al., 1995).
References
O Aprelikova, Y Xiong, ET Liu, "Both p16 and p21 families of cyclin-dependent kinase (CDK) inhibitors block the phosphorylation of
cyclin-dependent kinases by the CDK-activating kinase", J Biol Chem, 270, 1995, 18195-7.
Reaction
5.2.1.8 Phosphorylation of proteins involved in G1/S transition by active Cyclin E:Cdk2 complexes
Description
The G1/S transition is facilitated by Cyclin E:Cdk2-mediated phoshorylation of proteins including Rb and Cyclin Kinase Inhibitors (CKIs).
References
RA Woo, RY Poon, "Cyclin-dependent kinases and S phase control in mammalian cells", Cell Cycle, 2, 2003, 316-24.
5.2.1.8.1 Cyclin E:Cdk2-mediated phosphorylation of Rb
Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-10-10.
Reviewers
Coqueret, O, 2006-10-06.
Description
Cyclin E forms a complex with cdk2 and collaborates with the cyclin D-dependent kinases in phosphorylating Rb.
Reaction
5.2.1.9 Ubiquitin-Dependent Degradation of Cyclin E
Description
Failure to appropriately regulate cyclin E accumulation can lead to accelerated S phase entry, genetic instability, and tumorigenesis. The amount
of cyclin E protein in the cell is controlled by ubiquitin-mediated proteolysis (see Woo, 2003).
References
RA Woo, RY Poon, "Cyclin-dependent kinases and S phase control in mammalian cells", Cell Cycle, 2, 2003, 316-24.
5.2.1.9.1 Ubiquitin-Dependent Degradation of Cyclin E1
Description
Cyclin E is ubiquitinated by the SCFFbw7 ubiquitin ligase and degraded by the proteasome (Koepp et al., 2001; Clurman et al., 1996).
References
BE Clurman, RJ Sheaff, K Thress, M Groudine, JM Roberts, "Turnover of cyclin E by the ubiquitin-proteasome pathway is regulated by cdk2
binding and cyclin phosphorylation", Genes Dev, 10, 1996, 1979-90.
DM Koepp, LK Schaefer, X Ye, K Keyomarsi, C Chu, JW Harper, SJ Elledge, "Phosphorylation-dependent ubiquitination of cyclin E by the
SCFFbw7 ubiquitin ligase", Science, 294, 2001, 173-7.
5.2.1.9.2 Ubiquitin-Dependent Degradation of Cyclin E2
Description
This pathway will be covered in a future release.
5.2.2 G1/S-Specific Transcription
Description
The E2F family of transcription factors regulate the transition from the G1 to the S phase in the cell cycle. E2F activity is regulated by members
of the retinoblastoma protein (pRb) family, resulting in the tight control of the expression of E2F-responsive genes. Phosphorylation of pRb by
cyclin D?CDK complexes releases pRb from E2F inducing E2F-targeted genes such as cyclin E.
5.2.3 Activation of the pre-replicative complex
Description
In S. cerevisiae, two ORC subunits, Orc1 and Orc5, both bind ATP, and Orc1 in addition has ATPase activity. Both ATP binding and ATP
hydrolysis appear to be essential functions in vivo. ATP binding by Orc1 is unaffected by the association of ORC with origin DNA (ARS)
sequences, but ATP hydrolysis is ARS-dependent, being suppressed by associated double-stranded DNA and stimulated by associated
single-stranded DNA. These data are consistent with the hypothesis that ORC functions as an ATPase switch, hydrolyzing bound ATP and
changing state as DNA unwinds at the origin immediately before replication. It is attractive to speculate that ORC likewise functions as a switch
as human pre-replicative complexes are activated, but human Orc proteins are not well enough characterized to allow the model to be critically
tested. mRNAs encoding human orthologs of all six Orc proteins have been cloned, and ATP-binding amino acid sequence motifs have been
identified in Orc1, Orc4, and Orc5. Interactions among proteins expressed from the cloned genes have been characterized, but the ATP-binding
and hydrolyzing properties of these proteins and complexes of them have not been determined.
References
DG Lee, SP Bell, "ATPase switches controlling DNA replication initiation.", Curr Opin Cell Biol, 12, 2000, 280-5.
RD Klemm, SP Bell, "ATP bound to the origin recognition complex is important for preRC formation.", Proc Natl Acad Sci U S A, 98, 2001,
8361-7.
T Tugal, XH Zou-Yang, K Gavin, D Pappin, B Canas, R Kobayashi, T Hunt, B Stillman, "The Orc4p and Orc5p subunits of the Xenopus and
human origin recognition complex are related to Orc1p and Cdc6p.", J Biol Chem, 273, 1999, 32421-9.
M Fujita, Y Ishimi, H Nakamura, T Kiyono, T Tsurumi, "Nuclear organization of DNA replication initiation proteins in mammalian cells.", J Biol
Chem, 277, 2002, 10354-61.
DG Lee, AM Makhov, RD Klemm, SP Bell, "Regulation of origin recognition complex conformation and ATPase activity: differential effects of
single-stranded and double-stranded DNA binding.", EMBO J, 19, 2000, 4774-82.
SK Dhar, L Delmolino, A Dutta, "Architecture of the human origin recognition complex.", J Biol Chem, 276, 2001, 29067-71.
SG Prasanth, KV Prasanth, B Stillman, "Orc6 involved in DNA replication, chromosome segregation, and cytokinesis.", Science, 297, 2002,
1026-31.
RD Klemm, RJ Austin, SP Bell, "Coordinate binding of ATP and origin DNA regulates the ATPase activity of the origin recognition complex.",
Cell, 88, 1997, 493-502.
S Vashee, P Simancek, MD Challberg, TJ Kelly, "Assembly of the human origin recognition complex.", J Biol Chem, 276, 2001, 26666-73.
KA Gavin, M Hidaka, B Stillman, "Conserved initiator proteins in eukaryotes.", Science, 270, 1996, 1667-71.
DG Quintana, KC Thome, ZH Hou, AH Ligon, CC Morton, A Dutta, "ORC5L, a new member of the human origin recognition complex, is deleted
in uterine leiomyomas and malignant myeloid diseases.", J Biol Chem, 273, 1998, 27137-45.
DG Quintana, Zh Hou, KC Thome, M Hendricks, P Saha, A Dutta, "Identification of HsORC4, a member of the human origin of replication
recognition complex.", J Biol Chem, 272, 1997, 28247-51.
S Pinto, DG Quintana, P Smith, RM Mihalek, ZH Hou, S Boynton, CJ Jones, M Hendricks, K Velinzon, JA Wohlschlegel, RJ Austin, WS Lane, T
Tully, A Dutta, "latheo encodes a subunit of the origin recognition complex and disrupts neuronal proliferation and adult olfactory memory when
mutant.", Neuron, 23, 1999, 45-54.
5.2.3.1 Mcm10 associates with the pre-replicative complex, stabilizing Mcm2-7
Description
MCM10 is required for human DNA replication. In S. cerevisiae, Mcm10, like Mcm2-7, is required for minichromosome maintenance, but Mcm10
has no sequence homology with these other proteins (Merchant et al., 1997). Genetic studies have demonstrated that Mcm10 is required for
DNA replication in S. pombe (Aves et al., 1998) and S. cerevisiae cells (Homesley et al., 2000) and immunodepletion of XlMcm10 interferes with
DNA replication in Xenopus egg extracts (Wohlschlegel et al., 2002). Human Mcm10 interacts with chromatin in G1 phase and then dissociates
during G2 phase. In S. cerevisiae, Mcm10 has been shown to localize to origins during G1 (Ricke and Bielinsky, 2004), and it may stabilize the
association of Mcm2-7 with the pre-replicative complex (Sawyer et al., 2004). This timing of association is consistent with studies that
demonstrate that, in Xenopus egg extracts, Mcm10 is required for association of Cdc45, but not Mcm2-7 with chromatin. Biochemical evidence
that Mcm10 plays a direct role in the activation of the pre-replicative complex includes the requirement for SpMcm10 in the phosphorylation of
the Mcm2-7 complex by DDK (Lee et al., 2004) and the fact that SpMcm10 binds and stimulates DNA polymerase alpha activity (Fien et al.,
2004).
References
JK Lee, YS Seo, J Hurwitz, "The Cdc23 (Mcm10) protein is required for the phosphorylation of minichromosome maintenance complex by the
Dfp1-Hsk1 kinase", Proc Natl Acad Sci U S A, 100, 2003, 2334-9.
JA Wohlschlegel, SK Dhar, TA Prokhorova, A Dutta, JC Walter, "Xenopus Mcm10 binds to origins of DNA replication after Mcm2-7 and
stimulates origin binding of Cdc45.", Mol Cell, 9, 2002, 233-40.
L Homesley, M Lei, Y Kawasaki, S Sawyer, T Christensen, BK Tye, "Mcm10 and the MCM2-7 complex interact to initiate DNA synthesis and to
release replication factors from origins.", Genes Dev, 14, 2000, 913-26.
SJ Aves, N Tongue, AJ Foster, EA Hart, "The essential schizosaccharomyces pombe cdc23 DNA replication gene shares structural and
functional homology with the Saccharomyces cerevisiae DNA43 (MCM10) gene", Curr Genet, 34, 1998, 164-71.
SL Sawyer, IH Cheng, W Chai, BK Tye, "Mcm10 and Cdc45 cooperate in origin activation in Saccharomyces cerevisiae", J Mol Biol, 340, 2004,
195-202.
RM Ricke, AK Bielinsky, "Mcm10 regulates the stability and chromatin association of DNA polymerase-alpha", Mol Cell, 16, 2004, 173-85.
AM Merchant, Y Kawasaki, Y Chen, M Lei, BK Tye, "A lesion in the DNA replication initiation factor Mcm10 induces pausing of elongation forks
through chromosomal replication origins in Saccharomyces cerevisiae", Mol Cell Biol, 17, 1997, 3261-71.
M Izumi, K Yanagi, T Mizuno, M Yokoi, Y Kawasaki, KY Moon, J Hurwitz, F Yatagai, F Hanaoka, "The human homolog of Saccharomyces
cerevisiae Mcm10 interacts with replication factors and dissociates from nuclease-resistant nuclear structures in G(2) phase.", Nucleic Acids
Res, 28, 2000, 4769-77.
K Fien, YS Cho, JK Lee, S Raychaudhuri, I Tappin, J Hurwitz, "Primer utilization by DNA polymerase alpha-primase is influenced by its
interaction with Mcm10p", J Biol Chem, 279, 2004, 16144-53.
M Lei, BK Tye, "Initiating DNA synthesis: from recruiting to activating the MCM complex.", J Cell Sci, 114, 2001, 1447-54.
Reaction
5.2.3.2 Cdt1 is displaced from the pre-replicative complex.
Description
At the beginning of this reaction, 1 molecule of 'Mcm10:pre-replicative complex' is present. At the end of this reaction, 1 molecule of
'Mcm10:active pre-replicative complex', and 1 molecule of 'CDT1' are present.
This reaction takes place in the 'nucleus'.
References
SP Bell, A Dutta, "DNA replication in eukaryotic cells.", Annu Rev Biochem, 71, 2002, 333-74.
Reaction
5.2.3.3 CDK and DDK associate with the Mcm10:pre-replicative complex
Description
At the beginning of this reaction, 1 molecule of 'Mcm10:active pre-replicative complex', 1 molecule of 'DDK', and 1 molecule of 'CDK' are
present. At the end of this reaction, 1 molecule of 'CDK:DDK:Mcm10:pre-replicative complex' is present.

References
H Kumagai, N Sato, M Yamada, D Mahony, W Seghezzi, E Lees, K Arai, H Masai, "A novel growth- and cell cycle-regulated protein, ASK,
activates human Cdc7-related kinase and is essential for G1/S transition in mammalian cells.", Mol Cell Biol, 19, 1999, 5083-95.
W Jiang, D McDonald, TJ Hope, T Hunter, "Mammalian Cdc7-Dbf4 protein kinase complex is essential for initiation of DNA replication.", EMBO
J, 18, 1999, 5703-13.
Reaction
5.2.3.4 Mcm2-7 is phosphorylated by DDK
Description
At the beginning of this reaction, 1 molecule of 'Mcm2-7 complex', and 1 molecule of 'ATP' are present. At the end of this reaction, 1 molecule of
'phosphorylated Mcm2-7 complex', and 1 molecule of 'ADP' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'kinase activity' of 'DDK'.
References
H Masai, E Matsui, Z You, Y Ishimi, K Tamai, K Arai, "Human Cdc7-related kinase complex. In vitro phosphorylation of MCM by concerted
actions of Cdks and Cdc7 and that of a critical threonine residue of Cdc7 by Cdks.", J Biol Chem, 275, 2000, 29042-52.
Reaction
5.2.3.5 Cdc45 associates with the pre-replicative complex at the origin
Description
Once the Mcm2-7 complex has been assembled onto the origin of replication, the next step is the assembly of Cdc45, an essential replication
protein, in late G1. The assembly of Cdc45 onto origins of replication forms a complex distinct from the pre-replicative complex, sometimes
called the pre-initiation complex. The assembly of Cdc45 onto origins correlates with the time of initiation. Like the Mcm2-7 proteins, Cdc45
binds specifically to origins in the G1 phase of the cell cycle and then to non-origin DNA during S phase and is therefore thought to travel with
the replication fork. Indeed, S. cerevisiae Cdc45 is required for DNA replication elongation as well as replication initiation. Cdc45 is required for
the association of alpha DNA polymerase:primase with chromatin. Based on this observation and the observation that in S. cerevisiae, cCdc45
has been found in large complexes with some components of Mcm2-7 complex, it has been suggested that Cdc45 plays a scaffolding role at the
replication fork, coupling Pol-alpha:primase to the replication fork through the helicase. Association of Cdc45 with origin DNA is regulated in the
cell cycle and its association is dependent on the activity of cyclin-dependent kinases but not the Cdc7/Dbf4 kinase. In Xenopus egg extracts,
association of Cdc45 with chromatin is dependent on Xmus101. TopBP1, the human homolog of Xmus1, is essential for DNA replication and
interacts with DNA polymerase epsilon, one of the polymerases involved in replicating the genome. TopBP1 homologs have been found in S.
cerevisiae and S. pombe. Sld3, an additional protein required for Cdc45 association with chromatin in S. cerevisiae and S. pombe, has no known
human homolog.
References
OM Aparicio, AM Stout, SP Bell, "Differential assembly of Cdc45p and DNA polymerases at early and late origins of DNA replication.", Proc Natl
Acad Sci U S A, 96, 1999, 9130-5.
JA Tercero, K Labib, JF Diffley, "DNA synthesis at individual replication forks requires the essential initiation factor Cdc45p.", EMBO J, 19, 2000,
2082-93.
K Yamane, X Wu, J Chen, "A DNA damage-regulated BRCT-containing protein, TopBP1, is required for cell survival.", Mol Cell Biol, 22, 2001,
555-66.
L Zou, B Stillman, "Formation of a preinitiation complex by S-phase cyclin CDK-dependent loading of Cdc45p onto chromatin.", Science, 280,
1998, 593-6.
P Saha, KC Thome, R Yamaguchi, Z Hou, S Weremowicz, A Dutta, "The human homolog of Saccharomyces cerevisiae CDC45.", J Biol Chem,
273, 1998, 18205-9.
L Zou, B Stillman, "Assembly of a complex containing Cdc45p, replication protein A, and Mcm2p at replication origins controlled by S-phase
cyclin-dependent kinases and Cdc7p-Dbf4p kinase.", Mol Cell Biol, 20, 2000, 3086-96.
Y Kamimura, YS Tak, A Sugino, H Araki, "Sld3, which interacts with Cdc45 (Sld4), functions for chromosomal DNA replication in Saccharomyces
cerevisiae.", EMBO J, 20, 2001, 2097-107.
S Dalton, L Whitbread, "Cell cycle-regulated nuclear import and export of Cdc47, a protein essential for initiation of DNA replication in budding
yeast.", Proc Natl Acad Sci U S A, 92, 1995, 2514-8.
I Kukimoto, H Igaki, T Kanda, "Human CDC45 protein binds to minichromosome maintenance 7 protein and the p70 subunit of DNA polymerase
alpha.", Eur J Biochem, 265, 1999, 936-43.
OM Aparicio, DM Weinstein, SP Bell, "Components and dynamics of DNA replication complexes in S. cerevisiae: redistribution of MCM proteins
and Cdc45p during S phase.", Cell, 91, 1997, 59-69.
RA Van Hatten, AV Tutter, AH Holway, AM Khederian, JC Walter, WM Michael, "The Xenopus Xmus101 protein is required for the recruitment of
Cdc45 to origins of DNA replication.", J Cell Biol, 159, 2002, 541-7.
Reaction
5.2.3.6 DNA Replication Factor A (RPA) associates with the pre-replicative complex at the origin
Description
After pre-RC assembly and Cdc45 association with the origin of replication, Replication Protein A (RPA) also associates with chromatin. RPA is
a heterotrimeric complex containing p70, p34, and p11 subunits, and also is required for DNA recombination and DNA repair. The p70 subunit of
RPA binds to the primase subunits of Pol alpha:primase. The p70 and p34 subunits of RPA are phosphorylated in a cell cycle-dependent
manner. RPA is a single-strand DNA (ssDNA) binding protein and its association with chromatin at this stage suggests that DNA is partially
unwound. This suggestion has been confirmed by detection of ssDNA in budding yeast origins of replication using chemical methods.
References
S Din, SJ Brill, MP Fairman, B Stillman, "Cell-cycle-regulated phosphorylation of DNA replication factor A from human and yeast cells.", Genes
Dev, 4, 1990, 968-77.
Biochem, 66, 1997, 61-92.
I Dornreiter, LF Erdile, IU Gilbert, D von Winkler, TJ Kelly, "Interaction of DNA polymerase alpha-primase with cellular replication protein A and
SV40 T antigen.", EMBO J, 11, 1992, 769-76.
J Walter, J Newport, "Initiation of eukaryotic DNA replication: origin unwinding and sequential chromatin association of Cdc45, RPA, and DNA
polymerase alpha.", Mol Cell, 5, 2000, 617-27.
Reaction
5.2.3.7 DNA polymerase alpha:primase binds at the origin
Description
DNA polymerase alpha:primase is comprised of four subunits, p180, p70, p58, and p49. The two primase subunits, p58 and p49, form a tight
complex. The p49 subunit contains the DNA primase activity and one role of p58 appears to be tethering p49 to p180, the DNA polymerase
catalytic subunit. The fourth subunit, p70, binds p180 and may tether the DNA polymerase alpha:primase complex to Cdc45.
References
DN Frick, CC Richardson, "DNA primases.", Annu Rev Biochem, 70, 2002, 39-80.
WC Copeland, TS Wang, "Enzymatic characterization of the individual mammalian primase subunits reveals a biphasic mechanism for initiation
of DNA replication.", J Biol Chem, 268, 1994, 26179-89.
Reaction
5.2.3.8 DNA polymerase epsilon binds at the origin
Description
At the beginning of this reaction, 1 molecule of 'origin of replication', and 1 molecule of 'DNA polymerase epsilon' are present. At the end of this
reaction, 1 molecule of 'DNA polymerase epsilon:origin complex' is present.
References
Reaction
5.2.4 E2F mediated regulation of DNA replication
Authors
Description
Progression through G1 and G1 to S-phase transition that initiates DNA synthesis involve many complexes that are regulated by Rb:E2F
pathway. Rb:E2F pathway plays a key role in gene expression regulation in proliferating and differentiated cells. As a repressor, E2F remains
bound to Rb; it can activate the expression of S-phase genes involved in DNA replication after the phosphorylation of Rb.
E2F proteins regulate expression of genes involved in various processes thereby forming interlinks between cell cycle, DNA synthesis, DNA
damage recognition etc.
In this module, activation of replication related genes by E2F1 and two ways by which E2F1 regulates DNA replication initiation are annotated.
Detailed connection between the E2F protein function and various pathways will be covered in future releases of Reactome.
References
H Cam, BD Dynlacht, "Emerging roles for E2F: beyond the G1/S transition and DNA replication.", Cancer Cell, 3, 2003, 311-6.
C Gutierrez, E Ramirez-Parra, MM Castellano, JC del Pozo, "G(1) to S transition: more than a cell cycle engine switch.", Curr Opin Plant Biol, 5,
2002, 480-6.
O Stevaux, NJ Dyson, "A revised picture of the E2F transcriptional network and RB function.", Curr Opin Cell Biol, 14, 2002, 684-91.
5.2.4.1 E2F transcriptional targets at G1/S
Authors
Description
E2F1 binds to E2F binding sites on the genome activating the synthesis of the target proteins. For annotation purposes, The reactions regulated
by E2F1 are grouped under this pathway and information about the target genes alone are displayed for annotation purposes.
5.2.4.1.1 Activation of Cdc45 by E2F1
Authors
Description
E2F1 activation of CDC45 is shown in mouse cells by using human e2f1 construct.
References
Y Arata, M Fujita, K Ohtani, S Kijima, JY Kato, "Cdk2-dependent and -independent pathways in E2F-mediated S phase induction", J Biol Chem,
275, 2000, 6337-45.
Reaction
5.2.4.1.2 Activation of Cdt1 by E2F1
Description
At the end of this reaction, 1 molecule of 'CDT1' is present.
References
K Yoshida, I Inoue, "Regulation of Geminin and Cdt1 expression by E2F transcription factors.", Oncogene, 23, 2004, 3802-12.
Reaction
5.2.4.1.3 Activation of Cyclin E by E2F1
Authors
Description
Cyclin E is transcriptionally regulated by E2F1. Cyclin E protein plays important role in the transition of G1 in S-phase by associating with Cdk2.
References
K Ohtani, J DeGregori, JR Nevins, "Regulation of the cyclin E gene by transcription factor E2F1.", Proc Natl Acad Sci U S A, 92, 1996,
12146-50.
Reaction
5.2.4.1.4 Activation of PCNA by E2F1
Authors
Description
E2F activation of PCNA has also been demonstrated in some human cells by using recombinant adenovirus constructs. It has also been
demonstrated in Drosophila by DeGregori et al (1995).
References
J DeGregori, T Kowalik, JR Nevins, "Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory
genes", Mol Cell Biol, 15, 1995, 4215-24.
Reaction
5.2.4.1.5 Activation of PolA catalytic subunit by E2F1
Authors
Description
E2F activation of Polymerase A (Pol A) has also been demonstrated in some human cells. It has also been demonstrated in Drosophila by
Ohtani and Nevins (1994).
References
K Ohtani, JR Nevins, "Functional properties of a Drosophila homolog of the E2F1 gene.", Mol Cell Biol, 14, 1994, 1603-12.
Reaction
5.2.4.1.6 Activation of Rir2 by E2F1
Authors
Description
Rir2 protein is involved in dNTP level regulation and activation of these enzymes results in higher levels of dNTPs in anticipation of S-phase.
E2F activation of Rir2 has been shown also in Drosophila by Duronio and O'Farrell (1994).
References
RJ Duronio, PH O'Farrell, "Developmental control of a G1-S transcriptional program in Drosophila.", Development, 120, 1994, 1503-15.
Reaction
5.2.4.1.7 Activation of Cdc25a (string) by E2F1

Authors
Description
This reaction has been inferred from a similar event in Drosophila as observed by Reis and Edgar (2004). E2F1 activates string (cdc25) that in
turn activates Cyclin B/Cdk1. A similar phenomenon has been observed in mouse NIH 3T3 cells by Chen and Prywes (1999) and in Rat1 cells
by Vigo et al (1999).
References
X Chen, R Prywes, "Serum-induced expression of the cdc25A gene by relief of E2F-mediated repression", Mol Cell Biol, 19, 1999, 4695-702.
T Reis, BA Edgar, "Negative regulation of dE2F1 by cyclin-dependent kinases controls cell cycle timing.", Cell, 117, 2004, 253-64.
E Vigo, H Muller, E Prosperini, G Hateboer, P Cartwright, MC Moroni, K Helin, "CDC25A phosphatase is a target of E2F and is required for
efficient E2F-induced S phase", Mol Cell Biol, 19, 1999, 6379-95.
Source reaction
This reaction was inferred from the corresponding reaction "Activation of String (Cdc25a) by dE2F1" in species Drosophila melanogaster.
T Reis, BA Edgar, "Negative regulation of dE2F1 by cyclin-dependent kinases controls cell cycle timing.", Cell, 117, 2004, 253-64.
Reaction
5.2.4.1.8 Activation of ORC1 by E2F1
Authors
Description
It has been observed in Drosophila that E2F1 regulates expression of Orc1, stimulates ORC1-6 complex formation and binding to origin of
replication (Asano and Wharton, 1999). Orc1-6 recruit Cdc6 and Cdt1 that are required to recruit the MCM2-7 replication helicases. E2F1
regulation incorporates a feedback mechanism where in Geminin can inhibit MCM2-7 recruitment on ORC1-6 complex by interacting with
Cdc6/Cdt1 .
References
M Asano, RP Wharton, "E2F mediates developmental and cell cycle regulation of ORC1 in Drosophila.", EMBO J, 18, 1999, 2435-48.
K Ohtani, J DeGregori, G Leone, DR Herendeen, TJ Kelly, JR Nevins, "Expression of the HsOrc1 gene, a human ORC1 homolog, is regulated
by cell proliferation via the E2F transcription factor.", Mol Cell Biol, 16, 1997, 6977-84.
Reaction
5.2.4.1.9 Activation of TK2 (Dnk1) by E2F1
Authors
Description
This event is inferred from an event annotated in Drosophila.
Source reaction
This reaction was inferred from the corresponding reaction "Activation of Dnk1" in species Drosophila melanogaster.
RJ Duronio, PH O'Farrell, "Developmental control of a G1-S transcriptional program in Drosophila.", Development, 120, 1994, 1503-15.
Reaction
5.2.4.1.10 Activation of Thymidylate synthetase by E2F1
Description
At the end of this reaction, 1 molecule of 'Thymidylate synthase' is present.
References
Reaction
5.2.4.1.11 Activation of Dihydrofoloate reductase by E2F1
Description
At the end of this reaction, 1 molecule of 'Dihydrofolate reductase' is present.

References
Reaction
5.2.4.1.12 Activation of Cdc2 by E2F1
Description
At the end of this reaction, 1 molecule of 'Cdc2' is present.
References
Reaction
5.2.4.1.13 Activation of Cyclin A1 by E2F1

Description
At the end of this reaction, 1 molecule of 'Cyclin A1' is present.
References
Reaction
5.2.4.1.14 CDC6 protein is synthesized under the control of E2F transcription factors
Description
References
Z Yan, J DeGregori, R Shohet, G Leone, B Stillman, JR Nevins, RS Williams, "Cdc6 is regulated by E2F and is essential for DNA replication in
mammalian cells.", Proc Natl Acad Sci U S A, 95, 1998, 3603-8.
K Ohtani, A Tsujimoto, M Ikeda, M Nakamura, "Regulation of cell growth-dependent expression of mammalian CDC6 gene by the cell cycle
transcription factor E2F.", Oncogene, 17, 1998, 1777-85.
Reaction
5.2.4.1.15 Activation of Emi1 by E2F1
Authors
Lorca, T, Castro, A, 2006-01-26.
Editors
Matthews, L, 2006-02-17.
Description
Emi1 accumulates at the G1Ã¢Ë†â€™S transition and is a target of E2F-1.
References
Reaction
5.2.4.2 Inhibition of replication initiation of damaged DNA by Rb/E2F1
5.2.4.2.1 Detection of damage during initiation of DNA synthesis in S-phase
Description
At the beginning of this reaction, 1 molecule of 'RNA primer-DNA primer:origin duplex' is present. At the end of this reaction, 1 molecule of 'DNA
polymerase alpha:primase', and 1 molecule of 'RNA primer-DNA primer:origin duplex with DNA damage' are present.

Reaction
5.2.4.2.2 Pp2a mediated localization of Rb protein in chromatin
Description
At the beginning of this reaction, 1 molecule of 'phospho-Retinoblastoma protein', and 1 molecule of 'RNA primer-DNA primer:origin duplex with
DNA damage' are present. At the end of this reaction, 1 molecule of 'Rb1:RNA primer-DNA primer:origin duplex with DNA damage' is present.
This reaction takes place in the 'nucleus' and is mediated by the 'phosphoprotein phosphatase activity' of 'PP2A'.
References
D Avni, H Yang, F Martelli, F Hofmann, WM ElShamy, S Ganesan, R Scully, DM Livingston, "Active localization of the retinoblastoma protein in
chromatin and its response to S phase DNA damage.", Mol Cell, 12, 2003, 735-46.
Reaction
5.2.4.2.3 Replication initiation regulation by Rb1/E2F1
Authors
Description
This set of events is inferred from annotated events in Drosophila.
Rb1 is normally hyperphosphorylated by CycD/CDK4/CDK6 and Cyclin E/CDK2 for transition into S-phase. PP2A can then reverse this reaction,
in this case, in response to DNA damage induced checkpoint.
Source reaction
This reaction was inferred from the corresponding reaction "Replication initiation regulation by E2F1/Rbf1" in species Drosophila melanogaster.
G Bosco, W Du, TL Orr-Weaver, "DNA replication control through interaction of E2F-RB and the origin recognition complex.", Nat Cell Biol, 3,
2001, 289-95.
Reaction
5.2.4.3 E2F-enabled inhibition of pre-replication complex formation
Authors
Description
Under specific conditions, Cyclin B, a mitotic cyclin, can inhibit the functions of pre-replicative complex. E2F1 activates Cdc25A protein which
regulates Cyclin B in a positive manner. Cyclin B/Cdk1 function is restored which leads to the disruption of pre-replicative complex. This
phenomenon has been demonstrated by Bosco et al (2001) in Drosophila.
References
G Bosco, W Du, TL Orr-Weaver, "DNA replication control through interaction of E2F-RB and the origin recognition complex.", Nat Cell Biol, 3,
2001, 289-95.
BK Kennedy, DA Barbie, M Classon, E Harlow, NJ Dyson, "Nuclear organization of DNA replication in primary mammalian cells", Genes Dev,
14, 2000, 2855-68.
5.2.4.3.1 Activation of Cyclin B/Cdk1 by Cdc25a (string) protein

Description
At the end of this reaction, 1 molecule of 'Cyclin B:Cdk1 complex', and 1 molecule of 'Cdc25A' are present.
References
N Mailand, AV Podtelejnikov, A Groth, M Mann, J Lukas, "Regulation of G(2)/M events by Cdc25A through phosphorylation-dependent
modulation of its stability", EMBO J, 21, 2002, 5911-20.
Reaction
5.2.4.3.2 Association of Cyclin B/Cdk1 with replicative origin inhibits pre-RC formation
Authors
Description
This event is inferred from the fission yeast.Cyclin B activity is thought to inhibit pre-RC formation by first associating with ORC during DNA
replication.
Source reaction
This reaction was inferred from the corresponding reaction "Association of Cdc13/Cdk1 with replicative origin" in species Schizosaccharomyces
pombe.
J Wuarin, V Buck, P Nurse, JB Millar, "Stable association of mitotic cyclin B/Cdc2 to replication origins prevents endoreduplication.", Cell, 111,
2002, 419-31.
Reaction
5.3 S Phase
Description
DNA synthesis occurs in the S phase, or the synthesis phase, of the cell cycle. The cell duplicates its hereditary material, and two copies of the
chromosome are formed. As DNA replication continues, the E type cyclins shared by the G1 and S phases, are destroyed and the levels of the
mitotic cyclins rise.
5.3.1 Cyclin A:Cdk2-associated events at S phase entry
Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-09-29.
Reviewers
Coqueret, O, 2006-10-06.
Description
Cyclin A:Cdk2 plays a key role in S phase entry by phosphorylation of proteins including Cdh1, Rb, p21 and p27. During G1 phase of the cell
cycle, cyclin A is synthesized and associates with Cdk2. After forming in the cytoplasm, the Cyclin A:Cdk2 complexes are translocated to the
nucleus (Jackman et al.,2002). Prior to S phase entry, the activity of Cyclin A:Cdk2 complexes is negatively regulated through Tyr 15
phosphorylation of Cdk2 (Gu et al., 1995) and also by the association of the cyclin kinase inhibitors (CKIs), p27 and p21. Phosphorylation of
cyclin-dependent kinases (CDKs) by the CDK-activating kinase (CAK) is required for the activation of the CDK2 kinase activity (Aprelikova et al.,
1995). The entry into S phase is promoted by the removal of inhibitory Tyr 15 phosphates from the Cdk2 subunit of Cyclin A:Cdk2 complex by
the Cdc25 phosphatases (Blomberg and Hoffmann, 1999) and by SCF(Skp2)-mediated degradation of p27/p21 (see Ganoth et al., 2001). While
Cdk2 is thought to play a primary role in regulating entry into S phase, recent evidence indicates that Cdk1 is equally capable of promoting entry
into S phase and the initiation of DNA replication (see Bashir and Pagano, 2005). Thus, Cdk1 complexes may also play a significant role at this
point in the cell cycle.
References
ligase", Nature, 428, 2004, 190-3.
Y Gu, J Rosenblatt, DO Morgan, "Cell cycle regulation of CDK2 activity by phosphorylation of Thr160 and Tyr15", EMBO J, 11, 1992,
3995-4005.
5.3.1.1 Formation of Cyclin A:Cdk2 complexes
Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-09-28.
Reviewers
Coqueret, O, 2006-10-06.
Description
During G1 phase of the cell cycle, cyclin A is synthesized and associates with Cdk2.
References
M Pagano, R Pepperkok, F Verde, W Ansorge, G Draetta, "Cyclin A is required at two points in the human cell cycle", EMBO J, 11, 1992,
961-71.
Reaction
5.3.1.2 Translocation of Cyclin A:Cdk2 complexes to the nucleus
Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-09-28.
Reviewers
Coqueret, O, 2006-10-06.
Description
After forming in the cytoplasm, the Cyclin A:Cdk2 complexes are translocated to the nucleus.
References
Reaction
5.3.1.3 Inactivation of Cyclin A:Cdk2 complexes by p27/p21
Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-09-28.
Reviewers
Coqueret, O, 2006-10-06.
Description
premature entry into S phase (see Guardavaccaro and Pagano, 2006).
References
Reaction
5.3.1.4 SCF(Skp2)-mediated degradation of p27/p21
Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-09-19.
Reviewers
Coqueret, O, 2006-10-06.
Description
premature entry into S phase (see Guardavaccaro and Pagano, 2006). These two CKIs are degraded in late G1 phase by the ubiquitin pathway
(Pagano et al., 1995; Bloom et al., 2003) involving the ubiquitin ligase SCF(Skp2) (Tsvetkov et al., 1999; Carrano et al., 1999; Sutterluty et al.,
1999, Bornstein et al., 2003) and the cell-cycle regulatory protein Cks1 (Ganoth et al., 2001; Spruck et al 2001; Bornstein et al., 2003).
Recognition of p27 by SCF(Skp2) and its subsequent ubiquitination is dependent upon Cyclin E/A:Cdk2- mediated phosphorylated at Thr 187 of
p27 (Montagnoli et al., 1999). There is evidence that Cyclin A/B:Cdk1 complexes can also bind and phosphorylate p27 on Th187 (Nakayama et
al., 2004). Degradation of polyubiquitinated p27 by the 26S proteasome promotes the activity of CDKs in driving cells into S phase. (Montagnoli
et al., 1999; Tsvetkov et al., 1999, Carrano et al 1999). The mechanism of SCF(Skp2)-mediated degradation of p21 is similar to that of p27 in
terms of its requirements for the presence of Cks1 and of Cdk2/cyclin E/A (Bornstein et al.,2003; Wang et al., 2005). In addition, as observed for
p27, p21 phosphorylation at a specific site (Ser130) stimulates its ubiquitination. In contrast to p27, however, ubiquitination of p21 can take place
in the absence of phosphorylation, although with less efficiency (Bornstein et al.,2003). SCF(Skp2)-mediated degradation of p27/p21 continues
from late G1 through M-phase. During G0 and from early G1 to G1/S, Skp2 is degraded by the anaphase promoting complex/Cyclosome and its
activator Cdh1 [APC/C(Cdh1)] (Bashir et al, 2004; Wei et al, 2004). The tight regulation of APC/C(Cdh1) activity ensures the timely elimination
Skp2 and, thus, plays a critical role in controlling the G1/S transition. APC/C(Cdh1) becomes active in late M-phase by the association of
unphosphorylated Cdh1 with the APC/C. APC/C(Cdh1) remains active until the G1/S phase at which time it interacts with the inhibitory protein,
Emi1 (Hsu et al., 2002). Inhibition of APC/C(Cdh1) activity results in an accumulation of cyclins, which leads to the phosphorylation and
consequently to a further inactivation of Cdh1 at G1/S (Lukas et al., 1999). Finally, to make the inactivation of APC/C(Cdh1) permanent, Cdh1
and its E2, namely Ubc10, are eliminated in an auto-ubiquitination event (Listovsky et al., 2004; Rape and Kirschner, 2004). At G1/S, Skp2
reaccumulates as Cdh1 is inactivated, thus allowing the ubiquitination of p21 and p27 and resulting in a further increase in CDK activity.
References
ligase", Nature, 428, 2004, 190-3.
W Wei, NG Ayad, Y Wan, GJ Zhang, MW Kirschner, Jr Kaelin WG, "Degradation of the SCF component Skp2 in cell-cycle phase G1 by the
anaphase-promoting complex", Nature, 428, 2004, 194-8.
Biochemistry, 44, 2005, 14553-64.
1999, 193-9.
H Sutterluty, E Chatelain, A Marti, C Wirbelauer, M Senften, U Muller, W Krek, "p45SKP2 promotes p27Kip1 degradation and induces S phase
in quiescent cells", Nat Cell Biol, 1, 1999, 207-14.
J Bloom, V Amador, F Bartolini, G DeMartino, M Pagano, "Proteasome-mediated degradation of p21 via N-terminal ubiquitinylation", Cell, 115,
2003, 71-82.
M Rape, MW Kirschner, "Autonomous regulation of the anaphase-promoting complex couples mitosis to S-phase entry", Nature, 432, 2004,
588-95.
5.3.1.4.1 Cyclin E/A:Cdk2-mediated phosphorylation of p27/p21
Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-09-19.
Reviewers
Coqueret, O, 2006-10-06.
Description
References
1999, 193-9.
Reaction
5.3.1.4.1.1 Cyclin A:Cdk2 mediated phosphorylation of p27/p21
Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-09-28.
Reviewers
Coqueret, O, 2006-10-06.
Description
Recognition of p27 by SCF(Skp2) and the subsequent ubiquitination of p27 is dependent upon Cyclin E/A:Cdk2- mediated phosphorylated of
p27 at Thr 187 (Montagnoli et al., 1999). p21 is also phosphorylation at a specific site (Ser130) by Cyclin E/A:Cdk2 stimulating its ubiquitination.
Unlike p27, however, p21 ubiquitination can take place in the absence of phosphorylation, although with less efficiency (Bornstein et al.,2003).
References
Reaction
5.3.1.4.1.2 Cyclin E:Cdk2- mediated phosphorylation of p27/p21
Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-09-29.
Reviewers
Coqueret, O, 2006-10-06.
Description
References
1999, 193-9.
Reaction
5.3.1.4.2 Association of Cks1 with SCF(Skp2) complex
Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-09-19.
Reviewers
Coqueret, O, 2006-10-06.
Description
The accessory protein, Cks1 promotes efficient interaction between phosphorylated p27 and the SCF (Skp2) complex (Ganoth et al., 2001;
Spruck et al., 2001). Cks1 binds to Skp2 in the leucine-rich repeat (LRR) domain and C-terminal tail (Hao et al., 2005). The phosphorylated
Thr187 side chain of p27 associates with a phosphate binding site on Cks1, and the side chain containing Glu185 is positioned in the interface
between Skp2 and Cks1 where it interacts with both (Hao et al., 2005).
References
Reaction
5.3.1.4.3 Binding of phospho-p27/p21:Cdk2:Cyclin E/A to the SCF(Skp2):Cks1 complex
Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-09-19.
Reviewers
Coqueret, O, 2006-10-06.
Description
The association of Cks1 with both Skp2 and phosphorylated p27 promotes a tight interaction between p27 and the SCF complex (Hao et al.,
2005).
References
B Hao, N Zheng, BA Schulman, G Wu, JJ Miller, M Pagano, NP Pavletich, "Structural basis of the Cks1-dependent recognition of p27(Kip1) by
the SCF(Skp2) ubiquitin ligase", Mol Cell, 20, 2005, 9-19.
XH Zhu, H Nguyen, HD Halicka, F Traganos, A Koff, "Noncatalytic requirement for cyclin A-cdk2 in p27 turnover", Mol Cell Biol, 24, 2004,
6058-66.
Reaction
5.3.1.4.4 Ubiquitination of phospho-p27/p21
Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-09-19.
Reviewers
Coqueret, O, 2006-10-06.
Description
Once in tight contact with the SCF (Skp2):Cks1 complex, phosphorylated p27/p21 is ubiquitinated.
References
Reaction
5.3.1.4.5 Degradation of ubiquitinated p27/p21 by the 26S proteasome
Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-09-19.
Reviewers
Coqueret, O, 2006-10-06.
Description
Following ubiquitination by the SCF(Skp2):Cks1 complex, phospho-p27/p21 is degraded by the 26S proteasome.
References
1999, 193-9.
Reaction
5.3.1.5 Phosphorylation of Cyclin A:Cdk2 at Tyr 15
Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-09-28.
Reviewers
Coqueret, O, 2006-10-06.
Description
The CDK activity of the Cyclin A:Cdk2 complex is inhibited by phosphorylation at Tyr 15, presumably by the Wee1 kinase.
References
Y Gu, J Rosenblatt, DO Morgan, "Cell cycle regulation of CDK2 activity by phosphorylation of Thr160 and Tyr15", EMBO J, 11, 1992,
3995-4005.
Reaction
5.3.1.6 Cdc25A mediated dephosphorylation of Cyclin A:phospho-Cdk2

Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-09-28.
Reviewers
Coqueret, O, 2006-10-06.
Description
Cdc25A, and probably Cdc25B, regulate the entry into S phase cell cycle by removing inhibitory phosphates from the Cdk2 subunit of Cyclin
A:Cdk2.
References
Reaction
5.3.1.7 CAK-mediated phosphorylation of Cyclin A:Cdk2
Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-09-28.
Reviewers
Coqueret, O, 2006-10-06.
Description
Phosphorylation of cyclin-dependent kinases (CDKs) by the CDK-activating kinase (CAK) is required for the activation of the CDK kinase activity.
The association of p21/p27 with the Cyclin A/E:Cdk2 complex prevents CAK mediated phosphorylation of Cdk2 (Aprelikova et al., 1995).
References
Reaction
5.3.1.8 Phosphorylation of proteins involved in the G1/S transition by Cyclin A:Cdk2
Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-09-28.
Reviewers
Coqueret, O, 2006-10-06.
Description
Active Cyclin A:Cdk2 complexes phosphorylate and inactivate proteins required for maintaining the G1/S phase including: Cdh1, Rb, p21 and
p27. All this creates auto-amplification loops that render Cdk2 increasingly more active. In G2, Cdk2, in association with cyclin A, phosphorylates
E2F1 and E2F3 resulting in the inactivation and possibly degradation of these two transcription factors (Dynlacht et al., 1994; Krek et al., 1994).
References
J Mitra, GH Enders, J Azizkhan-Clifford, KL Lengel, "Dual regulation of the anaphase promoting complex in human cells by cyclin A-Cdk2 and
cyclin A-Cdk1 complexes", Cell Cycle, 5, 2006, 661-6.
Reaction
5.3.2 Synthesis of DNA
Description
The actual synthesis of DNA occurs in the S phase of the cell cycle. This includes the initiation of DNA replication, when the first nucleotide of
the new strand is laid down during the synthesis of the primer. The DNA replication preinitiation events begin in late M or early G1 phase.
5.3.2.1 DNA replication initiation
Description
DNA polymerases are not capable of de novo DNA synthesis and require synthesis of a primer, usually by a DNA-dependent RNA polymerase
(primase) to begin DNA synthesis. In eukaryotic cells, the primer is synthesized by DNA polymerase alpha:primase. First, the DNA primase
portion of this complex synthesizes approximately 6-10 nucleotides of RNA primer and then the DNA polymerase portion synthesizes an
additional 20 nucleotides of DNA.
References
TS Wang, SZ Hu, D Korn, "DNA primase from KB cells. Characterization of a primase activity tightly associated with immunoaffinity-purified DNA
polymerase-alpha.", J Biol Chem, 259, 1984, 1854-65.
5.3.2.1.1 The primase component of DNA polymerase:primase synthesizes a 6-10 nucleotide RNA primer at the origin
Description
At the beginning of this reaction, 1 molecule of 'DNA polymerase alpha:primase:DNA polymerase alpha:origin complex', and 1 molecule of 'NTP'
are present. At the end of this reaction, 1 molecule of 'DNA polymerase epsilon', and 1 molecule of 'RNA primer:origin duplex:DNA polymerase
alpha:primase complex' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'DNA-directed RNA polymerase activity' of 'DNA polymerase alpha:primase'.
References
Reaction
5.3.2.1.2 The polymerase component of DNA polymerase alpha:primase synthesizes a 20-nucleotide primer at the origin
Description
At the beginning of this reaction, 1 molecule of 'dTTP', 1 molecule of 'dGTP', 1 molecule of 'dATP', 1 molecule of 'RNA primer:origin duplex:DNA
polymerase alpha:primase complex', and 1 molecule of 'dCTP' are present. At the end of this reaction, 1 molecule of 'RNA primer-DNA
primer:origin duplex' is present.
This reaction takes place in the 'nucleus' and is mediated by the 'DNA-directed DNA polymerase activity' of 'DNA polymerase alpha:primase'.
Reaction
5.3.2.2 Switching of origins to a post-replicative state
5.3.2.2.1 Orc1 removal from chromatin
References
CB Wenger, MF Roberts, JA Stolwijk, ER Nadel, "Forearm blood flow during body temperature transients produced by leg exercise.", J Appl
Physiol, 38, 1975, 58-63.
EM Ladenburger, C Keller, R Knippers, "Identification of a binding region for human origin recognition complex proteins 1 and 2 that coincides
with an origin of DNA replication.", Mol Cell Biol, 22, 2002, 1036-48.
J MÃ©ndez, XH Zou-Yang, SY Kim, M Hidaka, WP Tansey, B Stillman, "Human origin recognition complex large subunit is degraded by
ubiquitin-mediated proteolysis after initiation of DNA replication.", Mol Cell, 9, 2002, 481-91.
CJ Li, ML DePamphilis, "Mammalian Orc1 protein is selectively released from chromatin and ubiquitinated during the S-to-M transition in the cell
division cycle.", Mol Cell Biol, 22, 2001, 105-16.
5.3.2.2.1.1 Orc1 is phosphorylated by cyclin A/CDK2

Description
At the beginning of this reaction, 1 molecule of 'ATP', and 1 molecule of 'pre-replicative complex' are present. At the end of this reaction, 1
molecule of 'phosphorylated Orc1', 1 molecule of 'pre-replicative complex (Orc1-minus)', and 1 molecule of 'ADP' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'kinase activity' of 'Cyclin A:Cdk2 complex'.
References
Reaction
5.3.2.2.1.2 Phosphorylated Orc1 is ubiquitinated while still associated with chromatin
Description
At the beginning of this reaction, 1 molecule of 'ubiquitin', and 1 molecule of 'phosphorylated Orc1' are present. At the end of this reaction, 1
molecule of 'ubiquitinated Orc1' is present.
References
Reaction
5.3.2.2.1.3 Ubiquitinated Orc1 enters the cytosol
Description
In this reaction, 1 molecule of 'ubiquitinated Orc1' is translocated from nucleoplasm to cytosol.
This movement of the molecule occurs through the 'nuclear pore'.
References
Reaction
5.3.2.2.1.4 Ubiquitinated Orc1 is degraded by the proteasome
Description
At the beginning of this reaction, 1 molecule of 'ubiquitinated Orc1' is present. At the end of this reaction, 1 molecule of 'ubiquitin' is present.
References
Reaction
5.3.2.2.2 CDK-mediated phosphorylation and removal of Cdc6
Description
As cells enter S phase, HsCdc6p is phosphorylated by CDK promoting its export from the nucleus (see Bell and Dutta 2002).
References
J MÃ©ndez, B Stillman, "Chromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during
the cell cycle: assembly of prereplication complexes in late mitosis.", Mol Cell Biol, 20, 2000, 8602-12.
5.3.2.2.2.1 Cdc6 protein is phosphorylated by CDK
Description
At the beginning of this reaction, 1 molecule of 'CDC6', and 1 molecule of 'ATP' are present. At the end of this reaction, 1 molecule of 'ADP', and
1 molecule of 'phosphorylated Cdc6' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'kinase activity' of 'CDK'.
References
W Jiang, NJ Wells, T Hunter, "Multistep regulation of DNA replication by Cdk phosphorylation of HsCdc6.", Proc Natl Acad Sci U S A, 96, 1999,
6193-8.
Reaction
5.3.2.2.2.2 Phosphorylated Cdc6 is exported from the nucleus
Description
In this reaction, 1 molecule of 'phosphorylated Cdc6' is translocated from nucleoplasm to cytosol.
References
6193-8.
Reaction
5.3.2.2.2.3 Cytoplasmic phosphorylated Cdc6 is ubiquitinated by the anaphase-promoting complex
Description
At the beginning of this reaction, 1 molecule of 'phosphorylated Cdc6', 1 molecule of 'ubiquitin', and 1 molecule of 'ATP' are present. At the end
of this reaction, 1 molecule of 'ubiquitinated Cdc6' is present.
This reaction takes place in the 'cytosol' and is mediated by the 'endopeptidase activity' of 'anaphase-promoting complex (APC)'.
References
BO Petersen, C Wagener, F Marinoni, ER Kramer, M Melixetian, EL Denchi, C Gieffers, C Matteucci, JM Peters, K Helin, "Cell cycle- and cell
growth-regulated proteolysis of mammalian CDC6 is dependent on APC-CDH1.", Genes Dev, 14, 2000, 2330-43.
Reaction
5.3.2.2.2.4 Ubiquitinated Cdc6 is degraded by the proteasome
Description
At the beginning of this reaction, 1 molecule of 'ubiquitinated Cdc6' is present. At the end of this reaction, 1 molecule of 'ubiquitin' is present.
References
Reaction
5.3.2.2.3 Mcm4,6,7 trimer forms and associates with the replication fork
Description
At the start of the elongation phase of DNA replication, the Mcm2-7 complex may re-arrange to function as the replicative helicase associated
with the replication fork. In general, a replicative helicase is associated with the replication fork and unwinds DNA ahead of the polymerase. In
yeast, the Mcm proteins associate with origin DNA in G1 phase and then exit the origin upon replication initiation, consistent with moving out of
the origin with the replication fork. The Mcm2-7 complex is a ring-shaped hexamer. Complexes of Mcm4, Mcm6 and Mcm7 proteins from
humans or S. pombe display a modest ATP-dependent helicase activity in vitro. Consistent with the hypothesis that eukaryotic Mcm complexes
function as helicases, an archaeal Mcm homolog is a ring-shaped double hexamer that has a processive DNA unwinding activity. Mcm proteins
may have additional functions during elongation, as uninterrupted function of all six is required for replication fork progression in budding yeast.
Mcm4,6,7 helicase activity may be negatively regulated in two ways. Mcm2, Mcm4, Mcm6, and Mcm7 also form a stable complex which,
however, has no helicase activity, suggesting that Mcm2 inhibits DNA unwinding by Mcm4,6,7. In addition, phosphorylation of human Mcm4,6,7
complex by CDK inhibits its helicase activity.
References
Z You, Y Komamura, Y Ishimi, "Biochemical analysis of the intrinsic Mcm4-Mcm6-mcm7 DNA helicase activity.", Mol Cell Biol, 19, 2000,
8003-15.
JP Chong, MK Hayashi, MN Simon, RM Xu, B Stillman, "A double-hexamer archaeal minichromosome maintenance protein is an
ATP-dependent DNA helicase.", Proc Natl Acad Sci U S A, 97, 2000, 1530-5.
Y Ishimi, "A DNA helicase activity is associated with an MCM4, -6, and -7 protein complex.", J Biol Chem, 272, 1997, 24508-13.
D Schaarschmidt, EM Ladenburger, C Keller, R Knippers, "Human Mcm proteins at a replication origin during the G1 to S phase transition.",
Nucleic Acids Res, 30, 2002, 4176-85.
Z Kelman, JK Lee, J Hurwitz, "The single minichromosome maintenance protein of Methanobacterium thermoautotrophicum DeltaH contains
DNA helicase activity.", Proc Natl Acad Sci U S A, 96, 2000, 14783-8.
M Sato, T Gotow, Z You, Y Komamura-Kohno, Y Uchiyama, N Yabuta, H Nojima, Y Ishimi, "Electron microscopic observation and
single-stranded DNA binding activity of the Mcm4,6,7 complex.", J Mol Biol, 300, 2000, 421-31.
BK Tye, "MCM proteins in DNA replication.", Annu Rev Biochem, 68, 2000, 649-86.
Y Ishimi, Y Komamura-Kohno, "Phosphorylation of Mcm4 at specific sites by cyclin-dependent kinase leads to loss of Mcm4,6,7 helicase
activity.", J Biol Chem, 276, 2001, 34428-33.
Y Ishimi, Y Komamura-Kohno, Z You, A Omori, M Kitagawa, "Inhibition of Mcm4,6,7 helicase activity by phosphorylation with cyclin A/Cdk2.", J
Biol Chem, 275, 2000, 16235-41.
K Labib, JA Tercero, JF Diffley, "Uninterrupted MCM2-7 function required for DNA replication fork progression.", Science, 288, 2000, 1643-7.
Reaction
5.3.2.3 DNA strand elongation
Authors
Tom, S, Bambara, RA, 2003-06-05.
Description
Accurate and efficient genome duplication requires coordinated processes to replicate two template strands at eucaryotic replication forks.
Knowledge of the fundamental reactions involved in replication fork progression is derived largely from biochemical studies of the replication of
simian virus and from yeast genetic studies. Since duplex DNA forms an anti-parallel structure, and DNA polymerases are unidirectional, one of
the new strands is synthesized continuously in the direction of fork movement. This strand is designated as the leading strand. The other strand
grows in the direction away from fork movement, and is called the lagging strand. Several specific interactions among the various proteins
involved in DNA replication underlie the mechanism of DNA synthesis, on both the leading and lagging strands, at a DNA replication fork. These
interactions allow the replication enzymes to cooperate in the replication process.
References
GS Brush, TJ Kelly, B Stillman, "Identification of eukaryotic DNA replication proteins using simian virus 40 in vitro replication system.", Methods
Enzymol, 262, 1996, 522-48.
R Ayyagari, KJ Impellizzeri, BL Yoder, SL Gary, PM Burgers, "A mutational analysis of the yeast proliferating cell nuclear antigen indicates
distinct roles in DNA replication and DNA repair.", Mol Cell Biol, 15, 1995, 4420-9.
ME Budd, JL Campbell, "A yeast replicative helicase, Dna2 helicase, interacts with yeast FEN-1 nuclease in carrying out its essential function.",
Mol Cell Biol, 17, 1997, 2136-42.
RA Bambara, RS Murante, LA Henricksen, "Enzymes and reactions at the eukaryotic DNA replication fork.", J Biol Chem, 272, 1997, 4647-50.
J Hurwitz, AD Kwong, SH Lee, "The in vitro replication of DNA containing the SV40 origin.", J Biol Chem, 265, 1990, 18043-6.
5.3.2.3.1 Unwinding of DNA
Authors
Tye, BK, 2006-03-17.
Editors
Description
DNA Replication is regulated accurately and precisely by various protein complexes. Many members of the MCM protein family are assembled
into the pre-Replication Complexes (pre-RC) at the end of M phase of the cell cycle. DNA helicase activity of some of the MCM family proteins
are important for the unwinding of DNA and initiation of replication processes. This section contains four events which have been proved in
different eukaryotic experimental systems to involve various proteins for this essential step during DNA Replication.
References
D Maiorano, O Cuvier, E Danis, M Mechali, "MCM8 is an MCM2-7-related protein that functions as a DNA helicase during replication elongation
and not initiation", Cell, 120, 2005, 315-28.
5.3.2.3.1.1 MCM2-7 mediated fork unwinding
Authors
Tye, BK, 2005-11-29.
Editors
Description
In budding yeast, all MCM proteins have been proved to be essential for elongation. The active form of this protein complex may be a
heterohexamer. A subcomplex of MCM proteins consisting fo MCM4,6, and -7 has a weak helicase activity that may contribute to DNA
unwinding.
References
Source reaction
This reaction was inferred from the corresponding reaction "Yeast Mcm2-7 mediated fork unwinding" in species Saccharomyces cerevisiae.
DL Kaplan, MJ Davey, M O'Donnell, "Mcm4,6,7 uses a "pump in ring" mechanism to unwind DNA by steric exclusion and actively
translocate along a duplex", J Biol Chem, 278, 2003, 49171-82.
JK Lee, J Hurwitz, "Processive DNA helicase activity of the minichromosome maintenance proteins 4, 6, and 7 complex requires forked DNA
structures", Proc Natl Acad Sci U S A, 98, 2001, 54-9.
BK Tye, S Sawyer, "The hexameric eukaryotic MCM helicase: building symmetry from nonidentical parts", J Biol Chem, 275, 2000, 34833-6.
Reaction
5.3.2.3.1.2 MCM8 mediated fork unwinding
Authors
Tye, BK, 2005-11-29.
Editors
Description
The MCM2-7 related protein, MCM8, is required to replicate chromosomal DNA in Xenopus egg extracts. MCM8 binds chromatin upon initiation
of DNA synthesis. It may function as an helicase in the elongation step.
References
Source reaction
This reaction was inferred from the corresponding reaction "Xenopus Mcm8 mediated fork unwinding" in species Xenopus laevis.
Reaction
5.3.2.3.1.3 Formation of GINS complex
Description
At the beginning of this reaction, 1 molecule of 'PSF3p', 1 molecule of 'SLD5P', 1 molecule of 'PSF2p', and 1 molecule of 'PSF1p' are present. At
the end of this reaction, 1 molecule of 'GINS complex' is present.
Reaction
5.3.2.3.1.4 Multiple proteins are localized at replication fork
Authors
Tye, BK, 2006-03-17.
Editors
Description
By applying the chromatin immunoprecipitation technique to paused forks, certain proteins like DNA pol alpha, DNA pol delta, DNA pol epsilon,
MCM2-7, CDC45, GINS and MCM10 were identified. By uncoupling a helicase at the site using a polymerase inhibitor, MCM2-7, GINS complex
and CDC45 alone were found to be enriched at the paused fork suggesting these proteins may form a part of an "unwindosome" at the
replicating fork.
References
M Pacek, AV Tutter, Y Kubota, H Takisawa, JC Walter, "Localization of MCM2-7, Cdc45, and GINS to the site of DNA unwinding during
eukaryotic DNA replication", Mol Cell, 21, 2006, 581-7.
Source reaction
This reaction was inferred from the corresponding reaction "Multiple Xenopus proteins are localized at replication fork" in species Xenopus
laevis.
Reaction
5.3.2.3.2 Leading Strand Synthesis
Description
The processive complex is responsible for synthesizing at least 5-10 kb of DNA in a continuous manner during leading strand synthesis. The
incorporation of nucleotides by pol delta is quite accurate. However, incorporation of an incorrect nucleotide does occur occasionally.
Misincorporated nucleotides are removed by the 3' to 5' exonucleolytic proofreading capability of pol delta.
References
T Matsumoto, T Eki, J Hurwitz, "Studies on the initiation and elongation reactions in the simian virus 40 DNA replication system.", Proc Natl Acad
Sci U S A, 87, 1991, 9712-6.
MY Lee, CK Tan, AG So, KM Downey, "Purification of deoxyribonucleic acid polymerase delta from calf thymus: partial characterization of
physical properties.", Biochemistry, 19, 1980, 2096-101.
R Hindges, U HÃ¼bscher, "DNA polymerase delta, an essential enzyme for DNA transactions.", Biol Chem, 378, 1997, 345-62.
5.3.2.3.2.1 Polymerase switching
Description
After the primers are synthesized, Replication Factor C binds to the 3'-end of the initiator DNA to trigger polymerase switching. The
non-processive nature of pol alpha catalytic activity and the tight binding of Replication Factor C to the primer-template junction presumably lead
to the turnover of the pol alpha:primase complex. After the Pol alpha-primase primase complex is displaced from the primer, the proliferating cell
nuclear antigen (PCNA) binds to form a "sliding clamp" structure. Replication Factor C then dissociates, and DNA polymerase delta binds and
catalyzes the processive synthesis of DNA.
References
SH Lee, AD Kwong, ZQ Pan, J Hurwitz, "Studies on the activator 1 protein complex, an accessory factor for proliferating cell nuclear
antigen-dependent DNA polymerase delta.", J Biol Chem, 266, 1991, 594-602.
T Tsurimoto, B Stillman, "Replication factors required for SV40 DNA replication in vitro. II. Switching of DNA polymerase alpha and delta during
initiation of leading and lagging strand synthesis.", J Biol Chem, 266, 1991, 1961-8.
T Tsurimoto, B Stillman, "Functions of replication factor C and proliferating-cell nuclear antigen: functional similarity of DNA polymerase
accessory proteins from human cells and bacteriophage T4.", Proc Natl Acad Sci U S A, 87, 1990, 1023-7.
5.3.2.3.2.1.1 RFC binding displaces Pol Alpha
Description
Once the RNA-DNA primer is synthesized, replication factor C (RFC) initiates a reaction called "polymerase switching"; pol delta, the processive
enzyme replaces pol alpha, the priming enzyme. RFC binds to the 3'-end of the RNA-DNA primer on the Primosome, to displace the pol alpha
primase complex. The binding of RFC triggers the binding of the primer recognition complex.
References
G Maga, M Stucki, S Spadari, U HÃ¼bscher, "DNA polymerase switching: I. Replication factor C displaces DNA polymerase alpha prior to
PCNA loading.", J Mol Biol, 295, 2000, 791-801.
R Mossi, RC Keller, E Ferrari, U HÃ¼bscher, "DNA polymerase switching: II. Replication factor C abrogates primer synthesis by DNA
polymerase alpha at a critical length.", J Mol Biol, 295, 2000, 803-14.
Reaction
5.3.2.3.2.1.2 Loading of PCNA - Sliding Clamp Formation
Description
The binding of the primer recognition complex involves the loading of proliferating cell nuclear antigen (PCNA). Replication Factor C transiently
opens the PCNA toroid in an ATP-dependent reaction, and then allows PCNA to re-close around the double helix adjacent to the primer
terminus. This leads to the formation of the "sliding clamp".
References
T Tsurimoto, T Melendy, B Stillman, "Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the
SV40 DNA replication origin.", Nature, 346, 1990, 534-9.
R Mossi, U HÃ¼bscher, "Clamping down on clamps and clamp loaders--the eukaryotic replication factor C.", Eur J Biochem, 254, 1998, 209-16.
Reaction
5.3.2.3.2.1.3 RFC dissociates after sliding clamp formation
Description
Replication factor C is proposed to dissociate from PCNA following sliding clamp formation, and the DNA toroid alone tethers pol delta to the
DNA.
References
VN Podust, N Tiwari, S Stephan, "Replication factor C disengages from proliferating cell nuclear antigen (PCNA) upon sliding clamp formation,
and PCNA itself tethers DNA polymerase delta to DNA.", J Biol Chem, 273, 1998, 31992-9.
Reaction
5.3.2.3.2.1.4 Formation of Processive Complex
Description
The loading of proliferating cell nuclear antigen (PCNA) leads to recruitment of pol delta. Human PCNA is a homotrimer of 36 kDa subunits that
form a toroidal structure. The loading of PCNA by RFC is a key event in the transition from the priming mode to the extension mode of DNA
synthesis. The processive complex is composed of the pol delta holoenzyme and PCNA.
References
SH Lee, J Hurwitz, "Mechanism of elongation of primed DNA by DNA polymerase delta, proliferating cell nuclear antigen, and activator 1.", Proc
Natl Acad Sci U S A, 87, 1990, 5672-6.
WC Brown, JL Campbell, "Interaction of proliferating cell nuclear antigen with yeast DNA polymerase delta.", J Biol Chem, 268, 1993, 21706-10.
SJ Zhang, XR Zeng, P Zhang, NL Toomey, RY Chuang, LS Chang, MY Lee, "A conserved region in the amino terminus of DNA polymerase
delta is involved in proliferating cell nuclear antigen binding.", J Biol Chem, 270, 1995, 7988-92.
PM Burgers, "Saccharomyces cerevisiae replication factor C. II. Formation and activity of complexes with the proliferating cell nuclear antigen
and with DNA polymerases delta and epsilon.", J Biol Chem, 266, 1991, 22698-706.
Reaction
5.3.2.3.2.2 Processive synthesis on the leading strand
Description
Polymerase switching is a key event that allows the processive synthesis of DNA by the pol delta and PCNA complex. Polymerase delta
possesses polymerization and proofreading activities, which increases the overall fidelity of DNA replication. The pol delta holoenzyme is a
heterotetrameric complex that contains p125, p66, p50, and p12 subunits, in human cells.
References
L Liu, J Mo, EM Rodriguez-Belmonte, MY Lee, "Identification of a fourth subunit of mammalian DNA polymerase delta.", J Biol Chem, 275, 2000,
18739-44.
Reaction
5.3.2.3.3 Lagging Strand Synthesis
Description
Due to the antiparallel nature of DNA, DNA polymerization is unidirectional, and one strand is synthesized discontinuously. This strand is called
the lagging strand. Although the polymerase switching on the lagging strand is very similar to that on the leading strand, the processive
synthesis on the two strands proceeds quite differently. Short DNA fragments, about 100 bases long, called Okazaki fragments are synthesized
on the RNA-DNA primers first. Strand-displacement synthesis occurs, whereby the primer-containing 5'-terminus of the adjacent Okazaki
fragment is folded into a single-stranded flap structure. This flap structure is removed by endonucleases, and the adjacent Okazaki fragments
are joined by DNA ligase.
References
5.3.2.3.3.1 Polymerase switching
Description
References
5.3.2.3.3.1.1 RFC binding displaces Pol Alpha
Description
References
Reaction
5.3.2.3.3.1.2 Loading of PCNA - Sliding Clamp Formation
Description
References
Reaction
5.3.2.3.3.1.3 RFC dissociates after sliding clamp formation
Description
DNA.
References
Reaction
5.3.2.3.3.1.4 Formation of Processive Complex
Description
References
Natl Acad Sci U S A, 87, 1990, 5672-6.
Reaction
5.3.2.3.3.2 Processive synthesis on the lagging strand
Description
The key event that allows the processive synthesis on the lagging strand, is polymerase switching from pol alpha to pol delta, as on the leading
strand. However, the processive synthesis on the lagging strand proceeds very differently. DNA synthesis is discontinuous, and involves the
formation of short fragments called the Okazaki fragments. During the synthesis of Okazaki fragments, the RNA primer is folded into a
single-stranded flap, which is removed by endonucleases. This is followed by the ligation of adjacent Okazaki fragments.
References
5.3.2.3.3.2.1 Formation of Okazaki fragments
Description
After RFC initiates the assembly of the primer recognition complex, the complex of pol delta and PCNA is responsible for incorporating the
additional nucleotides prior to the position of the next downstream initiator RNA primer. On the lagging strand, short discontinuous segments of
DNA, called Okazaki fragments, are synthesized on RNA primers. The average length of the Okazaki fragments is 100 nucleotides. Polymerase
switching is a key event that allows the processive synthesis of DNA by the pol delta and PCNA complex.
References
Natl Acad Sci U S A, 87, 1990, 5672-6.
T Nethanel, T Zlotkin, G Kaufmann, "Assembly of simian virus 40 Okazaki pieces from DNA primers is reversibly arrested by ATP depletion.", J
Virol, 66, 1992, 6634-40.
S Waga, G Bauer, B Stillman, "Reconstitution of complete SV40 DNA replication with purified replication factors.", J Biol Chem, 269, 1994,
10923-34.
Reaction
5.3.2.3.3.2.2 Formation of the Flap Intermediate
Description
When the polymerase delta:PCNA complex reaches a downstream Okazaki fragment, strand displacement synthesis occurs. The primer
containing 5'-terminus of the downstream Okazaki fragment is folded into a single-stranded flap.
References
G Maga, G Villani, V Tillement, M Stucki, GA Locatelli, S Spadari, U HÃ¼bscher, "Okazaki fragment processing: modulation of the strand
displacement activity of DNA polymerase delta by the concerted action of replication protein A, proliferating cell nuclear antigen, and flap
endonuclease-1.", Proc Natl Acad Sci U S A, 98, 2001, 14298-303.
VN Podust, LM Podust, F MÃ¼ller, U HÃ¼bscher, "DNA polymerase delta holoenzyme: action on single-stranded DNA and on double-stranded
DNA in the presence of replicative DNA helicases.", Biochemistry, 34, 1995, 5003-10.
SH Bae, KH Bae, JA Kim, YS Seo, "RPA governs endonuclease switching during processing of Okazaki fragments in eukaryotes.", Nature, 412,
2001, 456-61.
Reaction
5.3.2.3.3.2.3 Removal of the Flap Intermediate
Description
Two endonucleases, Dna2 and flap endonuclease 1 (FEN-1), are responsible for resolving the nascent flap structure. The Dna2
endonuclease/helicase in yeast is a monomer of approximately 172 kDa. Human FEN-1 is a single polypeptide of approximately 42 kDa.
Replication Protein A regulates the switching of endonucleases during the removal of the displaced flap.
References
ME Budd, JL Campbell, "A yeast gene required for DNA replication encodes a protein with homology to DNA helicases.", Proc Natl Acad Sci U S
A, 92, 1995, 7642-6.
JJ Harrington, MR Lieber, "The characterization of a mammalian DNA structure-specific endonuclease.", EMBO J, 13, 1994, 1235-46.
JJ Harrington, MR Lieber, "Functional domains within FEN-1 and RAD2 define a family of structure-specific endonucleases: implications for
nucleotide excision repair.", Genes Dev, 8, 1994, 1344-55.
5.3.2.3.3.2.3.1 RPA binds to the Flap
Description
The first step in the removal of the flap intermediate is the binding of Replication Protein A (RPA) to the long flap structure. RPA is a eukaryotic
single-stranded DNA binding protein.
References
2001, 456-61.
Reaction
5.3.2.3.3.2.3.2 Recruitment of Dna2 endonuclease
Description
After RPA binds the long flap, it recruits the Dna2 endonuclease. Dna2 endonuclease removes most of the flap, but the job of complete removal
of the flap is then completed by FEN-1.
References
2001, 456-61.
Reaction
5.3.2.3.3.2.3.3 Removal of RNA primer and dissociation of RPA and Dna2
Description
The Dna2 endonuclease removes the initiator RNA along with several downstream deoxyribonucleotides. The cleavage of the single-stranded
RNA substrate results in the disassembly of RPA and Dna2. The current data for the role of the Dna2 endonuclease has been derived from
studies with yeast and Xenopus Dna2.
References
2001, 456-61.
ME Budd, W Choe, JL Campbell, "The nuclease activity of the yeast DNA2 protein, which is related to the RecB-like nucleases, is essential in
vivo.", J Biol Chem, 275, 2000, 16518-29.
Reaction
5.3.2.3.3.2.3.4 Removal of remaining Flap
Description
The remaining flap, which is too short to support RPA binding, is then processed by FEN-1. There is evidence that binding of RPA to the
displaced end of the RNA-containing Okazaki fragment prevents FEN-1 from accessing the substrate. FEN-1 is a structure-specific
endonuclease that cleaves near the base of the flap at a position one nucleotide into the annealed region Biochemical studies have shown that
the preferred substrate for FEN-1 consists of a one-nucleotide 3'-tail on the upstream primer in addition to the 5'-flap of the downstream primer.
References
Y Xu, ND Grindley, CM Joyce, "Coordination between the polymerase and 5'-nuclease components of DNA polymerase I of Escherichia coli.", J
Biol Chem, 275, 2000, 20949-55.
HI Kao, LA Henricksen, Y Liu, RA Bambara, "Cleavage specificity of Saccharomyces cerevisiae flap endonuclease 1 suggests a double-flap
structure as the cellular substrate.", J Biol Chem, 277, 2002, 14379-89.
MW Kaiser, N Lyamicheva, W Ma, C Miller, B Neri, L Fors, VI Lyamichev, "A comparison of eubacterial and archaeal structure-specific
5'-exonucleases.", J Biol Chem, 274, 1999, 21387-94.
MR Lieber, "The FEN-1 family of structure-specific nucleases in eukaryotic DNA replication, recombination and repair.", Bioessays, 19, 1997,
233-40.
RS Murante, JA Rumbaugh, CJ Barnes, JR Norton, RA Bambara, "Calf RTH-1 nuclease can remove the initiator RNAs of Okazaki fragments by
endonuclease activity.", J Biol Chem, 271, 1996, 25888-97.
JJ Harrington, MR Lieber, "DNA structural elements required for FEN-1 binding.", J Biol Chem, 270, 1995, 4503-8.
Reaction
5.3.2.3.3.2.4 Joining of adjacent Okazaki fragments
Description
Removal of the flap by FEN-1 leads to the generation of a nick between the 3'-end of the upstream Okazaki fragment and the 5'-end of the
downstream Okazaki fragment. DNA ligase I then seals the nicks between adjacent processed Okazaki fragments to generate intact
double-stranded DNA.
References
DS Levin, AE McKenna, TA Motycka, Y Matsumoto, AE Tomkinson, "Interaction between PCNA and DNA ligase I is critical for joining of Okazaki
fragments and long-patch base-excision repair.", Curr Biol, 10, 2000, 919-22.
S Waga, B Stillman, "The DNA replication fork in eukaryotic cells.", Annu Rev Biochem, 67, 1998, 721-51.
JJ Turchi, RA Bambara, "Completion of mammalian lagging strand DNA replication using purified proteins.", J Biol Chem, 268, 1993, 15136-41.
Reaction
5.3.3 Ubiquitin-dependent degradation of Cyclin D
Description
Cyclin D turnover is regulated by ubiquitination and proteasomal degradation which are positively regulated by cyclin D phosphorylation on
threonine-286 (Diehl et al., 1997).
References
JA Diehl, F Zindy, CJ Sherr, "Inhibition of cyclin D1 phosphorylation on threonine-286 prevents its rapid degradation via the ubiquitin-proteasome
pathway", Genes Dev, 11, 1997, 957-72.
5.3.3.1 Ubiquitin-dependent degradation of Cyclin D1
Description
After the Cyclin D serves the role of mediating reactions by Cdk4 and Cdk6, it is shuttled to the cytoplasm and degraded in a ubiquitin-dependent
manner. Whether Cdk4 and Cdk6 are truly redundant is a topic still under investigation, although both the kinases are required for normal cell
cycle progression.
Destruction of the D type cyclins accompanies the end of the G1 phase, and the E type cyclins are involved in transition of the cell from G1 to S
phase.
5.3.3.1.1 Phosphorylation of Cyclin D1 at T286 by glycogen synthase kinase-3 beta
Description
At the beginning of this reaction, 1 molecule of 'Cyclin D1:Cdk4', and 1 molecule of 'ATP' are present. At the end of this reaction, 1 molecule of
'phospho(T286)-Cyclin D1:Cdk4', and 1 molecule of 'ADP' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'kinase activity' of 'glycogen synthase kinase-3 beta'.
References
S Benzeno, F Lu, M Guo, O Barbash, F Zhang, JG Herman, PS Klein, A Rustgi, JA Diehl, "Identification of mutations that disrupt
phosphorylation-dependent nuclear export of cyclin D1", Oncogene, 2006.
Source reaction
This reaction was inferred from the corresponding reaction "Phosphorylation of Cyclin D1 on Thr-286 by GSK-3 beta" in species Mus musculus.
JA Diehl, M Cheng, MF Roussel, CJ Sherr, "Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization",
Genes Dev, 12, 1998, 3499-511.
Reaction
5.3.3.1.2 Relocalization of nuclearly localized phospho-(T286):cyclin D1:Cdk4 to cytoplasm
Description
In this reaction, 1 molecule of 'phospho(T286)-Cyclin D1:Cdk4' is translocated from nucleoplasm to cytosol.
Reaction
5.3.3.1.3 Relocalization of nuclearly localized Cyclin D1 to the cytoplasm
Description
In this reaction, 1 molecule of 'phospho(T286)-Cyclin D1' is translocated from nucleoplasm to cytosol.

Reaction
5.3.3.1.4 Ubiquitination of Cyclin D1
Authors
Matthews, L, 2006-04-14.
Editors
Matthews, L, 2006-04-14.
Description
Cyclin D is targeted for degradation by multi-ubiquitination.
Reaction
5.3.3.1.5 Proteasome mediated degradation of Cyclin D1
Editors
Matthews, L, 2006-04-14.
Description
Phosphorylated Cyclin D1 is degraded during S phase by the 26S proteasome allowing for efficient DNA synthesis.
References
Y Guo, K Yang, J Harwalkar, JM Nye, DR Mason, MD Garrett, M Hitomi, DW Stacey, "Phosphorylation of cyclin D1 at Thr 286 during S phase
leads to its proteasomal degradation and allows efficient DNA synthesis", Oncogene, 24, 2005, 2599-612.
Reaction
5.3.4 S-specific transcription in mitotic cell cycle
Description
S phase-specific transcription will be described in a future release.

5.4 G2 Phase
Description
This is one of two 'gap' phases in the standard eukaryotic mitotic cell cycle. It is the interval between the completion of DNA synthesis and the
beginning of mitosis. Protein synthesis occurs in this phase, following DNA replication in the S phase. This is the time when the cell stockpiles on
the cytoplasmic contents, before mitosis and cytokinesis occurs.
5.4.1 G2-specific transcription in mitotic cell cycle
Description
G2-specific transcription will be described in a future release.
5.4.2 Phosphorylation of E2F1/E2F3 by Cyclin A:phosph-Cdk2(Thr 160)

Authors
Pagano, M, 2006-09-19.
Editors
Matthews, L, 2006-09-28.
Reviewers
Coqueret, O, 2006-10-06.
Description
In G2 Cdk2, in association with cyclin A, phosphorylates E2F1 and E2F3 resulting in the inactivation and possibly degradation of these two
transcription factors (Dynlacht et al., 1994; Krek et al., 1994).
References
W Krek, ME Ewen, S Shirodkar, Z Arany, Jr Kaelin WG, DM Livingston, "Negative regulation of the growth-promoting transcription factor E2F-1
by a stably bound cyclin A-dependent protein kinase", Cell, 78, 1994, 161-72.
BD Dynlacht, O Flores, JA Lees, E Harlow, "Differential regulation of E2F transactivation by cyclin/cdk2 complexes", Genes Dev, 8, 1994,
1772-86.
Reaction
5.5 G2/M Transition
Reviewers
Lorca, T, 2005-10-10.
Description
Cyclin A can also form complexes with Cdc2 (Cdk1). Together with three B-type cyclins, Cdc2 (Cdk1) regulates the transition from G2 into
mitosis. These complexes are activated by dephosphorylation of T14 and Y15. Cyclin A, B - Cdc2 complexes phosphorylate several proteins
involved in mitotic spindle structure and function, the breakdown of the nuclear envelope, and topological changes in chromosomes allowing
resolution of their entanglement and condensation that is necessary for the ~2 meters of DNA to be segregated at mitosis.
5.5.1 Cyclin A/B1 associated events during G2/M transition
Description
Cell cycle progression is regulated by cyclin-dependent protein kinases at both the G1/S and the G2/M transitions by cyclin-dependent protein
kinases. The G2/M transition is regulated through the phosphorylation of nuclear lamins and histones (reviewed in Sefton, 2001).
References
BM Sefton, "Overview of protein phosphorylation", Curr Protoc Cell Biol, 14, 2001, Unit 14.1.
5.5.1.1 Formation of Cyclin A:Cdc2 complexes
Description
Cyclin A is synthesized and associates with Cdc2 in G1. Cyclin dependent kinases are themselves catalytically inactive due to the fact that their
active sites are blocked by a portion of the CDK molecule itself. Binding to their corresponding cyclin partner results in a conformational change
that partially exposes the active site.
References
J Pines, T Hunter, "Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport", J Cell Biol,
115, 1991, 1-17.
Reaction
5.5.1.2 Myt-1 mediated phosphorylation of Cyclin A:Cdc2
Description
References
1997, 22300-6.
Source reaction
This reaction was inferred from the corresponding reaction "Myt-1 mediated phosphorylation of Cyclin B:Cdc2 complexes" in species Homo
sapiens.
1997, 22300-6.
Reaction
5.5.1.3 Translocation of Cyclin A:phospho-Cdc2 (Thr 14) to the nucleus
Description
Cyclin A:Cdc2 complexes translocate to the nucleus in G1 and may associate with condensing chromosomes in prophase (Pines and Hunter
1991).
References
115, 1991, 1-17.
Reaction
5.5.1.4 CAK-mediated phosphorylation of Cyclin A:Cdc2 complexes
Description
Full activity of most CDKs is dependent on CAK mediated phosphorylation at a conserved residue (Thr 161 in Cdc2). This modification is thought
to improve substrate binding. High affinity binding of Cyclin A within the Cyclin A:Cdc2 complex requires this phosphorylation (Desai et al 1995).
References
1995, 345-50.
Reaction
5.5.1.5 Wee1-mediated phosphorylation of Cyclin A:phospho-Cdc2 complexes
Description
The human Wee1 kinase phosphorylates Cdc2 on tyrosine 15 inactivating the cyclin:CDK complex (Watanabe et al., 1995).
References
N Watanabe, M Broome, T Hunter, "Regulation of the human WEE1Hu CDK tyrosine 15-kinase during the cell cycle", EMBO J, 14, 1995,
1878-91.
Source reaction
This reaction was inferred from the corresponding reaction "Wee1- mediated phosphorylation of Cyclin B1:phospho-Cdc2 complexes" in species
Homo sapiens.
Reaction
5.5.1.6 Dephosphorylation of nuclear Cyclin A:phosph-Cdc2 complexes
Description
Activation of the mitotic cyclin:Cdc2 complexes at mitosis requires the removal of the inhibitory phosphate groups on Cdc2. This
dephosphorylation is achieved by the activity of the Cdc25 family of phosphatases. The Cdc25 members, Cdc25A, Cdc25B, and Cdc25C are
kept inactive during interphase and are activated at the G2/M transition (see Wolfe and Gould 2004)
References
BA Wolfe, KL Gould, "Inactivating Cdc25, mitotic style", Cell Cycle, 3, 2004, 601-3.
Source reaction
This reaction was inferred from the corresponding reaction "Dephosphorylation of cytoplasmic Cyclin B1:phospho-Cdc2 (Thr 14, Tyr 15)
complexes by Cdc25 phosphatases" in species Homo sapiens.
I Hoffmann, PR Clarke, MJ Marcote, E Karsenti, G Draetta, "Phosphorylation and activation of human cdc25-C by cdc2--cyclin B and its
involvement in the self-amplification of MPF at mitosis", EMBO J, 12, 1993, 53-63.
R Honda, Y Ohba, A Nagata, H Okayama, H Yasuda, "Dephosphorylation of human p34cdc2 kinase on both Thr-14 and Tyr-15 by human
cdc25B phosphatase", FEBS Lett, 318, 1993, 331-4.
A Lindqvist, H Kallstrom, C Karlsson Rosenthal, "Characterisation of Cdc25B localisation and nuclear export during the cell cycle and in
response to stress", J Cell Sci, 117, 2004, 4979-90.
M Jackman, C Lindon, EA Nigg, J Pines, "Active cyclin B1-Cdk1 first appears on centrosomes in prophase", Nat Cell Biol, 5, 2003, 143-8.
2000, 1425-9.
U Strausfeld, A Fernandez, JP Capony, F Girard, N Lautredou, J Derancourt, JC Labbe, NJ Lamb, "Activation of p34cdc2 protein kinase by
microinjection of human cdc25C into mammalian cells. Requirement for prior phosphorylation of cdc25C by p34cdc2 on sites phosphorylated at
mitosis.", J Biol Chem, 269, 1994, 5989-6000.
Reaction
5.5.1.7 Phosphorylation of proteins involved in the G2/M transition by Cyclin A:Cdc2 complexes
Description
Cyclin A:Cdc2 complexes are detected in the nucleus earlier that cyclin B1:Cdc2 complexes and may play a role in the initial events in prophase.
Inactivation of Cdc25B by proteasome-mediated degradation is dependent upon cyclin A:Cdc2-mediated phosphorylation (Cans et al, 1999)
References
C Cans, B Ducommun, V Baldin, "Proteasome-dependent degradation of human CDC25B phosphatase", Mol Biol Rep, 26, 1999, 53-7.
5.5.1.7.1 Phosphorylation of proteins involved in G2/M transition by active Cyclin A1:Cdc2 complexes
Description
At the beginning of this reaction, 1 molecule of 'ATP', and 1 molecule of 'G2/M transition protein' are present. At the end of this reaction, 1
molecule of 'ADP', and 1 molecule of 'phospho-G2/M transition protein' are present.
This reaction takes place in the 'nucleoplasm' and is mediated by the 'cyclin-dependent protein kinase activity' of 'Cyclin A1:Cdc2'.
Reaction
5.5.1.7.2 Phosphorylation of proteins involved in G2/M transition by active Cyclin A2:Cdc2 complexes
Description
At the beginning of this reaction, 1 molecule of 'ATP', and 1 molecule of 'G2/M transition protein' are present. At the end of this reaction, 1
molecule of 'ADP', and 1 molecule of 'phospho-G2/M transition protein' are present.
This reaction takes place in the 'nucleoplasm' and is mediated by the 'cyclin-dependent protein kinase activity' of 'Cyclin A2:Cdc2'.
References
M Xu, KA Sheppard, CY Peng, AS Yee, H Piwnica-Worms, "Cyclin A/CDK2 binds directly to E2F-1 and inhibits the DNA-binding activity of
E2F-1/DP-1 by phosphorylation.", Mol Cell Biol, 14, 1994, 8420-31.
Reaction
5.5.1.8 Formation of Cyclin B:Cdc2 complexes
Description
Cyclin dependent kinases are themselves catalytically inactive due to the fact that their active site is blocked by a portion of the Cdk molecule
itself. Binding to their corresponding cyclin partner results in conformational change that partially exposes the active site.
References
115, 1991, 1-17.
Reaction
5.5.1.9 Myt-1 mediated phosphorylation of Cyclin B:Cdc2 complexes
Description
References
1997, 22300-6.
Reaction
5.5.1.10 Translocation of Cyclin B1:phospho-Cdc2 complexes to the nucleus
Description
During interphase, cyclin B1:Cdc2 shuttles continuously in and out of the nucleus. Cyclin B1:Cdc2 is transported into the nucleus by an unusual
mechanism that requires importin b but not importin a or Ran. Dissociation of the cyclin-B1:Cdc2:importin complex in the nucleus requires ATP
and involves other yet unidentified nuclear factors (Takizawa et al.,1991).
References
CG Takizawa, K Weis, DO Morgan, "Ran-independent nuclear import of cyclin B1-Cdc2 by importin beta", Proc Natl Acad Sci U S A, 96, 1999,
7938-43.
A Hagting, C Karlsson, P Clute, M Jackman, J Pines, "MPF localization is controlled by nuclear export", EMBO J, 17, 1998, 4127-38.
J Yang, ES Bardes, JD Moore, J Brennan, MA Powers, S Kornbluth, "Control of cyclin B1 localization through regulated binding of the nuclear
export factor CRM1", Genes Dev, 12, 1998, 2131-43.
Reaction
5.5.1.11 CAK-mediated phosphorylation of Cyclin B1:Cdc2 complexes
Description
Full activity of most CDKs is dependent on CAK mediated phosphorylation at a conserved residue (Thr 161 in Cdc2). This modification is thought
to improve substrate binding. Cyclin B:Cdc2 complexes have considerably low activity in the absence of CAK mediated phosphorylation (Desai
et al 1995).
References
1995, 345-50.
Reaction
5.5.1.12 Wee1- mediated phosphorylation of Cyclin B1:phospho-Cdc2 complexes
Description
References
Reaction
5.5.1.13 Translocation of Cyclin B1:phospho-Cdc2 to the cytoplasm

Description
During interphase, cyclin B1 shuttles continuously in and out of the nucleus. The cyclin B cytoplasmic retention sequence (CRS), which is
responsible for its interphase cytoplasmic localization, functions as a nuclear export sequence (Yang et al., 1998).
References
Reaction
5.5.1.14 Translocation of Cdc25B to the cytoplasm
Description
Cdc25B shuttles between the nucleus and the cytoplasm. Translocation out of the nucleus involves a nuclear export sequence in the N-terminus
of Cdc25B (Lindqvist et al., 2004).
References
Reaction
5.5.1.15 Dephosphorylation of cytoplasmic Cyclin B1:phospho-Cdc2 (Thr 14, Tyr 15) complexes by Cdc25 phosphatases
Description
Activation of the mitotic cyclin:Cdc2 complexes at mitosis requires the removal of the inhibitory phosphate groups on Cdc2. This
dephosphorylation is achieved by the activity of the Cdc25 family of phosphatases. The Cdc25 members, Cdc25A, Cdc25B, and Cdc25C are
kept inactive during interphase and are activated at the G2/M transition. Cyclin B1:Cdc2 itself appears to participate in the full activation of
Cdc25 in a process that involves an amplication loop (see Wolfe and Gould, 2004). The initial activation of the cyclin B1-Cdc2 complex occurs in
the cytoplasm in prophase (Jackman et al., 2003). Cdc25B, which is present at highest concentrations in the cytoplasm at this time, is thought to
trigger the activation of cyclin B1-Cdc2 (Lindqvist et al. 2004; Honda et al., 1993). Active cyclin B:Cdc2 then phosphorylates and activates
Cdc25C and stabilizes Cdc25A (Strausfeld et al., 1994; Hoffman et al.,1993; Mailand et al, 2002). This creates positive feedback loops that
allows Cdc25A and Cdc25C to dephosphorylate and further activate Cdc2.
References
2000, 1425-9.
Reaction
5.5.1.16 Phosphorylation of Cyclin B1 in the CRS domain
Description
At the onset of mitosis, cyclin B is phosphorylated in the CRS sequence which creates a nuclear import signal in the amino terminus. The
kinase(s) responsible for this phosphorylation are not yet known (Hagting et al., 1999).
References
A Hagting, M Jackman, K Simpson, J Pines, "Translocation of cyclin B1 to the nucleus at prophase requires a phosphorylation-dependent
nuclear import signal", Curr Biol, 9, 1999, 680-9.
Reaction
5.5.1.17 Translocation of CRS phosphorylated Cyclin B1:Cdc2 complexes
Description
The rapid translocation of cyclin B1:Cdc2 from the cytoplasm to the nucleus at the onset of mitosis is a result of an increase in the rate of import
and, likely, a decreased rate of export. The increased rate of nuclear import is dependent upon phosphorylation of the CRS which creates a
nuclear import signal in the amino terminus of cyclin B1 (Hagting et al, 1999).
References
Reaction
5.5.1.18 Translocation of Cdc25 to the nucleus
Description
The localization of the Cdc25A, B and C proteins is dynamic involving the shuttling of these proteins between the nucleus and the cytoplasm.
Sequences in these proteins mediate both nuclear export and import (Kallstrom et al., 2005; Lindqvist et al., 2004; Graves et al, 2001; Takizawa
and Morgan, 2000).
References
PR Graves, CM Lovly, GL Uy, H Piwnica-Worms, "Localization of human Cdc25C is regulated both by nuclear export and 14-3-3 protein
binding.", Oncogene, 20, 2001, 1839-51.
H Kallstrom, A Lindqvist, V Pospisil, A Lundgren, CK Rosenthal, "Cdc25A localisation and shuttling: characterisation of sequences mediating
nuclear export and import", Exp Cell Res, 303, 2005, 89-100.
CG Takizawa, DO Morgan, "Control of mitosis by changes in the subcellular location of cyclin-B1-Cdk1 and Cdc25C", Curr Opin Cell Biol, 12,
2000, 658-65.
Reaction
5.5.1.19 Dephosphorylation of nuclear Cyclin B1:phospho-Cdc2 (Thr 14, Tyr15) complexes by Cdc25 phosphatases
Description
Following its translocation to the nucleus, Cdc25 dephosphorylates and activates nuclear cyclin B1:Cdc2 complexes (Strausfeld et al., 1991).
References
U Strausfeld, JC Labbe, D Fesquet, JC Cavadore, A Picard, K Sadhu, P Russell, M Doree, "Dephosphorylation and activation of a
p34cdc2/cyclin B complex in vitro by human CDC25 protein", Nature, 351, 1991, 242-5.
Reaction
5.5.1.20 Phosphorylation of M phase proteins by active Cyclin B1:Cdc2 complexes
Description
A description of the mitotic proteins targeted by the mitotic cyclin:CDK complexes will be covered in a later release.
Reaction
5.5.2 Cyclin B2 mediated events
Description
The two B-type cyclins localize to different regions within
the cell and and are thought to have specific roles as CDK1-activating subunits (see Bellanger et al., 2007). Cyclin B1 is primarily cytoplasmic
during interphase and translocates into the nucleus at the onset of mitosis (Jackman et al., 1995; Hagting et al., 1999). Cyclin B2 colocalizes
with the Golgi apparatus and contributes to its fragmentation during mitosis (Jackman et al., 1995; Draviam et al., 2001).
References
VM Draviam, S Orrechia, M Lowe, R Pardi, J Pines, "The localization of human cyclins B1 and B2 determines CDK1 substrate specificity and
neither enzyme requires MEK to disassemble the Golgi apparatus", J Cell Biol, 152, 2001, 945-58.
M Jackman, M Firth, J Pines, "Human cyclins B1 and B2 are localized to strikingly different structures: B1 to microtubules, B2 primarily to the
Golgi apparatus", EMBO J, 14, 1995, 1646-54.
S Bellanger, A de Gramont, J Sobczak-ThÃ©pot, "Cyclin B2 suppresses mitotic failure and DNA re-replication in human somatic cells knocked
down for both cyclins B1 and B2", Oncogene, 26, 2007, 7175-84.
5.5.2.1 Dephosphorylation of cyclin B2:phospho-Cdc2 (Thr 14) by Cdc25
Description
At the beginning of this reaction, 1 molecule of 'Cyclin B2:phospho-Cdc2(Thr 14, Thr 161)', and 1 molecule of 'H2O' are present. At the end of
this reaction, 1 molecule of 'Cyclin B2:phospho-Cdc2(Thr 161)', and 1 molecule of 'Orthophosphate' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'phosphoprotein phosphatase activity' of 'Cdc25'.
Source reaction
This reaction was inferred from the corresponding reaction "Dephosphorylation of cytoplasmic Cyclin B1:phospho-Cdc2 (Thr 14, Tyr 15)
complexes by Cdc25 phosphatases" in species Homo sapiens.
2000, 1425-9.
Reaction
5.5.2.2 Phosphorylation of proteins involved in G2/M transition by active Cyclin B2:Cdc2 complexes
Description
Substrate specificity of cyclin B:Cdk1 complexes is primarily conferred by their subcellular localization (Draviam et al., 2001).
Cyclin B1 is primarily cytoplasmic but shuttles continuously between the nucleus and the cytoplasm during interphase (Hagting et al. 1998 Down;
Toyoshima et al. 1998 Down; Yang et al. 1998 Down). At the end of prophase, it abruptly translocates into the nucleus (Furuno et al. 1999
Down; Hagting et al. 1999 Down) and then associates with mitotic apparatus (Pines and Hunter 1991 Down; Hagting et al. 1998 Down; Clute
and Pines 1999 Down). Cyclin B2 is primarily associated with the Golgi apparatus during interphase and mitosis (Jackman et al. 1995 Down;
Brandeis et al. 1998 Down). Cyclin B1â€"CDK1 promotes chromosome condensation, reorganization microtubule reorgnization, and
disassembly of the nuclear lamina and the Golgi apparatus. Cyclin B2â€"CDK1 functions in disassembly of the Golgi apparatus (Draviam et al.,
2001).
References
VM Draviam, S Orrechia, M Lowe, R Pardi, J Pines, "The localization of human cyclins B1 and B2 determines CDK1 substrate specificity and
neither enzyme requires MEK to disassemble the Golgi apparatus", J Cell Biol, 152, 2001, 945-58.
M Brandeis, I Rosewell, M Carrington, T Crompton, MA Jacobs, J Kirk, J Gannon, T Hunt, "Cyclin B2-null mice develop normally and are fertile
whereas cyclin B1-null mice die in utero", Proc Natl Acad Sci U S A, 95, 1998, 4344-9.
M Jackman, M Firth, J Pines, "Human cyclins B1 and B2 are localized to strikingly different structures: B1 to microtubules, B2 primarily to the
Golgi apparatus", EMBO J, 14, 1995, 1646-54.
F Toyoshima, T Moriguchi, A Wada, M Fukuda, E Nishida, "Nuclear export of cyclin B1 and its possible role in the DNA damage-induced G2
checkpoint", EMBO J, 17, 1998, 2728-35.
115, 1991, 1-17.
P Clute, J Pines, "Temporal and spatial control of cyclin B1 destruction in metaphase", Nat Cell Biol, 1, 1999, 82-7.
Reaction
5.5.3 G2/M-specific transcription in mitotic cell cycle
Description
G2/M phase-specific transcription will be described in a future release.

5.5.4 Polo-like kinase mediated events
Authors
Lee, KS, 2004-12-08.
Editors
Description
At mitotic entry, Plk1 phosphorylates and activates Cdc25C phosphatase, whereas it phosphorylates and down-regulates Wee1A. Plk1 also
phosphorylates and inhibits Myt1 activity. Cyclin B1-bound Cdc2, which is the target of Cdc25C, Wee1A, and Myt1, functions in a feedback loop
and phosphorylates the latter components (Cdc25C, Wee1A, Myt1). The Cdc2- dependent phosphorylation provides docking sites for the
polo-box domain of Plk1, thus promoting the Plk1-dependent regulation of these components and, as a result, activation of Cdc2-Cyclin B1.
References
N Watanabe, H Arai, Y Nishihara, M Taniguchi, N Watanabe, T Hunter, H Osada, "M-phase kinases induce phospho-dependent ubiquitination of
somatic Wee1 by SCFbeta-TrCP", Proc Natl Acad Sci U S A, 101, 2004, 4419-24.
N Sagata, "The Polo-like kinase Plx1 interacts with and inhibits Myt1 after fertilization of Xenopus eggs", EMBO J, 24, 2005, 1057-67.
5.5.4.1 Inactivation of Wee1 kinase
Authors
Watanabe, N, Hunter, T, 2008-05-07.
Editors
Description
*Plk1 is shown to phosphorylate Wee1A, an event that is likely critical for recognition and ubiquitination of Wee1A by SCF and therefore for the
subsequent degradation of Wee1A . **Plk1 phosphorylates Wee1A at S53, creating the second phosphodegron, PD53. ** Evidence also exists
in budding yeast that the budding yeast polo homolog Cdc5 directly phosphorylates and down-regulate the budding yeast Wee1 ortholog Swe1.
Thus, polo kinase-dependent phosphorylation and degradation of Wee1A (or Swe1) is likely conserved throughout evolution and is critical for
normal mitotic entry.
References
N Watanabe, H Arai, Y Nishihara, M Taniguchi, N Watanabe, T Hunter, H Osada, "M-phase kinases induce phospho-dependent ubiquitination of
somatic Wee1 by SCFbeta-TrCP", Proc Natl Acad Sci U S A, 101, 2004, 4419-24.
LL Parker, SA Walter, PG Young, H Piwnica-Worms, "Phosphorylation and inactivation of the mitotic inhibitor Wee1 by the nim1/cdr1 kinase",
Nature, 363, 1993, 736-8.
K Sakchaisri, S Asano, LR Yu, MJ Shulewitz, CJ Park, JE Park, YW Cho, TD Veenstra, J Thorner, KS Lee, "Coupling morphogenesis to mitotic
entry", Proc Natl Acad Sci U S A, 101, 2004, 4124-9.
TR Coleman, Z Tang, "Negative regulation of the wee1 protein kinase by direct action of the nim1/cdr1 mitotic inducer", Cell, 72, 1993, 919-29.
Reaction
5.5.4.2 Activation of Cdc25C
Authors
Lee, KS, 2004-12-08.
Editors
Description
It has been shown that Xenopus polo homolog,Plx1, directly phosphorylates and activates Cdc25C, which in turn dephosphoryates and activates
Cdc2. This step is critical for the onset of mitosis. Since Plx1-dependent Cdc25C phosphorylation occurs in the absence of Cdc2 activity, it is
likely that Plx1 is a triggering kinase, which leads to the activation of Cdc2 and therefore the normal onset of mitosis.
References
A Kumagai, "Purification and molecular cloning of Plx1, a Cdc25-regulatory kinase from Xenopus egg extracts", Science, 273, 1996, 1377-80.
YW Qian, E Erikson, FE Taieb, JL Maller, "The polo-like kinase Plx1 is required for activation of the phosphatase Cdc25C and cyclin B-Cdc2 in
Xenopus oocytes", Mol Biol Cell, 12, 2001, 1791-9.
F Toyoshima-Morimoto, E Taniguchi, E Nishida, "Plk1 promotes nuclear translocation of human Cdc25C during prophase", EMBO Rep, 3, 2002,
341-8.
Reaction
5.5.4.3 Inactivation of Myt1 kinase
Authors
Lee, KS, 2004-12-08.
Editors
Description
At mitotic entry Plk1 phosphorylates and inhibits Myt1 activity. Cyclin B1-bound Cdc2, which is the target of Myt1, functions in a feedback loop
and phosphorylates and further inhibits Myt1.
References
H Nakajima, F Toyoshima-Morimoto, E Taniguchi, E Nishida, "Identification of a consensus motif for Plk (Polo-like kinase) phosphorylation
reveals Myt1 as a Plk1 substrate", J Biol Chem, 278, 2003, 25277-80.
N Sagata, "The Polo-like kinase Plx1 interacts with and inhibits Myt1 after fertilization of Xenopus eggs", EMBO J, 24, 2005, 1057-67.
Reaction
5.6 M Phase
Description
Mitosis, or the M phase, involves nuclear division and cytokinesis, where two identical daughter cells are produced. Mitosis involves prophase,
prometaphase, metaphase, anaphase, and telophase. Finally, cytokinesis leads to cell division. The phase between two M phases is called the
interphase; it encompasses the G1, S, and G2 phases of the cell cycle.
The detailed annotation of the M phase will be completed in a later version of GK.
5.6.1 Mitotic Prophase
Description
During the prophase, the chromatin in the nucleus condenses, and the nucleolus disappears. Centrioles begin moving to the opposite poles or
ends, of the cell. Some of the fibers that extend from the centromeres, cross the cell to form the mitotic spindle.
5.6.1.1 Golgi Cisternae Pericentriolar Stack Reorganization
Description
The pericentriolar stacks of Golgi cisternae undergo extensive fragmentation and reorganization.
5.6.1.1.1 Activation of the Anaphase Promoting Complex (APC) by Plk1
Authors
Lee, KS, 2004-12-08.
Editors
Description
During the early stages of mitosis, mitotic kinases including Cdc2 and Plk1 promote reorganization of pericentriolar stacks of Golgi cisternae by
phosphorylating GRASP65 (Golgi REasembly Stacking Protein of 65 kDa). Cdc2-Cyclin B1 phosphorylates GRASP65 at the time of mitotic
entry,a step that promotes the interaction between GRASP65 and the polo-box domain of Plk1 and the subsequent Plk1-dependent GRASP65
phosphorylation.
References
CY Lin, ML Madsen, FR Yarm, YJ Jang, X Liu, RL Erikson, "Peripheral Golgi protein GRASP65 is a target of mitotic polo-like kinase (Plk) and
Cdc2", Proc Natl Acad Sci U S A, 97, 2000, 12589-94.
C Preisinger, R Korner, M Wind, WD Lehmann, R Kopajtich, FA Barr, "Plk1 docking to GRASP65 phosphorylated by Cdk1 suggests a
mechanism for Golgi checkpoint signalling", EMBO J, 24, 2005, 753-65.
C Sutterlin, CY Lin, Y Feng, DK Ferris, RL Erikson, V Malhotra, "Polo-like kinase is required for the fragmentation of pericentriolar Golgi stacks
during mitosis", Proc Natl Acad Sci U S A, 98, 2001, 9128-32.
Reaction
5.6.2 Mitotic Prometaphase
Description
The dissolution of the nuclear membrane marks the beginning of the prometaphase. Kinetochores are created when proteins attach to the
centromeres. Microtubules then attach at the kinetochores, and the chromosomes begin to move.
5.6.2.1 Phosphorylation of the SA2 Cohesion Complex
Authors
Lee, KS, 2004-12-08.

Editors
Description
Prior to anaphase onset, sister-chromatids are held together by cohesin complexes. Plk1-dependent phosphorylation of the cohesin subunit SA2
promotes dissociation of cohesins in early mitosis.
References
I Sumara, E Vorlaufer, PT Stukenberg, O Kelm, N Redemann, EA Nigg, JM Peters, "The dissociation of cohesin from chromosomes in prophase
is regulated by Polo-like kinase", Mol Cell, 9, 2002, 515-25.
NC Hornig, F Uhlmann, "Preferential cleavage of chromatin-bound cohesin after targeted phosphorylation by Polo-like kinase", EMBO J, 23,
2004, 3144-53.
S Hauf, IC Waizenegger, JM Peters, "Cohesin cleavage by separase required for anaphase and cytokinesis in human cells", Science, 293,
2001, 1320-3.
S Hauf, E Roitinger, B Koch, CM Dittrich, K Mechtler, JM Peters, "Dissociation of cohesin from chromosome arms and loss of arm cohesion
during early mitosis depends on phosphorylation of SA2", PLoS Biol, 3, 2005, e69.
Reaction
5.6.3 Mitotic Metaphase
Description
Metaphase is marked by the formation of the metaphase plate. The metaphase plate is formed when the spindle fibers align the chromosomes
along the middle of the cell. Such an organization helps to ensure that later, when the chromosomes are separated, each new nucleus that is
formed receives one copy of each chromosome.
5.6.4 Mitotic Metaphase/Anaphase Transition
Description
The metaphase to anaphase transition during mitosis is triggered by the destruction of mitotic cyclins.
5.6.4.1 Down Regulation of Emi1 through Phosphorylation of Emi1
Authors
Lee, KS, 2004-12-08.
Editors
Description
During the early stages of mitosis, Cdc2 and Plk1 cooperate to phosphorylate Emi1 and this modification induces Emi1 degradation through a
Skp1-Cullin1 F-box protein (SCF) ubiquitin ligase-mediated proteolysis. Degradation of Emi1 permits activation of anaphase promoting complex
and thereby the onset of anaphase.
References
DV Hansen, AV Loktev, KH Ban, "Plk1 regulates activation of the anaphase promoting complex by phosphorylating and triggering
SCFbetaTrCP-dependent destruction of the APC Inhibitor Emi1", Mol Biol Cell, 15, 2004, 5623-34.
Y Moshe, J Boulaire, M Pagano, A Hershko, "Role of Polo-like kinase in the degradation of early mitotic inhibitor 1, a regulator of the anaphase
promoting complex/cyclosome", Proc Natl Acad Sci U S A, 101, 2004, 7937-42.
Reaction
5.6.4.2 Phosphorylation of the Scc1:Cohesion Complex
Authors
Lee, KS, 2004-12-08.
Editors
Description
Plk1-dependent phosphorylation of the cohesin subunit Scc1 promotes cleavage of Scc1 prior to the onset of anaphase. Destruction of securin
by APC/Cdc20-dependent proteolysis permits separase to cleave Scc1, resulting in sister-chromatid separation.
References
G Alexandru, F Uhlmann, K Mechtler, MA Poupart, K Nasmyth, "Phosphorylation of the cohesin subunit Scc1 by Polo/Cdc5 kinase regulates
sister chromatid separation in yeast", Cell, 105, 2001, 459-72.
NC Hornig, F Uhlmann, "Preferential cleavage of chromatin-bound cohesin after targeted phosphorylation by Polo-like kinase", EMBO J, 23,
2004, 3144-53.
S Hauf, IC Waizenegger, JM Peters, "Cohesin cleavage by separase required for anaphase and cytokinesis in human cells", Science, 293,
2001, 1320-3.
S Hauf, E Roitinger, B Koch, CM Dittrich, K Mechtler, JM Peters, "Dissociation of cohesin from chromosome arms and loss of arm cohesion
during early mitosis depends on phosphorylation of SA2", PLoS Biol, 3, 2005, e69.
Reaction
5.6.5 Mitotic Anaphase
Description
In Anaphase, the paired chromosomes separate at the kinetochores, and move to the opposite sides of the cell. The movement of the
chromosomes is facilitated by a combination of kinetochore movement along the spindle microtubules and through the physical interaction of
polar microtubules.
5.6.6 Mitotic Telophase /Cytokinesis
Description
In this final phase of mitosis, new membranes are formed around the two chromatids and two daughter cells are formed. The chromosomes and
the spindle fibers disperse, and the fiber ring around the center of the cell, composed of actin, contracts, pinching the cell into two daughter cells.
5.6.6.1 Regulation of MKLP-1 by phosphorylation
Description
At the beginning of this reaction, 1 molecule of 'MKLP-1' is present. At the end of this reaction, 1 molecule of 'phospho-MKLP-1' is present.
This reaction takes place in the 'nucleus' and is mediated by the 'kinase activity' of 'Serine/threonine-protein kinase PLK (EC 2.7.1.-) (PLK-1)
(Serine- threonine protein kinase 13) (STPK13)'.
References
X Liu, T Zhou, R Kuriyama, RL Erikson, "Molecular interactions of Polo-like-kinase 1 with the mitotic kinesin-like protein CHO1/MKLP-1", J Cell
Sci, 117, 2004, 3233-46.
Reaction
5.6.6.2 Regulation of MKLP-2 by phosphorylation
Description
At the beginning of this reaction, 1 molecule of 'MKLP-2' is present. At the end of this reaction, 1 molecule of 'phospho-MKLP-2' is present.
References
R Neef, C Preisinger, J Sutcliffe, R Kopajtich, EA Nigg, TU Mayer, FA Barr, "Phosphorylation of mitotic kinesin-like protein 2 by polo-like kinase 1
is required for cytokinesis", J Cell Biol, 162, 2003, 863-75.
Reaction
5.6.6.3 Regulation of NudC by phosphorylation
Description
At the beginning of this reaction, 1 molecule of 'NudC' is present. At the end of this reaction, 1 molecule of 'phospho-NudC' is present.
References
JP Aumais, SN Williams, W Luo, M Nishino, KA Caldwell, GA Caldwell, SH Lin, LY Yu-Lee, "Role for NudC, a dynein-associated nuclear
movement protein, in mitosis and cytokinesis", J Cell Sci, 116, 2003, 1991-2003.
T Zhou, JP Aumais, X Liu, LY Yu-Lee, RL Erikson, "A role for Plk1 phosphorylation of NudC in cytokinesis", Dev Cell, 5, 2003, 127-38.
Reaction
5.7 M/G1 Transition
Description
Finally, progression out of mitosis and division of the cell into two daughters (cytokinesis) requires the inactivation of Cyclin B - Cdc2 by
ubiquitin-dependent proteolysis of Cyclin A and B, which is regulated by a large E3 ubiquitin ligase complex known as the Anaphase Promoting
Complex (APC).
The detailed annotation of the M/G1 transition will be completed in a later version of GK.
5.7.1 Assembly of the pre-replicative complex
Authors
Davey, MJ, O'Donnell, M, Tye, BK, 2006-03-17.
Description
DNA replication pre-initiation in eukaryotic cells begins with the formation of the pre-replicative complex (pre-RC) during the late M phase and
continues in the G1 phase of the mitotic cell cycle, a process also called DNA replication origin licensing. The association of initiation proteins
(ORC, Cdc6, Cdt1, Mcm2-7) with the origin of replication in both S. cerevisiae and humans has been demonstrated by chromatin
immunoprecipitation experiments. In S. cerevisiae, pre-replicative complexes are assembled from late M to G1. In mammalian cells as well,
pre-replicative complexes are assembled from late M to G1, as shown by biochemical fractionation and immunostaining. There are significant
sequence similarities among some of the proteins in the pre-replicative complex. The ORC subunits Orc1, Orc4 and Orc5 are homologous to
one another and to Cdc6. The six subunits of the Mcm2-7 complex are homologous to one another. In addition, Orc1, Orc4, Orc5, Cdc6, and the
Mcm2-7 subunits, are members of the AAA+ superfamily of ATPases. Since the initial identification of these pre-RC components other factors
that participate in this complex have been found, including Cdt1 in human, Xenopus, S. pombe, and S. cerevisiae cells.
References
C Liang, B Stillman, "Persistent initiation of DNA replication and chromatin-bound MCM proteins during the cell cycle in cdc6 mutants.", Genes
Dev, 11, 1998, 3375-86.
AF Neuwald, L Aravind, JL Spouge, EV Koonin, "AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and
disassembly of protein complexes.", Genome Res, 9, 1999, 27-43.
DS Dimitrova, TA Prokhorova, JJ Blow, IT Todorov, DM Gilbert, "Mammalian nuclei become licensed for DNA replication during late telophase.",
J Cell Sci, 115, 2002, 51-9.
T Tanaka, D Knapp, K Nasmyth, "Loading of an Mcm protein onto DNA replication origins is regulated by Cdc6p and CDKs.", Cell, 90, 1997,
649-60.
5.7.1.1 Assembly of the ORC complex at the origin of replication
Authors
Davey, MJ, O'Donnell, M, 2003-06-05.
Description
In Saccharomyces cerevisiae, the entire ORC complex is constitutively bound to the origin of replication. However, in mammalian cells, Orc1,
and possibly other Orc subunits, dissociate from origins of replication, while Orc2 remains stably associated with chromatin across the cell cycle.
The first step in formation of the pre-RC is assembly of the hexameric Origin Recognition Complex (ORC) at the origin of replication. Recent
work on human (Hs) ORC has suggested that the heterohexamer assembles in an ordered manner. HsOrc2 appears to be constitutively bound
to origins of replication. HsOrc2 and HsOrc3 form a complex that interacts with HsOrc4 and HsOrc5. Recruitment of HsOrc5 into the complex is
important for the association of HsOrc1. HsOrc6 is also capable of interacting with the core HsOrc2:3:4:5 complex, but not smaller complexes.
Interestingly, HsOrc1 and a subunit of the Mcm2-7 complex (HsMcm2) interact with a histone acetyltransferase, HsHBO1.
References
Physiol, 38, 1975, 58-63.
DG Lee, SP Bell, "Architecture of the yeast origin recognition complex bound to origins of DNA replication.", Mol Cell Biol, 17, 1997, 7159-68.
JF Diffley, JH Cocker, SJ Dowell, A Rowley, "Two steps in the assembly of complexes at yeast replication origins in vivo.", Cell, 78, 1994,
303-16.
TW Burke, JG Cook, M Asano, JR Nevins, "Replication factors MCM2 and ORC1 interact with the histone acetyltransferase HBO1.", J Biol
Chem, 276, 2001, 15397-408.
M Iizuka, B Stillman, "Histone acetyltransferase HBO1 interacts with the ORC1 subunit of the human initiator protein.", J Biol Chem, 274, 1999,
23027-34.
SP Bell, B Stillman, "ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex.", Nature, 357, 1992, 128-34.
5.7.1.1.1 Orc3 associates with Orc2 constitutively bound at origins of replication
Description
At the beginning of this reaction, 1 molecule of 'Orc2:origin', and 1 molecule of 'Orc3' are present. At the end of this reaction, 1 molecule of
'Orc3:Orc2:origin' is present.
References
M Ritzi, M Baack, C Musahl, P Romanowski, RA Laskey, R Knippers, "Human minichromosome maintenance proteins and human origin
recognition complex 2 protein on chromatin.", J Biol Chem, 273, 1998, 24543-9.
Reaction
5.7.1.1.2 Orc5 associates with Orc3:Orc2:origin complexes

Description
At the beginning of this reaction, 1 molecule of 'Orc3:Orc2:origin', and 1 molecule of 'Orc5' are present. At the end of this reaction, 1 molecule of
'Orc5:Orc3:Orc2:origin' is present.
References
Reaction
5.7.1.1.3 Orc4 associates with Orc5:Orc3:Orc2:origin complexes
Description
At the beginning of this reaction, 1 molecule of 'Orc4', and 1 molecule of 'Orc5:Orc3:Orc2:origin' are present. At the end of this reaction, 1
molecule of 'Orc4:Orc5:Orc3:Orc2:origin' is present.
References
Reaction
5.7.1.1.4 Orc1 associates with Orc4:Orc5:Orc3:Orc2:origin complexes
Description
At the beginning of this reaction, 1 molecule of 'Orc4:Orc5:Orc3:Orc2:origin', and 1 molecule of 'Orc1' are present. At the end of this reaction, 1
molecule of 'Orc1:Orc4:Orc5:Orc3:Orc2:origin' is present.
References
Reaction
5.7.1.1.5 Orc6 associates with Orc1:Orc4:Orc5:Orc3:Orc2:origin complexes, forming ORC:origin complexes
Description
At the beginning of this reaction, 1 molecule of 'Orc1:Orc4:Orc5:Orc3:Orc2:origin', and 1 molecule of 'Orc6' are present. At the end of this
reaction, 1 molecule of 'ORC complex bound to origin' is present.
References
Reaction
5.7.1.2 Association of MCM8 with ORC:origin complex
Authors
Tye, BK, 2006-03-17.
Editors
Description
The MCM2-7 complex, an essential component of the pre-replication complex, recruits CDC6 and CDT1 proteins to the origin. MCM8, another
member of the MCM family has been found to bind to chromatin during early G1 phase. MCM8 interacts specifically with the ORC2 protein.
References
M Volkening, I Hoffmann, "Involvement of human MCM8 in prereplication complex assembly by recruiting hcdc6 to chromatin", Mol Cell Biol, 25,
2005, 1560-8.
Reaction
5.7.1.3 CDC6 association with the ORC:origin complex

Authors
Description
Cdc6 is a regulator of DNA replication initiation in both yeasts and human cells, but its mechanism of action differs between the two systems.
Genetic studies in budding yeast (S. cerevisiae) and fission yeast (S. pombe) indicate that the normal function of Cdc6 protein is required to
restrict DNA replication to once per cell cycle. Specifically, Cdc6 may function as an ATPase switch linked to Mcm2-7 association with the
Cdt1:Cdc6:ORC:origin complex. In S. cerevisiae, Cdc6 protein is expressed late in the M phase of the cell cycle and, in cells with a prolonged
G1 phase, late in G1. This protein has a short half-life, and is destroyed by ubiquitin-mediated proteolysis, mediated by the SCF complex.
Human Cdc6 protein levels are reduced early in G1 but otherwise are constant throughout the cell cycle. Some reports have suggested that after
cells enter S phase, Cdc6 is phosphorylated, excluded from the nucleus and subject to ubiquitination and degradation. Replenishing Cdc6
protein levels during G1 appears to be regulated by E2F transcription factors.
References
P Saha, J Chen, KC Thome, SJ Lawlis, ZH Hou, M Hendricks, JD Parvin, A Dutta, "Human CDC6/Cdc18 associates with Orc1 and cyclin-cdk
and is selectively eliminated from the nucleus at the onset of S phase.", Mol Cell Biol, 18, 1998, 2758-67.
LS Drury, G Perkins, JF Diffley, "The Cdc4/34/53 pathway targets Cdc6p for proteolysis in budding yeast.", EMBO J, 16, 1997, 5966-76.
BO Petersen, J Lukas, CS SÃ¸rensen, K Helin, "Phosphorylation of mammalian CDC6 by cyclin A/CDK2 regulates its subcellular localization.",
EMBO J, 18, 1999, 396-410.
LS Drury, G Perkins, JF Diffley, "The cyclin-dependent kinase Cdc28p regulates distinct modes of Cdc6p proteolysis during the budding yeast
cell cycle.", Curr Biol, 10, 2000, 231-40.
S Piatti, C Lengauer, K Nasmyth, "Cdc6 is an unstable protein whose de novo synthesis in G1 is important for the onset of S phase and for
preventing a 'reductional' anaphase in the budding yeast Saccharomyces cerevisiae.", EMBO J, 14, 1995, 3788-99.
6193-8.
G Perkins, LS Drury, JF Diffley, "Separate SCF(CDC4) recognition elements target Cdc6 for proteolysis in S phase and mitosis.", EMBO J, 20,
2001, 4836-45.
Description
References
Reaction
5.7.1.3.2 CDC6 association with ORC:origin complexes mediated by MCM8
Authors
Description
At the beginning of this reaction, 1 molecule of 'ORC:origin', and 1 molecule of 'CDC6' are present. At the end of this reaction, 1 molecule of
'CDC6:ORC:origin complex' is present.

References
2005, 1560-8.
Reaction
5.7.1.4 CDT1 association with the CDC6:ORC:origin complex
Description
Initiation protein Cdt1 was first identified in X. laevis, where it has been shown to be the second component of licensing factor (RLF-B) and in S.
pombe. Cdt1 homologs have been identified in D. melanogaster, humans, and S. cerevisiae. Genetic studies in S. pombe have shown that
binding of Cdc6 to chromatin requires the prior binding of Cdc18, the S. pombe homolog of Cdc6. In humans, the function of CDT1 is regulated
during the cell cycle by its tight association with an inhibitory factor, geminin.
References
JA Wohlschlegel, BT Dwyer, SK Dhar, C Cvetic, JC Walter, A Dutta, "Inhibition of eukaryotic DNA replication by geminin binding to Cdt1.",
Science, 290, 2000, 2309-12.
D Maiorano, J Moreau, M MÃ©chali, "XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis.", Nature, 404, 2000,
622-5.
TJ McGarry, MW Kirschner, "Geminin, an inhibitor of DNA replication, is degraded during mitosis.", Cell, 93, 1998, 1043-53.
A Dutta, SP Bell, "Initiation of DNA replication in eukaryotic cells.", Annu Rev Cell Dev Biol, 13, 1998, 293-332.
S Tanaka, JF Diffley, "Interdependent nuclear accumulation of budding yeast Cdt1 and Mcm2-7 during G1 phase.", Nat Cell Biol, 4, 2002,
198-207.
H Nishitani, Z Lygerou, T Nishimoto, P Nurse, "The Cdt1 protein is required to license DNA for replication in fission yeast.", Nature, 404, 2000,
625-8.
S Tada, A Li, D Maiorano, M MÃ©chali, JJ Blow, "Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by
geminin.", Nat Cell Biol, 3, 2001, 107-13.
AJ Whittaker, I Royzman, TL Orr-Weaver, "Drosophila double parked: a conserved, essential replication protein that colocalizes with the origin
recognition complex and links DNA replication with mitosis and the down-regulation of S phase transcripts.", Genes Dev, 14, 2000, 1765-76.
5.7.1.4.1 The geminin component of geminin:Cdt1 complexes is ubiquitinated, releasing Cdt1
Description
At the beginning of this reaction, 1 molecule of 'ubiquitin', and 1 molecule of 'Cdt1:geminin' are present. At the end of this reaction, 1 molecule of
'geminin:ubiquitin complex', and 1 molecule of 'Cdt1' are present.
References
Science, 290, 2000, 2309-12.
Reaction
5.7.1.4.2 Ubiquitinated geminin is degraded by the proteasome
Description
At the beginning of this reaction, 1 molecule of 'geminin:ubiquitin complex' is present. At the end of this reaction, 1 molecule of 'ubiquitin' is
present.
References
Science, 290, 2000, 2309-12.
Reaction
5.7.1.4.3 Free CDT1 associates with CDC6:ORC:origin complexes
Description
At the beginning of this reaction, 1 molecule of 'CDT1', and 1 molecule of 'CDC6:ORC:origin complex' are present. At the end of this reaction, 1
molecule of 'CDT1:CDC6:ORC:origin complex' is present.
References
Science, 290, 2000, 2309-12.
Reaction
5.7.1.5 Mcm2-7 associates with the Cdt1:CDC6:ORC:origin complex, forming the pre-replicative complex (preRC)
Description
Genetic studies in S. cerevisiae indicate that wild-type Cdc6 function is required for correctly timed loading of Mcm2-7 onto ORC. Biochemical
studies indicate that the human and Xenopus Cdc6 proteins likewise are required for Mcm2-7 loading, and that they are ATPase switches.
Specifically, Cdc6 may function as a clamp loader, assembling Mcm2-7 onto DNA in an ATP-dependent reaction. All known Cdc6 proteins have
the Walker A and Walker B sequence motifs characteristic of the AAA+ superfamily of ATPases. As expected for an AAA+ protein, human Cdc6
binds and slowly hydrolyzes ATP in vitro. ATP hydrolysis was disrupted by mutations of the Walker B motif, while both binding and hydrolysis
were disrupted by Walker A mutations. Microinjection of either mutant protein into HeLa cells blocked their progression through S phase. Both
wild-type and mutant proteins can dimerize in vitro, and studies with Xenopus egg extracts suggest that Cdc6 functions in vivo as a dimer or
larger multimer. In Xenopus extracts depleted of Cdc6 and reconstituted with either mutant protein, recruitment of Mcm2-7 to chromatin failed.
References
NS Frolova, N Schek, N Tikhmyanova, TR Coleman, "Xenopus Cdc6 performs separate functions in initiating DNA replication.", Mol Biol Cell,
13, 2002, 1298-312.
Dev, 11, 1998, 3375-86.
U Herbig, CA Marlar, "The Cdc6 nucleotide-binding site regulates its activity in DNA replication in human cells.", Mol Biol Cell, 10, 1999,
2631-45.
MJ Davey, M O'Donnell, "Mechanisms of DNA replication.", Curr Opin Chem Biol, 4, 2000, 581-6.
Reaction
5.8 APC/C-mediated degradation of cell cycle proteins
Authors
Editors
Matthews, L, 2006-01-30.
Reviewers
Peters, JM, 2006-03-27.
Description
The Anaphase Promoting Complex or Cyclosome (APC/C) functions during mitosis to promote sister chromatid separation and mitotic exit
through the degradation of mitotic cyclins and securin. This complex is also active in interphase insuring the appropriate length of the G1 phase
(reviewed in Peters, 2002). The APC/C contains at least 12 subunits and functions as an ubiquitin-protein ligase (E3) promoting the
multiubiquitination of its target proteins (see Gieffers et al., 2001).
In the ubiquitination reaction, ubiquitin is activated by the formation of a thioester bond with the (E1) ubiquitin activating enzyme then transferred
to a cysteine residue within the ubiquitin conjugating enzyme (E2) and ultimately to a lysine residue within the target protein, with the aid of
ubiquitin-protein ligase activity of the APC/C. The ubiquitin chains generated are believed to target proteins for destruction by the 26S
proteasome (Reviewed in Peters, 1994 )
The activity of the APC/C is highly periodic during the cell cycle and is controlled by a combination of regulatory events. The APC/C is activated
by phosphorylation and the regulated recruitment of activating subunits and is negatively regulated by sequestration by kinetochore-associated
checkpoint proteins. The Emi1 protein associates with Cdh1 and Cdc20, inhibiting the APC/C between G1/S and prophase. RSSA1 may play a
similar role in ihibiting the APC during early mitosis.
Following phosphorylation of the APC/C core subunits by mitotic kinases, the activating subunit, Cdc20 is recruited to the APC/C and is
responsible for mitotic activities, including the initiation of sister chromatid separation and the timing of exit from mitosis (See Zachariae and
Nasmyth, 1999). Substrates of the Cdc20:APC/C complex, which are recognized by a motif known as the destruction box (D box) include Cyclin
A, Nek2, Securin and Cyclin B. Degradation of Securin and Cyclin B does not occur until the mitotic spindle checkpoint has been satisfied (see
Castro et al. 2005).
Cdc20 is degraded late in mitosis (Reviewed in Owens and Hoyt, 2005). At this time the activating subunit, Cdh1, previously maintained in an
inactive phosphorylated state by mitotic kinases, is dephosphorylated and associates with and activates the APC/C. The APC/C:Cdh1 complex
recognizes substrates containing a D box, a KEN box (Pfleger and Kirschner, 2000) or a D box activated (DAD) domain (Castro et al., 2002)
sequence and promotes the ordered degration of mitotic cyclins and other mitotic proteins culminating with its own ubiquitin-conjugating enzyme
(E2) subunit UbcH10 (Rape et al., 2006). This ordered degradation promotes the stability of Cyclin A at the end of G1. This stabilization, in turn,
promotes the phosphorylation of Cdh1 and its abrupt dissociation from the APC/C, allowing accumulation of cyclins for the next G1/S transition
(Sorensen et al., 2001).
References
JM Peters, "The anaphase-promoting complex: proteolysis in mitosis and beyond", Mol Cell, 9, 2002, 931-43.
A Castro, C Bernis, S Vigneron, JC Labbe, T Lorca, "The anaphase-promoting complex: a key factor in the regulation of cell cycle", Oncogene,
24, 2005, 314-25.
C Gieffers, P Dube, JR Harris, H Stark, JM Peters, "Three-dimensional structure of the anaphase-promoting complex", Mol Cell, 7, 2001,
907-13.
JM Peters, "Proteasomes: protein degradation machines of the cell", Trends Biochem Sci, 19, 1994, 377-82.
TJ Owens, MA Hoyt, "The D box asserts itself", Mol Cell, 18, 2005, 611-2.
TK Fung, RY Poon, "A roller coaster ride with the mitotic cyclins", Semin Cell Dev Biol, 16, 2005, 335-42.
CM Pfleger, MW Kirschner, "The KEN box: an APC recognition signal distinct from the D box targeted by Cdh1", Genes Dev, 14, 2000, 655-65.
W Zachariae, K Nasmyth, "Whose end is destruction: cell division and the anaphase-promoting complex.", Genes Dev, 13, 1999, 2039-58.
M Rape, SK Reddy, MW Kirschner, "The processivity of multiubiquitination by the APC determines the order of substrate degradation", Cell, 124,
2006, 89-103.
JL Burton, MJ Solomon, "D box and KEN box motifs in budding yeast Hsl1p are required for APC-mediated degradation and direct binding to
Cdc20p and Cdh1p", Genes Dev, 15, 2001, 2381-95.
CS Sorensen, C Lukas, ER Kramer, JM Peters, J Lukas, "A conserved cyclin-binding domain determines functional interplay between
anaphase-promoting complex-Cdh1 and cyclin A-Cdk2 during cell cycle progression", Mol Cell Biol, 21, 2001, 3692-703.
5.8.1 Regulation of APC/C activators between G1/S and early anaphase
Reviewers
Peters, JM, 2006-03-27.
Description
The APC/C is activated by either Cdc20 or Cdh1. While both activators associate with the APC/C, they do so at different points in the cell cycle
and their binding is regulated differently (see Zachariae and Nasmyth, 1999). Cdc20, whose protein levels increase as cells enter into mitosis
and decrease upon mitotic exit, only associates with the APC/C during M phase. Cdh1 associates with the APC/C in G1. This interaction is
inhibited at other times by Cdk1 phosphorylation.
References
JM Peters, "The anaphase-promoting complex: proteolysis in mitosis and beyond", Mol Cell, 9, 2002, 931-43.
24, 2005, 314-25.
JW Harper, JL Burton, MJ Solomon, "The anaphase-promoting complex: it's not just for mitosis any more.", Genes Dev, 16, 2002, 2179-206.
BA Buschhorn, JM Peters, "How APC/C orders destruction", Nat Cell Biol, 8, 2006, 209-11.
5.8.1.1 Association of Cyclin A:Cdk2 with Cdh1
Authors
Editors
Matthews, L, 2006-10-10.
Reviewers
Peters, JM, 2006-10-10.
Description
Cyclin A-Cdk2 prevents unscheduled APC reactivation during S phase by binding and subsequently phosphorylating Cdh1.
Phosphorylation-dependent dissociation of the Cdh1-activating subunit inhibits the APC/C.
References
CS Sorensen, C Lukas, ER Kramer, JM Peters, J Lukas, "A conserved cyclin-binding domain determines functional interplay between
anaphase-promoting complex-Cdh1 and cyclin A-Cdk2 during cell cycle progression", Mol Cell Biol, 21, 2001, 3692-703.
Reaction
5.8.1.2 Phosphorylation of Cdh1 by Cyclin A:Cdk2
Authors

Editors
Matthews, L, 2006-01-30.
Reviewers
Peters, JM, 2006-03-27.
Description
At the G1/S transition, the Cdh1 subunit of the APC:Cdh1 complex is phosphorylated by Cyclin A:Cdk2 and dissociates from APC/C. This
inactivates APC/C and permits the accumulation of cell cycle proteins required for DNA synthesis and entry into mitosis.
References
Reaction
5.8.1.3 Dissociation of phospho-Cdh1 from the APC/C complex
Authors
Editors
Matthews, L, 2006-01-30.
Reviewers
Peters, JM, 2006-03-27.

Description
Following its phosphorylation, Cdh1 dissociates from the APC/C, rendering the APC/C inactive. This allows the stabilization of proteins required
for subsequent cell cycle progression.
References
Reaction
5.8.1.4 Association of Emi1 with Cdh1
Authors
Editors
Matthews, L, 2006-01-30.
Reviewers
Peters, JM, 2006-03-27.
Description
Emi1 promotes the accumulation of Cyclin A and entry into S phase by associating with and inhibiting the APC/C:Cdh1 complex at G1/S.
References
Reaction
5.8.1.5 Association of Emi1 with Cdc20
Authors
Editors
Matthews, L, 2006-01-30.
Reviewers
Peters, JM, 2006-03-27.
Description
In addition to its association with Cdh1 in G1 phase, Emi1 further contributes the inactivation of the APC/C between G2 and prophase by
associating with another APC/C activator, Cdc20.
References
MS Song, SJ Song, NG Ayad, JS Chang, JH Lee, HK Hong, H Lee, N Choi, J Kim, H Kim, JW Kim, EJ Choi, MW Kirschner, DS Lim, "The
tumour suppressor RASSF1A regulates mitosis by inhibiting the APC-Cdc20 complex", Nat Cell Biol, 6, 2004, 129-37.
Reaction
5.8.1.6 Phosphorylation of Emi1
Authors
Reviewers
Peters, JM, 2006-03-27.
Description
The phosphorylation of Emi1, which is required for its degradation in mitosis, appears to involve both Plk1 and Cdk1.
5.8.1.6.1 Phosphorylation of the Emi1 DSGxxS degron by Cyclin B:Cdc2
Authors
Editors
Matthews, L, 2006-02-17.
Reviewers
Peters, JM, 2006-03-27.
Description
Emi1 is also believed to be phosphorylated by Cyclin B:Cdc2 on a CDK consensus site at Ser 182. While Plk1 mediated phosphorylation of Emi1
at the DSGxxS (ÃŽÂ²TrCP recognition) motif is essential for Emi1 destruction in mitosis, Cdk phosphorylation has been shown to play an
important regulatory role.
References
F Margottin-Goguet, JY Hsu, A Loktev, HM Hsieh, JD Reimann, PK Jackson, "Prophase destruction of Emi1 by the SCF(betaTrCP/Slimb)
ubiquitin ligase activates the anaphase promoting complex to allow progression beyond prometaphase", Dev Cell, 4, 2003, 813-26.
Reaction
5.8.1.6.2 Phosphorylation of the Emi1 DSGxxS degron by Plk1
Authors
Editors
Matthews, L, 2006-02-17.
Reviewers
Peters, JM, 2006-03-27.
Description
The phosphorylation of Emi1 by Plk1 is believed to be involved in the degradation of Emi1 during mitosis. Plk1 phosphorylates serine residues in
the DSGxxS degron sequence of Emi1 recruiting the SCF(betaTrCP) ubiquitin ligase.
References
Reaction
5.8.1.7 SCF-beta-TrCP mediated degradation of Emi1
Authors
Editors
Matthews, L, 2006-01-30.
Reviewers
Peters, JM, 2006-03-27.
Description
Emi1 destruction in early mitosis requires the SCFÃŽÂ²TrCP ubiquitin ligase complex. Binding of ÃŽÂ²TrCP to Emi1 occurs in late prophase and
requires phosphorylation at the DSGxxS consensus motif as well as Cdk mediated phosphorylation. A two-step mechanism has been proposed
in which the phosphorylation of Emi1 by Cdc2 occurs after the G2-M transition followed soon after by binding of ÃŽÂ²TrCP to the DSGxxS
phosphorylation sites. Emi1 is then poly-ubiquitinated and degraded by the 26S proteasome.
References
5.8.1.7.1 Phosphorylated Emi1 binds the beta-TrCP in the SCF complex
Authors
Editors
Matthews, L, 2006-02-17.
Reviewers
Peters, JM, 2006-03-27.
Description
Cdk mediated phosphorylation of Emi1 is believed to promotes its phospho- Ser145-Ser149 dependent association with beta-TrCP.
References
E Latres, DS Chiaur, M Pagano, "The human F box protein beta-Trcp associates with the Cul1/Skp1 complex and regulates the stability of
beta-catenin", Oncogene, 18, 1999, 849-54.
Reaction
5.8.1.7.2 Ubiquitination of Emi1 by SCF-beta-TrCP

Authors
Editors
Matthews, L, 2006-01-30.
Reviewers
Peters, JM, 2006-03-27.
Description
Following its association with SCF-ÃŽÂ²TrCP, phospho-Emi1 is poly-ubiquitinated.
References
Reaction
5.8.1.7.3 SCF-mediated degradation of Emi1
Authors

Editors
Matthews, L, 2006-01-30.
Reviewers
Peters, JM, 2006-03-27.
Description
Multiubiquitinated Emi1 is degraded by the 26S proteasome.
References
Reaction
5.8.1.8 Phosphorylation of Cdh1 by Cyclin B1:Cdc2
Authors
Editors
Matthews, L, 2006-01-30.
Reviewers
Peters, JM, 2006-03-27.
Description
At the onset of mitosis, Cdh1 is phosphorylated by Cyclin B-Cdc2 resulting in a conformational change that prevents Cdh1 from activating the
APC/C.
References
J Bembenek, H Yu, "Regulation of the anaphase-promoting complex by the dual specificity phosphatase human Cdc14a", J Biol Chem, 276,
2001, 48237-42.
Reaction
5.8.1.9 Inhibition of the proteolytic activity of APC/C required for the onset of anaphase by mitotic spindle checkpoint components
Authors
Yen, T, 2004-05-05.
Reviewers
Peters, JM, 2006-03-27.
Description
The target of the mitotic checkpoint is the Anaphase Promoting Complex/Cyclosome (APC/C) an E3 ubiquitin ligase that targets proteins whose
destruction is essential for mitotic exit. Currently, there are two proposed mechanism by which inhibition of the APC/C is achieved. These
mechanisms differ depending on the mechanism of signal transduction. The APC/C may be inhibited directly by association with the Mitotic
Checkpoint Complex (MCC) or through the sequestration of its activator, Cdc20.
References
Cycle Res, 5, 2003, 431-9.
5.8.1.9.1 Inactivation of APC/C via CDC20 sequestration
Authors
Yen, T, 2004-05-05.
Editors
Matthews, L, 2006-02-17.
Reviewers
Peters, JM, 2006-03-27.
Description
In the sequestration model, the Mad2 molecules that dissociate from unattached kinetochores are perceived to bind to Cdc20, a protein that
recruits specific substrates to the APC/C. Consequently, Mad2 indirectly inhibits the APC/C by sequestering its activator, Cdc20. This requires
interaction between Mad1 and Mad2. Cdc20 and Mad1 bind to the same site on Mad2.
Reaction
5.8.1.9.2 Inactivation of APC/C via direct inhibition of the APC/C complex
Authors
Yen, T, 2004-05-05.
Reviewers
Peters, JM, 2006-03-27.
Description
In the direct inhibition model, the cytosolic Mitotic Checkpoint Complex, consisting of hBUBR1, hBUB3, Cdc20 and Mad2, directly inhibits APC/C
by binding to it.
References
MAD2", J Cell Biol, 154, 2001, 925-36.
Cycle Res, 5, 2003, 431-9.
G Fang, H Yu, MW Kirschner, "The checkpoint protein MAD2 and the mitotic regulator CDC20 form a ternary complex with the
anaphase-promoting complex to control anaphase initiation", Genes Dev, 12, 1998, 1871-83.
5.8.1.9.2.1 Formation of the MCC complex
Authors
Yen, T, 2004-05-05.
Reviewers
Peters, JM, 2006-03-27.
Description
Upon release from the kinetochore, Mad2 associates with Cdc20, hBUBR1, and hBUB3 to form the Mitotic Checkpoint Complex (MCC).
Assembly of this complex does not depend on kinetochores but this complex can only inhibit APC/C that has undergone mitotic modifications.
References
MAD2", J Cell Biol, 154, 2001, 925-36.
Reaction
5.8.1.9.2.2 Binding of the MCC complex to the APC/C complex
Authors
Yen, T, 2004-05-05.
Editors
Matthews, L, 2006-03-07.
Reviewers
Peters, JM, 2006-03-27.
Description
In the direct inhibition model, association of the MCC with APCC results in the inactivation of APC/C. However, the affinity between MCC and
APC/C is not high, so that the inhibition is readily reversible. The role of unattached kinetochores is to sensitize the APC/C to prolonged
inhibition by the MCC.
References
MAD2", J Cell Biol, 154, 2001, 925-36.
Reaction
5.8.2 Activation of APC/C and APC/C:Cdc20 mediated degradation of mitotic proteins
Authors

Reviewers
Peters, JM, 2006-03-27.
Description
APC/C:Cdc20 is first activated at the prometaphaseÃ¢â‚¬" metaphase transition through phosphorylation of core subunits of the APC/C by
mitotic kinases as well as recruitment of the APC/C activator protein Cdc20. APC/C:Cdc20 promotes the multiubiquitination and ordered
degradation of Cyclin A and Nek2 degradation in prometaphase followed by Cyclin B and securin in metaphase (Reviewed in Castro et al.,
2005).
References
24, 2005, 314-25.
5.8.2.1 Phosphorylation of the APC/C
Reviewers
Peters, JM, 2006-03-27.
Description
Phosphorylation of APC subunits is required for Cdc20 mediated activation by of the APC/C at the metaphase anaphase transition (Kramer et
al., 2000). While the kinases responsible for phosphorylation in vivo have not been determined with certainty, both Plk1 and Cyclin B:Cdc2 have
been implicated in this process.
References
ER Kramer, N Scheuringer, AV Podtelejnikov, M Mann, JM Peters, "Mitotic regulation of the APC activator proteins CDC20 and CDH1", Mol Biol
Cell, 11, 2000, 1555-69.
5.8.2.1.1 Free APC/C phosphorylated by Plk1
Authors

Editors
Matthews, L, 2006-01-30.
Reviewers
Peters, JM, 2006-03-27.
Description
Phosphorylation of the APC/C is believed to be required for its activation. While the identity of the essential phosphorylation sites and the
kinase(s) responsible are not known with certainty, in vitro studies have shown that the Apc1 and the tetratricopeptide repeat (TPR) subunits
Cdc27, Cdc16, Cdc23 and Apc7 are phosphorylated and that the Cdk1 and Plk1 kinases may play a role.
References
C Kraft, F Herzog, C Gieffers, K Mechtler, A Hagting, J Pines, JM Peters, "Mitotic regulation of the human anaphase-promoting complex by
phosphorylation", EMBO J, 22, 2003, 6598-609.
A Golan, Y Yudkovsky, A Hershko, "The cyclin-ubiquitin ligase activity of cyclosome/APC is jointly activated by protein kinases Cdk1-cyclin B
and Plk", J Biol Chem, 277, 2002, 15552-7.
Reaction
5.8.2.1.2 Free APC/C phosphorylated by Cyclin B:Cdc2
Authors

Editors
Matthews, L, 2006-01-30.
Reviewers
Peters, JM, 2006-03-27.
Description
Phosphorylation of the APC/C is believed to be required for its activation. While the identity of the essential phosphorylation sites and the
kinase(s) responsible are not known with certainty, in vitro studies have shown that the Apc1 and the tetratricopeptide repeat (TPR) subunits
Cdc27, Cdc16, Cdc23 and Apc7 are phosphorylated and that the Cdk1 and Plk1 kinases may play a role.
References
C Kraft, F Herzog, C Gieffers, K Mechtler, A Hagting, J Pines, JM Peters, "Mitotic regulation of the human anaphase-promoting complex by
phosphorylation", EMBO J, 22, 2003, 6598-609.
A Golan, Y Yudkovsky, A Hershko, "The cyclin-ubiquitin ligase activity of cyclosome/APC is jointly activated by protein kinases Cdk1-cyclin B
and Plk", J Biol Chem, 277, 2002, 15552-7.
Reaction
5.8.2.2 APC/C:Cdc20 mediated degradation of mitotic proteins
Reviewers
Peters, JM, 2006-03-27.

Description
Following phosphorylation of the APC/C core subunits by mitotic kinases, the activating protein, Cdc20 is recruited to the APC and promotes the
multiubiquitination and subsequent degradation of the mitotic cyclins (Cyclin A and Cyclin B) as well as the protein securin which functions in
sister chromatid cohesion. Timely degradation of these proteins is essential for sister chromatid separation and the proper timing of exit from
mitosis (See Zachariae and Nasmyth, 1999). Cdc20 is degraded late in mitosis (Reviewed in Owens and Hoyt, 2005)
References
TJ Owens, MA Hoyt, "The D box asserts itself", Mol Cell, 18, 2005, 611-2.
5.8.2.2.1 Activation of APC/C:Cdc20 by dissociation of Cdc20:phospho-APC/C from Cdc20:phospho-APC/C:Mad2:Bub3:BubR1
Authors

Editors
Reviewers
Peters, JM, 2006-03-27.
Description
One model ( the direct inhibition model) describing the inhibition of the APC/C during the mitotic spindle checkpoint suggests that the association
of the hBUBR1:hBUB3:MAD2*:CDC20 mitotic checkpoint complex (MCC) with APC/C results in the inactivation of APC/C. The affinity between
MCC and APC/C is not high, thus inhibition is readily reversible when the mitotic spindle checkpoint has been satisfied.
References
MAD2", J Cell Biol, 154, 2001, 925-36.
Reaction
5.8.2.2.2 APC/C:Cdc20 mediated degradation of Cyclin B
Authors
Editors
Matthews, L, 2006-01-30.
Reviewers
Peters, JM, 2006-03-27.

Description
The degradation of cyclin B1, which appears to occur at the mitotic spindle, is delayed until the metaphase /anaphase transition by the spindle
assembly checkpoint and is required in order for sister chromatids to separate (Geley et al. 2001;Hagting et al, 2002).
References
A Hagting, N Den Elzen, HC Vodermaier, IC Waizenegger, JM Peters, J Pines, "Human securin proteolysis is controlled by the spindle
checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1", J Cell Biol, 157, 2002, 1125-37.
S Geley, E Kramer, C Gieffers, J Gannon, JM Peters, T Hunt, "Anaphase-promoting complex/cyclosome-dependent proteolysis of human cyclin
A starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint", J Cell Biol, 153, 2001, 137-48.
5.8.2.2.2.1 Association of Cyclin B:Cdc2 with Cdc20:APC/C complex
Authors
Editors
Matthews, L, 2006-01-30.
Reviewers
Peters, JM, 2006-03-27.
Description
Cyclin B is believed to be recognized by the APC/C:Cdc20 complex through its D-box sequence.
Reaction
5.8.2.2.2.2 Ubiquitination of Cyclin B by phospho-APC/C:Cdc20 complex
Authors
Editors
Matthews, L, 2006-01-30.
Reviewers
Peters, JM, 2006-03-27.
Description
At the beginning of this reaction, 1 molecule of 'Cdc20:phospho-APC/C:Cyclin B:Cdc2 complex', and 3 molecules of 'ubiquitin' are present. At the
end of this reaction, 1 molecule of 'multiubiquitinated Cyclin B:Cdc2:Cdc20:phospho-APC/C complex' is present.
This reaction takes place in the 'cytosol' and is mediated by the 'ubiquitin-protein ligase activity' of 'Cdc20:Phospho-APC/C'.
References
Reaction
5.8.2.2.2.3 Degradation of multiubiquitinated Cyclin B

Authors
Editors
Matthews, L, 2006-01-30.
Reviewers
Peters, JM, 2006-03-27.
Description
Mulitubiquitinated Cyclin B is targeted for degradation by the 26S proteasome.
References
Reaction
5.8.2.2.3 APC/C:Cdc20 mediated degradation of Securin
Authors
Editors
Matthews, L, 2006-01-30.
Reviewers
Peters, JM, 2006-03-27.
Description
The separation of sister chromatids in anaphase requires the destruction of the anaphase inhibitor, securin. Securin associates with and
inactivates the protease, separase. Separase cleaves the cohesin subunit, Scc1 that is responsible for the cohesion of sister chromatids
(reviewed in Nasmyth et al., 2000). Securin destruction begins at metaphase after the mitotic spindle checkpoint has been satisfied (Hagting et
al., 2002).
References
K Nasmyth, JM Peters, F Uhlmann, "Splitting the chromosome: cutting the ties that bind sister chromatids", Science, 288, 2000, 1379-85.
5.8.2.2.3.1 Association of Securin with Cdc20:APC/C complex
Authors

Editors
Matthews, L, 2006-01-30.
Reviewers
Peters, JM, 2006-03-27.
Description
Securin is thought to be recognized by the APC/C:Cdc20 complex through its conserved D-box sequence.
References
Reaction
5.8.2.2.3.2 Ubiquitination of Securin by phospho-APC/C:Cdc20 complex
Authors
Editors
Matthews, L, 2006-01-30.
Reviewers
Peters, JM, 2006-03-27.

Description
Securin is ubiquitinated by APC/C:Cdc20 (Hagting et al., 2002).
References
Reaction
5.8.2.2.3.3 Degradation of multiubiquitinated Securin
Authors
Editors
Matthews, L, 2006-01-30.
Reviewers
Peters, JM, 2006-03-27.
Description
Following ubiquitination, securin is degraded by the 26S proteasome.

References
Reaction
5.8.3 Conversion from APC/C:Cdc20 to APC/C:Cdh1 in late anaphase
Authors
Editors
Matthews, L, 2006-02-17.
Reviewers
Peters, JM, 2006-03-27.
Description
The activity of the APC/C must be appropriately regulated during the cell cycle to ensure the timely degradation of its substrates. Of particular
importance is the conversion from APC/C:Cdc20 to APC/C:Cdh1 in late anaphase. Phosphorylation of both the APC/C complex and Cdh1
regulate this conversion. During mitosis, several APC/C subunits are phosphorylated increasing the activity of APC/C:Cdc20. However,
phosphorylation of Cdh1 by mitotic Cyclin:Cdk complexes prevents it from activating the APC/C. Dephosphorylation of Cdh1 in late anaphase by
Cdc14a results in the activation of APC/C:Cdh1 (reviewed in Castro et al, 2005).
References
24, 2005, 314-25.
5.8.3.1 Dephosphorylation of phospho-Cdh1
Authors
Editors
Matthews, L, 2006-02-17.
Reviewers
Peters, JM, 2006-03-27.
Description
Phosphorylation by mitotic kinases is believed to alter the conformation of Cdh1 preventing it from associating with the APC/C. Cdc14 is thought
to contribute to the dephosphorylation of Cdh1 in late mitosis. Dephosphorylated Cdh1 then associates with and activates the APC/C.
References
J Bembenek, H Yu, "Regulation of the anaphase-promoting complex by the dual specificity phosphatase human Cdc14a", J Biol Chem, 276,
2001, 48237-42.
Reaction
5.8.3.2 Dissociation of Cdc20 from APC/C complex

Authors
Editors
Matthews, L, 2006-01-30.
Reviewers
Peters, JM, 2006-03-27.
Description
In late mitosis, Cdc20 dissociates from the APC/C and is replaced by the activator Cdh1.
Reaction
5.8.3.3 Association of Cdh1 with the APC/C
Authors
Editors
Matthews, L, 2006-01-30.
Reviewers
Peters, JM, 2006-03-27.

Description
Following its dephosphorylation in late mitosis, Cdh1 replaces Cdc20 as the APC/C activator.
References
ER Kramer, N Scheuringer, AV Podtelejnikov, M Mann, JM Peters, "Mitotic regulation of the APC activator proteins CDC20 and CDH1", Mol Biol
Cell, 11, 2000, 1555-69.
Reaction
5.8.4 APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late
mitosis/early G1
Authors
Editors
Matthews, L, 2006-01-30.
Reviewers
Peters, JM, 2006-03-27.
Description
From late mitosis through G1 phase APC/C:Cdh1 insures the continued degradation of the mitotic proteins and during mitotic exit and G1 its
substrates include Cdc20, Plk1, Aurora A, Cdc6 and Geminin (see Castro et al., 2005). Rape et al. have recently demonstrated that the order in
which APC/C targeted proteins are degraded is determined by the processivity of multiubiquitination of these substrates. Processive substrates
acquire a polyubiquitin chain upon binding to the APC/C once and are degraded. Distributive substrates bind, dissociate and reassociate with the
APC/C multiple times before acquiring an ubiquitin chain of sufficient length to insure degradation. In addition, distributive substrates that
dissociate from the APC/C with short ubiquitin chains are targeted for deubiquitination (Rape et al., 2006).
References
24, 2005, 314-25.
M Rape, SK Reddy, MW Kirschner, "The processivity of multiubiquitination by the APC determines the order of substrate degradation", Cell, 124,
2006, 89-103.
5.8.4.1 Association of cell cycle proteins with the APC/C:Cdh1 complex
Authors
Editors
Matthews, L, 2006-02-17.
Reviewers
Peters, JM, 2006-03-27.
Description
The APC/C:Cdh1 complex recognizes substrates containing a D box, a KEN box (Pfleger and Kirschner, 2000) or a D box activated (DAD)
domain (Castro et al., 2002).
References
CM Pfleger, MW Kirschner, "The KEN box: an APC recognition signal distinct from the D box targeted by Cdh1", Genes Dev, 14, 2000, 655-65.
Reaction
5.8.4.2 Ubiquitination of cell cycle proteins targeted by the APC/C:Cdh1complex
Authors
Editors
Matthews, L, 2006-01-30.
Reviewers
Peters, JM, 2006-03-27.
Description
At the beginning of this reaction, 3 molecules of 'ubiquitin', and 1 molecule of 'cell cycle proteins:phospho-APC/C:Cdh1 complex' are present. At
the end of this reaction, 1 molecule of 'multiubiquitinated cell cycle protein:APC/C:Cdh1 complex' is present.
This reaction takes place in the 'cytosol' and is mediated by the 'ubiquitin-protein ligase activity' of 'cell cycle proteins:phospho-APC/C:Cdh1
complex'.
Reaction
5.8.4.3 Degradation of multiubiquitinated cell cycle proteins
Authors
Editors
Matthews, L, 2006-01-30.
Reviewers
Peters, JM, 2006-03-27.
Description
Cell cycle proteins mulitubiquitinated by the APC/C are targeted for degradation by the 26S proteasome.
Reaction
5.8.5 Autodegradation of Cdh1 by Cdh1:APC/C
Authors
Editors
Matthews, L, 2006-01-30.
Reviewers
Peters, JM, 2006-03-27.
Description
Cdh1 is degraded by the APC/C during in G1 and G0. This auto-regulation may contribute to reducing the levels of Cdh1 levels during G1 and
G0 (Listovsky et al., 2004).
References
5.8.5.1 Multiubiquitination of APC/C-associated Cdh1
Authors
Editors
Matthews, L, 2006-02-17.
Reviewers
Peters, JM, 2006-03-27.

Description
Cdh1 is multiubiquitinated by the APC/C:Cdh1 complex prior to degradation by the 26S proteasome.
References
Reaction
5.8.5.2 Degradation of multiubiquitinated Cdh1
Authors
Matthews, L, 2006-01-30.
Editors
Matthews, L, 2006-01-30.
Reviewers
Peters, JM, 2006-03-27.
Description
At the beginning of this reaction, 1 molecule of 'multiubiquitinated Cdh1 associated with APC/C' is present. At the end of this reaction, 1 molecule
of 'phosphorylated anaphase promoting complex (APC/C)', and 3 molecules of 'ubiquitin' are present.
This reaction takes place in the 'nucleoplasm' and is mediated by the 'endopeptidase activity' of '26S proteasome'.
References
Reaction
6 DNA Repair
Authors
Hoeijmakers, JH, Lees-Miller, S, Thompson, L, Gopinathrao, G, Matthews, L, Schultz, R, Pegg, A, 2003-07-10.
Editors
Matthews, L, Gopinathrao, G, 0000-00-00.
Reviewers
Khanna, KK, Lindahl, T, West, SC, Wood, RD, 0000-00-00.
Description
DNA repair is a phenomenal multi-enzyme, multi-pathway system required to ensure the integrity of the cellular genome. These cellular
mechanisms that must cope with the plethora of DNA base pair adducts that arise.
DNA damage can arise spontaneously in the cellular milieu through chemical alteration of base nucleotides or as a consequence of errors during
DNA replication. For example, it is well known that normal cellular pH and temperature offer an environment, which is hostile to the integrity of
DNA and its nucleotide components. Additionally, DNA damage may be induced in response to environmental exposures, including exposure to
physical agents such as ionizing or ultraviolet (UV) radiation. Finally, specific chemical agents are known to alkylate or cross-link DNA bases,
produce bulky adducts on DNA bases, or break DNA phosphate-sugar backbone.
The pioneering work from a number of laboratories have elucidated the basic mechanisms underlying distinct DNA repair pathways that include
nucleotide excision repair (NER), base excision repair (BER), DNA strand break repair (DSBR), direct reversal of DNA damage, and the
replication past DNA lesions by specialized DNA bypass polymerases (bypass replication). Defects in most of these repair pathways have been
associated with one or more specific human diseases. Additionally, the repair of damaged DNA is intimately associated with a number of other
distinct cellular processes such as DNA replication, DNA recombination, cell cycle checkpoint arrest, and other basic cellular mechanisms as
outlined herein.
6.1 Base Excision Repair
Authors
Joshi-Tope, G, 2003-07-14.
Editors
Joshi-Tope, G, 0000-00-00.
Description
Of the three major pathways involved in the repair of nucleotide damage in DNA, base excision repair (BER) involves the greatest number of
individual enzymatic activities. This is the consequence of the numerous individual glycosylases, each of which recognizes and removes a
specific modified base(s) from DNA. BER is responsible for the repair of the most prevalent types of DNA lesions, alterations, which arise
intracellularly as a consequence of normal mitochondrial metabolism or by oxidative free radicals resulting from ionizing radiation, lipid
peroxidation or activated phagocytic cells. BER is a two-step process initiated by one of the DNA glycosylases that recognizes a specific
modified base(s) and removes that base through the catalytic cleavage of the glycosylic bond, leaving an abasic site without disruption of the
phosphate-sugar DNA backbone. Subsequently, abasic sites are resolved by a series of enzymes that cleave the backbone, synthesize the
replacement residue(s), and ligate the DNA strand. BER may occur by either a single-nucleotide replacement pathway or a multiple-nucleotide
patch replacement pathway, depending on the structure of the terminal sugar phosphate residue. The glycosylases found in human cells
recognize "foreign adducts" and not standard functional modifications such as DNA methylation.
References
T Lindahl, RD Wood, "Quality control by DNA repair", Science, 286, 1999, 1897-905.
BA Sokhansanj, GR Rodrigue, JP Fitch, 3rd Wilson DM, "A quantitative model of human DNA base excision repair. I. Mechanistic insights",
6.1.1 Base-Excision Repair, AP Site Formation
Description
Base excision repair is initiated by DNA glycosylases that hydrolytically cleave of the base-deoxyribose glycosyl bond of a damaged nucleotide
residue releasing the damaged base (Lindahl and Wood, 1999).
References
6.1.1.1 Depurination
Authors
Matthews, L, 2004-02-09.
Description
Depurination of a damaged nucleotide is mediated by a purine-specific DNA glycosylase. The glycosylase cleaves the N-C1' glycosidic bond
between the damaged DNA base and the deoxyribose sugar generating a free base and an apurinic (AP) site.
6.1.1.1.1 Recognition and association of DNA glycosylase with site containing an affected purine
Authors
Matthews, L, 2004-02-09.
Description
The recognition and cleavage of an altered base by a DNA glycosylase is thought to involve the diffusion of the enzyme along the minor grove of
the DNA molecule. The enzyme presumably compresses the backbone of the affected DNA strand at the site of damage. This compression is
thought to result in an outward rotation of the damaged residue into a "pocket" of the enzyme that recognizes and cleaves the altered base
(Parikh et al., 1998).
References
SS Parikh, CD Mol, G Slupphaug, S Bharati, HE Krokan, JA Tainer, "Base excision repair initiation revealed by crystal structures and binding
kinetics of human uracil-DNA glycosylase with DNA", EMBO J, 17, 1998, 5214-26.
6.1.1.1.1.1 MPG glycosylase mediated recognition and binding of 3-methyladenine
Description
At the beginning of this reaction, 1 molecule of 'Double-strand DNA containing a 3-methyladenine', and 1 molecule of 'MPG glycosylase' are
present. At the end of this reaction, 1 molecule of 'MPG glycosylase:3-methyladenine complex' is present.
Reaction
6.1.1.1.1.2 MPG glycosylase mediated recognition and binding of ethenoadenine

Description
At the beginning of this reaction, 1 molecule of 'Double-strand DNA containing an ethenoadenine ', and 1 molecule of 'MPG glycosylase' are
present. At the end of this reaction, 1 molecule of 'MPG glycosylase:ethenoadenine complex' is present.
Reaction
6.1.1.1.1.3 MPG glycosylase mediated recognition and binding of hypoxanthine
Description
At the beginning of this reaction, 1 molecule of 'MPG glycosylase', and 1 molecule of 'Double-strand DNA containing a hypoxanthine' are
present. At the end of this reaction, 1 molecule of 'MPG glycosylase:hypoxanthine complex' is present.
Reaction
6.1.1.1.1.4 MYH glycosylase mediated recognition and binding of an adenine opposite to an 8-oxo guanine
Description
At the beginning of this reaction, 1 molecule of 'Double-strand DNA containing an adenine opposite to an 8-oxo guanine', and 1 molecule of
'MYH glycosylase' are present. At the end of this reaction, 1 molecule of 'MYH glycosylase:adenine complex' is present.

Reaction
6.1.1.1.1.5 hOGG1 glycosylase mediated recognition and binding of a formamidopyrimidine
Description
At the beginning of this reaction, 1 molecule of 'Double-strand DNA containing a formamidopyrimidine ', and 1 molecule of 'hOGG1 glycosylase'
are present. At the end of this reaction, 1 molecule of 'hOGG1 glycosylase: formamidopyrimidine complex' is present.
Reaction
6.1.1.1.1.6 hOGG1 glycosylase mediated recognition and binding of an 8-oxo guanine opposite to a cytosine
Description
At the beginning of this reaction, 1 molecule of 'Double-strand DNA containing an 8-oxo guanine opposite to a cytosine', and 1 molecule of
'hOGG1 glycosylase' are present. At the end of this reaction, 1 molecule of 'hOGG1 glycosylase:8-oxo guanine complex' is present.
Reaction
6.1.1.1.2 Cleavage of the damaged purine
Description
Damaged purines are first cleaved by purine-specific glycosylases (see Lindahl and Wood 1999).
References
6.1.1.1.2.1 Cleavage 8-oxo guanine by MYH glycosylase
Description
At the beginning of this reaction, 1 molecule of 'MYH glycosylase:adenine complex' is present. At the end of this reaction, 1 molecule of 'DNA
containing an MYH-bound apurinic/apyrimidinic site', and 1 molecule of '8-oxoguanine' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'DNA N-glycosylase activity' of 'MYH glycosylase'.
References
Reaction
6.1.1.1.2.2 Cleavage of 3-methyladenine by MPG glycosylase
Description
At the beginning of this reaction, 1 molecule of 'MPG glycosylase:3-methyladenine complex' is present. At the end of this reaction, 1 molecule of
'DNA containing an MPG-bound apurinic/apyrimidinic site', and 1 molecule of '3-Methyladenine' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'DNA N-glycosylase activity' of 'MPG glycosylase'.
References
Reaction
6.1.1.1.2.3 Cleavage of 8-oxo guanine by hOGG1 glycosylase
Description
At the beginning of this reaction, 1 molecule of 'hOGG1 glycosylase:8-oxo guanine complex' is present. At the end of this reaction, 1 molecule of
'8-oxoguanine', and 1 molecule of 'DNA containing an hOGG1-bound apurinic/apyrimidinic site' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'purine-specific oxidized base lesion DNA N-glycosylase activity' of 'hOGG1
glycosylase'.
References
Reaction
6.1.1.1.2.4 Cleavage of ethenoadenine by MPG glycosylase
Description
At the beginning of this reaction, 1 molecule of 'MPG glycosylase:ethenoadenine complex' is present. At the end of this reaction, 1 molecule of
'DNA containing an MPG-bound apurinic/apyrimidinic site', and 1 molecule of 'ethenoadenine' are present.
References
Reaction
6.1.1.1.2.5 Cleavage of formamidopyrimidine by hOGG1 glycosylase
Description
At the beginning of this reaction, 1 molecule of 'hOGG1 glycosylase: formamidopyrimidine complex' is present. At the end of this reaction, 1
molecule of 'DNA containing an hOGG1-bound apurinic/apyrimidinic site', and 1 molecule of 'formamidopyrimidine' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'purine-specific oxidized base lesion DNA N-glycosylase activity' of 'hOGG1
glycosylase'.
References
Reaction
6.1.1.1.2.6 Cleavage of hypoxanthine by MPG glycosylase
Description
At the beginning of this reaction, 1 molecule of 'MPG glycosylase:hypoxanthine complex' is present. At the end of this reaction, 1 molecule of
'Hypoxanthine', and 1 molecule of 'DNA containing an MPG-bound apurinic/apyrimidinic site' are present.
References
Reaction
6.1.1.2 Depyrimidination
Authors
Matthews, L, 2004-02-09.
Description
Depyrimidination of a damaged nucleotide is mediated by a pyrimidine-specific DNA glycosylase. The glycosylase cleaves the N-C1' glycosidic
bond between the damaged DNA base and the deoxyribose sugar generating a free base and an apyrimidinic (AP) site.
References
6.1.1.2.1 Recognition and association of DNA glycosylase with site containing an affected pyrimidine
Description
Base excision repair is initiated by a DNA glycosylase which first recognizes and removes a damaged or incorrect (e.g. mismatched) base.
References
6.1.1.2.1.1 UNG glycosylase mediated recognition and binding of a 5-hydroxyuracil opposite to a guanine
Description
At the beginning of this reaction, 1 molecule of 'Double-strand DNA containing 5-hydroxyuracil opposite to a guanine', and 1 molecule of 'UNG2'
are present. At the end of this reaction, 1 molecule of 'UNG2 glycosylase:5-hydroxyuracil complex' is present.
Reaction
6.1.1.2.1.2 UNG glycosylase mediated recognition and binding of an uracil opposite to a guanine
Description
At the beginning of this reaction, 1 molecule of 'Double-strand DNA containing an uracil opposite to a guanine', and 1 molecule of 'UNG2' are
present. At the end of this reaction, 1 molecule of 'UNG2 glycosylase:uracil complex' is present.

Reaction
6.1.1.2.1.3 TDG glycosylase mediated recognition and binding of an ethenocytosine
Description
At the beginning of this reaction, 1 molecule of 'Double-strand DNA containing an ethenocytosine', and 1 molecule of 'G/T mismatch-specific
thymine DNA glycosylase (EC 3.2.2.-)' are present. At the end of this reaction, 1 molecule of 'TDG glycosylase:ethenocytosine complex' is
present.
Reaction
6.1.1.2.1.4 TDG glycosylase mediated recognition and binding of an thymine opposite to a guanine
Description
At the beginning of this reaction, 1 molecule of 'Double-strand DNA containing a thymine opposite to a guanine', and 1 molecule of 'G/T
mismatch-specific thymine DNA glycosylase (EC 3.2.2.-)' are present. At the end of this reaction, 1 molecule of 'TDG glycosylase:thymine
complex' is present.
This reaction takes place in the 'nucleus' and is mediated by the 'DNA N-glycosylase activity' of 'G/T mismatch-specific thymine DNA glycosylase
(EC 3.2.2.-)'.
Reaction
6.1.1.2.1.5 TDG glycosylase mediated recognition and binding of an uracil opposite to a guanine
Description
At the beginning of this reaction, 1 molecule of 'Double-strand DNA containing an uracil opposite to a guanine', and 1 molecule of 'G/T
mismatch-specific thymine DNA glycosylase (EC 3.2.2.-)' are present. At the end of this reaction, 1 molecule of 'TDG glycosylase:uracil complex'
is present.
Reaction
6.1.1.2.1.6 hSMUG1 glycosylase mediated recognition and binding of uracil within single-stranded DNA
Description
At the beginning of this reaction, 1 molecule of 'hSMUG1 glycosylase', and 1 molecule of 'single-stranded DNA containing an uracil' are present.
At the end of this reaction, 1 molecule of 'hSMUG1glycosylase:uracil complex' is present.
Reaction
6.1.1.2.1.7 MBD4 glycosylase mediated recognition and binding of a thymine opposite to a guanine at CpG sequences
Description
At the beginning of this reaction, 1 molecule of 'Double-strand DNA containing a thymine opposite to a guanine at CpG sequences', and 1
molecule of 'MBD4 glycosylase' are present. At the end of this reaction, 1 molecule of 'MBD4 glycosylase:thymine complex' is present.
Reaction
6.1.1.2.1.8 MBD4 glycosylase mediated recognition and binding of an uracil opposite to a guanine at CpG sequences
Description
At the beginning of this reaction, 1 molecule of 'Double-strand DNA containing an uracil opposite to a guanine at CpG sequences', and 1
molecule of 'MBD4 glycosylase' are present. At the end of this reaction, 1 molecule of 'MBD4 glycosylase:uracil complex' is present.
Reaction
6.1.1.2.1.9 hNTH1 glycosylase mediated recognition and binding of cytosine glycol

Description
At the beginning of this reaction, 1 molecule of 'hNTH1', and 1 molecule of 'Double-strand DNA containing a cytosine glycol' are present. At the
end of this reaction, 1 molecule of 'hNTH1 glycosylase:cytosine glycol complex' is present.
Reaction
6.1.1.2.1.10 hNTH1 glycosylase mediated recognition and binding of dihydrouracil
Description
At the beginning of this reaction, 1 molecule of 'hNTH1', and 1 molecule of 'Double-strand DNA containing a dihydrouracil' are present. At the
end of this reaction, 1 molecule of 'hNTH1 glycosylase:dihydrouracil complex' is present.
Reaction
6.1.1.2.1.11 hNTH1 glycosylase mediated recognition and binding of formamidopyrimidine
Description
At the beginning of this reaction, 1 molecule of 'hNTH1', and 1 molecule of 'Double-strand DNA containing a formamidopyrimidine ' are present.
At the end of this reaction, 1 molecule of 'hNTH1 glycosylase: foramidopyrimidine complex' is present.

Reaction
6.1.1.2.1.12 hNTH1 glycosylase mediated recognition and binding of thymine glycol
Description
At the beginning of this reaction, 1 molecule of 'hNTH1', and 1 molecule of 'Double-strand DNA containing a thymine glycol ' are present. At the
end of this reaction, 1 molecule of 'hNTH1 glycosylase:thymine glycol' is present.
Reaction
6.1.1.2.2 Cleavage of the damaged pyrimidine
Description
Damaged pyrimidines are first cleaved by pyrimide-specific glycosylases (see Lindahl and Wood 1999).
References
6.1.1.2.2.1 Cleavage of uracil by UNG2 glycosylase

Description
At the beginning of this reaction, 1 molecule of 'UNG2 glycosylase:uracil complex' is present. At the end of this reaction, 1 molecule of 'DNA
containing UNG2-bound apurinic/apyrimidinic site', and 1 molecule of 'Uracil' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'uracil DNA N-glycosylase activity' of 'UNG2'.
References
Reaction
6.1.1.2.2.2 Cleavage of 5-hydroxyluracil by UNG2 glycosylase
Description
At the beginning of this reaction, 1 molecule of 'UNG2 glycosylase:5-hydroxyuracil complex' is present. At the end of this reaction, 1 molecule of
'DNA containing UNG2-bound apurinic/apyrimidinic site', and 1 molecule of '5-hydroxyuracil' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'uracil DNA N-glycosylase activity' of 'UNG2'.
References
Reaction
6.1.1.2.2.3 Cleavage of uracil by TDG glycosylase
Description
At the beginning of this reaction, 1 molecule of 'TDG glycosylase:uracil complex' is present. At the end of this reaction, 1 molecule of 'DNA
containing a TDG-bound apurinic/apyrimidinic site', and 1 molecule of 'Uracil' are present.
References
Reaction
6.1.1.2.2.4 Cleavage of thymine by TDG glycosylase
Description
At the beginning of this reaction, 1 molecule of 'TDG glycosylase:thymine complex' is present. At the end of this reaction, 1 molecule of 'DNA
containing a TDG-bound apurinic/apyrimidinic site', and 1 molecule of 'Thymine' are present.
(EC 3.2.2.-)'.
References
Reaction
6.1.1.2.2.5 Cleavage of uracil by hSMUG1 glycosylase
Description
At the beginning of this reaction, 1 molecule of 'hSMUG1glycosylase:uracil complex' is present. At the end of this reaction, 1 molecule of 'Uracil',
and 1 molecule of 'DNA containing an hSMUG1-bound apurinic/apyrimidinic site' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'single-strand selective uracil DNA N-glycosylase activity' of 'hSMUG1
glycosylase'.
References
Reaction
6.1.1.2.2.6 Cleavage of thymine glycol by hNTH1 glycosylase
Description
At the beginning of this reaction, 1 molecule of 'hNTH1 glycosylase:thymine glycol' is present. At the end of this reaction, 1 molecule of 'Thymine
glycol', and 1 molecule of 'DNA containing a hNTH1-bound apurinic/apyrimidinic site' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'pyrimidine-specific oxidized base lesion DNA N-glycosylase activity' of 'hNTH1'.
References
Reaction
6.1.1.2.2.7 Cleavage of cytosine glycol by hNTH1 glycosylase
Description
At the beginning of this reaction, 1 molecule of 'hNTH1 glycosylase:cytosine glycol complex' is present. At the end of this reaction, 1 molecule of
'cytosine glycol', and 1 molecule of 'DNA containing a hNTH1-bound apurinic/apyrimidinic site' are present.
References
Reaction
6.1.1.2.2.8 Cleavage of dihydrouracil by hNTH1 glycosylase
Description
At the beginning of this reaction, 1 molecule of 'hNTH1 glycosylase:dihydrouracil complex' is present. At the end of this reaction, 1 molecule of
'DNA containing a hNTH1-bound apurinic/apyrimidinic site', and 1 molecule of '5,6-Dihydrouracil' are present.
Reaction
6.1.1.2.2.9 Cleavage of formamidopyrimidine by hNTH1 glycosylase
Description
At the beginning of this reaction, 1 molecule of 'hNTH1 glycosylase: foramidopyrimidine complex' is present. At the end of this reaction, 1
molecule of 'formamidopyrimidine', and 1 molecule of 'DNA containing a hNTH1-bound apurinic/apyrimidinic site' are present.
Reaction
6.1.1.2.2.10 Cleavage of uracil by MBD4 glycosylase
Description
At the beginning of this reaction, 1 molecule of 'MBD4 glycosylase:uracil complex' is present. At the end of this reaction, 1 molecule of 'Uracil',
and 1 molecule of 'DNA containing an MBD4-bound apurinic/apyrimidinic site' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'DNA N-glycosylase activity' of 'MBD4 glycosylase'.
Reaction
6.1.1.2.2.11 Cleavage of thymine by MBD4 glycosylase

Description
At the beginning of this reaction, 1 molecule of 'MBD4 glycosylase:thymine complex' is present. At the end of this reaction, 1 molecule of 'DNA
containing an MBD4-bound apurinic/apyrimidinic site', and 1 molecule of 'Thymine' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'DNA N-glycosylase activity' of 'MBD4 glycosylase'.
Reaction
6.1.1.2.2.12 Cleavage of ethenocytosine by TDG glycosylase
Description
At the beginning of this reaction, 1 molecule of 'TDG glycosylase:ethenocytosine complex' is present. At the end of this reaction, 1 molecule of
'DNA containing a TDG-bound apurinic/apyrimidinic site', and 1 molecule of 'ethenocytosine' are present.
(EC 3.2.2.-)'.
Reaction
6.1.2 Resolution of Abasic Sites (AP sites)

Description
Resolution of AP sites can occur through the single-nucleotide replacement pathway or through the multiple-nucleotide patch replacement
pathway.
6.1.2.1 Resolution of AP sites via the single-nucleotide replacement pathway
Authors
Matthews, L, 2004-02-09.
Description
The single-nucleotide replacement pathway of base excision repair appears to facilitate the repair of most damaged bases. Following DNA
glycosylase mediated cleavage of the damaged base, the endonuclease, APE1 is recruited to the site of damage where it cleaves the 5' side of
the base-free deoxyribose residue. DNA polymerase, POL Beta then displaces the DNA glycosylase and cleaves the 3' side of the sugar
phosphate. APE1 is subsequently released, the XRCC1:LIG3 complex is recruited and POL Beta mediates the synthesis of the replacement
residue. Following LIG3 ligase mediated ligation of the replaced residue, the XRCC1:LIG3 complex dissociates (see Lindahl and Wood, 1999).
An alternative BER pathway is employed when the structure of the terminal sugar phosphate is such that it can not be cleaved through the AP
lyase activity of POL Beta.
References
6.1.2.1.1 Base-free sugar-phosphate removal via the single-nucleotide replacement pathway
Description
Base-free sugar-phosphate removal via the single-nucleotide replacement pathway requires displacement of DNA glycosylase by APE1, APE1
mediated endonucleolytic cleavage at the 5' side of the base-free deoxyribose residue, recruitment of POL Beta to the AP site and Excision of
the abasic sugar phosphate (dRP) residue at the strand break (Lindahl and Wood, 1999).
References
6.1.2.1.1.1 Displacement of DNA glycosylase by APE1
Description
Following cleavage of the damaged base, DNA glycosylase is displaced by APE1.
References
6.1.2.1.1.1.1 Displacement of UNG2 glycosylase by APE1 at the AP site
Description
At the beginning of this reaction, 1 molecule of 'DNA containing UNG2-bound apurinic/apyrimidinic site', and 1 molecule of 'APE1' are present. At
the end of this reaction, 1 molecule of 'DNA containing an APE1-bound AP site', and 1 molecule of 'UNG2' are present.
Reaction
6.1.2.1.1.1.2 Displacement of TDG glycosylase by APE1 at the AP site
Description
At the beginning of this reaction, 1 molecule of 'DNA containing a TDG-bound apurinic/apyrimidinic site', and 1 molecule of 'APE1' are present.
At the end of this reaction, 1 molecule of 'DNA containing an APE1-bound AP site', and 1 molecule of 'G/T mismatch-specific thymine DNA
glycosylase (EC 3.2.2.-)' are present.
Reaction
6.1.2.1.1.1.3 Displacement of hSMUG1 glycosylase by APE1 at the AP site
Description
At the beginning of this reaction, 1 molecule of 'DNA containing an hSMUG1-bound apurinic/apyrimidinic site', and 1 molecule of 'APE1' are
present. At the end of this reaction, 1 molecule of 'hSMUG1 glycosylase', and 1 molecule of 'DNA containing an APE1-bound AP site' are
present.
Reaction
6.1.2.1.1.1.4 Displacement of hNTH1 glycosylase by APE1 at the AP site
Description
At the beginning of this reaction, 1 molecule of 'DNA containing a hNTH1-bound apurinic/apyrimidinic site', and 1 molecule of 'APE1' are present.
At the end of this reaction, 1 molecule of 'hNTH1', and 1 molecule of 'DNA containing an APE1-bound AP site' are present.
Reaction
6.1.2.1.1.1.5 Displacement of MBD4 glycosylase by APE1 at the AP site
Description
At the beginning of this reaction, 1 molecule of 'DNA containing an MBD4-bound apurinic/apyrimidinic site', and 1 molecule of 'APE1' are
present. At the end of this reaction, 1 molecule of 'MBD4 glycosylase', and 1 molecule of 'DNA containing an APE1-bound AP site' are present.
Reaction
6.1.2.1.1.1.6 Displacement of hOGG1 glycosylase by APE1 at the AP site
Description
At the beginning of this reaction, 1 molecule of 'DNA containing an hOGG1-bound apurinic/apyrimidinic site', and 1 molecule of 'APE1' are
present. At the end of this reaction, 1 molecule of 'hOGG1 glycosylase', and 1 molecule of 'DNA containing an APE1-bound AP site' are present.
Reaction
6.1.2.1.1.1.7 Displacement of MYH glycosylase by APE1 at the AP site
Description
At the beginning of this reaction, 1 molecule of 'DNA containing an MYH-bound apurinic/apyrimidinic site', and 1 molecule of 'APE1' are present.
At the end of this reaction, 1 molecule of 'MYH glycosylase', and 1 molecule of 'DNA containing an APE1-bound AP site' are present.
Reaction
6.1.2.1.1.1.8 Displacement of MPG glycosylase by APE1 at the AP site
Description
At the beginning of this reaction, 1 molecule of 'DNA containing an MPG-bound apurinic/apyrimidinic site', and 1 molecule of 'APE1' are present.
At the end of this reaction, 1 molecule of 'MPG glycosylase', and 1 molecule of 'DNA containing an APE1-bound AP site' are present.

Reaction
6.1.2.1.1.2 APE1 mediated endonucleolytic cleavage at the 5' side of the base-free deoxyribose residue
Description
At the beginning of this reaction, 1 molecule of 'DNA containing an APE1-bound AP site' is present. At the end of this reaction, 1 molecule of
'APE1-bound DNA strand break containing an incision 5' to an AP site' is present.
This reaction takes place in the 'nucleus' and is mediated by the 'endodeoxyribonuclease activity' of 'APE1'.
References
Y Masuda, RA Bennett, B Demple, "Dynamics of the interaction of human apurinic endonuclease (Ape1) with its substrate and product.", J Biol
Chem, 273, 1998, 30352-9.
Reaction
6.1.2.1.1.3 Recruitment of POL Beta to the AP site

Description
At the beginning of this reaction, 1 molecule of 'APE1-bound DNA strand break containing an incision 5' to an AP site', and 1 molecule of 'DNA
polymerase beta (EC 2.7.7.7)' are present. At the end of this reaction, 1 molecule of 'POL Beta: APE1-bound DNA strand break containing
incision 5' to AP site ' is present.
References
RA Bennett, DM Wilson, D Wong, B Demple, "Interaction of human apurinic endonuclease and DNA polymerase beta in the base excision repair
pathway.", Proc Natl Acad Sci U S A, 94, 1997, 7166-9.
Reaction
6.1.2.1.1.4 Excision of the abasic sugar phosphate (dRP) residue at the strand break
Description
At the beginning of this reaction, 1 molecule of 'POL Beta: APE1-bound DNA strand break containing incision 5' to AP site ' is present. At the end
of this reaction, 1 molecule of 'POL Beta-bound DNA strand break containing gap left by excised residue', 1 molecule of 'APE1', and 1 molecule
of 'Sugar phosphate' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'DNA-(apurinic or apyrimidinic site) lyase activity' of 'DNA polymerase beta (EC
2.7.7.7)'.
Reaction
6.1.2.1.2 Recruitment of LIG3:XRRC1 complex to the site of repair by POL Beta
Description
At the beginning of this reaction, 1 molecule of 'POL Beta-bound DNA strand break containing gap left by excised residue', and 1 molecule of
'LIG3:XRRC1 complex ' are present. At the end of this reaction, 1 molecule of 'LIG3:XRCC1:POL Beta complex at site of base repair' is present.
References
Y Kubota, RA Nash, A Klungland, P SchÃ¤r, DE Barnes, T Lindahl, "Reconstitution of DNA base excision-repair with purified human proteins:
interaction between DNA polymerase beta and the XRCC1 protein.", EMBO J, 15, 1997, 6662-70.
Reaction
6.1.2.1.3 Resynthesis of excised residue
Authors
Matthews, L, 2004-01-29.
Description
DNA polymerase Beta mediates DNA synthesis that fills the gap left by the excised residue using the undamaged strand as a template.
References
Reaction
6.1.2.1.4 DNA ligation via the single-nucleotide replacement pathway
Description
At the beginning of this reaction, 1 molecule of 'LIG3:XRCC1:POL Beta:DNA strand break containing replaced unligated residue' is present. At
the end of this reaction, 1 molecule of 'LIG3:XRRC1-bound DNA strand containing the ligated residue' is present.
This reaction takes place in the 'nucleus' and is mediated by the 'DNA ligase activity' of 'DNA ligase III '.
References
Reaction
6.1.2.1.5 Dissociation of LIG3:XRCC1 complex from site of BER
Description
At the beginning of this reaction, 1 molecule of 'LIG3:XRRC1-bound DNA strand containing the ligated residue' is present. At the end of this
reaction, 1 molecule of 'LIG3:XRRC1 complex ', and 1 molecule of 'DNA strand containing replaced ligated residue' are present.

Reaction
6.1.2.2 Resolution of AP sites via the multiple-nucleotide patch replacement pathway
Authors
Matthews, L, 2004-02-09.
Description
While the single-nucleotide replacement pathway appears to facilitate the repair of most damaged bases, an alternative BER pathway is evoked
when the structure of the terminal sugar phosphate is such that it can not be cleaved through the AP lyase activity of DNA polymerase, POL
Beta. Under these circumstances, a short stretch of residues containing the abasic site is excised and replaced (Dianov et al., 1999). Following
DNA glycosylase mediated cleavage of the damaged base, the endonuclease APE1 is recruited to the site of damage where it cleaves the 5'
side of the base-free deoxyribose residue. POL Beta then displaces the DNA glycosylase and synthesizes the first replacement residue. DNA
polymerase, POL Delta replaces POL Beta and mediates the synthesis of several additional residues resulting in the displacement of a DNA flap
containing the abasic sugar phosphate and 3Ã¢â‚¬â„¢ flanking residues. The flap structure is recognized and cleaved by the flap endonuclease,
FEN1 and the replacement residues are then ligated by the DNA ligase, LIG1 (Klungland and Lindahl, 1997; Matsumoto et al., 1999).
References
GL Dianov, BR Jensen, MK Kenny, VA Bohr, "Replication protein A stimulates proliferating cell nuclear antigen-dependent repair of abasic sites
in DNA by human cell extracts.", Biochemistry, 38, 1999, 11021-5.
A Klungland, T Lindahl, "Second pathway for completion of human DNA base excision-repair: reconstitution with purified proteins and
requirement for DNase IV (FEN1).", EMBO J, 16, 1997, 3341-8.
Y Matsumoto, K Kim, J Hurwitz, R Gary, DS Levin, AE Tomkinson, MS Park, "Reconstitution of proliferating cell nuclear antigen-dependent
repair of apurinic/apyrimidinic sites with purified human proteins.", J Biol Chem, 274, 1999, 33703-8.
6.1.2.2.1 Removal of DNA patch containing abasic residue
Description
During removal of DNA patch containing abasic residue, DNA glycosylase is displaced by APE1 which endonucleolytically cleaves the 5' side of
the base-free deoxyribose residue. POL Beta is recruited to the AP site where it mediates incorporation of the first replacement nucleotide.
Association of POL delta with the AP site displaces POL Beta. DNA strand displacement synthesis then follows and flap structures are cleaved.
6.1.2.2.1.1 Displacement of DNA glycosylase by APE1
Description
Following cleavage of the damaged base, DNA glycosylase is displaced by APE1.
References
6.1.2.2.1.1.1 Displacement of UNG2 glycosylase by APE1 at the AP site
Description
At the beginning of this reaction, 1 molecule of 'DNA containing UNG2-bound apurinic/apyrimidinic site', and 1 molecule of 'APE1' are present. At
the end of this reaction, 1 molecule of 'DNA containing an APE1-bound AP site', and 1 molecule of 'UNG2' are present.

Reaction
6.1.2.2.1.1.2 Displacement of TDG glycosylase by APE1 at the AP site
Description
At the beginning of this reaction, 1 molecule of 'DNA containing a TDG-bound apurinic/apyrimidinic site', and 1 molecule of 'APE1' are present.
At the end of this reaction, 1 molecule of 'DNA containing an APE1-bound AP site', and 1 molecule of 'G/T mismatch-specific thymine DNA
glycosylase (EC 3.2.2.-)' are present.
Reaction
6.1.2.2.1.1.3 Displacement of hSMUG1 glycosylase by APE1 at the AP site
Description
At the beginning of this reaction, 1 molecule of 'DNA containing an hSMUG1-bound apurinic/apyrimidinic site', and 1 molecule of 'APE1' are
present. At the end of this reaction, 1 molecule of 'hSMUG1 glycosylase', and 1 molecule of 'DNA containing an APE1-bound AP site' are
present.
Reaction
6.1.2.2.1.1.4 Displacement of hNTH1 glycosylase by APE1 at the AP site
Description
At the beginning of this reaction, 1 molecule of 'DNA containing a hNTH1-bound apurinic/apyrimidinic site', and 1 molecule of 'APE1' are present.
At the end of this reaction, 1 molecule of 'hNTH1', and 1 molecule of 'DNA containing an APE1-bound AP site' are present.

Reaction
6.1.2.2.1.1.5 Displacement of MBD4 glycosylase by APE1 at the AP site
Description
At the beginning of this reaction, 1 molecule of 'DNA containing an MBD4-bound apurinic/apyrimidinic site', and 1 molecule of 'APE1' are
present. At the end of this reaction, 1 molecule of 'MBD4 glycosylase', and 1 molecule of 'DNA containing an APE1-bound AP site' are present.
Reaction
6.1.2.2.1.1.6 Displacement of hOGG1 glycosylase by APE1 at the AP site
Description
At the beginning of this reaction, 1 molecule of 'DNA containing an hOGG1-bound apurinic/apyrimidinic site', and 1 molecule of 'APE1' are
present. At the end of this reaction, 1 molecule of 'hOGG1 glycosylase', and 1 molecule of 'DNA containing an APE1-bound AP site' are present.
Reaction
6.1.2.2.1.1.7 Displacement of MYH glycosylase by APE1 at the AP site
Description
At the beginning of this reaction, 1 molecule of 'DNA containing an MYH-bound apurinic/apyrimidinic site', and 1 molecule of 'APE1' are present.
At the end of this reaction, 1 molecule of 'MYH glycosylase', and 1 molecule of 'DNA containing an APE1-bound AP site' are present.

Reaction
6.1.2.2.1.1.8 Displacement of MPG glycosylase by APE1 at the AP site
Description
At the beginning of this reaction, 1 molecule of 'DNA containing an MPG-bound apurinic/apyrimidinic site', and 1 molecule of 'APE1' are present.
At the end of this reaction, 1 molecule of 'MPG glycosylase', and 1 molecule of 'DNA containing an APE1-bound AP site' are present.
Reaction
6.1.2.2.1.2 APE1 mediated endonucleolytic cleavage at the 5' side of the base-free deoxyribose residue
Description
At the beginning of this reaction, 1 molecule of 'DNA containing an APE1-bound AP site' is present. At the end of this reaction, 1 molecule of
'APE1-bound DNA strand break containing an incision 5' to an AP site' is present.
This reaction takes place in the 'nucleus' and is mediated by the 'endodeoxyribonuclease activity' of 'APE1'.
References
Y Masuda, RA Bennett, B Demple, "Dynamics of the interaction of human apurinic endonuclease (Ape1) with its substrate and product.", J Biol
Chem, 273, 1998, 30352-9.
Reaction
6.1.2.2.1.3 Recruitment of POL Beta to the AP site

Description
At the beginning of this reaction, 1 molecule of 'APE1-bound DNA strand break containing an incision 5' to an AP site', and 1 molecule of 'DNA
polymerase beta (EC 2.7.7.7)' are present. At the end of this reaction, 1 molecule of 'POL Beta: APE1-bound DNA strand break containing
incision 5' to AP site ' is present.
References
RA Bennett, DM Wilson, D Wong, B Demple, "Interaction of human apurinic endonuclease and DNA polymerase beta in the base excision repair
pathway.", Proc Natl Acad Sci U S A, 94, 1997, 7166-9.
Reaction
6.1.2.2.1.4 POL Beta mediated incorporation of the first replacement nucleotide
Description
At the beginning of this reaction, 1 molecule of 'dNTP', and 1 molecule of 'POL Beta: APE1-bound DNA strand break containing incision 5' to AP
site ' are present. At the end of this reaction, 1 molecule of 'pyrophosphate', and 1 molecule of 'POL Beta: APE1-bound 5' incised DNA strand
break containing first resynthesized base ' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'beta DNA polymerase activity' of 'DNA polymerase beta (EC 2.7.7.7)'.
References
AJ Podlutsky, II Dianova, VN Podust, VA Bohr, GL Dianov, "Human DNA polymerase beta initiates DNA synthesis during long-patch repair of
reduced AP sites in DNA.", EMBO J, 20, 2001, 1477-82.
Reaction
6.1.2.2.1.5 POL delta associates with AP site displacing POL Beta
Description
At the beginning of this reaction, 1 molecule of 'POL Beta: APE1-bound 5' incised DNA strand break containing first resynthesized base ', and 1
molecule of 'DNA Polymerase delta tetramer' are present. At the end of this reaction, 1 molecule of 'DNA polymerase beta (EC 2.7.7.7)', and 1
molecule of 'POL Delta:APE1-bound 5' incised DNA strand break containing first resynthesized nucleotide' are present.
References
Reaction
6.1.2.2.1.6 DNA strand displacement synthesis
Description
At the beginning of this reaction, 1 molecule of 'POL Delta:APE1-bound 5' incised DNA strand break containing first resynthesized nucleotide',
and 1 molecule of 'Deoxynucleoside triphosphate' are present. At the end of this reaction, 1 molecule of 'pyrophosphate', and 1 molecule of
'single-stranded DNA flap structure at the site of damaged residue' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'DNA-directed DNA polymerase activity' of 'DNA Polymerase delta tetramer'.
References
TA Ranalli, S Tom, RA Bambara, "AP endonuclease 1 coordinates flap endonuclease 1 and DNA ligase I activity in long patch base excision
repair.", J Biol Chem, 277, 2002, 41715-24.
Reaction
6.1.2.2.1.7 Cleavage of flap structures
Authors
Matthews, L, 2004-01-29.
Description
FEN1 cleaves the dRp group at the AP site and the 3'-adjacent nucleotide(s).
References
A Klungland, T Lindahl, "Second pathway for completion of human DNA base excision-repair: reconstitution with purified proteins and
requirement for DNase IV (FEN1).", EMBO J, 16, 1997, 3341-8.
Reaction
6.1.2.2.2 Ligation of DNA at sites of patch replacement
Description
At the beginning of this reaction, 1 molecule of 'DNA containing unligated replacement-synthesized patch' is present. At the end of this reaction,
1 molecule of 'Ligated patch-repaired DNA' is present.
This reaction takes place in the 'nucleus' and is mediated by the 'DNA ligase activity' of 'DNA ligase I '.
References
Reaction
6.2 DNA Damage Bypass
Authors
Description
In addition to various processes for removing damaging lesions from the DNA, cells have developed specific mechanisms for tolerating
unexcised damages during the replication of the genome. Such processes are collectively called DNA damage bypass pathways. Several
proteins including novel Y-family polymerases that have been recently identified in multitude of organisms are involved in this process.
Translesion synthesis (TLS) or replicative bypass of damaged bases that are known to arrest high fidelity, highly processive polymerases
involved in DNA replication is carried out by error-prone polymerases Pol zeta, Pol eta and Rev3 protein among others. TLS is implicated in UV
and chemical induced mutagenesis of normal human cells where lesions in the replicating genome are carried over to the newly formed
daughter cells.
All these 3 enzymes are found to lack 3Ã¢â‚¬â„¢->5Ã¢â‚¬â„¢ exonuclease activity, while exhibiting low fidelity, weak processivity and sufficient
polymerase activities. An outline of the bypass synthesis by these 3 enzymes is annotated here. Complete details of damage recognition and
discrimination, initiation of specific polymerase activity and the finer mechanisms are yet to be elucidated.
References
EC Friedberg, PL Fischhaber, C Kisker, "Error-prone DNA polymerases: novel structures and the benefits of infidelity.", Cell, 107, 2001, 9-12.
AR Lehmann, "Replication of UV-damaged DNA: new insights into links between DNA polymerases, mutagenesis and human disease.", Gene,
253, 2000, 1-12.
F Zhu, M Zhang, "DNA polymerase zeta: new insight into eukaryotic mutagenesis and mammalian embryonic development.", World J
Gastroenterol, 9, 2003, 1165-9.
6.2.1 Translesion synthesis by DNA polymerases bypassing lesion on DNA template
Description
Ubiquitous environmental and endogenous genotoxic agents cause lesions that can interfere with normal DNA metabolism including DNA
replication, eventually resulting in mutations that lead to carcinogenesis and/or cell death. While cells posses repair mechanisms like nucleotide
excision and base excision repair pathways to maintain the integrity of the genome, not all lesions on the genome can be repaired efficiently by
these processes in time for DNA replication, and some types of lesion are repaired very inefficiently. To prevent acute cell death through
arrested DNA replication at unrepaired lesions, cells have a mechanism, referred to as translesion synthesis, which allows DNA synthesis to
proceed past lesions (Masutani et al., 2000).
References
C Masutani, R Kusumoto, S Iwai, F Hanaoka, "Mechanisms of accurate translesion synthesis by human DNA polymerase eta.", EMBO J, 19,
2000, 3100-9.
6.2.1.1 Translesion synthesis by HREV1
Authors
Description
HREV1 encodes a template dependent dCMP transferase activity that can insert a C residue opposite an abasic site.
References
PE Gibbs, XD Wang, Z Li, TP McManus, WG McGregor, CW Lawrence, VM Maher, "The function of the human homolog of Saccharomyces
cerevisiae REV1 is required for mutagenesis induced by UV light.", Proc Natl Acad Sci U S A, 97, 2000, 4186-91.
W Lin, H Xin, Y Zhang, X Wu, F Yuan, "The human REV1 gene codes for a DNA template-dependent dCMP transferase.", Nucleic Acids Res,
27, 1999, 4468-75.
6.2.1.1.1 Binding of HREV1 to lesioned DNA template
Description
At the beginning of this reaction, 1 molecule of 'damaged DNA substrate ', and 1 molecule of 'HREV1 protein' are present. At the end of this
reaction, 1 molecule of 'HREV1:damaged DNA template complex' is present.
Reaction
6.2.1.1.2 Misinsertion of bases opposite to the lesion by HREV1
Description
At the beginning of this reaction, 1 molecule of 'HREV1:damaged DNA template complex' is present. At the end of this reaction, 1 molecule of
'HREV1:lesioned DNA template with misinserted bases' is present.
This reaction takes place in the 'nucleus' and is mediated by the 'DNA-directed DNA polymerase activity' of 'HREV1:damaged DNA template
complex'.
Reaction
6.2.1.1.3 Elongation by HREV1 protein

Description
At the beginning of this reaction, 1 molecule of 'HREV1:lesioned DNA template with misinserted bases' is present. At the end of this reaction, 1
molecule of 'HREV1 protein', and 1 molecule of 'Elongated DNA template with bypassed lesion' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'DNA-directed DNA polymerase activity' of 'HREV1:lesioned DNA template with
misinserted bases'.
Reaction
6.2.1.2 Translesion synthesis by Pol eta
Authors
Description
Pol eta consists of 713 amino acids and can bypass thymidine-thymidine dimers adding two AÃ¢â‚¬â„¢s correctly opposite to the lesion.
Mutations in Pol eta gene result in the loss of this bypass activity in accounting for the XP variant phenotype (XPV) among human xeroderma
pigmentosum disorder patients. Pol eta can carry out TLS past various UV and chemical induced lesions via two steps: a. preferential
incorporation of correct bases opposite to the lesion
b. conditional elongation only at the sites where such correct bases are inserted.
References
C Masutani, M Araki, A Yamada, R Kusumoto, T Nogimori, T Maekawa, S Iwai, F Hanaoka, "Xeroderma pigmentosum variant (XP-V) correcting
protein from HeLa cells has a thymine dimer bypass DNA polymerase activity.", EMBO J, 18, 1999, 3491-501.
C Masutani, R Kusumoto, S Iwai, F Hanaoka, "Mechanisms of accurate translesion synthesis by human DNA polymerase eta.", EMBO J, 19,
2000, 3100-9.
6.2.1.2.1 Binding of Pol eta to lesioned DNA template

Description
At the beginning of this reaction, 1 molecule of 'damaged DNA substrate ', and 1 molecule of 'Pol eta protein' are present. At the end of this
reaction, 1 molecule of 'Pol eta:damaged DNA template complex' is present.
Reaction
6.2.1.2.2 Insertion of correct bases opposite to the lesion by Pol eta
Description
At the beginning of this reaction, 1 molecule of 'Pol eta:damaged DNA template complex' is present. At the end of this reaction, 1 molecule of
'Pol eta:lesioned DNA template inserted with correct base complement' is present.
This reaction takes place in the 'nucleus' and is mediated by the 'eta DNA polymerase activity' of 'Pol eta:damaged DNA template complex'.
Reaction
6.2.1.2.3 Elongation by Pol eta
Description
At the beginning of this reaction, 1 molecule of 'Pol eta:lesioned DNA template inserted with correct base complement' is present. At the end of
this reaction, 1 molecule of 'Pol eta protein', and 1 molecule of 'Elongated DNA template with bypassed lesion' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'eta DNA polymerase activity' of 'Pol eta:lesioned DNA template inserted with
correct base complement'.
Reaction
6.2.1.3 Translesion synthesis by Pol zeta
Authors
Description
Pol zeta consists of two proteins: HREV3 with polymerase activity and HREV7, an accessory factor with unidentified activity. It has the ability to
bypass pyrimidine dimers and other adducts in DNA.
References
W Lin, X Wu, "A full-length cDNA of hREV3 is predicted to encode DNA polymerase zeta for damage-induced mutagenesis in humans.", Mutat
Res, 433, 1999, 89-98.
PE Gibbs, WG McGregor, VM Maher, P Nisson, CW Lawrence, "A human homolog of the Saccharomyces cerevisiae REV3 gene, which
encodes the catalytic subunit of DNA polymerase zeta.", Proc Natl Acad Sci U S A, 95, 1998, 6876-80.
6.2.1.3.1 Binding of Pol zeta to lesioned DNA template
Description
At the beginning of this reaction, 1 molecule of 'damaged DNA substrate ', and 1 molecule of 'Pol zeta complex' are present. At the end of this
reaction, 1 molecule of 'Pol zeta:damaged DNA template complex' is present.
Reaction
6.2.1.3.2 Elongation by Pol zeta complex
Description
At the beginning of this reaction, 1 molecule of 'dNTP', and 1 molecule of 'Pol zeta:damaged DNA template complex' are present. At the end of
this reaction, 1 molecule of 'Pol zeta complex', and 1 molecule of 'Elongated DNA template with bypassed lesion' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'DNA-directed DNA polymerase activity' of 'Pol zeta:damaged DNA template
complex'.
Reaction
6.3 DNA Damage Reversal
Authors
Pegg, A, 2004-02-04.
Editors
Joshi-Tope, G, 0000-00-00.
Description
The direct reversal of DNA damage by dealkylation will be annotated in a subsequent GK release
6.3.1 MGMT/hAGT mediated DNA Damage Reversal
Authors
Pegg, A, 2004-02-04.
Editors
Joshi-Tope, G, 0000-00-00.
Description
Reactive cellular catabolites can cause DNA damage by methylation of the O-6-guanine. O-6-methylguanine can pair ambiguously with both C
and T, and can cause transition mutations. Active reversal of such damage can be facilitated by the 6-O-methylguanine-DNA methyltransferase
(MGMT).
MGMT removes the methyl group from the guanine and transfers it to the cysteine residue at position 145 on the protein itself. MGMT thus
methylated is not regenerated, as the S-methylcysteine is very stable. This is an energetically expensive approach to DNA repair as one entire
protein molecule is sacrificed per lesion that is corrected in this manner.
References
S Mitra, B Kaina, "Regulation of repair of alkylation damage in mammalian genomes.", Prog Nucleic Acid Res Mol Biol, 44, 1993, 109-42.
Reaction
6.3.2 Reversal of Alkylation Damage By DNA Dioxygenases
Authors
Description
DNA in cells is susceptible to different types of cytotoxic and mutagenic damages caused by alkylating agents. These genotoxic chemicals
generate major lesions like 1-methyladenine and 3-methylcytosine in single-stranded DNA and 3-methyladenine and O6-methylguanine in
double-stranded DNA . Cells have inbuilt repair mechanisms against such toxic molecules. For example, 3-methyladeninie-DNA glycosylases
excise some methylated bases while MGMT/hAGT protein transfers alkyl groups from others lesions onto cysteine residues. E.coli AlkB protein
has a unique function wherein 1-methyladenine and 3-methylcytosine are demethylated by a combination of oxidative decarboxylation and
hydroxylation activities. AlkB and its human orthologs, ABH2 and ABH3 belong to alpha-ketoglutarate deoxygenase family of enzymes that
oxidize chemically inert compounds in the presence of alpha-ketoglutarate, oxygen and ferrous ions. As a byproduct of these chemical reactions,
formaldehyde is released in the case of methylated lesions and acetaldehyde in the case 1-ethyladenine in DNA. CO2 and succinate are also
released in an intermediate step not shown in the following illustration.Unlike other mechanisms which involve some kind of nuclease activities,
this type of repair mechanism leaves the repaired bases intact by just removing the reactive alkyl groups that get bound to the bases thereby
effecting accurate restoration of damaged DNA sequences.
References
T Duncan, SC Trewick, P Koivisto, PA Bates, T Lindahl, B Sedgwick, "Reversal of DNA alkylation damage by two human dioxygenases.", Proc
Natl Acad Sci U S A, 99, 2002, 16660-5.
B Sedgwick, "Repairing DNA-methylation damage.", Nat Rev Mol Cell Biol, 5, 2004, 148-57.
SC Trewick, TF Henshaw, RP Hausinger, T Lindahl, B Sedgwick, "Oxidative demethylation by Escherichia coli AlkB directly reverts DNA base
damage.", Nature, 419, 2002, 174-8.
6.3.2.1 ABH2 mediated Reversal of Alkylation Damage
Description
AlkB is an alpha-ketoglutarate- and Fe(II)-dependent dioxygenase that oxidizes the relevant methyl groups and releases them as formaldehyde.
The human homologs of both these enzymes remove 1-methyladenine and 3-methylcytosine from methylated polynucleotides in an
alpha-ketoglutarate-dependent reaction. They act by direct damage reversal with the regeneration of the unsubstituted bases. E.coli AlkB, and
human ABH2, and ABH3 can also repair 1-ethyladenine residues in DNA with the release of acetaldehyde (Duncan et al., 2002).
References
Natl Acad Sci U S A, 99, 2002, 16660-5.
DH Lee, SG Jin, S Cai, Y Chen, GP Pfeifer, TR O'Connor, "Repair of methylation damage in DNA and RNA by mammalian AlkB homologues", J
Biol Chem, 280, 2005, 39448-59.
6.3.2.1.1 Oxidative demethylation of 1-MeA damaged DNA By ABH2
Description
At the beginning of this reaction, 1 molecule of 'Oxygen', 1 molecule of '2-Oxoglutarate', and 1 molecule of 'Alkylated DNA with 1-methyladenine'
are present. At the end of this reaction, 1 molecule of 'Formaldehyde', 1 molecule of 'Repaired DNA after oxidative dealkylation', 1 molecule of
'CO2', and 1 molecule of 'Succinate' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'damaged DNA binding activity' of 'ABH2 protein'.
References
Natl Acad Sci U S A, 99, 2002, 16660-5.
Reaction
6.3.2.1.2 Oxidative demethylation of 1-EtA damaged DNA By ABH2
Description
At the beginning of this reaction, 1 molecule of 'Oxygen', 1 molecule of 'Alkylated DNA with 1-ethyladenine', and 1 molecule of '2-Oxoglutarate'
are present. At the end of this reaction, 1 molecule of 'Repaired DNA after oxidative dealkylation', 1 molecule of 'CO2', 1 molecule of 'Succinate',
and 1 molecule of 'Acetaldehyde' are present.
References
Natl Acad Sci U S A, 99, 2002, 16660-5.
Reaction
6.3.2.2 ABH3 mediated Reversal of Alkylation Damage
Description
AlkB is an alpha-ketoglutarate- and Fe(II)-dependent dioxygenase that oxidizes the relevant methyl groups and releases them as formaldehyde.
The human homologs of both these enzymes remove 1-methyladenine and 3-methylcytosine from methylated polynucleotides in an
alpha-ketoglutarate-dependent reaction. They act by direct damage reversal with the regeneration of the unsubstituted bases. E.coli AlkB, and
human ABH2, and ABH3 can also repair 1-ethyladenine residues in DNA with the release of acetaldehyde (Duncan et al., 2002).
References
Natl Acad Sci U S A, 99, 2002, 16660-5.
DH Lee, SG Jin, S Cai, Y Chen, GP Pfeifer, TR O'Connor, "Repair of methylation damage in DNA and RNA by mammalian AlkB homologues", J
Biol Chem, 280, 2005, 39448-59.
6.3.2.2.1 Oxidative demethylation of 1-MeA damaged DNA By ABH3
Description
At the beginning of this reaction, 1 molecule of 'Oxygen', 1 molecule of '2-Oxoglutarate', and 1 molecule of 'Alkylated DNA with 1-methyladenine'
References
Natl Acad Sci U S A, 99, 2002, 16660-5.
Reaction
6.3.2.2.2 Oxidative demethylation of 3-MeC damaged DNA By ABH3
Description
At the beginning of this reaction, 1 molecule of 'Alkylated DNA with 3-methylcytosine', 1 molecule of 'Oxygen', and 1 molecule of '2-Oxoglutarate'
References
Natl Acad Sci U S A, 99, 2002, 16660-5.
Reaction
6.3.2.2.3 Oxidative demethylation of 1-EtA damaged DNA By ABH3
Description
At the beginning of this reaction, 1 molecule of 'Oxygen', 1 molecule of 'Alkylated DNA with 1-ethyladenine', and 1 molecule of '2-Oxoglutarate'
are present. At the end of this reaction, 1 molecule of 'Repaired DNA after oxidative dealkylation', 1 molecule of 'CO2', 1 molecule of 'Succinate',
and 1 molecule of 'Acetaldehyde' are present.
References
Natl Acad Sci U S A, 99, 2002, 16660-5.
Reaction
6.4 Double-Strand Break Repair
Authors
Lees-Miller, S, Thompson, L, 2003-07-14.
Editors
Matthews, L, 0000-00-00.
Description
Numerous types of DNA damage can occur within a cell due to the endogenous production of oxygen free radicals, normal alkylation reactions,
or exposure to exogenous radiations and chemicals. Double-strand breaks (DSBs), one of the most dangerous type of DNA damage along with
interstrand crosslinks, are caused by ionizing radiation or certain chemicals such as bleomycin, and occur normally during the processes of DNA
replication, meiotic exchange, and V(D)J recombination.
Two distinct mechanisms for DSB repair are the error-free homologous recombination repair (HRR) pathway and the error-prone
nonhomologous end-joining (NHEJ) pathway. The choice of pathway may be determined by whether the DNA region has already replicated and
the precise nature of the break. NHEJ functions at all stages of the cell cycle, but plays the predominant role in both the G1 phase and in
S-phase regions of DNA that have not yet replicated (Rothkamm et al. 2003). HRR functions primarily in repairing both one-sided DSBs that
arise at DNA replication forks and two-sided DSBs arising in S or G2-phase chromatid regions that have replicated.
References
K Rothkamm, I KrÃ¼ger, LH Thompson, M LÃ¶brich, "Pathways of DNA double-strand break repair during the mammalian cell cycle.", Mol Cell
Biol, 23, 2003, 5706-15.
6.4.1 Homologous Recombination Repair
Authors
Thompson, L, 2003-07-14.
Editors
Matthews, L, 0000-00-00.
Reviewers
West, SC, 0000-00-00.
Description
The HRR pathway is an "error free" DNA repair mechanism that utilizes information encoded by homologous sequence to repair double-strand
breaks (DSBs). HRR acts on DSBs occurring within replicated DNA (replication-independent DSBs) or on DSBs that are generated at broken
replication forks (replication-dependent DSBs). Repair by homologous recombination involves processing of the ends of the DNA double-strand
break, homologous DNA pairing and strand exchange, repair DNA synthesis, and resolution of the heteroduplex molecules.
References
LH Thompson, D Schild, "Recombinational DNA repair and human disease.", Mutat Res, 509, 2002, 49-78.
L Thompson, L Limoli, "Origin, Recognition, Signaling and Repair of DNA Double-Strand Breaks in Mammalian Cells", Damage Surveillance and
Repair (Keith Caldecott, editor), 2003, 1-40.
KK Khanna, "DNA double-strand breaks: signaling, repair and the cancer connection.", Nat Genet, 27, 2001, 247-54.
LH Thompson, D Schild, "Homologous recombinational repair of DNA ensures mammalian chromosome stability", Mutat Res, 477, 2001,
131-53.
D Ristic, M Modesti, R Kanaar, C Wyman, "Rad52 and Ku bind to different DNA structures produced early in double-strand break repair.",
6.4.1.1 Homologous recombination repair of replication-independent double-strand breaks
Description
Homologous recombination repair of replication-independent double-strand breaks requires the activation of ATM followed by ATM mediated
phosphorylation of DNA repair proteins. DNA repair and signaling proteins are then recruited to double-strand breaks. The ends of the DNA
double strand breaks must be resectioned and RPA complexes associate with the resulting ssDNA. Homologous DNA pairing and strand
exchange then occurs followed by DNA repair synthesis and resolution of D-loop structures.
6.4.1.1.1 ATM mediated response to DNA double-strand break
Authors
Matthews, L, 2003-11-18.
Description
Detection of DNA double-strand breaks involves sensor proteins that become activated setting off of signaling cascades. This signaling leads to
the recruitment of repair proteins and subsequent repair of the damaged DNA. ATM is one of the primary candidate sensors in double-strand
break repair.
References
H Beamish, P Kedar, H Kaneko, P Chen, T Fukao, C Peng, S Beresten, N Gueven, D Purdie, S Lees-Miller, N Ellis, N Kondo, MF Lavin,
"Functional link between BLM defective in Bloom's syndrome and the ataxia-telangiectasia-mutated protein, ATM.", J Biol Chem, 277, 2002,
30515-23.
6.4.1.1.1.1 Intermolecular autophosphorylation of ATM within dimeric ATM complexes
Authors
Matthews, L, 2003-11-18.
Description
ATM is present in unirradiated cells as a dimer or higher-order multimer. Following irradiation, ATM undergoes rapid intermolecular
autophosphorylation of serine 1981.
References
CJ Bakkenist, MB Kastan, "DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation", Nature, 421, 2003,
499-506.
Reaction
6.4.1.1.1.2 Dissociation of dimeric phospho-ATM complexes
Authors
Matthews, L, 2003-12-10.
Description
Autophosphorylation of ATM results in the dissociation of the dimeric/multimeric ATM complex and initiation of ATM kinase activity.
Autophosphorylation does not directly regulate ATM kinase activity. Instead, it is the resulting dissociation of ATM oligomers that allows
substrates access to the ATM kinase domain.
References
499-506.
Reaction
6.4.1.1.1.3 ATM mediated phosphorylation of repair proteins
Authors
Matthews, L, 2003-11-18.
Description
Following the detection of DSBs, ATM mediates the phosphorylation of proteins involved in DNA repair (Thompson and Schild, 2002).
References
LH Thompson, D Schild, "Recombinational DNA repair and human disease.", Mutat Res, 509, 2002, 49-78.
6.4.1.1.1.3.1 Phosphorylation of histone H2AX at Serine-139 by ATM at the site of DSB
Authors
Matthews, L, 2003-08-11.
Description
H2AX phosphorylation (producing the gamma-H2AX protein form) occurs within 1-3 minutes of DNA damage (Rogakou et al,1998) and is
promoted by MDC1/NFBD1 (Stewart et al., 2003). gamma H2AX is one of the first proteins to appear at the site of damage, localizing to a region
of about 2 Mbp surrounding the site of the double-strand break (Rogakou et al,1998). gamma-H2AX appears to play an essential role in
recruiting other repair proteins including Rad50, Rad51 and BRCA1(Paull et al., 2000) (Stewart et al., 2003).
References
EP Rogakou, DR Pilch, AH Orr, VS Ivanova, WM Bonner, "DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139.",
J Biol Chem, 273, 1998, 5858-68.
GS Stewart, B Wang, CR Bignell, AM Taylor, SJ Elledge, "MDC1 is a mediator of the mammalian DNA damage checkpoint.", Nature, 421, 2003,
961-6.
TT Paull, EP Rogakou, V Yamazaki, CU Kirchgessner, M Gellert, WM Bonner, "A critical role for histone H2AX in recruitment of repair factors to
nuclear foci after DNA damage.", Curr Biol, 10, 2000, 886-95.
Reaction
6.4.1.1.1.3.2 Phosphorylation of BRCA1 at multiple sites by ATM
Authors
Matthews, L, 2003-11-18.
Description
ATM mediated phosphorylation of BRCA1 at Ser 1189, Ser 1542 and Ser 1524 (Cortez et al., 1999) is regulated by MDC1 (Lou et al., 2003).
BRCA1 plays an important role in the response to double-strand breaks, participating in genome surveillance, DNA repair, and cell cycle
checkpoint arrests. BRCA1 is required for ATM-dependent phosphorylation of NBS1 following exposure to ionizing radiation (Foray et al., 2003).
BRCA1 also interacts with the MRE11-RAD50-NBS1 complex which has been implicated early in HRR during resection of the double-strand
break.
References
N Foray, D Marot, A Gabriel, V Randrianarison, M Perricaudet, A Ashworth, P Jeggo, "A subset of ATM- and ATR-dependent phosphorylation
events requires the BRCA1 protein.", EMBO J, 22, 2003, 2860-71.
D Cortez, Y Wang, J Qin, SJ Elledge, "Requirement of ATM-dependent phosphorylation of brca1 in the DNA damage response to double-strand
breaks", Science, 286, 1999, 1162-6.
Reaction
6.4.1.1.1.3.3 Phosphorylation of NBS1 by ATM
Authors
Matthews, L, 2003-11-18.
Description
NSB1 is a component of the MRE11/RAD50/NBS1 complex which acts early in HRR during resection of the double-strand break. In addition,
NBS1 is required for activation of the S-phase checkpoint in response to ionizing radiation (IR), ATM-dependent activation of CHK2 and cell
survival after exposure to IR.
References
JH Lee, B Xu, CH Lee, JY Ahn, MS Song, H Lee, CE Canman, JS Lee, MB Kastan, DS Lim, "Distinct functions of Nijmegen breakage syndrome
in ataxia telangiectasia mutated-dependent responses to DNA damage.", Mol Cancer Res, 1, 2003, 674-81.
Reaction
6.4.1.1.1.3.4 Phosphorylation of MDC1/NFBD1 by ATM (within 2 c-term BRCT domains)
Authors
Matthews, L, 2003-11-18.
Description
ATM mediated phosphorylation of MDC1/NFBD1 may be involved in the rapid relocalization of MDC1/NFBD1 to nuclear foci that contain the
MRE11 complex, phosphorylated histone H2AX and 53BP1 (Goldberg et al., 2003). NFBD1 may also function in recruiting DNA checkpoint
signaling and repair proteins to the sites of DNA damage (Xu and Stern., 2003 and Stewart et al.,2003).
References
M Goldberg, M Stucki, D D'Amours, D Rahman, D Pappin, "MDC1 is required for the intra-S-phase DNA damage checkpoint.", Nature, 421,
2003, 952-6.
X Xu, DF Stern, "NFBD1/MDC1 regulates ionizing radiation-induced focus formation by DNA checkpoint signaling and repair factors.", FASEB J,
17, 2003, 1842-8.
961-6.
Reaction
6.4.1.1.2 Recruitment of repair and signaling proteins to double-strand breaks
Authors
Matthews, L, 2003-11-18.
Description
Following exposure to ionizing radiation, a number of recombination/repair proteins and complexes relocalize to nuclear foci that are believed to
correspond to the sites of double-strand breaks. These proteins include gamma-H2AX, ATM, RAD51, BRCA1, BRCA2, NBS1, RPA, and
MDC1/NFBD1 and the MRE11-RAD50-NBS1. Gamma-H2AX, the phosphorylated form of H2AX, is one of the first proteins to appear within
nuclear foci and plays an essential role in recruiting other repair proteins (Paull et al., 2000).
One model illustrating the organization of the recruited repair proteins (Van den Bosch et al., 2003) is shown here. (A) Undamaged section of a
chromosome, showing two chromatin loops and an inactive ATM dimer. (B,C) Induction of a DNA double-stranded break (DSB), modification of
chromatin, activation of ATM and recruitment of both ATM and MRE11/RAD50/NBS1(MRN). The possibility that MRN binds before ATM is
shown, but the activation of ATM appears to require the MDC1 and the MRN complex (Uziel, et al 2003; Mochan et al. 2003). The thin black line
indicates modified chromatin. (D,E) Following DNA damage, a wave of H2AX phosphorylation occurs and the mediator proteins (mediator of
DNA damage checkpoint protein 1 (MDC1), p53-binding protein 1(53BP1), and breast-cancer-associated protein 1(BRCA1)) are recruited to the
growing focus where they are phosphorylated in an ATM-dependent manner. The molecular architecture of the focus is unknown. (F)
Disassembly of the focus, ATM inactivation and chromatin remodeling. The model suggests at least two distinct forms of soluble ATM: an
inactive oligomer and an active monomer, and at least two distinct active, insoluble forms: one directly at the lesion and another integral to the
growing focus. Note that the MRN complex is also a component of the growing focus, but for clarity, has been omitted here. Complex persistent
lesions are thought to be more difficult to repair, and this is reflected in the size attained by the growing focus.
References
T Uziel, Y Lerenthal, L Moyal, Y Andegeko, L Mittelman, Y Shiloh, "Requirement of the MRN complex for ATM activation by DNA damage.",
EMBO J, 22, 2003, 5612-21.
M van den Bosch, RT Bree, NF Lowndes, "The MRN complex: coordinating and mediating the response to broken chromosomes.", EMBO Rep,
4, 2003, 844-9.
TA Mochan, M Venere, RA DiTullio, TD Halazonetis, "53BP1 and NFBD1/MDC1-Nbs1 function in parallel interacting pathways activating
ataxia-telangiectasia mutated (ATM) in response to DNA damage.", Cancer Res, 63, 2003, 8586-91.
TT Paull, EP Rogakou, V Yamazaki, CU Kirchgessner, M Gellert, WM Bonner, "A critical role for histone H2AX in recruitment of repair factors to
nuclear foci after DNA damage.", Curr Biol, 10, 2000, 886-95.
6.4.1.1.2.1 MDC1/NFBD1associates with gamma H2AX at nuclear foci
Authors
Matthews, L, 2003-11-18.
Description
Recruitment of MDC1 to nuclear foci is mediated by H2AX. MDC1 forms complexes with phosphorylated H2AX, promotes efficient
phosphorylation of H2AX and controls the formation of BRCA1 and MRN foci (Stewart et al.,2003).
References
M Goldberg, M Stucki, D D'Amours, D Rahman, D Pappin, "MDC1 is required for the intra-S-phase DNA damage checkpoint.", Nature, 421,
2003, 952-6.
961-6.
Reaction
6.4.1.1.2.2 phospho-ATM (Serine 1981) associates with DNA at the site of double-strand breaks
Description
At the beginning of this reaction, 1 molecule of 'MDC1/NFBD1:gamma-H2AX complex', and 1 molecule of 'phospho-ATM (Ser 1981)' are
present. At the end of this reaction, 1 molecule of 'ATM associated with DNA double-strand break ends' is present.
References
499-506.
Reaction
6.4.1.1.2.3 53BP1 associates with gamma H2AX at nuclear foci
Description
At the beginning of this reaction, 1 molecule of '53BP1', and 1 molecule of 'gamma H2AX:MDC1/NFBD1 complex at site of DNA double-strand
break' are present. At the end of this reaction, 1 molecule of '53BP1:H2AX complex at site of double-strand break' is present.
References
499-506.
Reaction
6.4.1.1.2.4 MRN complex relocalizes to nuclear foci
Authors
Matthews, L, 2003-11-23.
Description
MRN complexes localize to nuclear foci in response to double-strand breaks and have been implicated in DNA end-processing during
homologous recombination repair (Trujillo et al., 1998). The MRN complex may function as a bridge between double-strand break ends through
interactions between the coiled-coil domains of RAD50 (Hoptner et al., 2002).
References
BE Nelms, RS Maser, JF MacKay, MG Lagally, JH Petrini, "In situ visualization of DNA double-strand break repair in human fibroblasts.",
Science, 280, 1998, 590-2.
KP Hopfner, L Craig, G Moncalian, RA Zinkel, T Usui, BA Owen, A Karcher, B Henderson, JL Bodmer, CT McMurray, JP Carney, JH Petrini, JA
Tainer, "The Rad50 zinc-hook is a structure joining Mre11 complexes in DNA recombination and repair.", Nature, 418, 2002, 562-6.
KM Trujillo, SS Yuan, EY Lee, P Sung, "Nuclease activities in a complex of human recombination and DNA repair factors Rad50, Mre11, and
p95.", J Biol Chem, 273, 1998, 21447-50.
6.4.1.1.2.4.1 Association of gamma-H2AX with NBS1
Authors
Matthews, L, 2003-08-11.
Description
NBS1 binds to H2AX and functions in the relocalization of MRE11/RAD50 nuclease complex to the double-strand break (Kobayashi, J. et al.,
2002).
References
J Kobayashi, H Tauchi, S Sakamoto, A Nakamura, K Morishima, S Matsuura, T Kobayashi, K Tamai, K Tanimoto, K Komatsu, "NBS1 localizes
to gamma-H2AX foci through interaction with the FHA/BRCT domain.", Curr Biol, 12, 2002, 1846-51.
Reaction
6.4.1.1.2.4.2 Assembly of the RAD50-MRE11-NBS1 complex at DNA double-strand breaks
Description
Assembly of the RAD50-MRE11-NBS1 complex at DNA double-strand breaks involves the formation of RAD50:MRE11 complex, association of
RAD50:MRE11 complex with NBS1 through MRE11 and association of the MRN with sites of DSB.
6.4.1.1.2.4.2.1 Formation of RAD50:MRE11 complex
Authors
Matthews, L, 2003-08-11.
Description
MRE11 has both manganese dependent ss DNA 3'-5' exonuclease and endonuclease activities. MRE11 associates with RAD50 resulting in
increased 3'-5' exonuclease activity.
Reaction
6.4.1.1.2.4.2.2 Association of RAD50:MRE11 complex with NBS1 via MRE11 interaction
Description
At the beginning of this reaction, 1 molecule of 'RAD50:MRE11 complex', and 1 molecule of 'NBS1' are present. At the end of this reaction, 1
molecule of 'MRE11:RAD50:NBS1 complex' is present.
Reaction
6.4.1.1.2.4.2.3 Association of MRN with sites of DSB
Description
At the beginning of this reaction, 1 molecule of 'MRE11:RAD50:NBS1 complex', and 1 molecule of 'gamma-H2AX:NBS1 complex at the site of
double-strand break' are present. At the end of this reaction, 1 molecule of 'DNA double-strand break ends: MRN complex' is present.
Reaction
6.4.1.1.2.5 BRCA1 associates with 53BP1at the site of DNA double-strand break
Description
At the beginning of this reaction, 1 molecule of '53BP1:H2AX complex at site of double-strand break', and 1 molecule of 'Breast cancer type 1
susceptibility protein' are present. At the end of this reaction, 1 molecule of 'BRCA1:53BP1 complex at site of DNA double-strand break' is
present.
References
SC West, "Molecular views of recombination proteins and their control.", Nat Rev Mol Cell Biol, 4, 2003, 435-45.
555-66.
B Wang, S Matsuoka, PB Carpenter, SJ Elledge, "53BP1, a mediator of the DNA damage checkpoint.", Science, 298, 2002, 1435-8.
Reaction
6.4.1.1.3 Processing of DNA double-strand break ends
Description
The processing of DNA double strand breaks requires resectioning of the broken ends followed by the association of RPA complexes with
ssDNA.
References
6.4.1.1.3.1 Resection of double-strand break ends
Authors
Matthews, L, 2003-08-11.
Description
In order for repair of the double-strand break to occur, the 5' ends of the break must first be resected to produce single-strands that invade
homologous duplex DNA. Although the enzymatic activities responsible for end-processing in humans are not known with certainty, the
MRE11-RAD50-NBS1 (MRN) complex has been implicated in this process (Thompson and Schild, 2001). The MRN complex may function as a
bridge between double-strand break ends through interactions between the coiled-coil domains of RAD50 (Hoptner et al., 2002).
References
DM Kolpashchikov, SN Khodyreva, DY Khlimankov, MS Wold, A Favre, OI Lavrik, "Polarity of human replication protein A binding to DNA.",
KP Hopfner, L Craig, G Moncalian, RA Zinkel, T Usui, BA Owen, A Karcher, B Henderson, JL Bodmer, CT McMurray, JP Carney, JH Petrini, JA
Tainer, "The Rad50 zinc-hook is a structure joining Mre11 complexes in DNA recombination and repair.", Nature, 418, 2002, 562-6.
KM Trujillo, SS Yuan, EY Lee, P Sung, "Nuclease activities in a complex of human recombination and DNA repair factors Rad50, Mre11, and
p95.", J Biol Chem, 273, 1998, 21447-50.
LH Thompson, D Schild, "Homologous recombinational repair of DNA ensures mammalian chromosome stability", Mutat Res, 477, 2001,
131-53.
Reaction
6.4.1.1.3.2 Association of RPA complexes with ssDNA
Authors
Matthews, L, 2003-09-10.
Description
Human replication protein A (RPA) is a single-stranded DNA (ssDNA) binding protein required for DNA replication, recombination, and repair.
RPA is a stable heterotrimer consisting of subunits with molecular masses of 14, 32 and 70 kDa (p14, p32 and p70, respectively). Association of
RPA with ssDNA is thought to contribute to both the protection and removal of secondary structure from single-stranded DNA.
References
MJ McIlwraith, E Van Dyck, JY Masson, AZ Stasiak, A Stasiak, SC West, "Reconstitution of the strand invasion step of double-strand break
repair using human Rad51 Rad52 and RPA proteins", J Mol Biol, 304, 2000, 151-64.
Reaction
6.4.1.1.4 Homologous DNA pairing and strand exchange

Authors
Matthews, L, 2003-11-23.
Description
Following double-strand break end-processing, the resulting single-stranded ends must locate and pair with complementary sequences within
homologous duplex DNA. RAD51, which has been implicated in both the search for homology between DNA strands and the formation of joint
molecules, plays an important role in these processes. RAD54, which has been shown to interact with RAD51, may play an important role in the
homologous DNA pairing reaction that occurs during strand invasion. Rad54 has DNA strand opening activities that are stimulated following
association with Rad51(Sigurdsson et al., 2002).
References
RC Gupta, E Folta-Stogniew, CM Radding, "Human Rad51 protein can form homologous joints in the absence of net strand exchange.", J Biol
Chem, 274, 1999, 1248-56.
S Sigurdsson, S Van Komen, G Petukhova, P Sung, "Homologous DNA pairing by human recombination factors Rad51 and Rad54.", J Biol
Chem, 277, 2002, 42790-4.
6.4.1.1.4.1 Presynaptic phase of homologous DNA pairing and strand exchange
Description
The presynaptic phase of homologous DNA pairing and strand exchange begins with the displacement of RPA from ssDNA, followed by the
association of RAD51 with BRCA2, formation of RAD52 heptameric ring structure complexes on ssDNA, association of RAD52 with the RPA
complex, association of RAD52 with ssDNA at resected ends of double-strand break and assembly of the RAD51-ssDNA nucleoprotein
complex.
References
6.4.1.1.4.1.1 Displacement of RPA from ssDNA
Description
RPA is displaced from single stranded DNA.

6.4.1.1.4.1.2 Association of RAD51 with BRCA2
Authors
Matthews, L, 2003-11-23.
Description
BRCA2 and RAD51 interact directly through the highly conserved BRC repeats in BRCA2 (Venkitaraman, 2002).
References
M Tarsounas, D Davies, SC West, "BRCA2-dependent and independent formation of RAD51 nuclear foci.", Oncogene, 22, 2003, 1115-23.
Reaction
6.4.1.1.4.1.3 Formation of RAD52 heptameric ring structure complexes on ssDNA
Authors
Matthews, L, 2003-09-10.
Description
RAD52 interacts with ssDNA forming heptameric ring structure complexes. The conformation of the RAD52-ssDNA complex is thought to place
the ssDNA on an exposed surface of the protein, in a configuration that may promote the DNA-DNA annealing of complementary DNA strands.
References
AZ Stasiak, E Larquet, A Stasiak, S MÃ¼ller, A Engel, E Van Dyck, SC West, EH Egelman, "The human Rad52 protein exists as a heptameric
ring.", Curr Biol, 10, 2000, 337-40.
MR Singleton, LM Wentzell, Y Liu, SC West, DB Wigley, "Structure of the single-strand annealing domain of human RAD52 protein.", Proc Natl
Acad Sci U S A, 99, 2002, 13492-7.
P Baumann, SC West, "Heteroduplex formation by human Rad51 protein: effects of DNA end-structure, hRP-A and hRad52.", J Mol Biol, 291,
1999, 363-74.
E Van Dyck, NM Hajibagheri, A Stasiak, SC West, "Visualisation of human rad52 protein and its complexes with hRad51 and DNA.", J Mol Biol,
284, 1999, 1027-38.
CA Parsons, P Baumann, E Van Dyck, SC West, "Precise binding of single-stranded DNA termini by human RAD52 protein.", EMBO J, 19,
2000, 4175-81.
W Kagawa, H Kurumizaka, R Ishitani, S Fukai, O Nureki, T Shibata, S Yokoyama, "Crystal structure of the homologous-pairing domain from the
human Rad52 recombinase in the undecameric form.", Mol Cell, 10, 2002, 359-71.
Reaction
6.4.1.1.4.1.4 Association of RAD52 with the RPA complex
Description
At the beginning of this reaction, 1 molecule of 'RAD52', and 1 molecule of 'RPA heterotrimer' are present. At the end of this reaction, 1 molecule
of 'RAD52:RPA complex' is present.
Reaction
6.4.1.1.4.1.5 Association of RAD52 with ssDNA at resected ends of double-strand break
Description
At the beginning of this reaction, 1 molecule of '3' overhanging DNA at resected DSB ends', and 1 molecule of 'RAD52' are present. At the end of
this reaction, 1 molecule of 'RAD52:DNA double-strand break' is present.

References
2000, 4175-81.
Reaction
6.4.1.1.4.1.6 Assembly of the RAD51-ssDNA nucleoprotein complex
Authors
Matthews, L, 2003-09-10.
Description
The RAD51-ssDNA nucleoprotein filament is a right-hand helical nucleoprotein filament referred to as the presynaptic filament. This
nucleoprotein complex contains a binding site for the double-stranded homologous DNA molecule with which it interacts during strand exchange.
Loading of RAD51 onto ssDNA is thought to be facilitated by recombination mediator RAD52 (McIlwraith et al., 2000), RAD51 paralogs:
RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3 (Sigurdsson et al., 2001), by the association of RAD52 with ssDNA (Parsons et al., 2000)
and by the association of RAD51 with BRCA2 (Tarsounas et al., 2003).
References
D Ristic, C Wyman, C Paulusma, R Kanaar, "The architecture of the human Rad54-DNA complex provides evidence for protein translocation
along DNA.", Proc Natl Acad Sci U S A, 98, 2001, 8454-60.
S Sigurdsson, S Van Komen, W Bussen, D Schild, JS Albala, P Sung, "Mediator function of the human Rad51B-Rad51C complex in
Rad51/RPA-catalyzed DNA strand exchange.", Genes Dev, 15, 2001, 3308-18.
2000, 4175-81.
M Tarsounas, D Davies, SC West, "BRCA2-dependent and independent formation of RAD51 nuclear foci.", Oncogene, 22, 2003, 1115-23.
6.4.1.1.4.1.6.1 Association of RAD51 with RAD52:DNA double-strand break ends
Authors
Matthews, L, 2003-11-23.
Description
Binding of the RAD51:RAD52 complex to ssDNA may provide nucleation sites promoting the association of free RAD51 with the growing
nucleoprotein filament. This interaction may also play a role in stimulating the strand transfer reactions mediated by RAD51 (Baumann, 1999).
References
P Baumann, SC West, "Heteroduplex formation by human Rad51 protein: effects of DNA end-structure, hRP-A and hRad52.", J Mol Biol, 291,
1999, 363-74.
Reaction
6.4.1.1.4.1.6.2 Association of RAD51 with the RPA complex

Authors
Matthews, L, 2003-11-23.
Description
The binding of RAD51 to ssDNA may be facilitated by association with RPA (Golub et al., 1998).
References
EI Golub, RC Gupta, T Haaf, MS Wold, CM Radding, "Interaction of human rad51 recombination protein with single-stranded DNA binding
protein, RPA.", Nucleic Acids Res, 26, 1999, 5388-93.
Reaction
6.4.1.1.4.1.6.3 Association of RAD51 with the resected ends of the DNA double-strand break
Description
At the beginning of this reaction, 1 molecule of 'RAD51', and 1 molecule of '3' overhanging DNA at resected DSB ends' are present. At the end of
this reaction, 1 molecule of 'RAD51:resected ends of DNA double-strand break' is present.
References
Reaction
6.4.1.1.4.2 Synaptic stable pairing/D-loop structure formation between recombining DNA molecules
Authors
Matthews, L, 2003-11-23.
Description
Stable synaptic pairing between recombining DNA molecules involves the invasion of homologous duplex DNA by the processed single-stranded
ends of the double-strand break, followed by displacement of the non-complimentary strand of the duplex. This results in the formation of a
D-loop structure.
6.4.1.1.4.2.1 Strand invasion

Authors
Matthews, L, 2003-11-23.
Description
Invasion of the heteroduplex DNA by RAD51 nucleoprotein filament involves the identification of a region of homology between these two
molecules. The mechanism by which this occurs is not yet known.
6.4.1.1.4.2.2 Heteroduplex formation
Authors
Matthews, L, 2003-11-23.
Description
RAD51 catalyses the invasion of the nucleoprotein filament into homologous double-stranded DNA. Watson-Crick hydrogen bonding between
the ssDNA within the nucleoprotein filament and the homologous strand of the invaded DNA duplex result in the displacement of the non-pairing
strand (strand displacement) and formation of the D-loop structure (Sung et al., 2003).
The RAD51 nucleoprotein filament has independent binding sites for the ssDNA strand upon which it assembles and the recombining
homologous duplex DNA. The repair proteins RPA, RAD52, and RAD54 appear to promote RAD51 mediated invasion and homologous DNA
pairing reaction (McIlwraith et al, 2000 and Sigurdsson et al., 2002). The conformation of the RAD52-ssDNA complex is thought to place the
ssDNA on an exposed surface of the protein, in a configuration that may promote the DNA-DNA annealing of complementary DNA strands.
RAD54 may function in the transient separation of the duplex DNA strands via its ATP hydrolysis-mediated DNA supercoiling function
(Sigurdsson et al., 2002).
References
P Sung, L Krejci, S Van Komen, MG Sehorn, "Rad51 recombinase and recombination mediators.", J Biol Chem, 278, 2003, 42729-32.
S Sigurdsson, S Van Komen, G Petukhova, P Sung, "Homologous DNA pairing by human recombination factors Rad51 and Rad54.", J Biol
Chem, 277, 2002, 42790-4.
6.4.1.1.4.3 Strand exchange/Branch migration
Authors
Matthews, L, 2003-11-23.
Description
Branch migration or strand exchange occurs as the complimentary duplex DNA strand is progressively taken up into the nucleoprotein filament
to base pair with the invading single-strand sequence (Sung et al., 2003).
References
P Sung, L Krejci, S Van Komen, MG Sehorn, "Rad51 recombinase and recombination mediators.", J Biol Chem, 278, 2003, 42729-32.
Reaction
6.4.1.1.5 DNA repair synthesis
Authors
Matthews, L, 2003-11-23.
Description
Following branch migration, the invading 3Ã¢â‚¬â„¢ resected ends of the double-strand break act as primers for repair DNA synthesis using the
complementary strand of the invaded duplex as a template.
Reaction
6.4.1.1.6 Resolution of D-loop structures
Authors
Matthews, L, 2003-08-11.
Description
Once repair synthesis has occurred, the D-loop structure may be resolved either through Holliday junction intermediates of through
synthesis-dependent strand-annealing.
References
F Prado, A Aguilera, "Control of cross-over by single-strand DNA resection.", Trends Genet, 19, 2003, 428-31.
6.4.1.1.6.1 Resolution of D-loop structures through Holliday junction intermediates
Authors
Matthews, L, 2003-11-24.
Description
Following the synthesis of new DNA at resected 5' ends of the DSB, the heterologous DNA molecules may be ligated resulting in the formation
of Holliday junctions. The Holliday structures are then cleaved resulting in reciprocal exchange of sequence between sister chromatids.
6.4.1.1.6.1.1 Ligation of DNA and formation of Holliday structures following repair synthesis
Authors
Matthews, L, 2003-08-11.
Description
Ligation of the crossed-strand intermediate results in the formation of Holliday structures with two crossovers. In humans, the identity of this
ligase activity involved in this process is not known.
Reaction
6.4.1.1.6.1.2 Cleavage of Holliday junctions
Authors
Matthews, L, 2003-09-10.
Description
The enzymatic activities that cleave Holliday junctions have not yet been identified in human. Resolution of the D-loop through cleavage of the
two Holliday junctions in the same orientation (i.e.: sites a and b or c and c) results in non-recombinant chromatids. Cleavage in opposite
orientation (a and c or b and d) does not produce recombinants.
Reaction
6.4.1.1.6.2 Resolution of D-loop structures through synthesis-dependent strand-annealing
Authors
Matthews, L, 2003-11-24.
Description
In the synthesis-dependant strand-annealing (SDSA) model of D-loop resolution, strand exchange intermediates revert (dissociate from the
heteroduplex), synthesis of the resected regions is completed and the newly synthesized ends are reannealed to the resected 5' end.
6.4.1.1.6.2.1 Dissociation of the extended strands
Authors
Matthews, L, 2003-11-23.
Description
Following repair synthesis, the extended strands may disassociate from their compliments within the duplex DNA and reanneal with their original
complimentary strands through sequence acquired in repair synthesis .
Reaction
6.4.1.1.6.2.2 Fill-in DNA synthesis
Authors
Matthews, L, 2003-11-23.
Description
DNA synthesis occurs to fill in remaining single-stranded gaps present in the reannealed DNA duplex.
Reaction
6.4.1.2 Homologous recombination repair of replication-dependent double-strand breaks
Authors
Matthews, L, 2003-11-18.
Description
Homologous recombination repair of replication-dependent DNA double-strand breaks will be described in a later version of GKB.
6.4.2 Nonhomologous End-joining (NHEJ)
Authors
Lees-Miller, S, 2003-07-14.
Editors
Joshi-Tope, G, 0000-00-00.
Reviewers
West, SC, 0000-00-00.
Description
The NHEJ pathway is initiated in response to the formation of a DNA double-strand break (DSB) induced by a DNA-damaging agent such as
ionizing radiation. First, the Ku70/80 heterodimer binds to the ends of the DSB. The catalytic subunit of the DNA-dependent protein kinase
(DNA-PKcs) is then recruited to DNA-bound Ku to form the DNA-PK holoenzyme. The ends of the break are brought together as two molecules
of DNA-PK (one at each end of the break) are joined in a synaptic complex. Other factors, such as polynucleotide kinase (PNK), Artemis, the
MRE complex, hTdp1 or the Werner Syndrome protein (WRN) may be required for processing of the DNA ends prior to end rejoining, but exactly
when processing takes place is not known. Following the formation of the synaptic complex, the XRCC4/DNA ligase IV complex is recruited.
Prior to end rejoining, protein factors must be removed from the DNA. This may involve DNA-PK autophosphorylation (Chan and Lees-Miller,
1996; Douglas et al., 2001; 2002; Merkle et al., 2002). After removal of the repair factors, the DNA ends are ligated and the DNA is repaired.
Both Mg-ATP and the protein kinase activity of DNA-PKcs are required for NHEJ (Kurimasa et al.,1999; Kienker et al, 2000; Baumann and West,
1998), probably through phosphorylation of DNA-PKcs and/or other proteins. In addition, inositol hexaphosphate (IP6) stimulates end joining in
vitro (Hanakahi et al., 2000) and binds to Ku, but its precise role in NHEJ is unknown.
References
DW Chan, SP Lees-Miller, "The DNA-dependent protein kinase is inactivated by autophosphorylation of the catalytic subunit.", J Biol Chem, 271,
1996, 8936-41.
P Douglas, GB Moorhead, R Ye, SP Lees-Miller, "Protein phosphatases regulate DNA-dependent protein kinase activity.", J Biol Chem, 276,
2001, 18992-8.
A Kurimasa, S Kumano, NV Boubnov, MD Story, CS Tung, SR Peterson, DJ Chen, "Requirement for the kinase activity of human
DNA-dependent protein kinase catalytic subunit in DNA strand break rejoining.", Mol Cell Biol, 19, 1999, 3877-84.
S Lees-Miller, K Meek, "Nonhomologous end joining: repair of DNA double strand breaks after ionizing radiation", Biochimie, 2003, (in press).
"Sensing and repairing DNA double-strand breaks.", Carcinogenesis, 23, 2002, 687-96.
MR Lieber, Y Ma, U Pannicke, K Schwarz, "Mechanism and regulation of human non-homologous DNA end-joining.", Nat Rev Mol Cell Biol, 4,
2003, 712-20.
LA Hanakahi, M Bartlet-Jones, C Chappell, D Pappin, SC West, "Binding of inositol phosphate to DNA-PK and stimulation of double-strand
break repair.", Cell, 102, 2000, 721-9.
P Baumann, SC West, "DNA end-joining catalyzed by human cell-free extracts.", Proc Natl Acad Sci U S A, 95, 1998, 14066-70.
LJ Kienker, EK Shin, K Meek, "Both V(D)J recombination and radioresistance require DNA-PK kinase activity, though minimal levels suffice for
V(D)J recombination.", Nucleic Acids Res, 28, 2000, 2752-61.
D Merkle, P Douglas, GB Moorhead, Z Leonenko, Y Yu, D Cramb, DP Bazett-Jones, SP Lees-Miller, "The DNA-dependent protein kinase
interacts with DNA to form a protein-DNA complex that is disrupted by phosphorylation.", Biochemistry, 41, 2002, 12706-14.
6.4.2.1 Association of Ku heterodimer with ends of DNA double-strand break
Authors
Matthews, L, 2003-09-07.
Description
Ionizing radiation (IR) induces single-strand breaks i.e. cleavage of the phosphodiester backbone. When two single-strand breaks occur within
approximately 10 base pairs, a DNA double-strand break results. IR-induced DSBs are complex DNA damage lesions, containing base damage
and frequently containing 5'-OH groups and 3'-hydroxy or phosphoglycolate groups that must be removed prior to ligation in the final step of
NHEJ (Friedberg et al, 1995; Nikjoo et al, 2001; Valerie and Povirk, 2003). The Ku70/80 heterodimer (Walker et al., 2001) binds to the ends of
the double-strand break. Ku can translocate inwards from the site of the break in an ATP-independent manner (reviewed in Dynan and Yoo,
1998).
References
WS Dynan, S Yoo, "Interaction of Ku protein and DNA-dependent protein kinase catalytic subunit with nucleic acids.", Nucleic Acids Res, 26,
1998, 1551-9.
K Valerie, LF Povirk, "Regulation and mechanisms of mammalian double-strand break repair.", Oncogene, 22, 2003, 5792-812.
C Walker, C Friedberg, W Siede, "DNA Repair and Mutagenesis", ASM Press, Washington, D.C, 1995.
H Nikjoo, P O'Neill, WE Wilson, DT Goodhead, "Computational approach for determining the spectrum of DNA damage induced by ionizing
radiation.", Radiat Res, 156, 2001, 577-83.
JR Walker, RA Corpina, J Goldberg, "Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair.", Nature,
412, 2001, 607-14.
Reaction
6.4.2.2 Autophosphorylation of DNA-PKcs
Authors
Matthews, L, 2003-10-06.
Description
Autophosphorylation of DNA-PKcs is required for NHEJ in vivo (Chan et al, 2002; Ding et al, 2003).
References
DW Chan, BP Chen, S Prithivirajsingh, A Kurimasa, MD Story, J Qin, DJ Chen, "Autophosphorylation of the DNA-dependent protein kinase
catalytic subunit is required for rejoining of DNA double-strand breaks.", Genes Dev, 16, 2002, 2333-8.
Q Ding, YV Reddy, W Wang, T Woods, P Douglas, DA Ramsden, SP Lees-Miller, K Meek, "Autophosphorylation of the catalytic subunit of the
DNA-dependent protein kinase is required for efficient end processing during DNA double-strand break repair.", Mol Cell Biol, 23, 2003, 5836-48.
Reaction
6.4.2.3 Association of DNA-PKcs with Ku-bound ends of DNA double-strand breaks
Authors
Matthews, L, 2003-09-07.
Description
DNA-PKcs is recruited to the Ku-DNA end complex (Gottlieb and Jackson, 1993), causing Ku to translocate inwards (away from the break)
approximately 10 bp (Yoo and Dynan, 1999). This forms the DNA-PK complex (DNA-PKcs plus Ku70/Ku80) at each end of the DNA double
strand break.
References
S Yoo, WS Dynan, "Geometry of a complex formed by double strand break repair proteins at a single DNA end: recruitment of DNA-PKcs
induces inward translocation of Ku protein.", Nucleic Acids Res, 27, 2000, 4679-86.
TM Gottlieb, "The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen.", Cell, 72, 1993, 131-42.
Reaction
6.4.2.4 Synapsis, or interaction between two DNA-PK:DNA complexes at opposing ends of DNA DSB
Authors
Matthews, L, 2003-09-07.
Description
Two DNA-PK-DNA complexes, one on either side of the break, interact to bring the DNA ends together in synaptic complex (DeFazio et al.,
2002).
References
LG DeFazio, RM Stansel, G Chu, "Synapsis of DNA ends by DNA-dependent protein kinase.", EMBO J, 21, 2002, 3192-200.
Reaction
6.4.2.5 Processing of DNA ends prior to end rejoining
Authors
Matthews, L, 2003-09-07.
Description
Ionizing radiation induced DNA double strand breaks often contain end groups that must be processed before the DNA can be rejoined. Possible
candidate proteins for the processing steps include Artemis (a nuclease) (Ma and Lieber, 2002); WRN (a helicase with exonuclease activity) (Li
and Comai, 2001; Yannone et al., 2002; Karmakar, et al., 2002a, 2002b); PNK (a 5'-OH kinase and 3'-phosphate phosphatase) (Chappell et al.,
2002); the Mre11/Rad50/Nbs1 nuclease complex (D' Amours and Jackson, 2002); or hTdp1 (tyrosyl-DNA phosphodiesterase, which removes
3'-glycolates)(Inamdar et al., 2002; Valerie and Povirk, 2003). Precisely when processing of the DNA ends occurs during NHEJ is not known.
References
P Karmakar, J Piotrowski, RM Brosh, JA Sommers, SP Miller, WH Cheng, CM Snowden, DA Ramsden, VA Bohr, "Werner protein is a target of
DNA-dependent protein kinase in vivo and in vitro, and its catalytic activities are regulated by phosphorylation.", J Biol Chem, 277, 2002,
18291-302.
D D'Amours, "The Mre11 complex: at the crossroads of dna repair and checkpoint signalling.", Nat Rev Mol Cell Biol, 3, 2002, 317-27.
KV Inamdar, JJ Pouliot, T Zhou, SP Lees-Miller, A Rasouli-Nia, LF Povirk, "Conversion of phosphoglycolate to phosphate termini on 3'
overhangs of DNA double strand breaks by the human tyrosyl-DNA phosphodiesterase hTdp1.", J Biol Chem, 277, 2002, 27162-8.
B Li, L Comai, "Requirements for the nucleolytic processing of DNA ends by the Werner syndrome protein-Ku70/80 complex.", J Biol Chem, 276,
2001, 9896-902.
K Valerie, LF Povirk, "Regulation and mechanisms of mammalian double-strand break repair.", Oncogene, 22, 2003, 5792-812.
P Karmakar, CM Snowden, DA Ramsden, VA Bohr, "Ku heterodimer binds to both ends of the Werner protein and functional interaction occurs
at the Werner N-terminus.", Nucleic Acids Res, 30, 2002, 3583-91.
Y Ma, U Pannicke, K Schwarz, MR Lieber, "Hairpin opening and overhang processing by an Artemis/DNA-dependent protein kinase complex in
nonhomologous end joining and V(D)J recombination.", Cell, 108, 2002, 781-94.
C Chappell, LA Hanakahi, F Karimi-Busheri, M Weinfeld, SC West, "Involvement of human polynucleotide kinase in double-strand break repair
by non-homologous end joining.", EMBO J, 21, 2002, 2827-32.
SM Yannone, S Roy, DW Chan, MB Murphy, S Huang, J Campisi, DJ Chen, "Werner syndrome protein is regulated and phosphorylated by
DNA-dependent protein kinase.", J Biol Chem, 276, 2001, 38242-8.
6.4.2.5.1 Removal of 3'-phosphoglycolate (PG) moiety from DSB ends
Authors
Description
At the beginning of this reaction, 1 molecule of 'DNA-PK synaptic complex' is present. At the end of this reaction, 1 molecule of 'DNA-PK:DNA
synaptic complex with ligatable ends' is present.
This reaction takes place in the 'nucleoplasm' and is mediated by the 'endonuclease activity' of 'nucleases removing 3' phosphoglycolate'.
References
KV Inamdar, JJ Pouliot, T Zhou, SP Lees-Miller, A Rasouli-Nia, LF Povirk, "Conversion of phosphoglycolate to phosphate termini on 3'
overhangs of DNA double strand breaks by the human tyrosyl-DNA phosphodiesterase hTdp1", J Biol Chem, 277, 2002, 27162-8.
Reaction
6.4.2.5.2 Removal of 3'-phosphate moiety from DSB ends
Authors
Description
At the beginning of this reaction, 1 molecule of 'DNA-PK synaptic complex' is present. At the end of this reaction, 1 molecule of 'DNA-PK:DNA
synaptic complex with ligatable ends' is present.
This reaction takes place in the 'nucleoplasm' and is mediated by the 'endonuclease activity' of 'unknown endonuclease'.
Reaction
6.4.2.6 Association of the XRCC4:DNA ligase IV complex with the DNA-PK:DNA synaptic complex
Authors
Matthews, L, 2003-09-07.
Description
A complex consisting of XRCC4 and DNA ligase IV is recruited to the DNA-PK-DNA complex (Critchlow and Jackson, 1997; Leber et al., 1998).
References
R Leber, TW Wise, R Mizuta, K Meek, "The XRCC4 gene product is a target for and interacts with the DNA-dependent protein kinase.", J Biol
Chem, 273, 1998, 1794-801.
SE Critchlow, RP Bowater, "Mammalian DNA double-strand break repair protein XRCC4 interacts with DNA ligase IV.", Curr Biol, 7, 1997,
588-98.
Reaction
6.4.2.7 Removal of repair proteins and ligation of the processed ends of the DNA double-strand break
Authors
Matthews, L, 2003-09-07.
Description
Presumably the proteins must be released from the DNA prior to end joining but precisely when this step occurs is not known. Mg-ATP and the
protein kinase activity of DNA-PKcs are required for repair of DNA double strand breaks (Kurimasa et al, 1999; Kienker et al, 2000; Baumann
and West, 1998). In vitro, DNA-PK undergoes autophosphorylation which correlates with loss of protein kinase activity and disruption of the
DNA-PKcs-Ku-DNA complex (Chan and Lees-Miller, 1996; Douglas et al, 2001; 2002; Merkle et al, 2002). Moreover, autophosphorylation of
DNA-PKcs is required for NHEJ in vivo (Chan et al, 2002; Ding et al, 2003), suggesting that autophosphorylation may serve as a mechanism to
remove DNA-PKcs from DNA prior to end joining.
References
DW Chan, SP Lees-Miller, "The DNA-dependent protein kinase is inactivated by autophosphorylation of the catalytic subunit.", J Biol Chem, 271,
1996, 8936-41.
P Douglas, GB Moorhead, R Ye, SP Lees-Miller, "Protein phosphatases regulate DNA-dependent protein kinase activity.", J Biol Chem, 276,
2001, 18992-8.
P Douglas, GP Sapkota, N Morrice, Y Yu, AA Goodarzi, D Merkle, K Meek, DR Alessi, SP Lees-Miller, "Identification of in vitro and in vivo
phosphorylation sites in the catalytic subunit of the DNA-dependent protein kinase.", Biochem J, 368, 2002, 243-51.
A Kurimasa, S Kumano, NV Boubnov, MD Story, CS Tung, SR Peterson, DJ Chen, "Requirement for the kinase activity of human
DNA-dependent protein kinase catalytic subunit in DNA strand break rejoining.", Mol Cell Biol, 19, 1999, 3877-84.
DW Chan, BP Chen, S Prithivirajsingh, A Kurimasa, MD Story, J Qin, DJ Chen, "Autophosphorylation of the DNA-dependent protein kinase
catalytic subunit is required for rejoining of DNA double-strand breaks.", Genes Dev, 16, 2002, 2333-8.
P Baumann, SC West, "DNA end-joining catalyzed by human cell-free extracts.", Proc Natl Acad Sci U S A, 95, 1998, 14066-70.
LJ Kienker, EK Shin, K Meek, "Both V(D)J recombination and radioresistance require DNA-PK kinase activity, though minimal levels suffice for
V(D)J recombination.", Nucleic Acids Res, 28, 2000, 2752-61.
Q Ding, YV Reddy, W Wang, T Woods, P Douglas, DA Ramsden, SP Lees-Miller, K Meek, "Autophosphorylation of the catalytic subunit of the
DNA-dependent protein kinase is required for efficient end processing during DNA double-strand break repair.", Mol Cell Biol, 23, 2003, 5836-48.
D Merkle, P Douglas, GB Moorhead, Z Leonenko, Y Yu, D Cramb, DP Bazett-Jones, SP Lees-Miller, "The DNA-dependent protein kinase
interacts with DNA to form a protein-DNA complex that is disrupted by phosphorylation.", Biochemistry, 41, 2002, 12706-14.
Reaction
6.5 Nucleotide Excision Repair
Authors
Hoeijmakers, JH, 2003-07-24.
Editors
Description
NER was first described in the model organism E. coli in the early 1960s as a process whereby bulky base damage is enzymatically removed
from DNA, facilitating the recovery of DNA synthesis and cell survival. Deficient NER processes have been identified from the cells of
cancer-prone patients with different variants of xeroderma pigmentosum (XP), trichothiodystrophy (TTD), and CockayneÃ¢â‚¬â„¢s syndrome.
These XP cells exhibited an ultraviolet radiation hypersensitivity leading to a hypermutability response to UV, offering a direct connection
between deficient NER, increased mutations, and cancer. While the NER pathway in prokaryotes is unique, the pathway utilized in yeast and
higher eukaryotes is highly conserved and includes a variety of proteins that interact to form complexes.
NER is involved in the repair of bulky adducts in DNA, such as UV-induced photo lesions [of both 6-4 photoproducts (6-4 pps) and cyclobutane
pyrimidine dimer (CPDs)], intrastrand cross-links, large chemical adducts formed from exposure to aflatoxin, benzopyrene and other genotoxic
agents. Specific proteins have been identified that participate in base damage recognition, cleavage of the damaged strand on both sides of the
lesion, excision of the oligonucleotide bearing the lesion, and accessory proteins necessary for efficient function. Polymerization and ligation
restore the strand to its original state. NER consists of two related pathways called global genomic repair (GG-NER) and transcription-coupled
NER (TC-NER). The pathways differ in the way in which DNA damage is initially recognized, but the majority of the participating molecules are
shared between these two branches of NER". GG-NER is considered to be transcription-independent, removing lesions from non-transcribed
regions of genome in addition to non-transcribed strands of transcribed regions. The preferential repair of UV-induced damage in transcribed
strands of active genes is known as Transcription-coupled NER (TC-NER).
Several of the proteins involved in NER are key components of the basal transcription complex TFIIH. NER proteins have also been shown to
interact with the 19S regulatory subunit of the proteasome, suggesting a role in cellular regulation signal pathways. The establishment of mutant
mouse models for NER genes and other DNA repair-related genes have been useful in demonstrating the associations between NER defects
and cancer.
References
M Christmann, MT Tomicic, WP Roos, B Kaina, "Mechanisms of human DNA repair: an update.", Toxicology, 193, 2003, 3-34.
EC Friedberg, "How nucleotide excision repair protects against cancer.", Nat Rev Cancer, 1, 2002, 22-33.
6.5.1 Global Genomic NER (GG-NER)

Authors
Description
GG-NER is considered to be transcription-independent, removing lesions from non-transcribed regions of genome in addition to non-transcribed
strands of transcribed regions.
The three events that characterize NER are well characterized in GG-NER: damage recognition, bimodal incision of DNA via repair protein
complex, resulting excision of DNA fragment with the lesion. Post-excision polymerization and ligation restore back the native chemistry and
configuration to the damaged DNA.
6.5.1.1 DNA Damage Recognition in GG-NER
Authors
Joshi-Tope, G, 2003-07-14.
Description
Several subunits of the NER multiprotein repair machinery are required for the recognition of damage in DNA. Mainly, XPC, HR23B, XPA and
RPA are implicated in this process. XPA and RPA are thought to be binding to the damage site after the binding of XPC-HR23B complex.
6.5.1.1.1 XPC binds to HR23B forming a heterodimeric complex
Authors
Description
XPC is mutated in individuals with xeroderma pigmentosum from genetic Complementation Group C (XP-C). It forms a tight heterodimeric
complex with human Rad 23B homolog, HR23B and is thought to bind to the damaged site with lesion first triggering subsequent reactions
Reaction
6.5.1.1.2 XPC:HR23B complex binds to damaged DNA site with lesion
Authors
Description
Disruption of normal Watson-Crick base pairing and altered chemistry in the damaged strand involving bases may act as signals of damage that
are recognized by XPC:HR23B complex.
Reaction
6.5.1.2 Formation of incision complex in GG-NER
Authors
Joshi-Tope, G, 2003-07-14.
Description
Binding of XPC complex to the damaged site on the DNA substrate is followed by XPA and RPA recruitment. XPA is a metalloprotein that binds
to different types of DNA damage. Binding of RPA, or Replicative protein A, a heterotrimer, initiates the formation of incision complex. This
includes a serial assembly of the rest of the NER machinery consisting of TFIIH, XPG etc. Action of helicases in TFIIH complex opens up a
bubble structure in the DNA template, exposing a fragment of DNA with lesion for endonuclease activity.
6.5.1.2.1 Recruitment of repair factors to form preincision complex
Authors
Joshi-Tope, G, 2003-07-14.
Description
Transcription factor II H (TFIIH) and XPG are added to the damaged site on the DNA to form a pre-incision complex along with lesioned DNA
template.
References
SJ AraÃºjo, RD Wood, "Protein complexes in nucleotide excision repair.", Mutat Res, 435, 1999, 23-33.
M Volker, MJ MonÃ©, P Karmakar, A van Hoffen, W Schul, W Vermeulen, JH Hoeijmakers, R van Driel, AA van Zeeland, LH Mullenders,
"Sequential assembly of the nucleotide excision repair factors in vivo.", Mol Cell, 8, 2001, 213-24.
Reaction
6.5.1.2.2 Formation of open bubble structure in DNA by helicases

Authors
Joshi-Tope, G, 2003-07-14.
Description
Two DNA helicases XPG and XPD, which are part of TFIIH, unwind DNA duplex around this lesion to form an open bubble structure that
exposes the damaged site.
Reaction
6.5.1.2.3 Binding of ERCC1-XPF to preincision complex
Authors
Description
ERCC1-XPF complex with 5Ã¢â‚¬â„¢ endonuclease activity binds to this pre-incision complex around the bubble structure to form an active
incision complex.
References
RD Wood, "DNA damage recognition during nucleotide excision repair in mammalian cells.", Biochimie, 81, 1999, 39-44.
Reaction
6.5.1.3 Dual incision reaction in GG-NER
Authors
Joshi-Tope, G, 2003-07-14.
Description
Dual incision at defined positions flanking the DNA damage is carried out by XPG (3' -incision) and ERCC1-XPF (5'-incision) complex. The
resulting excised fragment is ~27-30 bp long and contains the lesion.
References
6.5.1.3.1 3'- incision of DNA by XPG in GG-NER
Authors
Joshi-Tope, G, 2003-07-14.
Description
The cleavage of the damaged strand of DNA 3' to the site of damage occurs at the junction of single-stranded DNA and double-stranded DNA
that is formed when the DNA duplex is unwound. The incision is carried out by XPG.
References
Reaction
6.5.1.3.2 5'-incision of DNA by ERCC1-XPF in GG-NER
Authors
Joshi-Tope, G, 2003-07-14.
Description
The cleavage of the damaged strand of DNA 5' to the site of damage occurs at the junction of single-stranded DNA and double-stranded DNA
that is formed when the DNA duplex is unwound. The incision is carried out by ERCC1-XPF complex.
References
Reaction
6.5.1.4 Gap-filling DNA repair synthesis and ligation in GG-NER
Authors
Description
The resultant gap is filled by polymerase activities of Pol delta and Pol epsilon. Accessory replication protein complexes of RPA, PCNA and RFC
play a role in this synthesis. DNA Ligase 1 seals the gap restoring the covalent integrity of the repaired strand.
References
RD Wood, MK Shivji, "Which DNA polymerases are used for DNA-repair in eukaryotes?", Carcinogenesis, 18, 1997, 605-10.
6.5.1.4.1 Repair synthesis of patch ~27-30 bases long by DNA polymerase
Authors
Description
Repair synthesis is carried out by the DNA dependent DNA polymerases, delta and epsilon.
6.5.1.4.1.1 Repair synthesis of ~27-30 bases long patch by DNA Pol Epsilon
Description
At the beginning of this reaction, 1 molecule of 'dNTP', 1 molecule of 'incised DNA without lesion', 1 molecule of 'RPA heterotrimer', 1 molecule
of 'PCNA homotrimer', 1 molecule of 'RFC Heteropentamer', and 1 molecule of 'DNA polymerase epsilon' are present. At the end of this reaction,
1 molecule of 'incised DNA without lesion', 1 molecule of 'RPA heterotrimer', 1 molecule of 'PCNA homotrimer', 1 molecule of 'RFC
Heteropentamer', 1 molecule of 'newly synthesized DNA fragment ', and 1 molecule of 'DNA polymerase epsilon' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'DNA-directed DNA polymerase activity' of 'DNA polymerase epsilon'.
Reaction
6.5.1.4.1.2 Repair synthesis of patch ~27-30 bases long by DNA Pol Delta
Description
At the beginning of this reaction, 1 molecule of 'dNTP', 1 molecule of 'incised DNA without lesion', 1 molecule of 'RPA heterotrimer', 1 molecule
of 'PCNA homotrimer', 1 molecule of 'RFC Heteropentamer', and 1 molecule of 'DNA Polymerase delta tetramer' are present. At the end of this
reaction, 1 molecule of 'incised DNA without lesion', 1 molecule of 'RPA heterotrimer', 1 molecule of 'PCNA homotrimer', 1 molecule of 'RFC
Heteropentamer', 1 molecule of 'newly synthesized DNA fragment ', and 1 molecule of 'DNA Polymerase delta tetramer' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'delta DNA polymerase activity' of 'DNA Polymerase delta tetramer'.
Reaction
6.5.1.4.2 Ligation of newly synthesized repair patch to incised DNA in GG-NER
Authors
Description
DNA Ligase 1 ligates the newly synthesized fragment to the gap in the template DNA.
Reaction
6.5.2 Transcription-coupled NER (TC-NER)
Authors
Joshi-Tope, G, 2003-07-14.
Description
The preferential repair of UV-induced damage in transcribed strands of active genes is known as Transcription-coupled NER (TC-NER).
Impairment of the ability for TC-NER results in the onset of a severe hereditary disorder called CockayneÃ¢â‚¬â„¢s syndrome, an autosomal
recessive disease characterized by hypersensitivity to UV light . Many types of helix distorting lesions can block the movement of elongating
RNA Pol II leading to its stalling and subsequent triggering of repair mechanisms. The repair of DNA damage in active genes occurs much faster
compared to the repair in non-transcribed genomic regions (via GG-NER). Also, the non-transcribed strand of the same gene is repaired at a
similar rate as that of non-transcribed genomic regions.
The transcriptional responses to DNA damage in terms of stalling of Pol II, its displacement from the lesion site etc. are not well elucidated. The
following annotation in GK provides an overall picture of TC-NER and will be updated as and when new insights are obtained about these crucial
steps.
References
JQ Svejstrup, "Mechanisms of transcription-coupled DNA repair.", Nat Rev Mol Cell Biol, 3, 2002, 21-9.
I Mellon, VA Bohr, CA Smith, PC Hanawalt, "Preferential DNA repair of an active gene in human cells.", Proc Natl Acad Sci U S A, 83, 1987,
8878-82.
6.5.2.1 Formation of transcription-coupled NER (TC-NER) repair complex
Authors
Description
The Ã¢â‚¬Å"road blockÃ¢â‚¬Â• induced by the DNA damage to the transcription machinery triggers assembly of a transcription couple repair
complex, whose composition and function are yet to fully understood. Damage recognition is achieved by the arrest of Pol II active complex.
Subsequently various factors like CSA, CSB, TFIIH, XPG, XPF-ERCC1 complexes are recruited. The importance of active CSA and CSB are
known from the mutation profile of the patients bearing the corresponding disease phenotypes. CSB can act as a Ã¢â‚¬Ëœrepair-transcription
uncoupling factorÃ¢â‚¬â„¢ that may use its DNA translocase activity to remove the Pol II activity from the lesion site. TFIIS may cleave up to 35
nucleotides from the 3Ã¢â‚¬â„¢ end of the nascent RNA product leading to the enhanced access to damage site on the corresponding template
DNA.
6.5.2.1.1 Formation of active Pol II complex with lesioned DNA template

Authors
Description
An active Pol II complex consisting mainly of the Pol II holoenzyme transcribes the damaged DNA template.
Reaction
6.5.2.1.2 RNA Pol II is blocked by the lesion leading to reduced transcription
Authors
Description
At the site of damage, the Pol II complex is arrested resulting in reduced levels of transcription. Several models have been proposed to explain
the mechanism of this transcriptional downregulation. These include a. hyperphosphorylation of Pol II resulting in aborted entry to new
pre-initiation complexes b. sequestration of TATA-binding proteins (TBP) c. enhanced use of TFIIH complexes for repair purposes precluding
their use in transcription.
Reaction
6.5.2.1.3 Assembly of repair proteins at the site of Pol II blockage
Authors
Description
A proper assembly of repair complex may require displacement of Pol II from the damage site exposing a significant length of the corresponding
template DNA with the lesions. Speculations on the mode of this displacement of Pol II are available from experimental evidences: a. CSB
mediated dissociation of Pol II b. degradation of Pol II c. CSB mediated remodeling of damaged DNA-RNA PII interface etc.
The TC-repair complex now consists of damaged DNA template: nascent mRNA hybrid. The damage site needs to be exposed to subsequent
endonuclease activities.
Reaction
6.5.2.2 Dual incision reaction in TC-NER
Authors
Description
Dual incisions are carried out by XPG, and ERCC1-XPF complex as seen in GG-NER.
6.5.2.2.1 Displacement of stalled Pol II from the lesion site
Description
At the beginning of this reaction, 1 molecule of 'Transcription-coupled (TC) repair complex', and 1 molecule of 'Stalled Pol II complex with
damaged DNA hybrid' are present. At the end of this reaction, 1 molecule of 'Transcription-coupled (TC) repair complex', 1 molecule of 'Stalled
Pol II in TC-NER', and 1 molecule of 'damaged DNA substrate:nascent mRNA hybrid' are present.

Reaction
6.5.2.2.2 3' incision of the lesioned strand of DNA in TC-NER
Description
At the beginning of this reaction, 1 molecule of 'Transcription-coupled (TC) repair complex', and 1 molecule of 'damaged DNA substrate:nascent
mRNA hybrid' are present. At the end of this reaction, 1 molecule of 'Transcription-coupled (TC) repair complex', and 1 molecule of 'damaged
DNA substrate:nascent mRNA hybrid with 3' incision' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'endodeoxyribonuclease activity' of 'Transcription-coupled (TC) repair complex'.
References
AL Gnatt, P Cramer, J Fu, DA Bushnell, RD Kornberg, "Structural basis of transcription: an RNA polymerase II elongation complex at 3.3 A
resolution.", Science, 292, 2001, 1876-82.
Reaction
6.5.2.2.3 5' incision leading to excision of DNA fragment with lesion in TC-NER
Description
At the beginning of this reaction, 1 molecule of 'Transcription-coupled (TC) repair complex', and 1 molecule of 'damaged DNA substrate:nascent
mRNA hybrid with 3' incision' are present. At the end of this reaction, 1 molecule of 'Transcription-coupled (TC) repair complex', 1 molecule of
'excised DNA fragment with lesion', and 1 molecule of 'damaged DNA substrate:nascent mRNA hybrid with dual incisions' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'endodeoxyribonuclease activity' of 'Transcription-coupled (TC) repair complex'.
References
Reaction
6.5.2.3 Gap-filling DNA repair synthesis and ligation in TC-NER
Authors
Description
Polymerization is carried out by DNA polymerases, delta and epsilon.
6.5.2.3.1 Repair synthesis for gap-filling by DNA polymerase in TC-NER
6.5.2.3.1.1 Repair synthesis for gap-filling by DNA pol delta in TC-NER
Description
At the beginning of this reaction, 1 molecule of 'dNTP', 1 molecule of 'RPA heterotrimer', 1 molecule of 'PCNA homotrimer', 1 molecule of 'RFC
Heteropentamer', 1 molecule of 'DNA Polymerase delta tetramer', and 1 molecule of 'damaged DNA substrate:nascent mRNA hybrid with dual
incisions' are present. At the end of this reaction, 1 molecule of 'RPA heterotrimer', 1 molecule of 'PCNA homotrimer', 1 molecule of 'RFC
Heteropentamer', 1 molecule of 'newly synthesized DNA fragment ', 1 molecule of 'DNA Polymerase delta tetramer', and 1 molecule of 'damaged
DNA substrate:nascent mRNA hybrid with dual incisions' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'delta DNA polymerase activity' of 'DNA Polymerase delta tetramer'.
Reaction
6.5.2.3.1.2 Repair synthesis for gap-filling by DNA pol epsilon in TC-NER
Description
At the beginning of this reaction, 1 molecule of 'dNTP', 1 molecule of 'RPA heterotrimer', 1 molecule of 'PCNA homotrimer', 1 molecule of 'RFC
Heteropentamer', 1 molecule of 'DNA polymerase epsilon', and 1 molecule of 'damaged DNA substrate:nascent mRNA hybrid with dual incisions'
are present. At the end of this reaction, 1 molecule of 'RPA heterotrimer', 1 molecule of 'PCNA homotrimer', 1 molecule of 'RFC
Heteropentamer', 1 molecule of 'newly synthesized DNA fragment ', 1 molecule of 'DNA polymerase epsilon', and 1 molecule of 'damaged DNA
substrate:nascent mRNA hybrid with dual incisions' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'DNA-directed DNA polymerase activity' of 'DNA polymerase epsilon'.
Reaction
6.5.2.3.2 Ligation of newly synthesized repair patch to incised DNA in TC-NER

Description
At the beginning of this reaction, 1 molecule of 'newly synthesized DNA fragment ', 1 molecule of 'DNA ligase I ', and 1 molecule of 'damaged
DNA substrate:nascent mRNA hybrid with dual incisions' are present. At the end of this reaction, 1 molecule of 'repaired DNA template:nascent
mRNA hybrid', and 1 molecule of 'DNA ligase I ' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'DNA ligase activity' of 'DNA ligase I '.
Reaction
6.5.2.4 Resumption of transcription after TC-NER
Description
At the beginning of this reaction, 1 molecule of 'Stalled Pol II in TC-NER', and 1 molecule of 'repaired DNA template:nascent mRNA hybrid' are
present. At the end of this reaction, 1 molecule of 'Active Pol II complex with repaired DNA template:mRNA hybrid' is present.
Reaction
7 DNA Replication
Authors
Bambara, RA, Catlett, M, Davey, MJ, Forsburg, S, O'Donnell, M, Tom, S, Tye, BK, 2003-01-06.
Editors
D'Eustachio, P, Joshi-Tope, G, Nickerson, E, 0000-00-00.
Reviewers
Mendez, J, Aladjem, M, 0000-00-00.
Description
Studies in the past decade have suggested that the basic mechanism of DNA replication initiation is conserved in all kingdoms of life. Initiation in
unicellular eukaryotes, in particular Saccharomyces cerevisiae (budding yeast), is well understood, and has served as a model for studies of
DNA replication initiation in multicellular eukaryotes, including humans. In general terms, the first step of initiation is the binding of the replication
initiator to the origin of replication. The replicative helicase is then assembled onto the origin, usually by a helicase assembly factor. Either
shortly before or shortly after helicase assembly, some local unwinding of the origin of replication occurs in a region rich in adenine and thymine
bases (often termed a DNA unwinding element, DUE). The unwound region provides the substrate for primer synthesis and initiation of DNA
replication. The best-defined eukaryotic origins are those of S. cerevisiae, which have well-conserved sequence elements for initiator binding,
DNA unwinding and binding of accessory proteins. In multicellular eukaryotes, unlike S. cerevisiae, these loci appear not to be defined by the
presence of a DNA sequence motif. Indeed, choice of replication origins in a multicellular eukaryote may vary with developmental stage and
tissue type. In cell-free models of metazoan DNA replication, such as the one provided by Xenopus egg extracts, there are only limited DNA
sequence specificity requirements for replication initiation.
References
TJ Kelly, GW Brown, "Regulation of chromosome replication.", Annu Rev Biochem, 69, 2000, 829-80.
Y Marahrens, B Stillman, "A yeast chromosomal origin of DNA replication defined by multiple functional elements.", Science, 255, 1992, 817-23.
HM Mahbubani, T Paull, JK Elder, JJ Blow, "DNA replication initiates at multiple sites on plasmid DNA in Xenopus egg extracts.", Nucleic Acids
Res, 20, 1992, 1457-62.
DM Cimbora, M Groudine, "The control of mammalian DNA replication: a brief history of space and timing.", Cell, 104, 2001, 643-6.
O Hyrien, M MÃ©chali, "Chromosomal replication initiates and terminates at random sequences but at regular intervals in the ribosomal DNA of
Xenopus early embryos.", EMBO J, 12, 1993, 4511-20.
7.1 DNA Replication Pre-Initiation
Authors
Editors
Joshi-Tope, G, 0000-00-00.
Description
Although, DNA replication occurs in the S phase of the cell cycle, the formation of the DNA replication pre-initiation complex begins during late
G1/M transition.
7.1.1 Assembly of the pre-replicative complex
Authors
Description
DNA replication pre-initiation in eukaryotic cells begins with the formation of the pre-replicative complex (pre-RC) during the late M phase and
continues in the G1 phase of the mitotic cell cycle, a process also called DNA replication origin licensing. The association of initiation proteins
(ORC, Cdc6, Cdt1, Mcm2-7) with the origin of replication in both S. cerevisiae and humans has been demonstrated by chromatin
immunoprecipitation experiments. In S. cerevisiae, pre-replicative complexes are assembled from late M to G1. In mammalian cells as well,
pre-replicative complexes are assembled from late M to G1, as shown by biochemical fractionation and immunostaining. There are significant
sequence similarities among some of the proteins in the pre-replicative complex. The ORC subunits Orc1, Orc4 and Orc5 are homologous to
one another and to Cdc6. The six subunits of the Mcm2-7 complex are homologous to one another. In addition, Orc1, Orc4, Orc5, Cdc6, and the
Mcm2-7 subunits, are members of the AAA+ superfamily of ATPases. Since the initial identification of these pre-RC components other factors
that participate in this complex have been found, including Cdt1 in human, Xenopus, S. pombe, and S. cerevisiae cells.
References
Dev, 11, 1998, 3375-86.
AF Neuwald, L Aravind, JL Spouge, EV Koonin, "AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and
disassembly of protein complexes.", Genome Res, 9, 1999, 27-43.
DS Dimitrova, TA Prokhorova, JJ Blow, IT Todorov, DM Gilbert, "Mammalian nuclei become licensed for DNA replication during late telophase.",
J Cell Sci, 115, 2002, 51-9.
T Tanaka, D Knapp, K Nasmyth, "Loading of an Mcm protein onto DNA replication origins is regulated by Cdc6p and CDKs.", Cell, 90, 1997,
649-60.
7.1.1.1 Assembly of the ORC complex at the origin of replication
Authors
Davey, MJ, O'Donnell, M, 2003-06-05.
Description
In Saccharomyces cerevisiae, the entire ORC complex is constitutively bound to the origin of replication. However, in mammalian cells, Orc1,
and possibly other Orc subunits, dissociate from origins of replication, while Orc2 remains stably associated with chromatin across the cell cycle.
The first step in formation of the pre-RC is assembly of the hexameric Origin Recognition Complex (ORC) at the origin of replication. Recent
work on human (Hs) ORC has suggested that the heterohexamer assembles in an ordered manner. HsOrc2 appears to be constitutively bound
to origins of replication. HsOrc2 and HsOrc3 form a complex that interacts with HsOrc4 and HsOrc5. Recruitment of HsOrc5 into the complex is
important for the association of HsOrc1. HsOrc6 is also capable of interacting with the core HsOrc2:3:4:5 complex, but not smaller complexes.
Interestingly, HsOrc1 and a subunit of the Mcm2-7 complex (HsMcm2) interact with a histone acetyltransferase, HsHBO1.
References
Physiol, 38, 1975, 58-63.
DG Lee, SP Bell, "Architecture of the yeast origin recognition complex bound to origins of DNA replication.", Mol Cell Biol, 17, 1997, 7159-68.
JF Diffley, JH Cocker, SJ Dowell, A Rowley, "Two steps in the assembly of complexes at yeast replication origins in vivo.", Cell, 78, 1994,
303-16.
TW Burke, JG Cook, M Asano, JR Nevins, "Replication factors MCM2 and ORC1 interact with the histone acetyltransferase HBO1.", J Biol
Chem, 276, 2001, 15397-408.
M Iizuka, B Stillman, "Histone acetyltransferase HBO1 interacts with the ORC1 subunit of the human initiator protein.", J Biol Chem, 274, 1999,
23027-34.
SP Bell, B Stillman, "ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex.", Nature, 357, 1992, 128-34.
7.1.1.1.1 Orc3 associates with Orc2 constitutively bound at origins of replication
Description
At the beginning of this reaction, 1 molecule of 'Orc2:origin', and 1 molecule of 'Orc3' are present. At the end of this reaction, 1 molecule of
'Orc3:Orc2:origin' is present.
References
M Ritzi, M Baack, C Musahl, P Romanowski, RA Laskey, R Knippers, "Human minichromosome maintenance proteins and human origin
recognition complex 2 protein on chromatin.", J Biol Chem, 273, 1998, 24543-9.
Reaction
7.1.1.1.2 Orc5 associates with Orc3:Orc2:origin complexes

Description
At the beginning of this reaction, 1 molecule of 'Orc3:Orc2:origin', and 1 molecule of 'Orc5' are present. At the end of this reaction, 1 molecule of
'Orc5:Orc3:Orc2:origin' is present.
References
Reaction
7.1.1.1.3 Orc4 associates with Orc5:Orc3:Orc2:origin complexes
Description
At the beginning of this reaction, 1 molecule of 'Orc4', and 1 molecule of 'Orc5:Orc3:Orc2:origin' are present. At the end of this reaction, 1
molecule of 'Orc4:Orc5:Orc3:Orc2:origin' is present.
References
Reaction
7.1.1.1.4 Orc1 associates with Orc4:Orc5:Orc3:Orc2:origin complexes
Description
References
Reaction
7.1.1.1.5 Orc6 associates with Orc1:Orc4:Orc5:Orc3:Orc2:origin complexes, forming ORC:origin complexes
Description
At the beginning of this reaction, 1 molecule of 'Orc1:Orc4:Orc5:Orc3:Orc2:origin', and 1 molecule of 'Orc6' are present. At the end of this
reaction, 1 molecule of 'ORC complex bound to origin' is present.
References
Reaction
7.1.1.2 Association of MCM8 with ORC:origin complex
Authors
Tye, BK, 2006-03-17.
Editors
Description
The MCM2-7 complex, an essential component of the pre-replication complex, recruits CDC6 and CDT1 proteins to the origin. MCM8, another
member of the MCM family has been found to bind to chromatin during early G1 phase. MCM8 interacts specifically with the ORC2 protein.
References
2005, 1560-8.
Reaction
7.1.1.3 CDC6 association with the ORC:origin complex

Authors
Description
Cdc6 is a regulator of DNA replication initiation in both yeasts and human cells, but its mechanism of action differs between the two systems.
Genetic studies in budding yeast (S. cerevisiae) and fission yeast (S. pombe) indicate that the normal function of Cdc6 protein is required to
restrict DNA replication to once per cell cycle. Specifically, Cdc6 may function as an ATPase switch linked to Mcm2-7 association with the
Cdt1:Cdc6:ORC:origin complex. In S. cerevisiae, Cdc6 protein is expressed late in the M phase of the cell cycle and, in cells with a prolonged
G1 phase, late in G1. This protein has a short half-life, and is destroyed by ubiquitin-mediated proteolysis, mediated by the SCF complex.
Human Cdc6 protein levels are reduced early in G1 but otherwise are constant throughout the cell cycle. Some reports have suggested that after
cells enter S phase, Cdc6 is phosphorylated, excluded from the nucleus and subject to ubiquitination and degradation. Replenishing Cdc6
protein levels during G1 appears to be regulated by E2F transcription factors.
References
P Saha, J Chen, KC Thome, SJ Lawlis, ZH Hou, M Hendricks, JD Parvin, A Dutta, "Human CDC6/Cdc18 associates with Orc1 and cyclin-cdk
and is selectively eliminated from the nucleus at the onset of S phase.", Mol Cell Biol, 18, 1998, 2758-67.
LS Drury, G Perkins, JF Diffley, "The Cdc4/34/53 pathway targets Cdc6p for proteolysis in budding yeast.", EMBO J, 16, 1997, 5966-76.
BO Petersen, J Lukas, CS SÃ¸rensen, K Helin, "Phosphorylation of mammalian CDC6 by cyclin A/CDK2 regulates its subcellular localization.",
EMBO J, 18, 1999, 396-410.
LS Drury, G Perkins, JF Diffley, "The cyclin-dependent kinase Cdc28p regulates distinct modes of Cdc6p proteolysis during the budding yeast
cell cycle.", Curr Biol, 10, 2000, 231-40.
S Piatti, C Lengauer, K Nasmyth, "Cdc6 is an unstable protein whose de novo synthesis in G1 is important for the onset of S phase and for
preventing a 'reductional' anaphase in the budding yeast Saccharomyces cerevisiae.", EMBO J, 14, 1995, 3788-99.
6193-8.
G Perkins, LS Drury, JF Diffley, "Separate SCF(CDC4) recognition elements target Cdc6 for proteolysis in S phase and mitosis.", EMBO J, 20,
2001, 4836-45.
Description
References
Reaction
7.1.1.3.2 CDC6 association with ORC:origin complexes mediated by MCM8
Authors

Description
At the beginning of this reaction, 1 molecule of 'ORC:origin', and 1 molecule of 'CDC6' are present. At the end of this reaction, 1 molecule of
'CDC6:ORC:origin complex' is present.
References
2005, 1560-8.
Reaction
7.1.1.4 CDT1 association with the CDC6:ORC:origin complex
Description
Initiation protein Cdt1 was first identified in X. laevis, where it has been shown to be the second component of licensing factor (RLF-B) and in S.
pombe. Cdt1 homologs have been identified in D. melanogaster, humans, and S. cerevisiae. Genetic studies in S. pombe have shown that
binding of Cdc6 to chromatin requires the prior binding of Cdc18, the S. pombe homolog of Cdc6. In humans, the function of CDT1 is regulated
during the cell cycle by its tight association with an inhibitory factor, geminin.
References
Science, 290, 2000, 2309-12.
D Maiorano, J Moreau, M MÃ©chali, "XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis.", Nature, 404, 2000,
622-5.
A Dutta, SP Bell, "Initiation of DNA replication in eukaryotic cells.", Annu Rev Cell Dev Biol, 13, 1998, 293-332.
S Tanaka, JF Diffley, "Interdependent nuclear accumulation of budding yeast Cdt1 and Mcm2-7 during G1 phase.", Nat Cell Biol, 4, 2002,
198-207.
H Nishitani, Z Lygerou, T Nishimoto, P Nurse, "The Cdt1 protein is required to license DNA for replication in fission yeast.", Nature, 404, 2000,
625-8.
S Tada, A Li, D Maiorano, M MÃ©chali, JJ Blow, "Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by
geminin.", Nat Cell Biol, 3, 2001, 107-13.
AJ Whittaker, I Royzman, TL Orr-Weaver, "Drosophila double parked: a conserved, essential replication protein that colocalizes with the origin
recognition complex and links DNA replication with mitosis and the down-regulation of S phase transcripts.", Genes Dev, 14, 2000, 1765-76.
7.1.1.4.1 The geminin component of geminin:Cdt1 complexes is ubiquitinated, releasing Cdt1
Description

References
Science, 290, 2000, 2309-12.
Reaction
7.1.1.4.2 Ubiquitinated geminin is degraded by the proteasome
Description
At the beginning of this reaction, 1 molecule of 'geminin:ubiquitin complex' is present. At the end of this reaction, 1 molecule of 'ubiquitin' is
present.
References
Science, 290, 2000, 2309-12.
Reaction
7.1.1.4.3 Free CDT1 associates with CDC6:ORC:origin complexes
Description
At the beginning of this reaction, 1 molecule of 'CDT1', and 1 molecule of 'CDC6:ORC:origin complex' are present. At the end of this reaction, 1
molecule of 'CDT1:CDC6:ORC:origin complex' is present.
References
Science, 290, 2000, 2309-12.
Reaction
7.1.1.5 Mcm2-7 associates with the Cdt1:CDC6:ORC:origin complex, forming the pre-replicative complex (preRC)
Description
Genetic studies in S. cerevisiae indicate that wild-type Cdc6 function is required for correctly timed loading of Mcm2-7 onto ORC. Biochemical
studies indicate that the human and Xenopus Cdc6 proteins likewise are required for Mcm2-7 loading, and that they are ATPase switches.
Specifically, Cdc6 may function as a clamp loader, assembling Mcm2-7 onto DNA in an ATP-dependent reaction. All known Cdc6 proteins have
the Walker A and Walker B sequence motifs characteristic of the AAA+ superfamily of ATPases. As expected for an AAA+ protein, human Cdc6
binds and slowly hydrolyzes ATP in vitro. ATP hydrolysis was disrupted by mutations of the Walker B motif, while both binding and hydrolysis
were disrupted by Walker A mutations. Microinjection of either mutant protein into HeLa cells blocked their progression through S phase. Both
wild-type and mutant proteins can dimerize in vitro, and studies with Xenopus egg extracts suggest that Cdc6 functions in vivo as a dimer or
larger multimer. In Xenopus extracts depleted of Cdc6 and reconstituted with either mutant protein, recruitment of Mcm2-7 to chromatin failed.
References
NS Frolova, N Schek, N Tikhmyanova, TR Coleman, "Xenopus Cdc6 performs separate functions in initiating DNA replication.", Mol Biol Cell,
13, 2002, 1298-312.
Dev, 11, 1998, 3375-86.
U Herbig, CA Marlar, "The Cdc6 nucleotide-binding site regulates its activity in DNA replication in human cells.", Mol Biol Cell, 10, 1999,
2631-45.
MJ Davey, M O'Donnell, "Mechanisms of DNA replication.", Curr Opin Chem Biol, 4, 2000, 581-6.
Reaction
7.1.2 Activation of the pre-replicative complex
Description
In S. cerevisiae, two ORC subunits, Orc1 and Orc5, both bind ATP, and Orc1 in addition has ATPase activity. Both ATP binding and ATP
hydrolysis appear to be essential functions in vivo. ATP binding by Orc1 is unaffected by the association of ORC with origin DNA (ARS)
sequences, but ATP hydrolysis is ARS-dependent, being suppressed by associated double-stranded DNA and stimulated by associated
single-stranded DNA. These data are consistent with the hypothesis that ORC functions as an ATPase switch, hydrolyzing bound ATP and
changing state as DNA unwinds at the origin immediately before replication. It is attractive to speculate that ORC likewise functions as a switch
as human pre-replicative complexes are activated, but human Orc proteins are not well enough characterized to allow the model to be critically
tested. mRNAs encoding human orthologs of all six Orc proteins have been cloned, and ATP-binding amino acid sequence motifs have been
identified in Orc1, Orc4, and Orc5. Interactions among proteins expressed from the cloned genes have been characterized, but the ATP-binding
and hydrolyzing properties of these proteins and complexes of them have not been determined.
References
RD Klemm, SP Bell, "ATP bound to the origin recognition complex is important for preRC formation.", Proc Natl Acad Sci U S A, 98, 2001,
8361-7.
M Fujita, Y Ishimi, H Nakamura, T Kiyono, T Tsurumi, "Nuclear organization of DNA replication initiation proteins in mammalian cells.", J Biol
Chem, 277, 2002, 10354-61.
DG Lee, AM Makhov, RD Klemm, SP Bell, "Regulation of origin recognition complex conformation and ATPase activity: differential effects of
single-stranded and double-stranded DNA binding.", EMBO J, 19, 2000, 4774-82.
SG Prasanth, KV Prasanth, B Stillman, "Orc6 involved in DNA replication, chromosome segregation, and cytokinesis.", Science, 297, 2002,
1026-31.
RD Klemm, RJ Austin, SP Bell, "Coordinate binding of ATP and origin DNA regulates the ATPase activity of the origin recognition complex.",
Cell, 88, 1997, 493-502.
KA Gavin, M Hidaka, B Stillman, "Conserved initiator proteins in eukaryotes.", Science, 270, 1996, 1667-71.
DG Quintana, Zh Hou, KC Thome, M Hendricks, P Saha, A Dutta, "Identification of HsORC4, a member of the human origin of replication
recognition complex.", J Biol Chem, 272, 1997, 28247-51.
S Pinto, DG Quintana, P Smith, RM Mihalek, ZH Hou, S Boynton, CJ Jones, M Hendricks, K Velinzon, JA Wohlschlegel, RJ Austin, WS Lane, T
Tully, A Dutta, "latheo encodes a subunit of the origin recognition complex and disrupts neuronal proliferation and adult olfactory memory when
mutant.", Neuron, 23, 1999, 45-54.
7.1.2.1 Mcm10 associates with the pre-replicative complex, stabilizing Mcm2-7
Description
MCM10 is required for human DNA replication. In S. cerevisiae, Mcm10, like Mcm2-7, is required for minichromosome maintenance, but Mcm10
has no sequence homology with these other proteins (Merchant et al., 1997). Genetic studies have demonstrated that Mcm10 is required for
DNA replication in S. pombe (Aves et al., 1998) and S. cerevisiae cells (Homesley et al., 2000) and immunodepletion of XlMcm10 interferes with
DNA replication in Xenopus egg extracts (Wohlschlegel et al., 2002). Human Mcm10 interacts with chromatin in G1 phase and then dissociates
during G2 phase. In S. cerevisiae, Mcm10 has been shown to localize to origins during G1 (Ricke and Bielinsky, 2004), and it may stabilize the
association of Mcm2-7 with the pre-replicative complex (Sawyer et al., 2004). This timing of association is consistent with studies that
demonstrate that, in Xenopus egg extracts, Mcm10 is required for association of Cdc45, but not Mcm2-7 with chromatin. Biochemical evidence
that Mcm10 plays a direct role in the activation of the pre-replicative complex includes the requirement for SpMcm10 in the phosphorylation of
the Mcm2-7 complex by DDK (Lee et al., 2004) and the fact that SpMcm10 binds and stimulates DNA polymerase alpha activity (Fien et al.,
2004).
References
JK Lee, YS Seo, J Hurwitz, "The Cdc23 (Mcm10) protein is required for the phosphorylation of minichromosome maintenance complex by the
Dfp1-Hsk1 kinase", Proc Natl Acad Sci U S A, 100, 2003, 2334-9.
JA Wohlschlegel, SK Dhar, TA Prokhorova, A Dutta, JC Walter, "Xenopus Mcm10 binds to origins of DNA replication after Mcm2-7 and
stimulates origin binding of Cdc45.", Mol Cell, 9, 2002, 233-40.
L Homesley, M Lei, Y Kawasaki, S Sawyer, T Christensen, BK Tye, "Mcm10 and the MCM2-7 complex interact to initiate DNA synthesis and to
release replication factors from origins.", Genes Dev, 14, 2000, 913-26.
SJ Aves, N Tongue, AJ Foster, EA Hart, "The essential schizosaccharomyces pombe cdc23 DNA replication gene shares structural and
functional homology with the Saccharomyces cerevisiae DNA43 (MCM10) gene", Curr Genet, 34, 1998, 164-71.
SL Sawyer, IH Cheng, W Chai, BK Tye, "Mcm10 and Cdc45 cooperate in origin activation in Saccharomyces cerevisiae", J Mol Biol, 340, 2004,
195-202.
RM Ricke, AK Bielinsky, "Mcm10 regulates the stability and chromatin association of DNA polymerase-alpha", Mol Cell, 16, 2004, 173-85.
AM Merchant, Y Kawasaki, Y Chen, M Lei, BK Tye, "A lesion in the DNA replication initiation factor Mcm10 induces pausing of elongation forks
through chromosomal replication origins in Saccharomyces cerevisiae", Mol Cell Biol, 17, 1997, 3261-71.
M Izumi, K Yanagi, T Mizuno, M Yokoi, Y Kawasaki, KY Moon, J Hurwitz, F Yatagai, F Hanaoka, "The human homolog of Saccharomyces
cerevisiae Mcm10 interacts with replication factors and dissociates from nuclease-resistant nuclear structures in G(2) phase.", Nucleic Acids
Res, 28, 2000, 4769-77.
K Fien, YS Cho, JK Lee, S Raychaudhuri, I Tappin, J Hurwitz, "Primer utilization by DNA polymerase alpha-primase is influenced by its
interaction with Mcm10p", J Biol Chem, 279, 2004, 16144-53.
Reaction
Description
References
Reaction
7.1.2.3 CDK and DDK associate with the Mcm10:pre-replicative complex
Description
At the beginning of this reaction, 1 molecule of 'Mcm10:active pre-replicative complex', 1 molecule of 'DDK', and 1 molecule of 'CDK' are
present. At the end of this reaction, 1 molecule of 'CDK:DDK:Mcm10:pre-replicative complex' is present.
References
H Kumagai, N Sato, M Yamada, D Mahony, W Seghezzi, E Lees, K Arai, H Masai, "A novel growth- and cell cycle-regulated protein, ASK,
activates human Cdc7-related kinase and is essential for G1/S transition in mammalian cells.", Mol Cell Biol, 19, 1999, 5083-95.
W Jiang, D McDonald, TJ Hope, T Hunter, "Mammalian Cdc7-Dbf4 protein kinase complex is essential for initiation of DNA replication.", EMBO
J, 18, 1999, 5703-13.
Reaction
7.1.2.4 Mcm2-7 is phosphorylated by DDK
Description
At the beginning of this reaction, 1 molecule of 'Mcm2-7 complex', and 1 molecule of 'ATP' are present. At the end of this reaction, 1 molecule of
'phosphorylated Mcm2-7 complex', and 1 molecule of 'ADP' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'kinase activity' of 'DDK'.
References
H Masai, E Matsui, Z You, Y Ishimi, K Tamai, K Arai, "Human Cdc7-related kinase complex. In vitro phosphorylation of MCM by concerted
actions of Cdks and Cdc7 and that of a critical threonine residue of Cdc7 by Cdks.", J Biol Chem, 275, 2000, 29042-52.
Reaction
7.1.2.5 Cdc45 associates with the pre-replicative complex at the origin
Description
Once the Mcm2-7 complex has been assembled onto the origin of replication, the next step is the assembly of Cdc45, an essential replication
protein, in late G1. The assembly of Cdc45 onto origins of replication forms a complex distinct from the pre-replicative complex, sometimes
called the pre-initiation complex. The assembly of Cdc45 onto origins correlates with the time of initiation. Like the Mcm2-7 proteins, Cdc45
binds specifically to origins in the G1 phase of the cell cycle and then to non-origin DNA during S phase and is therefore thought to travel with
the replication fork. Indeed, S. cerevisiae Cdc45 is required for DNA replication elongation as well as replication initiation. Cdc45 is required for
the association of alpha DNA polymerase:primase with chromatin. Based on this observation and the observation that in S. cerevisiae, cCdc45
has been found in large complexes with some components of Mcm2-7 complex, it has been suggested that Cdc45 plays a scaffolding role at the
replication fork, coupling Pol-alpha:primase to the replication fork through the helicase. Association of Cdc45 with origin DNA is regulated in the
cell cycle and its association is dependent on the activity of cyclin-dependent kinases but not the Cdc7/Dbf4 kinase. In Xenopus egg extracts,
association of Cdc45 with chromatin is dependent on Xmus101. TopBP1, the human homolog of Xmus1, is essential for DNA replication and
interacts with DNA polymerase epsilon, one of the polymerases involved in replicating the genome. TopBP1 homologs have been found in S.
cerevisiae and S. pombe. Sld3, an additional protein required for Cdc45 association with chromatin in S. cerevisiae and S. pombe, has no known
human homolog.
References
OM Aparicio, AM Stout, SP Bell, "Differential assembly of Cdc45p and DNA polymerases at early and late origins of DNA replication.", Proc Natl
Acad Sci U S A, 96, 1999, 9130-5.
JA Tercero, K Labib, JF Diffley, "DNA synthesis at individual replication forks requires the essential initiation factor Cdc45p.", EMBO J, 19, 2000,
2082-93.
555-66.
L Zou, B Stillman, "Formation of a preinitiation complex by S-phase cyclin CDK-dependent loading of Cdc45p onto chromatin.", Science, 280,
1998, 593-6.
P Saha, KC Thome, R Yamaguchi, Z Hou, S Weremowicz, A Dutta, "The human homolog of Saccharomyces cerevisiae CDC45.", J Biol Chem,
273, 1998, 18205-9.
Y Kamimura, YS Tak, A Sugino, H Araki, "Sld3, which interacts with Cdc45 (Sld4), functions for chromosomal DNA replication in Saccharomyces
cerevisiae.", EMBO J, 20, 2001, 2097-107.
S Dalton, L Whitbread, "Cell cycle-regulated nuclear import and export of Cdc47, a protein essential for initiation of DNA replication in budding
yeast.", Proc Natl Acad Sci U S A, 92, 1995, 2514-8.
I Kukimoto, H Igaki, T Kanda, "Human CDC45 protein binds to minichromosome maintenance 7 protein and the p70 subunit of DNA polymerase
alpha.", Eur J Biochem, 265, 1999, 936-43.
RA Van Hatten, AV Tutter, AH Holway, AM Khederian, JC Walter, WM Michael, "The Xenopus Xmus101 protein is required for the recruitment of
Cdc45 to origins of DNA replication.", J Cell Biol, 159, 2002, 541-7.
Reaction
7.1.2.6 DNA Replication Factor A (RPA) associates with the pre-replicative complex at the origin
Description
After pre-RC assembly and Cdc45 association with the origin of replication, Replication Protein A (RPA) also associates with chromatin. RPA is
a heterotrimeric complex containing p70, p34, and p11 subunits, and also is required for DNA recombination and DNA repair. The p70 subunit of
RPA binds to the primase subunits of Pol alpha:primase. The p70 and p34 subunits of RPA are phosphorylated in a cell cycle-dependent
manner. RPA is a single-strand DNA (ssDNA) binding protein and its association with chromatin at this stage suggests that DNA is partially
unwound. This suggestion has been confirmed by detection of ssDNA in budding yeast origins of replication using chemical methods.
References
S Din, SJ Brill, MP Fairman, B Stillman, "Cell-cycle-regulated phosphorylation of DNA replication factor A from human and yeast cells.", Genes
Dev, 4, 1990, 968-77.
Biochem, 66, 1997, 61-92.
I Dornreiter, LF Erdile, IU Gilbert, D von Winkler, TJ Kelly, "Interaction of DNA polymerase alpha-primase with cellular replication protein A and
SV40 T antigen.", EMBO J, 11, 1992, 769-76.
Reaction
7.1.2.7 DNA polymerase alpha:primase binds at the origin
Description
DNA polymerase alpha:primase is comprised of four subunits, p180, p70, p58, and p49. The two primase subunits, p58 and p49, form a tight
complex. The p49 subunit contains the DNA primase activity and one role of p58 appears to be tethering p49 to p180, the DNA polymerase
catalytic subunit. The fourth subunit, p70, binds p180 and may tether the DNA polymerase alpha:primase complex to Cdc45.
References
WC Copeland, TS Wang, "Enzymatic characterization of the individual mammalian primase subunits reveals a biphasic mechanism for initiation
of DNA replication.", J Biol Chem, 268, 1994, 26179-89.
Reaction
7.1.2.8 DNA polymerase epsilon binds at the origin
Description
At the beginning of this reaction, 1 molecule of 'origin of replication', and 1 molecule of 'DNA polymerase epsilon' are present. At the end of this
reaction, 1 molecule of 'DNA polymerase epsilon:origin complex' is present.
References
Reaction
7.2 DNA replication initiation
Description
additional 20 nucleotides of DNA.
References
7.2.1 The primase component of DNA polymerase:primase synthesizes a 6-10 nucleotide RNA primer at the
origin
Description
At the beginning of this reaction, 1 molecule of 'DNA polymerase alpha:primase:DNA polymerase alpha:origin complex', and 1 molecule of 'NTP'
are present. At the end of this reaction, 1 molecule of 'DNA polymerase epsilon', and 1 molecule of 'RNA primer:origin duplex:DNA polymerase
alpha:primase complex' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'DNA-directed RNA polymerase activity' of 'DNA polymerase alpha:primase'.
References
Reaction
7.2.2 The polymerase component of DNA polymerase alpha:primase synthesizes a 20-nucleotide primer at
the origin
Description
At the beginning of this reaction, 1 molecule of 'dTTP', 1 molecule of 'dGTP', 1 molecule of 'dATP', 1 molecule of 'RNA primer:origin duplex:DNA
polymerase alpha:primase complex', and 1 molecule of 'dCTP' are present. At the end of this reaction, 1 molecule of 'RNA primer-DNA
primer:origin duplex' is present.
This reaction takes place in the 'nucleus' and is mediated by the 'DNA-directed DNA polymerase activity' of 'DNA polymerase alpha:primase'.
Reaction
7.3 Switching of origins to a post-replicative state
7.3.1 Orc1 removal from chromatin
References
Physiol, 38, 1975, 58-63.
7.3.1.1 Orc1 is phosphorylated by cyclin A/CDK2
Description
References
Reaction
7.3.1.2 Phosphorylated Orc1 is ubiquitinated while still associated with chromatin
Description
References
Reaction
7.3.1.3 Ubiquitinated Orc1 enters the cytosol
Description
References
Reaction
7.3.1.4 Ubiquitinated Orc1 is degraded by the proteasome
Description
References
Reaction
7.3.2 CDK-mediated phosphorylation and removal of Cdc6
Description
References
7.3.2.1 Cdc6 protein is phosphorylated by CDK
Description
References
6193-8.
Reaction
7.3.2.2 Phosphorylated Cdc6 is exported from the nucleus
Description
References
6193-8.
Reaction
7.3.2.3 Cytoplasmic phosphorylated Cdc6 is ubiquitinated by the anaphase-promoting complex
Description
References
Reaction
7.3.2.4 Ubiquitinated Cdc6 is degraded by the proteasome
Description
References
Reaction
7.3.3 Mcm4,6,7 trimer forms and associates with the replication fork
Description
References
8003-15.
Biol Chem, 275, 2000, 16235-41.
Reaction
7.4 DNA strand elongation
Authors
Tom, S, Bambara, RA, 2003-06-05.
Description
Accurate and efficient genome duplication requires coordinated processes to replicate two template strands at eucaryotic replication forks.
Knowledge of the fundamental reactions involved in replication fork progression is derived largely from biochemical studies of the replication of
simian virus and from yeast genetic studies. Since duplex DNA forms an anti-parallel structure, and DNA polymerases are unidirectional, one of
the new strands is synthesized continuously in the direction of fork movement. This strand is designated as the leading strand. The other strand
grows in the direction away from fork movement, and is called the lagging strand. Several specific interactions among the various proteins
involved in DNA replication underlie the mechanism of DNA synthesis, on both the leading and lagging strands, at a DNA replication fork. These
interactions allow the replication enzymes to cooperate in the replication process.
References
GS Brush, TJ Kelly, B Stillman, "Identification of eukaryotic DNA replication proteins using simian virus 40 in vitro replication system.", Methods
Enzymol, 262, 1996, 522-48.
R Ayyagari, KJ Impellizzeri, BL Yoder, SL Gary, PM Burgers, "A mutational analysis of the yeast proliferating cell nuclear antigen indicates
distinct roles in DNA replication and DNA repair.", Mol Cell Biol, 15, 1995, 4420-9.
ME Budd, JL Campbell, "A yeast replicative helicase, Dna2 helicase, interacts with yeast FEN-1 nuclease in carrying out its essential function.",
Mol Cell Biol, 17, 1997, 2136-42.
J Hurwitz, AD Kwong, SH Lee, "The in vitro replication of DNA containing the SV40 origin.", J Biol Chem, 265, 1990, 18043-6.
7.4.1 Unwinding of DNA
Authors
Tye, BK, 2006-03-17.
Editors
Description
DNA Replication is regulated accurately and precisely by various protein complexes. Many members of the MCM protein family are assembled
into the pre-Replication Complexes (pre-RC) at the end of M phase of the cell cycle. DNA helicase activity of some of the MCM family proteins
are important for the unwinding of DNA and initiation of replication processes. This section contains four events which have been proved in
different eukaryotic experimental systems to involve various proteins for this essential step during DNA Replication.
References
7.4.1.1 MCM2-7 mediated fork unwinding
Authors
Tye, BK, 2005-11-29.
Editors
Description
In budding yeast, all MCM proteins have been proved to be essential for elongation. The active form of this protein complex may be a
heterohexamer. A subcomplex of MCM proteins consisting fo MCM4,6, and -7 has a weak helicase activity that may contribute to DNA
unwinding.
References
Source reaction
This reaction was inferred from the corresponding reaction "Yeast Mcm2-7 mediated fork unwinding" in species Saccharomyces cerevisiae.
DL Kaplan, MJ Davey, M O'Donnell, "Mcm4,6,7 uses a "pump in ring" mechanism to unwind DNA by steric exclusion and actively
translocate along a duplex", J Biol Chem, 278, 2003, 49171-82.
JK Lee, J Hurwitz, "Processive DNA helicase activity of the minichromosome maintenance proteins 4, 6, and 7 complex requires forked DNA
structures", Proc Natl Acad Sci U S A, 98, 2001, 54-9.
BK Tye, S Sawyer, "The hexameric eukaryotic MCM helicase: building symmetry from nonidentical parts", J Biol Chem, 275, 2000, 34833-6.
Reaction
7.4.1.2 MCM8 mediated fork unwinding
Authors
Tye, BK, 2005-11-29.
Editors
Description
The MCM2-7 related protein, MCM8, is required to replicate chromosomal DNA in Xenopus egg extracts. MCM8 binds chromatin upon initiation
of DNA synthesis. It may function as an helicase in the elongation step.
References
Source reaction
This reaction was inferred from the corresponding reaction "Xenopus Mcm8 mediated fork unwinding" in species Xenopus laevis.
Reaction
7.4.1.3 Formation of GINS complex
Description
At the beginning of this reaction, 1 molecule of 'PSF3p', 1 molecule of 'SLD5P', 1 molecule of 'PSF2p', and 1 molecule of 'PSF1p' are present. At
the end of this reaction, 1 molecule of 'GINS complex' is present.
Reaction
7.4.1.4 Multiple proteins are localized at replication fork
Authors
Tye, BK, 2006-03-17.
Editors
Description
By applying the chromatin immunoprecipitation technique to paused forks, certain proteins like DNA pol alpha, DNA pol delta, DNA pol epsilon,
MCM2-7, CDC45, GINS and MCM10 were identified. By uncoupling a helicase at the site using a polymerase inhibitor, MCM2-7, GINS complex
and CDC45 alone were found to be enriched at the paused fork suggesting these proteins may form a part of an "unwindosome" at the
replicating fork.
References
Source reaction
This reaction was inferred from the corresponding reaction "Multiple Xenopus proteins are localized at replication fork" in species Xenopus
laevis.
Reaction
7.4.2 Leading Strand Synthesis
Description
The processive complex is responsible for synthesizing at least 5-10 kb of DNA in a continuous manner during leading strand synthesis. The
incorporation of nucleotides by pol delta is quite accurate. However, incorporation of an incorrect nucleotide does occur occasionally.
Misincorporated nucleotides are removed by the 3' to 5' exonucleolytic proofreading capability of pol delta.
References
T Matsumoto, T Eki, J Hurwitz, "Studies on the initiation and elongation reactions in the simian virus 40 DNA replication system.", Proc Natl Acad
Sci U S A, 87, 1991, 9712-6.
7.4.2.1 Polymerase switching
Description
References
7.4.2.1.1 RFC binding displaces Pol Alpha
Description
References
Reaction
7.4.2.1.2 Loading of PCNA - Sliding Clamp Formation
Description
References
Reaction
7.4.2.1.3 RFC dissociates after sliding clamp formation
Description
DNA.
References
Reaction
7.4.2.1.4 Formation of Processive Complex
Description
References
Natl Acad Sci U S A, 87, 1990, 5672-6.
Reaction
7.4.2.2 Processive synthesis on the leading strand
Description
Polymerase switching is a key event that allows the processive synthesis of DNA by the pol delta and PCNA complex. Polymerase delta
possesses polymerization and proofreading activities, which increases the overall fidelity of DNA replication. The pol delta holoenzyme is a
heterotetrameric complex that contains p125, p66, p50, and p12 subunits, in human cells.
References
L Liu, J Mo, EM Rodriguez-Belmonte, MY Lee, "Identification of a fourth subunit of mammalian DNA polymerase delta.", J Biol Chem, 275, 2000,
18739-44.
Reaction
7.4.3 Lagging Strand Synthesis
Description
References
7.4.3.1 Polymerase switching
Description
References
7.4.3.1.1 RFC binding displaces Pol Alpha
Description
References
Reaction
7.4.3.1.2 Loading of PCNA - Sliding Clamp Formation
Description
References
Reaction
7.4.3.1.3 RFC dissociates after sliding clamp formation
Description
DNA.
References
Reaction
7.4.3.1.4 Formation of Processive Complex
Description
References
Natl Acad Sci U S A, 87, 1990, 5672-6.
Reaction
7.4.3.2 Processive synthesis on the lagging strand
Description
The key event that allows the processive synthesis on the lagging strand, is polymerase switching from pol alpha to pol delta, as on the leading
strand. However, the processive synthesis on the lagging strand proceeds very differently. DNA synthesis is discontinuous, and involves the
formation of short fragments called the Okazaki fragments. During the synthesis of Okazaki fragments, the RNA primer is folded into a
single-stranded flap, which is removed by endonucleases. This is followed by the ligation of adjacent Okazaki fragments.
References
7.4.3.2.1 Formation of Okazaki fragments
Description
References
Natl Acad Sci U S A, 87, 1990, 5672-6.
Virol, 66, 1992, 6634-40.
10923-34.
Reaction
7.4.3.2.2 Formation of the Flap Intermediate
Description
References
2001, 456-61.
Reaction
7.4.3.2.3 Removal of the Flap Intermediate
Description
References
A, 92, 1995, 7642-6.
7.4.3.2.3.1 RPA binds to the Flap
Description
References
2001, 456-61.
Reaction
7.4.3.2.3.2 Recruitment of Dna2 endonuclease
Description
References
2001, 456-61.
Reaction
7.4.3.2.3.3 Removal of RNA primer and dissociation of RPA and Dna2
Description
References
2001, 456-61.
vivo.", J Biol Chem, 275, 2000, 16518-29.
Reaction
7.4.3.2.3.4 Removal of remaining Flap
Description
References
Biol Chem, 275, 2000, 20949-55.
233-40.
Reaction
7.4.3.2.4 Joining of adjacent Okazaki fragments
Description
double-stranded DNA.
References
Reaction
7.5 Regulation of DNA replication
Description
DNA replication is regulated at various levels via ORC proteins. This pathway includes annotation of individual events that lead to the regulation
of replication.
7.5.1 Association of licensing factors with the pre-replicative complex
Description
The eukaryotic six-subunit origin recognition complex (ORC) governs the initiation site of DNA replication and formation of the prereplication
complex.
7.5.1.1 Orc1 associates with Orc4:Orc5:Orc3:Orc2:origin complexes
Description
References
Reaction
7.5.1.2 CDC6 protein is synthesized under the control of E2F transcription factors
Description
References
Reaction
7.5.1.3 The geminin component of geminin:Cdt1 complexes is ubiquitinated, releasing Cdt1
Description
References
Science, 290, 2000, 2309-12.
Reaction
7.5.2 Removal of licensing factors from origins
Description
Licensing factors are removed from the origin by various means like biochemical modification (phosphorylation) or by physical association with
other proteins. This pathway includes the annotations of events in which the fates of different proteins at the origin are outlined.
7.5.2.1 Orc1 removal from chromatin
References
Physiol, 38, 1975, 58-63.
7.5.2.1.1 Orc1 is phosphorylated by cyclin A/CDK2
Description
References
Reaction
7.5.2.1.2 Phosphorylated Orc1 is ubiquitinated while still associated with chromatin
Description
References
Reaction
7.5.2.1.3 Ubiquitinated Orc1 enters the cytosol
Description
References
Reaction
7.5.2.1.4 Ubiquitinated Orc1 is degraded by the proteasome
Description
References
Reaction
Description
References
Reaction
7.5.2.3 Cdt1 associates with geminin
Description
At the beginning of this reaction, 1 molecule of 'geminin', and 1 molecule of 'CDT1' are present. At the end of this reaction, 1 molecule of
'Cdt1:geminin' is present.
References
Science, 290, 2000, 2309-12.
Reaction
7.5.2.4 CDK-mediated phosphorylation and removal of Cdc6
Description
References
7.5.2.4.1 Cdc6 protein is phosphorylated by CDK
Description
References
6193-8.
Reaction
7.5.2.4.2 Phosphorylated Cdc6 is exported from the nucleus
Description
References
6193-8.
Reaction
7.5.2.4.3 Cytoplasmic phosphorylated Cdc6 is ubiquitinated by the anaphase-promoting complex
Description
References
Reaction
7.5.2.4.4 Ubiquitinated Cdc6 is degraded by the proteasome
Description
References
Reaction
7.5.2.5 Mcm4,6,7 trimer forms and associates with the replication fork
Description
References
8003-15.
Biol Chem, 275, 2000, 16235-41.
Reaction
8 Electron Transport Chain
Authors
Jassal, B, 2005-04-21.
Reviewers
Ferguson, SJ, 2005-05-12.
Description
Mitochondria are often described as the "powerhouse" of a cell as it is here that energy is largely released from the oxidation of food. Reducing
equivalents generated from beta-oxidation of fatty acids and from the Krebs cycle enter the electron transport chain (also called the respiratory
chain). During a series of redox reactions, electrons travel down the chain releasing their energy in controlled steps. These reactions drive the
active transport of H+ ions from the mitochondrial matrix , through the inner membrane to the intermembrane space. The respiratory chain
consists of five main types of carrier; flavins, iron-sulfur centres, quinones, cytochromes (heme proteins) and copper. The two main reducing
equivalents entering the respiratory chain are NADH and FADH2. NADH is linked through the NADH-specific dehydrogenase whereas FADH2 is
reoxidised within succinate dehydrogenase and a ubiquinone reductase of the fatty acid oxidation pathway. Oxygen is the final acceptor of
electrons and with H+ ions, is converted to form water, the end product of aerobic cellular respiration.
A proton electrochemical gradient (often called protonmotive force) is established across the inner membrane, with positive charge in the
intermembrane space relative to the matrix. Protons driven by the proton-motive force, can enter ATP synthase thus returning to the
mitochondrial matrix. ATP synthases use this exergonic flow to form ATP in the matrix, a process called chemiosmotic coupling. A by-product of
this process is heat generation.
An antiport, ATP-ADP translocase, preferentially exports ATP from the matrix thereby maintaining a high ADP:ATP ratio in the matrix. The tight
coupling of electron flow to ATP synthesis means oxygen consumption is dependent on ADP availability (termed respiratory control). High ADP
(low ATP) increases electron flow thereby increasing oxygen consumption and low ADP (high ATP) decreases electron flow and thereby
decreases oxygen consumption. There are many inhibitors of mitochondrial ATP synthesis. Most act by either blocking the flow of electrons (eg
cyanide, carbon monoxide, rotenone) or uncoupling electron flow from ATP synthesis (eg dinitrophenol). Thermogenin is a natural protein found
in brown fat. Newborn babies have a large amount of brown fat and the heat generated by thermogenin is an alternative to ATP synthesis (and
thus electron flow only produces heat) and allows the maintenance of body temperature in newborns.
The electron transport chain is located in the inner mitochondrial membrane and comprises some 80 proteins organized in four enzymatic
complexes (I-IV). Complex V generates ATP but has no electron transfer activity. In addition to these 5 complexes, there are also two electron
shuttle molecules; Coenzyme Q (also known as ubiquinone, CoQ) and Cytochrome c (Cytc). These two molecules shuttle electrons between the
large complexes in the chain.
How many ATPs are generated by this process? Theoretically, for each glucose molecule, 32 ATPs can be produced. As electrons drop from
NADH to oxygen in the chain, the number of protons pumped out and returning through ATP synthase can produce 2.5 ATPs per electron pair.
For each pair donated by FADH2, only 1.5 ATPs can be formed. Twelve pairs of electrons are removed from each glucose molecule;
10 by NAD+ = 25 ATPs
2 by FADH2 = 3 ATPs.
Making a total of 28 ATPs. However, 2 ATPs are formed during the Krebs' cycle and 2 ATPs formed during glycolysis for each glucose molecule
therefore making a total ATP yield of 32 ATPs. In reality, the energy from the respiratory chain is used for other processes (such as active
transport of important ions and molecules) so under conditions of normal respiration, the actual ATP yield probably does not reach 32 ATPs.
The reducing equivalents that fuel the electron transport chain, namely NADH and FADH2, are produced by the Krebs cycle (TCA cycle) and the
beta-oxidation of fatty acids. At three steps in the Krebs cycle (isocitrate conversion to oxoglutarate; oxoglutarate conversion to succinyl-CoA;
Malate conversion to oxaloacetate), a pair of electrons (2e-) are removed and transferred to NAD+, forming NADH and H+. At a single step, a
pair of electrons are removed from succinate, reducing FAD to FADH2. From the beta-oxidation of fatty acids, one step in the process forms
NADH and H+ and another step forms FADH2.
Cytoplasmic NADH, generated from glycolysis, has to be oxidized to reform NAD+, essential for glycolysis, otherwise glycolysis would cease to
function. There is no carrier that transports NADH directly into the mitochondrial matrix and the inner mitochondrial membrane is impermeable to
NADH so the cell uses two shuttle systems to move reducing equivalents into the mitochondrion and regenerate cytosolic NAD+.
The first is the glycerol phosphate shuttle, which uses electrons from cytosolic NADH to produce FADH2 within the inner membrane. These
electrons then flow to Coenzyme Q. Complex I is bypassed so only 1.5 ATPs can be formed per NADH via this route. The overall balanced
equation, summing all the reactions in this system, is
NADHcytosol + H+cytosol + NAD+mito -> NAD+cytosol + NADHmito + H+mito
The malate-aspartate shuttle uses the oxidation of malate to generate NADH in the mitochondrial matrix. This NADH can then be fed directly to
complex I and thus can form 3 ATPs via the respiratory chain. The overall balanced equation is
NADHcytosol + H+cytosol + FADinner memb -> NAD+cytosol + FADH2 inner memb
Both of these shuttle systems regenerate cytosolic NAD+.
The entry point for NADH is complex I (NADH dehydrogenase) and the entry point for FADH2 is Coenzyme Q. The input of electrons from fatty
acid oxidation via ubiquinone is complicated and not shown in the diagram.
References
PD Barker, SJ Ferguson, "Still a puzzle: why is haem covalently attached in c-type cytochromes?", Structure Fold Des, 7, 1999, R281-90.
G Michal, "Biochemical Pathways - An Atlas of Biochemistry and Molecular Biology", 1999, 193-200.
DE Metzler, "Biochemistry - The Chemical Reactions of Living Cells 2nd Edn.", 2, 2003, 1018-84.
Y Hatefi, "The mitochondrial electron transport and oxidative phosphorylation system", Annu Rev Biochem, 54, 1985, 1015-69.
DL Nelson, MM Cox, "Lehninger - Principles of Biochemistry 4th Edn.", 2005, 690-750.
DG Nicholls, SJ Ferguson, "Bioenergetics 3", 2002, 89-134.
RK Murray, "Harper's Biochemistry 25th Edn.", 137-48.

8.1 NADH enters the respiratory chain at Complex I
Authors
Jassal, B, 2005-05-10.
Description
Complex I (NADH:ubiquinone oxidoreductase or NADH dehydrogenase) utilizes NADH formed from glycolysis and the TCA cycle to pump
protons out of the mitochondrial matrix. It is the largest enzyme complex in the electron transport chain, containing 45 subunits. Seven subunits
(ND1-6, ND4L) are encoded by mitochondrial DNA (Loeffen et al [1998]), the remainder are encoded in the nucleus. The enzyme has a FMN
prosthetic group and 8 Iron-Sulfur (Fe-S) clusters. The electrons from NADH oxidation pass through the flavin (FMN) and Fe-S clusters to
ubiquinone (CoQ). This electron transfer is coupled with the translocation of protons from the mitochondrial matrix to the intermembrane space.
For each electron transferred, 2 protons can be pumped out of the matrix. As there are 2 electrons transferred, 4 protons can be pumped out.
Complex I is made up of 3 sub-complexes - Iron-Sulfur protein fraction (IP), Flavoprotein fraction (FP) and the Hydrophobic protein fraction (HP),
probably arranged in an L-shaped structure with the IP and FP fractions protruding into the mitochondrial matrix and the HP arm lying within the
inner mitochondrial membrane. The overall reaction can be summed as below:
NADH + Ubiquinone + 5H+matrix -> NAD+ + Ubiquinol + 4H+memb. space
The electrons from complex I are transferred to ubiquinone (Coenzyme Q, CoQ), a small mobile carrier of electrons located within the inner
membrane. Ubiquinone is reduced to ubiquinol during this process.
References
T Yano, "The energy-transducing NADH: quinone oxidoreductase, complex I", Mol Aspects Med, 23, 2002, 345-68.
JL Loeffen, RH Triepels, LP van den Heuvel, M Schuelke, CA Buskens, RJ Smeets, JM Trijbels, JA Smeitink, "cDNA of eight nuclear encoded
subunits of NADH:ubiquinone oxidoreductase:", Biochem Biophys Res Commun, 253, 1998, 415-22.
J Smeitink, R Sengers, F Trijbels, L van den Heuvel, "Human NADH:ubiquinone oxidoreductase", J Bioenerg Biomembr, 33, 2001, 259-66.
J Hirst, J Carroll, IM Fearnley, RJ Shannon, JE Walker, "The nuclear encoded subunits of complex I from bovine heart mitochondria", Biochim
Biophys Acta, 1604, 2003, 135-50.
I Bourges, C Ramus, B Mousson de Camaret, R Beugnot, C Remacle, P Cardol, G Hofhaus, JP Issartel, "Structural organization of
mitochondrial human complex I: role of the ND4", Biochem J, 383, 2004, 491-9.
T Friedrich, B Bottcher, "The gross structure of the respiratory complex I: a Lego System", Biochim Biophys Acta, 1608, 2004, 1-9.
Reaction
8.2 Transfer of electrons through the succinate dehydrogenase complex
Authors
Jassal, B, 2005-06-10.
Description
This event is deduced on the basis of bovine experimental data.
Complex II (succinate dehydrogenase) transfers electrons from the TCA cycle to ubiquinone. The 6th step in the TCA cycle is where succinate is
dehydrogenated to fumarate with subsequent reduction of FAD to FADH2. FADH2 provides the electrons for the transport chain. Succinate
dehydrogenase belongs to subclass 1 of the SQR family (succinate:quinone reductase) (classified by Hagerhall, C and Hederstedt, L [1996]).
It consists of 4 subunits (referred to as A, B, C and D), all nuclear-encoded and is located on the matrix side of the inner mitochondrial
membrane. Subunits A and B are hydrophilic whereas subunits C and D are integral proteins of the inner membrane. SQRs usually contain 3
Fe-S clusters bound by the B subunit. Succinate dehydrogenase contains one [2Fe-2S] cluster, one [4Fe-4S] cluster and one [3Fe-4S] cluster.
Additionally, the A subunit has a covalently-bound FAD group. Reduced complex II has this FAD converted to FADH2. The electrons from
complex II are transferred to ubiquinone (also called Q, Coenzyme Q or CoQ), a small mobile carrier of electrons located within the inner
membrane. Ubiquinone is reduced to ubiquinol during this process.
References
C Hagerhall, L Hederstedt, "A structural model for the membrane-integral domain of succinate: quinone", FEBS Lett, 389, 1996, 25-31.
Source reaction
This reaction was inferred from the corresponding reaction "Transfer of electrons through the bovine succinate dehydrogenase complex" in
species Bos taurus.
BA Ackrell, MB Ball, EB Kearney, "Peptides from complex II active in reconstitution of succinate-ubiquinone", J Biol Chem, 255, 1980, 2761-9.
GY Lee, DY He, L Yu, CA Yu, "Identification of the ubiquinone-binding domain in QPs1 of", J Biol Chem, 270, 1995, 6193-8.
L Yu, CA Yu, "Quantitative resolution of succinate-cytochrome c reductase into", J Biol Chem, 257, 1982, 2016-21.
Reaction
8.3 Reducing equivalents from beta-oxidation of fatty acids transfer to ETF
Description
Electron transfer flavoprotein, ETF, a 63kDa heterodimer composed of alpha and beta subunit, binds one FAD and one AMP per dimer. ETF
resides on the matrix face of the mitochondrial inner membrane. Reducing equivalents from the beta-oxidation of fatty acyl CoAs are transferred
to ETF, reducing the ETF-bound FAD to FADH2.
Reaction
8.4 Transfer of electrons from ETF to ubiquinone by ETF-QO
Description
ETF-ubiquinone oxidoreductase (ETF-QO), catalyzes the re-oxidation of reduced ETF, with ubiquinone as the electron acceptor.
Reaction
8.5 Electron transfer from ubiquinol to cytochrome c of complex III
Authors
Jassal, B, 2005-06-14.
Description
The Protonmotive Q cycle is the mechanism by which complex III transfers electrons from ubiquinol to cytochrome c, linking this process to
translocation of protons across the membrane. This cycle is complicated by the fact that both ubiquinol is oxidised and ubiquinone is reduced
during this process. Through a complex series of electron transfers, Complex III consumes two molecules of ubiquinol (QH2) and two molecules
of oxidized cytochrome c, generates one molecule of ubiquinone (Q) and two molecules of reduced cytochrome c, regenerates one molecule of
ubiquinol (QH2), and mediates the translocation of two protons from the mitochondrial matrix to the mitochondrial intermembrane space.
The overall reaction can be summed up as below:
2QH2 + 2cyt c(ox.) + Q + 2H+matrix -> 2Q + 2cyt c(red.) + QH2 + 4H+memb. space
References
P Mitchell, "Protonmotive redox mechanism of the cytochrome b-c1 complex in the", FEBS Lett, 56, 1975, 1-6.
P Mitchell, "Possible molecular mechanisms of the protonmotive function of cytochrome", J Theor Biol, 62, 1976, 327-67.
Reaction
8.6 Electron transfer from reduced cytochrome c to molecular oxygen
Authors
Jassal, B, 2005-06-28.
Description
Complex IV (cytochrome oxidase) contains the hemeprotein cytochrome a and a3. It also contains copper atoms which undergo a transition from
Cu+ to Cu2+ during the transfer of electrons through the complex to molecular oxygen. A bimetallic centre containing a copper atom and a
heme-linked iron protein binds oxygen after 4 electrons have been picked up. Water, the final product of oxygen reduction, is then released.
Oxygen is the final electron acceptor in the respiratory chain. The overall reaction can be summed as below:
4Cyt c(red.) + 4H+scalar + 4H+matrix + O2 -> 4Cyt c(ox.) + 2H2O + 4H+memb. space
Four protons are taken up from the matrix side of the membrane to form the water (scalar protons). Wikstrom (1977) suggests 4 protons are
additionally transferred out from the matrix to the intermembrane space.
References
BE Schultz, SI Chan, "Structures and proton-pumping strategies of mitochondrial respiratory", Annu Rev Biophys Biomol Struct, 30, 2001, 23-65.
MK Wikstrom, "Proton pump coupled to cytochrome c oxidase in mitochondria", Nature, 266, 1977, 271-3.
BL Trumpower, RB Gennis, "Energy transduction by cytochrome complexes in mitochondrial and bacterial", Annu Rev Biochem, 63, 1994,
675-716.
Reaction
9 Gap junction trafficking and regulation
Authors
Gilleron, J, Segretain, D, Falk, MM, 2007-01-03.
Editors
Matthews, L, 2007-01-26.
Description
Gap junctions are clusters of intercellular channels connecting adjacent cells and permitting the direct exchange of ions and small molecules
between cells. These channels are composed of two hemichannels, or connexons, one located on each of the two neighboring cells. Each
connexon is composed of 6 trans-membrane protein subunits of the connexin (Cx) family. A gap of approximately 3 nm remains between the
adjacent cell membranes, but two connexons interact and dock head-to-head in the extra-cellular space forming a tightly sealed,
double-membrane intercellular channel (see Segretain and Falk, 2004). The activity of these intercellular channels is regulated, particularly by
intramolecular modifications such as phosphorylation which appears to regulate connexin turnover, gap junction assembly and the opening and
closure (gating) of gap junction channels.
References
R Bruzzone, TW White, DL Paul, "Connections with connexins: the molecular basis of direct intercellular signaling", Eur J Biochem, 238, 1996,
1-27.
M Piehl, C Lehmann, A Gumpert, JP Denizot, D Segretain, MM Falk, "Internalization of Large Double-Membrane Intercellular Vesicles by a
Clathrin-dependent Endocytic Process", Mol Biol Cell, 2007.
AM Simon, DA Goodenough, "Diverse functions of vertebrate gap junctions", Trends Cell Biol, 8, 1998, 477-83.
R Bruzzone, "Learning the language of cell-cell communication through connexin channels", Genome Biol, 2, 2001, REPORTS4027.
D Segretain, MM Falk, "Regulation of connexin biosynthesis, assembly, gap junction formation, and removal", Biochim Biophys Acta, 1662,
2004, 3-21.
DP Kelsell, J Dunlop, MB Hodgins, "Human diseases: clues to cracking the connexin code?", Trends Cell Biol, 11, 2001, 2-6.
VM Berthoud, PJ Minogue, JG Laing, EC Beyer, "Pathways for degradation of connexins and gap junctions", Cardiovasc Res, 62, 2004, 256-67.
DW Laird, M Castillo, L Kasprzak, "Gap junction turnover, intracellular trafficking, and phosphorylation of connexin43 in brefeldin A-treated rat
mammary tumor cells", J Cell Biol, 131, 1995, 1193-203.
JC Herve, N Bourmeyster, D Sarrouilhe, "Diversity in protein-protein interactions of connexins: emerging roles", Biochim Biophys Acta, 1662,
2004, 22-41.
9.1 Gap junction trafficking
Authors
Editors
Matthews, L, 2007-01-07.
Description
Gap junctions are intercellular communication channels formed from Cx (connexin) protein subunits (see Segretain and Falk 2004 and Evans et
al. 2006 for comprehensive reviews). Connexins are transported to the plasma membrane after oligomerizing into hexameric assemblies called
hemichannels (CxHcs) or connexons. Connexons dock head-to-head in the extracellular space with opposing hexameric channels located in the
plasma membranes of neighbouring cells. The double membrane channel or gap junction generated directly links the cytoplasms of interacting
cells and facilitates the integration and co-ordination of cellular signalling, metabolism, secretion and contraction. In addition to their role in
intercellular communication, connexon hemichannels coordinate the release of ATP, glutamate, NAD+ and prostaglandin E2 from the cells.
CxHcs open in response to various types of external changes, including mechanical, shear, ionic and ischaemic stress.
The trafficking of gap junctions involves (1) synthesis of connexin polypeptides at endoplasmic reticulum membranes, (2) oligomerization into
homomeric- and heteromeric gap junction connexons (hemi-channels), (3) passage through the Golgi stacks, (4) intracellular storage within
Trans Golgi membranes, (5) trafficking along microtubules, (6) insertion of connexons into the plasma membrane, (7) lateral diffusion of
connexons in the plasma membrane, (8) aggregation of individual gap junction channels into plaques, (9) stabilization of peripheral microtubule
plus-ends by binding to Cx43-based gap junctions, (10) internalization of the channel plaque leading to cytoplasmic annular junctions, and (11)
complete degradation via lysosomal and proteasomal pathways (see Segretain and Falk 2004). Aspects of gap assembly are described here.
Additional details of assembly, regulation and degradation will be covered in the next release.
References
WH Evans, E De Vuyst, L Leybaert, "The gap junction cellular internet: connexin hemichannels enter the signalling limelight", Biochem J, 397,
2006, 1-14.
2004, 3-21.
9.1.1 Gap junction assembly
Authors

Editors
Matthews, L, 2007-01-26, Joshi-Tope, G, 2004-04-22.
Description
The assembly of gap junctions of involves (1) synthesis of connexin polypeptides at endoplasmic reticulum membranes, (2) oligomerization into
homomeric- and heteromeric gap junction connexons (hemi-channels), (3) passage through the Golgi stacks, (4) intracellular storage within
Trans Golgi membranes, (5) trafficking along microtubules, (6) insertion of connexons into the plasma membrane, (7) lateral diffusion of
connexons in the plasma membrane, (8) aggregation of individual gap junction channels into plaques, and (9) stabilization of peripheral
microtubule plus-ends by binding to Cx43-based gap junctions (see Segretain and Falk, 2004.)
References
1-27.
2004, 3-21.
9.1.1.1 Connexin synthesis
Authors
Editors
Matthews, L, 2007-01-07.
Description
Connexins (Cxs) are encoded by a large gene family predicted to include at least 20 isoforms in humans. Most mammalian Cx genes consist of
two exons. The first consists of untranslated sequence, and the second contains the entire coding sequence as depicted in Figure 1 below.
Exceptionally, Cx36 and Cx45 contain 3 exons and 2 introns and the third exon contains the coding sequence (Belluardo et al. 1999 ; Jacob and
Beyer 2001). Connexins have been divided in two major subgroups, alpha and beta, according to their amino acid sequence similarity as shown
in Figure 2 below (see Bruzzone et al., 2001; Willecke et al., 2002). Alternative names and additional subgroups have been suggested as well.
Cx are synthesized by ribosomes in the endoplasmic reticulum (ER) membrane. All Cx proteins contain four trans-membrane domains (TM1 to
TM4), two extracellular loops (E1 and E2) and one cytoplasmic loop. The amino- and carboxyl termini are located in the cytosol (reviewed in
Segretain and Falk, 2004). After targeting to the ER, connexins are checked by a quality control system to prevent misfolded forms from
progressing through the secretory pathway. Aberrant proteins are removed by endoplasmic-reticulum-associated degradation (ERAD).
References
K Willecke, J Eiberger, J Degen, D Eckardt, A Romualdi, M Guldenagel, U Deutsch, G Sohl, "Structural and functional diversity of connexin
genes in the mouse and human genome", Biol Chem, 383, 2002, 725-37.
A Jacob, EC Beyer, "Mouse connexin 45: genomic cloning and exon usage", DNA Cell Biol, 20, 2001, 11-9.
MM Falk, NM Kumar, NB Gilula, "Membrane insertion of gap junction connexins: polytopic channel forming membrane proteins", J Cell Biol, 127,
1994, 343-55.
N Belluardo, A Trovato-Salinaro, G Mudo, YL Hurd, DF Condorelli, "Structure, chromosomal localization, and brain expression of human Cx36
gene", J Neurosci Res, 57, 1999, 740-52.
R Bruzzone, "Learning the language of cell-cell communication through connexin channels", Genome Biol, 2, 2001, REPORTS4027.
2004, 3-21.
S Ahmad, JA Diez, CH George, WH Evans, "Synthesis and assembly of connexins in vitro into homomeric and heteromeric functional gap
junction hemichannels", Biochem J, 1999, 247-53.
Reaction
9.1.1.2 Transport of connexins along the secretory pathway
Authors
Editors
Matthews, L, 2007-01-26.
Description
Connexins follow the classical secretory transport route from the ER to the plasma membrane: ER -> ERGIC -> Golgi -> TGN (Trans Golgi
Network) -> PM (Plasma Membrane). All connexins assemble or oligomerize into hexameric connexons. The site of assembly varies and
depends on Cx isoform, or cell type (see Koval et al., 2006).
Oligomerization of connexins has been observed during ER membrane insertion (Cx32), just after exit from the ER, in the ER-Golgi-intermediate
compartment (Cx26) and inside the Trans-Golgi Network (Cx43) (Falk et al. 1997; Ahmad et al. 1999; Musil and Goodenough 1993; Diez et al.
1999).
References
LS Musil, DA Goodenough, "Multisubunit assembly of an integral plasma membrane channel protein, gap junction connexin43, occurs after exit
from the ER", Cell, 74, 1993, 1065-77.
JA Diez, S Ahmad, WH Evans, "Assembly of heteromeric connexons in guinea-pig liver en route to the Golgi apparatus, plasma membrane and
gap junctions", Eur J Biochem, 262, 1999, 142-8.
M Koval, "Pathways and control of connexin oligomerization", Trends Cell Biol, 16, 2006, 159-66.
MM Falk, LK Buehler, NM Kumar, NB Gilula, "Cell-free synthesis and assembly of connexins into functional gap junction membrane channels",
EMBO J, 16, 1997, 2703-16.
Source pathway
This pathway was inferred from the corresponding pathway "Transport of connexins along the secretory pathway" in species Rattus norvegicus.
from the ER", Cell, 74, 1993, 1065-77.
M Koval, "Pathways and control of connexin oligomerization", Trends Cell Biol, 16, 2006, 159-66.
EMBO J, 16, 1997, 2703-16.
9.1.1.2.1 Transport of connexins to the ER-Golgi intermediate compartment
Authors
Editors
Matthews, L, 2007-01-07.
Description
Transport of connexins along the secretory pathway (including transit from the ER to the ERGIC where Cx32 is predicted to oligomerize) occurs
in vesicular transport containers.
Source reaction
This reaction was inferred from the corresponding reaction "Transport of connexins to the ER-Golgi intermediate compartment" in species Cavia
porcellus.
Reaction
9.1.1.2.2 Transport of connexins to the Trans-Golgi Network (TGN)
Authors
Editors
Matthews, L, 2007-01-07.
Description
Transport of connexins along the secretory pathway (including transit from the Golgi to the TGN where Cx43 is predicted to oligomerize) occurs
in vesicular transport containers.
Source reaction
This reaction was inferred from the corresponding reaction "Transport of Connexin to the Trans-Golgi network" in species Rattus norvegicus.
from the ER", Cell, 74, 1993, 1065-77.
Reaction
9.1.1.3 Oligomerization of connexins into connexons
Authors
Editors
Matthews, L, 2007-01-26.
Description
The mechanism of connexin assembly into connexons has been well characterized. Two different types of connexons can be formed. A
connexon containing six identical connexin molecules is referred to as an homomeric connexon, while a connexon containing at least two
different connexin molecules is referred to as an heteromeric connexon. The connexin molecules making up an heteromeric connexon appear to
belong to only one subgroup (alpha or beta); heteromeric connexons containing both alpha and beta subunits have not yet been observed.
Indeed, an intrinsic signal in four amino acid positions appears to confer different physicochemical characteristics to certain connexins in the
alpha and beta groups (LagrÃƒÂ©e et al., 2003). These intrinsic signals are Cx specific, however (see Gemel et al., 2006). Therefore, additional
yet unknown signals are required to regulate connexin compatibility and hetero-oligomerization.
The identification of the subcellular location at which gap junction assembly occurs has proven difficult. One explanation for this difficulty may be
that the location of oligomerization for each connexon varies depending upon Cx type or cell type. Oligomerization has been observed after ER
membrane insertion (Cx43, Cx32, Cx26) (Falk et al., 1997; Ahmad et al., 1999; Ahmad and Evans, 2002), in the ER-Golgi-intermediate
compartment (ERGIC) (Cx32) (Diez et al. 1999) and inside the trans-Goligi network (Cx43) (Musil and Goodenough, 1993).
References
from the ER", Cell, 74, 1993, 1065-77.
J Gemel, X Lin, RD Veenstra, EC Beyer, "N-terminal residues in Cx43 and Cx40 determine physiological properties of gap junction channels, but
do not influence heteromeric assembly with each other or with Cx26", J Cell Sci, 119, 2006, 2258-68.
V Lagree, K Brunschwig, P Lopez, NB Gilula, G Richard, MM Falk, "Specific amino-acid residues in the N-terminus and TM3 implicated in
channel function and oligomerization compatibility of connexin43", J Cell Sci, 116, 2003, 3189-201.
EMBO J, 16, 1997, 2703-16.
S Ahmad, WH Evans, "Post-translational integration and oligomerization of connexin 26 in plasma membranes and evidence of formation of
membrane pores: implications for the assembly of gap junctions", Biochem J, 365, 2002, 693-9.
9.1.1.3.1 Connexin oligomerization in endoplasmic reticulum membrane
Authors
Editors
Matthews, L, 2007-01-07.
Description
Studies using microsomes have revealed that oligomerization of connexins Cx26, Cx43, and Cx32 can occur after insertion of connexins in the
ER membrane (Falk et al. 1997; Ahmad et al. 1999, Ahmad and Evans, 2002).
References
EMBO J, 16, 1997, 2703-16.
S Ahmad, WH Evans, "Post-translational integration and oligomerization of connexin 26 in plasma membranes and evidence of formation of
membrane pores: implications for the assembly of gap junctions", Biochem J, 365, 2002, 693-9.
Reaction
9.1.1.3.2 Connexin oligomerization in ER-Golgi-Intermediate Compartment
Authors

Editors
Matthews, L, 2007-01-07.
Description
Oligomerization of connexins Cx32 and C26 has also been observed in the ER-Golgi-intermediate compartment (ERGIC) (Diez et al. 1999).
Heteromeric connexons containing both Cx32 and C26 have been observed. For the sake of simplicity, the connexon here is described as
containing equal numbers of Cx26 and Cx32 subunits, although the ratio may vary.
References
PE Martin, ET Mambetisaeva, DA Archer, CH George, WH Evans, "Analysis of gap junction assembly using mutated connexins detected in
Charcot-Marie-Tooth X-linked disease", J Neurochem, 74, 2000, 711-20.
Source reaction
This reaction was inferred from the corresponding reaction "Connexin oligomerization in ER-Golgi-intermediate compartment" in species Cavia
porcellus.
Reaction
9.1.1.3.3 Connexin oligomerization in Trans-Golgi Network (TGN)
Authors

Editors
Matthews, L, 2007-01-07.
Description
A study using cultured cells demonstrated connexon oligomerization from Cx43 subunits inside the Trans-Golgi Network after exit from the ER
(Musil and Goodenough 1993).
References
from the ER", Cell, 74, 1993, 1065-77.
Source reaction
This reaction was inferred from the corresponding reaction "Connexin oligomerization in the Trans-Golgi network" in species Rattus norvegicus.
from the ER", Cell, 74, 1993, 1065-77.
Reaction
9.1.1.4 Transport of connexons to the plasma membrane
Authors
Editors
Matthews, L, 2007-04-12.
Description
Following connexon oligomerization, the hemichannels must be transported to the plasma membrane. This has been shown to occur in transport
vesicles called "cargo containers". Most of post-Golgi cargo containers have a diameter of of 50- 200 nm (Lauf et al., 2002). Recently direct
transport of connexins to GJ assembly sides has been described (Shaw et al., 2007). Besides microtuble-dependent trafficking, a
microtubule-independent delivery pathway may exist as concluded from studies using the secretory transport inhibitor, Brefeldin A (Musil and
Goodenough 1993; De Sousa et al. 1993; Laird et al. 1995).
References
from the ER", Cell, 74, 1993, 1065-77.
PA De Sousa, G Valdimarsson, BJ Nicholson, GM Kidder, "Connexin trafficking and the control of gap junction assembly in mouse
preimplantation embryos", Development, 117, 1993, 1355-67.
U Lauf, BN Giepmans, P Lopez, S Braconnot, SC Chen, MM Falk, "Dynamic trafficking and delivery of connexons to the plasma membrane and
accretion to gap junctions in living cells", Proc Natl Acad Sci U S A, 99, 2002, 10446-51.
RM Shaw, AJ Fay, MA Puthenveedu, M von Zastrow, YN Jan, LY Jan, "Microtubule plus-end-tracking proteins target gap junctions directly from
the cell interior to adherens junctions", Cell, 128, 2007, 547-60.
9.1.1.4.1 Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane
Authors
Editors
Matthews, L, 2007-04-12.
Description
Through videomicroscopy, a saltatory transport of connexon vesicles along curvilinear microtubules from the Golgi to the plasma membrane has
been observed (Lauf et al., 2002). Such a transport system has been described for similar secretory vesicles (Toomre et al., 1999).
References
D Toomre, P Keller, J White, JC Olivo, K Simons, "Dual-color visualization of trans-Golgi network to plasma membrane traffic along microtubules
in living cells", J Cell Sci, 1999, 21-33.
9.1.1.4.1.1 Budding of connexon-containing transport vesicles from the Golgi
Authors
Editors
Matthews, L, 2007-01-07.
Description
Connexon-containing transport vesicles have been shown to emanate from the Golgi and deliver connexons to the plasma membrane (Lauf et
al., 2002).
References
Reaction
9.1.1.4.1.2 Association of Golgi transport vesicles with microtubules and transport to the plasma membrane
Authors
Editors
Matthews, L, 2007-01-07.
Description
One mechanism of transport of connexon-containing vesicles involves movement along microtubules (Segretain and Falk, 2004). Such a
transport system has been described for similar secretory vesicles (Toomre et al., 1999). Direct microtubule-dependent transport of connexons
to GJ-assembly sites has recently been reported as well (Shaw et al., 2007).
References
2004, 3-21.
D Toomre, P Keller, J White, JC Olivo, K Simons, "Dual-color visualization of trans-Golgi network to plasma membrane traffic along microtubules
in living cells", J Cell Sci, 1999, 21-33.
Reaction
9.1.1.4.2 Microtubule-independent trafficking of connexons to the plasma membrane
Authors
Editors
Matthews, L, 2007-04-12.
Description
Connexons may also traffic using a microtubule-independent mechanism. A few studies suggest that rough ER membranes can directly transfer
connexons to the plasma membrane (Martin et al. 2001; Bloom and Goldstein 1998). Other cytoskeletal components, such as actin filaments,
might be involved in the delivery of connexons to gap junction plaques (Thomas et al. 2001; Gilleron et al. 2006).
References
GS Bloom, LS Goldstein, "Cruising along microtubule highways: how membranes move through the secretory pathway", J Cell Biol, 140, 1998,
1277-80.
T Thomas, K Jordan, DW Laird, "Role of cytoskeletal elements in the recruitment of Cx43-GFP and Cx26-YFP into gap junctions", Cell Commun
Adhes, 8, 2001, 231-6.
J Gilleron, M Nebout, L Scarabelli, F Senegas-Balas, S Palmero, D Segretain, G Pointis, "A potential novel mechanism involving connexin 43
gap junction for control of sertoli cell proliferation by thyroid hormones", J Cell Physiol, 209, 2006, 153-61.
PE Martin, G Blundell, S Ahmad, RJ Errington, WH Evans, "Multiple pathways in the trafficking and assembly of connexin 26, 32 and 43 into gap
junction intercellular communication channels", J Cell Sci, 114, 2001, 3845-55.
Reaction
9.1.1.5 Docking of connexons into junctional, double-membrane spanning channels
Authors
Editors
Matthews, L, 2007-04-12.
Description
Docking of Cx43 at the plasma membrane may involve ZO-1 as well as alpha- and beta-catenin (Shaw et al., 2007). The role of ZO-1 in
regulating gap junction biology is unclear. Recent results indicate a role for ZO-1 in regulating gap junction plaque size (Hunter et al., 2007).
References
AW Hunter, RJ Barker, C Zhu, RG Gourdie, "Zonula occludens-1 alters connexin43 gap junction size and organization by influencing channel
accretion", Mol Biol Cell, 16, 2005, 5686-98.
Source reaction
This reaction was inferred from the corresponding reaction "Docking of connexons into junctional, double-membrane spanning channels" in
species Rattus norvegicus.
JC Wu, RY Tsai, TH Chung, "Role of catenins in the development of gap junctions in rat cardiomyocytes", J Cell Biochem, 88, 2003, 823-35.
Reaction
9.1.1.6 Insertion of connexons into the plasma membrane resulting in the formation of hemi-channels
Authors
Editors
Matthews, L, 2007-04-12.
Description
Insertion of a connexon into the cell membrane results in the formation of a hemi-channel. This channel permits direct exchanges between cell
cytoplasm and extracellular matrix (Figure 3). Freeze-fracture electron microscopy studies have revealed that cytoplasmic vesicles can fuse with
the plasma membrane to permit connexon insertion (Shivers and Bowman 1985).
References
G Gaietta, TJ Deerinck, SR Adams, J Bouwer, O Tour, DW Laird, GE Sosinsky, RY Tsien, MH Ellisman, "Multicolor and electron microscopic
imaging of connexin trafficking", Science, 296, 2002, 503-7.
S Boucher, SA Bennett, "Differential connexin expression, gap junction intercellular coupling, and hemichannel formation in NT2/D1 human
neural progenitors and terminally differentiated hNT neurons", J Neurosci Res, 72, 2003, 393-404.
F Zhang, B Crise, B Su, Y Hou, JK Rose, A Bothwell, K Jacobson, "Lateral diffusion of membrane-spanning and
glycosylphosphatidylinositol-linked proteins: toward establishing rules governing the lateral mobility of membrane proteins", J Cell Biol, 115,
1991, 75-84.
AP Quist, SK Rhee, H Lin, R Lal, "Physiological role of gap-junctional hemichannels. Extracellular calcium-dependent isosmotic volume
regulation.", J Cell Biol, 148, 2000, 1063-74.
EL Benedetti, I Dunia, M Recouvreur, P Nicolas, NM Kumar, H Bloemendal, "Structural organization of gap junctions as revealed by
freeze-fracture and SDS fracture-labeling", Eur J Cell Biol, 79, 2000, 575-82.
DF Hulser, B Rehkopf, O Traub, "Dispersed and aggregated gap junction channels identified by immunogold labeling of freeze-fractured
membranes", Exp Cell Res, 233, 1997, 240-51.
2004, 3-21.
RR Shivers, PD Bowman, "A freeze-fracture paradigm of the mechanism for delivery and insertion of gap junction particles into the plasma
membrane", J Submicrosc Cytol, 17, 1985, 199-203.
JE Contreras, HA Sanchez, EA Eugenin, D Speidel, M Theis, K Willecke, FF Bukauskas, MV Bennett, JC Saez, "Metabolic inhibition induces
opening of unapposed connexin 43 gap junction hemichannels and reduces gap junctional communication in cortical astrocytes in culture", Proc
Natl Acad Sci U S A, 99, 2002, 495-500.
SA John, R Kondo, SY Wang, JI Goldhaber, JN Weiss, "Connexin-43 hemichannels opened by metabolic inhibition", J Biol Chem, 274, 1999,
236-40.
Source reaction
This reaction was inferred from the corresponding reaction "Insertion of connexons in the plasma membrane forming hemi-channels" in species
Rattus norvegicus.
2004, 3-21.
Reaction
9.1.1.7 Formation of junctional channels
Authors
Editors
Matthews, L, 2007-04-12.
Description
Junctional channels are an assembly of two docked connexons on adjacent cells that permits direct communication of the cytoplasm in the two
cells as shown below. Proteins associated with GJs such as catenins (Wu et al., 2003, Shaw et al., 2007) and L-CAM (Musil et al., 1990) might
be required for connexon docking. Docking occurs through a tight interaction of the extracellular loops (Unger et al., 1999; Sosinsky and
Nicholson, 2005). Intramolecular disulfide bridges between the two extracellular loops (E1 and E2) of connexin polypeptides are important for the
correct three-dimensional structure of the extracellular loops (Foote et al., 1998)
References
VM Unger, NM Kumar, NB Gilula, M Yeager, "Three-dimensional structure of a recombinant gap junction membrane channel", Science, 283,
1999, 1176-80.
CI Foote, L Zhou, X Zhu, BJ Nicholson, "The pattern of disulfide linkages in the extracellular loop regions of connexin 32 suggests a model for
the docking interface of gap junctions", J Cell Biol, 140, 1998, 1187-97.
GI Fishman, AP Moreno, DC Spray, LA Leinwand, "Functional analysis of human cardiac gap junction channel mutants", Proc Natl Acad Sci U S
A, 88, 1991, 3525-9.
GE Sosinsky, BJ Nicholson, "Structural organization of gap junction channels", Biochim Biophys Acta, 1711, 2005, 99-125.
LS Musil, BA Cunningham, GM Edelman, DA Goodenough, "Differential phosphorylation of the gap junction protein connexin43 in junctional
communication-competent and -deficient cell lines", J Cell Biol, 111, 1990, 2077-88.
Reaction
9.1.1.8 Assembly of gap junction plaques
Authors
Editors
Matthews, L, 2007-03-27.
Description
Once transported to the plasma membrane, junctional channels aggregate into clusters forming gap junction plaques that may contain a few to
many thousands of individual channels and that vary in size from a few square nanometers to many square micrometers (Bruzzone et al. 1996;
Falk 2000; Severs et al. 2001). Gap junction plaques are involved in numerous processes including growth and differentiation (Loewenstein and
Rose 1992), pathological cell proliferation (Roger et al. 2004; Segretain et al. 2003) and spermatogenesis (Juneja et al. 1999; Plum et al. 2000).
The physiological importance of gap junction plaques is underscored by the diverse pathologies associated with connexin gene mutations (De
Maio et al. 2002). An arbitrary number (10) of channels is shown as aggregating in this reaction but the actual number may be hundreds to
thousands.
References
D Segretain, X Decrouy, J Dompierre, D Escalier, N Rahman, C Fiorini, B Mograbi, JP Siffroi, I Huhtaniemi, P Fenichel, G Pointis,
"Sequestration of connexin43 in the early endosomes: an early event of Leydig cell tumor progression", Mol Carcinog, 38, 2003, 179-87.
MM Falk, "Connexin-specific distribution within gap junctions revealed in living cells", J Cell Sci, 113, 2000, 4109-20.
M Wang, VM Berthoud, EC Beyer, "Connexin43 increases the sensitivity of prostate cancer cells to TNFalpha-induced apoptosis", J Cell Sci,
120, 2007, 320-9.
SC Juneja, KJ Barr, GC Enders, GM Kidder, "Defects in the germ line and gonads of mice lacking connexin43", Biol Reprod, 60, 1999, 1263-70.
1-27.
C Roger, B Mograbi, D Chevallier, JF Michiels, H Tanaka, D Segretain, G Pointis, P Fenichel, "Disrupted traffic of connexin 43 in human
testicular seminoma cells: overexpression of Cx43 induces membrane location and cell proliferation decrease", J Pathol, 202, 2004, 241-6.
A Plum, G Hallas, T Magin, F Dombrowski, A Hagendorff, B Schumacher, C Wolpert, J Kim, WH Lamers, M Evert, P Meda, O Traub, K Willecke,
"Unique and shared functions of different connexins in mice", Curr Biol, 10, 2000, 1083-91.
NJ Severs, S Rothery, E Dupont, SR Coppen, HI Yeh, YS Ko, T Matsushita, R Kaba, D Halliday, "Immunocytochemical analysis of connexin
expression in the healthy and diseased cardiovascular system", Microsc Res Tech, 52, 2001, 301-22.
WR Loewenstein, B Rose, "The cell-cell channel in the control of growth", Semin Cell Biol, 3, 1992, 59-79.
A De Maio, VL Vega, JE Contreras, "Gap junctions, homeostasis, and injury", J Cell Physiol, 191, 2002, 269-82.
Reaction
9.1.2 Gap junction degradation
Authors
Editors
Matthews, L, 2007-03-27.
Description
The half-life of Cx is very short (1 to 5h) compared to other junctional proteins (Laird et al., 1995 ; Fallon and Goudenough, 1981). Connexins are
targeted for degradation by the proteasome and the lysosome. Degradation appears to involve the phosphorylation of Connexins as well as their
interactions with other proteins (Piehl et al., 2007).
References
RF Fallon, DA Goodenough, "Five-hour half-life of mouse liver gap-junction protein", J Cell Biol, 90, 1981, 521-6.
9.1.2.1 Formation of annular gap junctions
Authors
Description
Gap junction plaque internalization and the disruption cell communication requires a reorganization of Cx molecular interactions. Proteins
including Dab-2, AP-2, Dynamin and Myosin VI associate with gap junction plaques permitting the internalisation of plaques after clathrin
association (Piehl et al., 2007).
References
K Jordan, R Chodock, AR Hand, DW Laird, "The origin of annular junctions: a mechanism of gap junction internalization", J Cell Sci, 114, 2001,
763-73.
PL Sorgen, HS Duffy, P Sahoo, W Coombs, M Delmar, DC Spray, "Structural changes in the carboxyl terminus of the gap junction protein
connexin43 indicates signaling between binding domains for c-Src and zonula occludens-1", J Biol Chem, 279, 2004, 54695-701.
D Segretain, C Fiorini, X Decrouy, N Defamie, JR Prat, G Pointis, "A proposed role for ZO-1 in targeting connexin 43 gap junctions to the
endocytic pathway", Biochimie, 86, 2004, 241-4.
9.1.2.1.1 Dab2 is recruited to the junctional plaques
Authors
Editors
Matthews, L, 2007-04-17.
Reviewers
Falk, MM, 2007-02-01.
Description
Dab2 is recruited to Cx43-based GJs possibly through a direct interaction between its N-terminal phosphotyrosine binding (PTB) domain and a
putative XPXY internalization motif found in the C-terminal tail of Cx43 as well as a number of other connexin family members (Piehl et al.,
2007).
References
Reaction
9.1.2.1.2 Dab2 interacts with clathrin
Authors
Editors
Matthews, L, 2007-04-17.
Reviewers
Falk, MM, 2007-02-01.
Description
The distal portion of Dab2 on its opposite end binds the globular N-terminal domain of clathrin heavy chains (Piehl et al., 2007).
References
Reaction
9.1.2.1.3 Dynamin is recruited to the gap junction plaque
Authors
Editors
Matthews, L, 2007-04-17.
Reviewers
Falk, MM, 2007-02-01.
Description
The GTPase dynamin, which functions in the completion of vesicle budding localizes in Cx43-based GJs and especially invaginating plaques
and AGJ vesicles (Piehl et al., 2007).
References
Reaction
9.1.2.2 Internalization of gap junction plaques
Authors

Editors
Matthews, L, 2007-04-12.
Reviewers
Falk, MM, 2007-02-01.
Description
GJ plaques, clusters of GJ channels, can be internalized to form large, double-membrane vesicles (aka AGJs). Internalized AGJ vesicles
subdivide into smaller vesicles that are subsequently degraded by endo/lysosomal pathways (Piehl et al., 2007).
References
Reaction
9.1.2.3 Lysosomal degradation of gap junction plaques
Authors
Editors
Matthews, L, 2007-04-23.
Description
Internalized GJ plaques are degraded by lysosomes. Lysosomal degradation appears to be the most common pathway of GJ degradation. (Qin
et al., 2003; Grinzberg and Gilula., 1979 ; Berthoud et al., 2004 ; and Leithe et al., 2006).
References
RD Ginzberg, NB Gilula, "Modulation of cell junctions during differentiation of the chicken otocyst sensory epithelium", Dev Biol, 68, 1979,
110-29.
H Qin, Q Shao, SA Igdoura, MA Alaoui-Jamali, DW Laird, "Lysosomal and proteasomal degradation play distinct roles in the life cycle of Cx43 in
gap junctional intercellular communication-deficient and -competent breast tumor cells", J Biol Chem, 278, 2003, 30005-14.
E Leithe, A Brech, E Rivedal, "Endocytic processing of connexin43 gap junctions: a morphological study", Biochem J, 393, 2006, 59-67.
VM Berthoud, PJ Minogue, JG Laing, EC Beyer, "Pathways for degradation of connexins and gap junctions", Cardiovasc Res, 62, 2004, 256-67.
Reaction
9.2 Regulation of gap junction activity
Authors
Editors
Matthews, L, 2007-04-17.
Description
Src is believed to suppress gap junction communication by phosphorylating Cx43.
9.2.1 c-src mediated regulation of Cx43 function and closure of gap junctions
Authors
Editors
Matthews, L, 2007-04-17.
Description
The kinases c-Src (Giepmans et al. 2001; Sorgen et al. 2004), PKc (Lin et al. 2003) and MAPK (Mograbi et al. 2003) play an essential role in the
phosphorylation of Cx which leads to its degradation. c-Src appears to associate with and phosphorylate Cx43 leading to closure of gap
junctions. Evidence suggests that v-src may activate MAPK, which in turn phosphorylates Cx43 on serine sites leading to channel gating (Zhou
et al. 1999).
References
D Lin, J Zhou, PS Zelenka, DJ Takemoto, "Protein kinase Cgamma regulation of gap junction activity through caveolin-1-containing lipid rafts",
Invest Ophthalmol Vis Sci, 44, 2003, 5259-68.
BN Giepmans, T Hengeveld, FR Postma, WH Moolenaar, "Interaction of c-Src with gap junction protein connexin-43. Role in the regulation of
cell-cell communication.", J Biol Chem, 276, 2001, 8544-9.
PL Sorgen, HS Duffy, P Sahoo, W Coombs, M Delmar, DC Spray, "Structural changes in the carboxyl terminus of the gap junction protein
connexin43 indicates signaling between binding domains for c-Src and zonula occludens-1", J Biol Chem, 279, 2004, 54695-701.
L Zhou, EM Kasperek, BJ Nicholson, "Dissection of the molecular basis of pp60(v-src) induced gating of connexin 43 gap junction channels", J
Cell Biol, 144, 1999, 1033-45.
B Mograbi, E Corcelle, N Defamie, M Samson, M Nebout, D Segretain, P Fenichel, G Pointis, "Aberrant Connexin 43 endocytosis by the
carcinogen lindane involves activation of the ERK/mitogen-activated protein kinase pathway", Carcinogenesis, 24, 2003, 1415-23.
9.2.1.1 Association of Cx43 with ZO-1
Authors
Editors
Matthews, L, 2007-01-07.
Description
Connexin-interacting proteins appear to function in regulating gap junction formation and communication. ZO-1 has been shown to alter the
membrane localization of Cx43 and plays a role in regulating Cx43-mediated gap junctional communication in osteoblastic cells (Laing et al.
2005). ZO-1 may function in the delivery of Cx43 from a lipid raft domain to gap junctional plaques, which may be an important regulatory step in
gap junction formation.
References
JG Laing, BC Chou, TH Steinberg, "ZO-1 alters the plasma membrane localization and function of Cx43 in osteoblastic cells", J Cell Sci, 118,
2005, 2167-76.
Source reaction
This reaction was inferred from the corresponding reaction "Association of Cx43 with ZO-1" in species Rattus norvegicus.
BN Giepmans, WH Moolenaar, "The gap junction protein connexin43 interacts with the second PDZ domain of the zona occludens-1 protein",
Curr Biol, 8, 1998, 931-4.
JG Laing, RN Manley-Markowski, M Koval, R Civitelli, TH Steinberg, "Connexin45 interacts with zonula occludens-1 and connexin43 in
osteoblastic cells", J Biol Chem, 276, 2001, 23051-5.
Reaction
9.2.1.2 c-src associates with Cx43 in gap junctions
Authors
Editors
Matthews, L, 2007-04-17.
Description
c-src has been shown to interact with Cx43 (Giepmans et al., 2001). Models describing v-src mediated Cx43 channel gating propose that the
initial interaction between v-src and Cx43 may occur via a SH3 domain interaction (see Lau 2005).
References
BN Giepmans, T Hengeveld, FR Postma, WH Moolenaar, "Interaction of c-Src with gap junction protein connexin-43. Role in the regulation of
cell-cell communication.", J Biol Chem, 276, 2001, 8544-9.
AF Lau, "c-Src: bridging the gap between phosphorylation- and acidification-induced gap junction channel closure", Sci STKE, 2005, 2005, pe33.
Reaction
9.2.1.3 Phosphorylation of Cx43 by c-src
Authors
Editors
Matthews, L, 2007-04-17.
Description
c-Src phosphorylates Cx43 on Tyr 265.
References
S Huang, T Dudez, I Scerri, MA Thomas, BN Giepmans, S Suter, M Chanson, "Defective activation of c-Src in cystic fibrosis airway epithelial
cells results in loss of tumor necrosis factor-alpha-induced gap junction regulation", J Biol Chem, 278, 2003, 8326-32.
Reaction
9.2.1.4 Closure of gap junction
Authors
Editors
Matthews, L, 2007-04-17.
Description
The closure of Cx43 gap junction channels is observed following src-mediated Cx43 phosphorylation.
References
F Spinella, L Rosano, V Di Castro, MR Nicotra, PG Natali, A Bagnato, "Endothelin-1 decreases gap junctional intercellular communication by
inducing phosphorylation of connexin 43 in human ovarian carcinoma cells", J Biol Chem, 278, 2003, 41294-301.
Reaction
10 Gene Expression
Authors
Kornblihtt, AR, Proudfoot, NJ, 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
Gene Expression covers the process of transcription of mRNA genes, the processing of pre-mRNA, and its subsequent translation to result in a
protein. The "expression" of non-protein-coding genes is not included in this section yet. However, the transcription of RNAs other than mRNA is
described in the section on transcription; in the sections 'RNA Polymerase I Transcription', and 'RNA Polymerase III Transcription'.
10.1 Generic Transcription Pathway
Reviewers
Freedman, LP, 2008-02-25.
Description
OVERVIEW OF TRANSCRIPTION REGULATION:
Detailed studies of gene transcription regulation in a wide variety of eukaryotic systems has revealed the general principles and mechanisms by
which cell- or tissue-specific regulation of differential gene transcription is mediated (reviewed in Naar, 2001. Kadonaga, 2004, Maston, 2006,
Barolo, 2002; Roeder, 2005, Rosenfeld, 2006). Of the three major classes of DNA polymerase involved in eukaryotic gene transcription,
Polymerase II generally regulates protein-encoding genes. Figure 1 shows a diagram of the various components involved in cell-specific
regulation of Pol-II gene transcription.
Core Promoter: Pol II-regulated genes typically have a Core Promoter where Pol II and a variety of general factors bind to specific DNA motifs:
i: the TATA box (TATA DNA sequence), which is bound by the "TATA-binding protein" (TBP).
ii: the Initiator motif (INR), where Pol II and certain other core factors bind, is present in many Pol II-regulated genes.
iii: the Downstream Promoter Element (DPE), which is present in a subset of Pol II genes, and where additional core factors bind.
The core promoter binding factors are generally ubiquitously expressed, although there are exceptions to this.
Proximal Promoter: immediately upstream (5') of the core promoter, Pol II target genes often have a Proximal Promoter region that spans up to
500 base pairs (b.p.), or even to 1000 b.p.. This region contains a number of functional DNA binding sites for a specific set of transcription
activator (TA) and transcription repressor (TR) proteins. These TA and TR factors are generally cell- or tissue-specific in expression, rather than
ubiquitous, so that the presence of their cognate binding sites in the proximal promoter region programs cell- or tissue-specific expression of the
target gene, perhaps in conjunction with TA and TR complexes bound in distal enhancer regions.
Distal Enhancer(s): many or most Pol II regulated genes in higher eukaryotes have one or more distal Enhancer regions which are essential for
proper regulation of the gene, often in a cell or tissue-specific pattern. Like the proximal promoter region, each of the distal enhancer regions
typically contain a cluster of binding sites for specific TA and/or TR DNA-binding factors, rather than just a single site.
Enhancers generally have three defining characteristics:
i: They can be located very long distances from the promoter of the target gene they regulate, sometimes as far as 100 Kb, or more.
ii: They can be either upstream (5') or downstream (3') of the target gene, including within introns of that gene.
iii: They can function in either orientation in the DNA.
Combinatorial mechanisms of transcription regulation: The specific combination of TA and TR binding sites within the proximal promoter and/or
distal enhancer(s) provides a "combinatorial transcription code" that mediates cell- or tissue-specific expression of the associated target gene.
Each promoter or enhancer region mediates expression in a specific subset of the overall expression pattern. In at least some cases, each
enhancer region functions completely independently of the others, so that the overall expression pattern is a linear combination of the expression
patterns of each of the enhancer modules.
Co-Activator and Co-Repressor Complexes: DNA-bound TA and TR proteins typically recruit the assembly of specific Co-Activator (Co-A) and
Co-Repressor (Co-R) Complexes, respectively, which are essential for regulating target gene transcription. Both Co-A's and Co-R's are
multi-protein complexes that contain several specific protein components.
Co-Activator complexes generally contain at lease one component protein that has Histone Acetyl Transferase (HAT) enzymatic activity. This
functions to acetylate Histones and/or other chromatin-associated factors, which typically increases that transcription activation of the target
gene. By contrast, Co-Repressor complexes generally contain at lease one component protein that has Histone De-Acetylase (HDAC)
enzymatic activity. This functions to de-acetylate Histones and/or other chromatin-associated factors. This typically increases the transcription
repression of the target gene.
Adaptor (Mediator) complexes: In addition to the co-activator complexes that assemble on particular cell-specific TA factors, - there are at least
two additional transcriptional co-activator complexes common to most cells. One of these is the Mediator complex, which functions as an
"adaptor" complex that bridges between the tissue-specific co-activator complexes assembled in the proximal promoter (or distal enhancers).
The human Mediator complex has been shown to contain at least 19 protein distinct components. Different combinations of these co-activator
proteins are also found to be components of specific transcription Co-Activator complexes, such as the DRIP, TRAP and ARC complexes
described below.
TBP/TAF complex: Another large Co-A complex is the "TBP-associated factors" (TAFs) that assemble on TBP (TATA-Binding Protein), which is
bound to the TATA box present in many promoters. There are at least 23 human TAF proteins that have been identified. Many of these are
ubiquitously expressed, but TAFs can also be expressed in a cell or tissue-specific pattern.
Specific Coactivator Complexes for DNA-binding Transcription Factors.
A number of specific co-activator complexes for DNA-binding transcription factors have been identified, including DRIP, TRAP, and ARC
(reviewed in Bourbon, 2004, Blazek, 2005, Conaway, 2005, and Malik, 2005). The DRIP co-activator complex was originally identified and
named as a specific complex associated with the Vitamin D Receptor member of the nuclear receptor family of transcription factors (Rachez,
1998). Similarly, the TRAP co-activator complex was originally identified as a complex that associates with the thyroid receptor (Yuan, 1998). It
was later determined that all of the components of the DRIP complex are also present in the TRAP complex, and the ARC complex (discussed
further below). For example, the DRIP205 and TRAP220 proteins were show to be identical, as were specific pairs of the other components of
these complexes (Rachez, 1999).
In addition, these various transcription co-activator proteins identified in mammalian cells were found to be the orthologues or homologues of the
Mediator ("adaptor") complex proteins (reviewed in Bourbon, 2004). The Mediator proteins were originally identified in yeast by Kornberg and
colleagues, as complexes associated with DNA polymerase (Kelleher, 1990). In higher organisms, Adapter complexes bridge between the basal
transcription factors (including Pol II) and tissue-specific transcription factors (TFs) bound to sites within upstream Proximal Promoter regions or
distal Enhancer regions (Figure 1). However, many of the Mediator homologues can also be found in complexes associated with specific
transcription factors in higher organisms. A unified nomenclature system for these adapter / co-activator proteins now labels them Mediator 1
through Mediator 31 (Bourbon, 2004). For example, the DRIP205 / TRAP220 proteins are now identified as Mediator 1 (Rachez, 1999), based
on homology with yeast Mediator 1.
Example Pathway: Specific Regulation of Target Genes During Notch Signaling:
One well-studied example of cell-specific regulation of gene transcription is selective regulation of target genes during Notch signaling. Notch
signaling was first identified in Drosophila, where it has been studied in detail at the genetic, molecular, biochemical and cellular levels (reviewed
in Justice, 2002; Bray, 2006; Schweisguth, 2004; Louvri, 2006). In Drosophila, Notch signaling to the nucleus is thought always to be mediated
by one specific DNA binding transcription factor, Suppressor of Hairless. In mammals, the homologous genes are called CBF1 (or RBPJkappa),
while in worms they are called Lag-1, so that the acronym "CSL" has been given to this conserved transcription factor family. There are at least
two human CSL homologues, which are now named RBPJ and RBPJL.
In Drosophila, Su(H) is known to be bifunctional, in that it represses target gene transcription in the absence of Notch signaling, but activates
target genes during Notch signaling. At least some of the mammalian CSL homologues are believed also to be bifunctional, and to mediate
target gene repression in the absence of Notch signaling, and activation in the presence of Notch signaling.
Notch Co-Activator and Co-Repressor complexes: This repression is mediated by at least one specific co-repressor complexes (Co-R) bound to
CSL in the absence of Notch signaling. In Drosophila, this co-repressor complex consists of at least three distinct co-repressor proteins:
Hairless, Groucho, and dCtBP (Drosophila C-terminal Binding Protein). Hairless has been show to bind directly to Su(H), and Groucho and
dCtBP have been shown to bind directly to Hairless (Barolo, 2002). All three of the co-repressor proteins have been shown to be necessary for
proper gene regulation during Notch signaling in vivo (Nagel, 2005).
In mammals, the same general pathway and mechanisms are observed, where CSL proteins are bifunctional DNA binding transcription factors
(TFs), that bind to Co-Repressor complexes to mediate repression in the absence of Notch signaling, and bind to Co-Activator complexes to
mediate activation in the presence of Notch signaling. However, in mammals, there may be multiple co-repressor complexes, rather than the
single Hairless co-repressor complex that has been observed in Drosophila.
During Notch signaling in all systems, the Notch transmembrane receptor is cleaved and the Notch intracellular domain (NICD) translocates to
the nucleus, where it there functions as a specific transcription co-activator for CSL proteins. In the nucleus, NICD replaces the Co-R complex
bound to CSL, thus resulting in de-repression of Notch target genes in the nucleus (Figure 2). Once bound to CSL, NICD and CSL proteins
recruit an additional co-activator protein, Mastermind, to form a CSL-NICD-Mam ternary co-activator (Co-A) complex. This Co-R complex was
initially thought to be sufficient to mediate activation of at least some Notch target genes. However, there now is evidence that still other
co-activators and additional DNA-binding transcription factors are required in at least some contexts (reviewed in Barolo, 2002).
Thus, CSL is a good example of a bifunctional DNA-binding transcription factor that mediates repression of specific targets genes in one context,
but activation of the same targets in another context. This bifunctionality is mediated by the association of specific Co-Repressor complexes vs.
specific Co-Activator complexes in different contexts, namely in the absence or presence of Notch signaling.
References
RG Roeder, "Transcriptional regulation and the role of diverse coactivators in animal cells", FEBS Lett, 579, 2005, 909-15.
NJ Justice, YN Jan, "Variations on the Notch pathway in neural development", Curr Opin Neurobiol, 12, 2002, 64-70.
C Rachez, BD Lemon, Z Suldan, V Bromleigh, M Gamble, AM NÃ¤Ã¤r, H Erdjument-Bromage, P Tempst, LP Freedman, "Ligand-dependent
transcription activation by nuclear receptors requires the DRIP complex", Nature, 398, 1999, 824-8.
GA Maston, SK Evans, MR Green, "Transcriptional regulatory elements in the human genome", Annu Rev Genomics Hum Genet, 7, 2006,
29-59.
AM NÃ¤Ã¤r, BD Lemon, R Tjian, "Transcriptional coactivator complexes", Annu Rev Biochem, 70, 2001, 475-501.
A Louvi, S Artavanis-Tsakonas, "Notch signalling in vertebrate neural development", Nat Rev Neurosci, 7, 2006, 93-102.
HM Bourbon, A Aguilera, AZ Ansari, FJ Asturias, AJ Berk, S Bjorklund, TK Blackwell, T Borggrefe, M Carey, M Carlson, JW Conaway, RC
Conaway, SW Emmons, JD Fondell, LP Freedman, T Fukasawa, CM Gustafsson, M Han, X He, PK Herman, AG Hinnebusch, S Holmberg, FC
Holstege, JA Jaehning, YJ Kim, L Kuras, A Leutz, JT Lis, M Meisterernest, AM Naar, K Nasmyth, JD Parvin, M Ptashne, D Reinberg, H Ronne, I
Sadowski, H Sakurai, M Sipiczki, PW Sternberg, DJ Stillman, R Strich, K Struhl, JQ Svejstrup, S Tuck, F Winston, RG Roeder, RD Kornberg, "A
unified nomenclature for protein subunits of mediator complexes linking transcriptional regulators to RNA polymerase II", Mol Cell, 14, 2004,
553-7.
JW Conaway, L Florens, S Sato, C Tomomori-Sato, TJ Parmely, T Yao, SK Swanson, CA Banks, MP Washburn, RC Conaway, "The
mammalian Mediator complex", FEBS Lett, 579, 2005, 904-8.
F Schweisguth, "Notch signaling activity", Curr Biol, 14, 2004, R129-38.
SJ Bray, "Notch signalling: a simple pathway becomes complex", Nat Rev Mol Cell Biol, 7, 2006, 678-89.
MG Rosenfeld, VV Lunyak, CK Glass, "Sensors and signals: a coactivator/corepressor/epigenetic code for integrating signal-dependent
programs of transcriptional response", Genes Dev, 20, 2006, 1405-28.
JT Kadonaga, "Regulation of RNA polymerase II transcription by sequence-specific DNA binding factors", Cell, 116, 2004, 247-57.
S Malik, RG Roeder, "Dynamic regulation of pol II transcription by the mammalian Mediator complex", Trends Biochem Sci, 30, 2005, 256-63.
S Barolo, JW Posakony, "Three habits of highly effective signaling pathways: principles of transcriptional control by developmental cell
signaling", Genes Dev, 16, 2002, 1167-81.
E Blazek, G Mittler, M Meisterernst, "The mediator of RNA polymerase II", Chromosoma, 113, 2005, 399-408.
10.1.1 Formation of ARC coactivator complex
Reviewers
Description
ARC co-activator complex and assembly
The ARC co-activator complex is a subset of 18 proteins from the set of at least 31 Mediator proteins that, in different combinations, form
"Adapter" complexes in human cells. Adapter complexes bridge between the basal transcription factors (including Pol II) and tissue-specific
transcription factors (TFs) bound to sites within upstream Proximal Promoter regions or distal Enhancer regions (reviewed in Maston, 2006 and
Naar, 2001).
The ARC complex was originally identified and named as a co-activator complex associated with transcription activator proteins (reviewed in
Malik, 2005 and references therein). It was subsequently determined that many of the components of the ARC complex are also in the DRIP
complex, and in the TRAP complex..
The ARC complex contains the following 14 proteins, which also are common to the DRIP and TRAP complexes: MED1, MED4, MED6, MED7,
MED10, MED12, MED13, MED14, MED16, MED17, MED23, MED24, CDK8, CycC.
The ARC complex also contains 4 additional, ARC-specific components, which are now called: MED8, MED15, MED25, and MED 26 in the
unified nomenclature scheme (Bourbon, 2004).
Mediator complex proteins in yeast, first identified by Kornberg and colleagues (Kelleher, 1990). The unified nomenclature system for these
adapter / co-activator proteins now labels them Mediator 1 through Mediator 31 (Bourbon, 2004).
The order of addition of the ARC proteins during complex assembly is not fully determined, and may vary in different cell contexts. Therefore,
ARC complex assembly is represented as a single reaction event, in which all 19 components assemble simultaneously into the ARC
co-activator complex.
References
3rd Kelleher RJ, PM Flanagan, RD Kornberg, "A novel mediator between activator proteins and the RNA polymerase II transcription apparatus",
Cell, 61, 1990, 1209-15.
C Rachez, Z Suldan, J Ward, CP Chang, D Burakov, H Erdjument-Bromage, P Tempst, LP Freedman, "A novel protein complex that interacts
with the vitamin D3 receptor in a ligand-dependent manner and enhances VDR transactivation in a cell-free system", Genes Dev, 12, 1998,
1787-800.
29-59.
CX Yuan, M Ito, JD Fondell, ZY Fu, RG Roeder, "The TRAP220 component of a thyroid hormone receptor- associated protein (TRAP)
coactivator complex interacts directly with nuclear receptors in a ligand-dependent fashion", Proc Natl Acad Sci U S A, 95, 1998, 7939-44.
553-7.
S Malik, RG Roeder, "Dynamic regulation of pol II transcription by the mammalian Mediator complex", Trends Biochem Sci, 30, 2005, 256-63.
Reaction
10.1.2 Formation of DRIP coactivator complex
Reviewers
Description
DRIP co-activator complex and assembly
The DRIP co-activator complex is a subset of 14 proteins from the set of at least 31 Mediator proteins that, in different combinations, form
"Adapter" complexes. Adapter complexes bridge between the basal transcription factors (including Pol II) and tissue-specific transcription factors
(TFs) bound to sites within upstream Proximal Promoter regions or distal Enhancer regions (reviewed in Maston, 2006 and Naar, 2001).
The DRIP complex was originally identified and named as a co-activator complex associated with the Vitamin D Receptor member of the nuclear
receptor family of transcription factors (Rachez, 1998). It was later determined that all of the components of the DRIP complex were also in the
TRAP complex, and the ARC complex.
The DRIP complex contains the following 14 proteins, which also are common to the ARC and TRAP complexes: MED1, MED4, MED6, MED7,
All of the DRIP adapter complex components are present in the ARC adapter complex, but the ARC complex also has 4 additional components
(Rachez, 1999). These ARC-specific components are now called: MED8, MED15, MED25, and MED 26 in the unified nomenclature scheme
(Bourbon, 2004).
Similarly, all 14 of the DRIP adapter complex components are present in the TRAP adapter complex, but the TRAP complex also has 4
additional components (Bourbon, 2004), These TRAP-specific components are now called: MED20, MED27, MED30, and MED 31 in the unified
nomenclature scheme.
Mediator complex identified in yeast, first identified by Kornberg and colleagues (Kelleher, 1990).
References
Cell, 61, 1990, 1209-15.
1787-800.
29-59.
AM NÃ¤Ã¤r, BD Lemon, R Tjian, "Transcriptional coactivator complexes", Annu Rev Biochem, 70, 2001, 475-501.
553-7.
Reaction
10.1.3 Formation of TRAP coactivator complex
Reviewers
Description
TRAP co-activator complex and assembly
The TRAP co-activator complex is a subset of 18 proteins from the set of at least 31 Mediator proteins that, in different combinations and in
different contexts, form specific co-activator or "Adapter" complexes in human cells. These complexes bridge between the basal transcription
factors (including Pol II) and tissue-specific transcription factors (TFs) bound to sites within upstream Proximal Promoter regions or distal
Enhancer regions (reviewed in Maston, 2006 and Naar, 2001).
The TRAP complex was originally identified and named as a co-activator complex associated with the Thyroid Hormone Receptor member of the
nuclear receptor family of transcription factors (Yuan, 1998). It was later determined that many of the components of the TRAP complex are also
in the DRIP complex, and in the ARC complex.
The TRAP complex contains the following 14 proteins, which also are common to the DRIP and ARC complexes: MED1, MED4, MED6, MED7,
The TRAP complex also contains 4 additional components, which are now called: MED20, MED27, MED30, and MED 31 in the unified
nomenclature scheme (Bourbon, 2004).
Mediator complex proteins in yeast, first identified by Kornberg and colleagues (Kelleher, 1990). The unified nomenclature system for these
adapter / co-activator proteins now labels them Mediator 1 through Mediator 31 (Bourbon, 2004).
The order of addition of the TRAP proteins during complex assembly is not fully determined, and may vary in different cell contexts. Therefore,
TRAP co-activator complex assembly is represented as a single reaction event, in which all 18 components assemble simultaneously into the
TRAP co-activator complex.
References
Cell, 61, 1990, 1209-15.
1787-800.
553-7.
Reaction
10.2 Formation and Maturation of mRNA Transcript
Authors
Editors
Joshi-Tope, G, 0000-00-00.
Description
Before a gene transcript is ready to be transported out of the nucleus, it has to undergo three major processing events to produce a fully
translatable mRNA. These comprise capping, splicing out of introns from within the body of the pre-mRNA, and the generation of a 3' end, by
cleavage, and except in the case of histone pre-mRNAs, polyadenylation. Although each of these reactions is a biochemically distinct process,
these processes are interlinked and hence, influence one another's specificity and efficiency. On the other hand, most mRNA processing
reactions occur co-transcriptionally. This is particularly important in very long genes where a strictly post-transcriptional processing would imply
the existence of extremely long primary transcript molecules that would be susceptible to degradation. The co-transcriptional nature of
pre-mRNA processing does not necessarily imply a functional coupling between the transcription and mRNA processing machineries. In some
cases it may simply reflect that processing reactions occur during transcription because they are relatively fast compared with the time it takes to
transcribe a gene to its end. In other cases a tight link exists between a particular processing reaction and the transcription process, due to the
ability of the carboxy-terminal (CTD) of RNA polymerase II largest subunit to bind or recruit processing factors. The CTD consists of 52 heptad
repeats (YSPTSPS). Specific phosphorylation/dephosphorylation patterns of serines 2 and 5 are critical for CTD function in coupling. CTD
deletions that do not inactivate transcription significantly decrease the efficiency of capping, splicing and polyadenylation.
The export of mRNA from the nucleus and mRNA splicing are also coupled. Mature mRNAs generated by splicing are more efficiently exported
than their identical counterparts transcribed from a complementary DNA (cDNA). This effect of splicing on export is due to the recruitment of the
mRNA export factor ALY to the mRNA during the splicing reaction, which in turn delivers the mRNP to the nuclear pore for export.
10.2.1 RNA Polymerase II Transcription Pre-Initiation
Authors
Reinberg, D, 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
Formation of the pre-initiation complex

References
G Orphanides, T Lagrange, D Reinberg, "The general transcription factors of RNA polymerase II.", Genes Dev, 10, 1997, 2657-83.
10.2.1.1 Recognition and Binding of Core Promoter Elements by TFIID
Authors
Reinberg, D, 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
Although TBP (TATA box binding factor) is necessary and sufficient for initiation of basal transcription, the other subunits of the general
transcription factor TFIID, the TBP-associated factors, are required for response to transcriptional activators. TBP binds to the TATA box (a core
promoter element), and bends the DNA 80 degrees toward the major groove. This conformation of TBP-TATA box provides the proper topology
for the binding of the general transcription factor TFIIB.
Transcriptional activators function by affecting the kinetics of binding of TBP to the promoter DNA.
References
N Hernandez, "TBP, a universal eukaryotic transcription factor?", Genes Dev, 7, 1993, 1291-308.
Reaction
10.2.1.2 Binding of TFIIA and TFIIB to the pol II promoter:TFIID complex

Authors
Reinberg, D, 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
The general transcription factor TFIIB is a single polypeptide of approximately 35 kDa. There is a Zn-binding domain near the N terminus of
TFIIB, and the C-terminal domain encompasses two imperfect repeats; between the N and C termini is a phylogenetically conserved region. The
C terminus interacts with TBP and RNA Polymerase II, whereas the N terminus interacts with factor TFIIF and RNA polymerase II. TFIIB is a
sequence-specific factor, and it interacts with the BRE element within the promoter.
TFIIB interacts with the Rpb1 subunit of RNA polymerase II to define transcription strat sites. Several activators directly bind TFIIB, and stimulate
transcription. The N-terminus and the C-terminus can participate in intramolecular interactions, and this can be disrupted by specific activators
by causing a conformational change in TFIIB.
TFIIA also binds the preinitiation complex along with TFIIB. However, TFIIA is not required for accurate initiation, but rather functions as a
coactivator of transcription.
References
Reaction
10.2.1.3 Recruitment of RNA Polymerase II Holoenzyme by TFIIF to the pol II promoter:TFIID:TFIIA:TFIIB complex
Authors
Reinberg, D, 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
The general transcription factor TFIIF has a high affinity for the RNA Polymerase II holoenzyme. TFIIF stabilizes the preinitiation complex, and
suppresses non-specific binding of RNA Pol II to DNA, and is thus critical for start site recognition.
References
Reaction
10.2.1.4 Binding of TFIIE to the growing preinitiation complex
Authors
Reinberg, D, 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
Factor TFIIE enters the preinitiation complex after TFIIF recruits RNA Polymerase II. TFIIE is composed of two subunits of 56 kDA and 34 kDa.
TFIIE facilitates the recruitment of factor TFIIH to the preinitiation complex, and it also stimulates the phosphorylation of the RNA Polymerase II
CTD by TFIIH.
References
Reaction
10.2.1.5 Formation of the closed pre-initiation complex
Authors
Reinberg, D, 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
The binding of TFIIH completes the assembly of the preinitiation complex (PIC) for RNA Polymerase II transcription. Although RNA polymerase
binds the TATA box on the promoter DNA, no initiation of transcription occurs until TFIIH is bound to the PIC. TFIIH is the only factor with known
enzymatic activities.
References
Reaction
10.2.2 RNA Polymerase II Transcription Initiation And Promoter Clearance
10.2.2.1 RNA Polymerase II Promoter Opening: First Transition
Authors
Timmers, H. T. M., 2003-09-11.

Editors
Joshi-Tope, G, 0000-00-00.
Description
After assembly of the complete RNA polymerase II-preinitiation complex, the next step is separation of the two DNA strands. This isomerization
step is known as the closed-to-open complex transition and occurs prior to the initiation of mRNA synthesis. In the RNA polymerase II system
this step requires the hydrolysis of ATP or dATP into Pi and ADP or dADP (in contrast to the other RNA polymerase systems) and is catalyzed
by the XPB subunit of TFIIH. The region of the promoter, which becomes single-stranded , spans from Ã¢â‚¬"10 to +2 relative to the transcription
start site.
Negative supercoiling in the promoter region probably induces transient opening events and can alleviate requirement of TFIIE, TFIIH and
ATP-hydrolysis for open complex formation. ATP is also used in this step by the cdk7-subunit of TFIIH to phosphorylate the heptad repeats of
the C-terminal domain of the largest subunit of RNA polymerase II (RPB1) on serine-2
References
FC Holstege, U Fiedler, HT Timmers, "Three transitions in the RNA polymerase II transcription complex during initiation.", EMBO J, 16, 1998,
7468-80.
D Bunick, R Zandomeni, S Ackerman, R Weinmann, "Mechanism of RNA polymerase II--specific initiation of transcription in vitro: ATP
requirement and uncapped runoff transcripts.", Cell, 29, 1983, 877-86.
F Tirode, D Busso, F Coin, JM Egly, "Reconstitution of the transcription factor TFIIH: assignment of functions for the three enzymatic subunits,
XPB, XPD, and cdk7.", Mol Cell, 3, 1999, 87-95.
JD Parvin, PA Sharp, "DNA topology and a minimal set of basal factors for transcription by RNA polymerase II.", Cell, 73, 1993, 533-40.
JA Goodrich, R Tjian, "Transcription factors IIE and IIH and ATP hydrolysis direct promoter clearance by RNA polymerase II.", Cell, 77, 1994,
145-56.
U Fiedler, HT Marc Timmers, "Peeling by binding or twisting by cranking: models for promoter opening and transcription initiation by RNA
polymerase II.", Bioessays, 22, 2000, 316-26.
W Wang, M Carey, JD Gralla, "Polymerase II promoter activation: closed complex formation and ATP-driven start site opening.", Science, 255,
1992, 450-3.
Reaction
10.2.2.2 RNA Polymerase II Transcription Initiation
Authors
Timmers, H. T. M., 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
Formation of the open complex exposes the template strand to the catalytic center of the RNA polymerase II enzyme. This facilitates formation
of the first phosphodiester bond, which marks transcription initiation. As a result of this, the TFIIB basal transcription factor dissociates from the
initiation complex.
The open transcription initiation complex is unstable and can revert to the closed state. Initiation at this stage requires continued
(d)ATP-hydrolysis by TFIIH. Dinucleotide transcripts are not stably associated with the transcription complex. Upon dissociation they form
abortive products. The transcription complex is also sensitive to inhibition by small oligo-nucleotides.
Dinucleotides complementary to position -1 and +1 in the template can also direct first phosphodiester bond formation. This reaction is
independent on the basal transcription factors TFIIE and TFIIH and does not involve Ã¢â‚¬Å"fullÃ¢â‚¬Â• open complex formation. This reaction
is sensitive to inhibition by single-stranded oligonucleotides.
References
7468-80.
JF Kugel, JA Goodrich, "Translocation after synthesis of a four-nucleotide RNA commits RNA polymerase II to promoter escape.", Mol Cell Biol,
22, 2002, 762-73.
L Zawel, KP Kumar, D Reinberg, "Recycling of the general transcription factors during RNA polymerase II transcription.", Genes Dev, 9, 1995,
1479-90.
DS Luse, GA Jacob, "Abortive initiation by RNA polymerase II in vitro at the adenovirus 2 major late promoter.", J Biol Chem, 262, 1987,
14990-7.
FC Holstege, PC van der Vliet, HT Timmers, "Opening of an RNA polymerase II promoter occurs in two distinct steps and requires the basal
transcription factors IIE and IIH.", EMBO J, 15, 1996, 1666-77.
10.2.2.2.1 NTP Binds Active Site of RNA Polymerase II
Description
At the beginning of this reaction, 1 molecule of 'pol II open pre-initiation complex', and 2 molecules of 'NTP' are present. At the end of this
reaction, 1 molecule of 'Pol II initiation complex' is present.

Reaction
10.2.2.2.2 Nucleophillic Attack by 3'-hydroxyl Oxygen of nascent transcript on the Alpha Phosphate of NTP
Description
At the beginning of this reaction, 1 molecule of 'Pol II initiation complex' is present. At the end of this reaction, 1 molecule of 'Pol II Initiation
complex with phosphodiester-PPi intermediate' is present.
Reaction
10.2.2.2.3 Newly Formed Phosphodiester Bond Stabilized and PPi Released
Description
At the beginning of this reaction, 1 molecule of 'Pol II Initiation complex with phosphodiester-PPi intermediate' is present. At the end of this
reaction, 1 molecule of 'pyrophosphate', and 1 molecule of 'pol II transcription complex' are present.
Reaction
10.2.2.3 RNA Polymerase II Promoter Escape
Authors
Timmers, H. T. M., 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
RNA Polymerase II promoter escape occurs after the first phosphodiester bond has been created.
References
7468-80.
10.2.2.3.1 Addition of the third nucleotide on the nascent transcript
Authors
Timmers, H. T. M., 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
Formation of the second phosphodiester bond creates a 3-nt product. This short transcript is still loosely associated with the RNA polymerase II
initiation complex and can dissociate to yield abortive products, which are not further extended. The transcription complex still requires continued
ATP-hydrolysis by TFIIH and remains sensitive to single-stranded oligo-nucleotide inhibition.
The open region (Ã¢â‚¬Å"transcription bubbleÃ¢â‚¬Â•) expands concomitant with the site of RNA-extension. In this case this region spans
positions -9 to +3.
References
7468-80.
22, 2002, 762-73.
Reaction
10.2.2.3.2 Addition of the fourth nucleotide on the Nascent Transcript: Second Transition
Authors
Timmers, H. T. M., 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
Formation of the third phosphodiester bond creates a 4-nt product. This commits the initiation complex to promoter escape. The short 4-nt
transcript is still loosely associated with the RNA polymerase II initiation complex and can dissociate to yield abortive products, which are not
further extended. Inhibition of ATP-hydrolysis by TFIIH does not lead to collapse of the open region any longer. The transcription complex has
lost the sensitivity to single-stranded oligo-nucleotide inhibition. However, ATP-hydrolysis and TFIIH are required for efficient promoter escape.
positions -9 to +4.
References
7468-80.
22, 2002, 762-73.
Reaction
10.2.2.3.3 Addition of Nucleotides 5 through 9 on the growing Transcript
Authors
Timmers, H. T. M., 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
Formation of the second phosphodiester bond creates a 3-nt product. This transcript is still loosely associated with the RNA polymerase II
initiation complex and can dissociate to yield abortive products, which are not further extended. At this stage pausing by RNA polymerase II may
result in repeated slippage and reextension of the nascent RNA. The transcription complex still requires continued ATP-hydrolysis by TFIIH for
efficient promoter escape. Basal transcription factor TFIIE dissociates from the initiation complex before position +10.
Basal transcription factor TFIIF may reassociate and can stimulate transcription elongation at multiple stages. The open region
(Ã¢â‚¬Å"transcription bubbleÃ¢â‚¬Â•) expands concomitant with the site of RNA-extension, eventually reaching an open region from -9 to +9.
References
7468-80.
A Dvir, RC Conaway, JW Conaway, "A role for TFIIH in controlling the activity of early RNA polymerase II elongation complexes.", Proc Natl
Acad Sci U S A, 94, 1997, 9006-10.
1479-90.
M Pal, DS Luse, "Strong natural pausing by RNA polymerase II within 10 bases of transcription start may result in repeated slippage and
reextension of the nascent RNA.", Mol Cell Biol, 22, 2001, 30-40.
Reaction
10.2.2.3.4 Addition of nucleotides 10 and 11 on the growing transcript: Third Transition
Authors
Timmers, H. T. M., 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
Formation of phosphodiester bonds nine and ten creates RNA products, which do not dissociate from the RNA pol II initiation complex. The
transcription complex has enter the productive elongation phase. TFIIH and ATP-hydrolysis are required for efficient promoter escape. The open
region (Ã¢â‚¬Å"transcription bubbleÃ¢â‚¬Â•) expands concomitant with the site of RNA-extension. The region upstream from the transcription
start site (-9 to -3) collapses to the double-stranded state. TFIIH remains associated to the RNA pol II initiation complex.
References
7468-80.
1479-90.
Reaction
10.2.2.3.5 Addition of nucleotides between position +11 and +30
Authors
Timmers, H. T. M., 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
RNA polymerase II transcription complexes are susceptible to transcriptional stalling and arrest, when extending nascent transcripts to 30-nt.
This susceptibility depends on presence on down-stream DNA, the particular DNA-sequence of the template and presence of transcription
factors. Transcription factor TFIIH remains associated to the RNA pol II elongation complex until position +30. At this stage transcription
elongation factor TFIIS can rescue stalled transcription elongation complexes. The transcription bubble varies between 13- and 22-nt in size.
References
M Pal, D McKean, DS Luse, "Promoter clearance by RNA polymerase II is an extended, multistep process strongly affected by sequence.", Mol
Cell Biol, 21, 2001, 5815-25.
U Fiedler, HT Timmers, "Analysis of the open region of RNA polymerase II transcription complexes in the early phase of elongation.", Nucleic
Acids Res, 29, 2001, 2706-14.
1479-90.
A Dvir, S Tan, JW Conaway, RC Conaway, "Promoter escape by RNA polymerase II. Formation of an escape-competent transcriptional
intermediate is a prerequisite for exit of polymerase from the promoter.", J Biol Chem, 272, 1997, 28175-8.
Reaction
10.2.3 mRNA Capping
Description
The 5'-ends of all eukaryotic pre-mRNAs studied thus far are converted to cap structures. The cap is thought to influence splicing of the first
intron, and is bound by 'cap-binding' proteins, CBP80 and CBP20, in the nucleus. The cap is important for translation initiation, and it also
interacts with the poly(A)terminus, via proteins, resulting in circularization of the mRNA to facilitate multiple rounds of translation. The cap is also
important for mRNA stability, protecting it from 5' to 3' nucleases, and is required for mRNA export to the cytoplasm.
The capping reaction usually occurs very rapidly on nascent transcripts; after the synthesis of only a few nucleotides by RNA polymerase II. The
capping reaction involves the conversion of the 5'-end of the nascent transcript from a triphosphate to a diphosphate by a RNA
5'-triphosphatase, followed by the addition of a guanosine monophosphate by the mRNA guanylyltransferase, to form a 5'-5'-triphosphate
linkage. This cap is then methylated by 2'-O-methyltransferases.
References
K Mizumoto, Y Kaziro, "Messenger RNA capping enzymes from eukaryotic cells.", Prog Nucleic Acid Res Mol Biol, 34, 1988, 1-28.
NJ Proudfoot, A Furger, MJ Dye, "Integrating mRNA processing with transcription.", Cell, 108, 2002, 501-12.
AJ Shatkin, JL Manley, "The ends of the affair: capping and polyadenylation.", Nat Struct Biol, 7, 2000, 838-42.
D Bentley, "Coupling RNA polymerase II transcription with pre-mRNA processing.", Curr Opin Cell Biol, 11, 1999, 347-51.
10.2.3.1 Capping complex formation
Authors
Buratowski, S, 2003-10-15.
Editors
Description
The capping enzyme binds the 5'-end of the nascent transcript soon after it is synthesized on the DNA template, and results in the formation of
the capping complex along with the C-terminal domain of RNA polymerase II, and Spt5.
Reaction
10.2.3.2 Hydrolysis of the 5'-end of the nascent transcript by the capping enzyme
Authors
Description
After the capping complex is formed, the RNA triphosphatase activity of the capping enzyme hydrolyzes the 5'-end phosphate group of the
nascent mRNA transcript to a diphosphate.
The RNA triphosphatase (RTP) domain of mammalian capping enzyme is a member of a superfamily of phosphatases that include the protein
tyrosine phosphatases, some lipid phosphatases, and several nucleic acid phosphatases. This family uses a conserved nucleophilic cysteine
residue to attack the target phosphate. A transient phospho-cysteinyl enzyme intermediate is then hydrolyzed to regenerate the enzyme active
site. It should be noted that while higher eukaryotic capping enzymes use PTP-like triphosphatase domains, the yeast triphosphatases are a
completely different class of enzymes. The yeast RTPs are metal-dependent phosphatases. RNA 5'-triphosphatase (RTP) catalyzed first
reaction can be represented as:pppN(pN)n + GTP -> ppN(pN)n + Pi; (n=20-25)
References
T Yamada-Okabe, R Doi, O Shimmi, M Arisawa, H Yamada-Okabe, "Isolation and characterization of a human cDNA for mRNA 5'-capping
enzyme.", Nucleic Acids Res, 26, 1998, 1700-6.
Reaction
10.2.3.3 Formation of the CE:GMP intermediate complex
Authors
Description
A highly conserved lysine within the guanylyltransferase (GT) site of the mRNA capping enzyme attacks the alpha-phosphate of GTP. An
enzyme-GMP covalent intermediate is formed.
References
Reaction
10.2.3.4 Transfer of GMP from the capping enzyme GT site to 5'-end of mRNA
Authors
Description
The diphosphate 5'-end of the mRNA is joined to the GMP, releasing it from the enzyme. At this time, it is unclear how the RNA diphosphate end
is transferred from the active site of the triphosphatase to the guanylyltransferase site. The covalent enzyme-GMP complex can form in the
absence of RNA.
Guanylyltransferase (GT) catalyzed second reaction can be represented as:ppN(pN)n + GTP -> GpppN(pN)n + PPi
References
Reaction
10.2.3.5 Dissociation of transcript with 5'-GMP from GT
Authors
Description
GMP capped mRNA transcript dissociates from GT for further modification.
Reaction
10.2.3.6 Methylation of GMP-cap by RNA Methyltransferase
Authors
Description
In the final step of the capping reaction, the methyltransferase takes a methyl group from S-adenosyl-methionine to the N7 position of the cap
guanine. N7G-methyltransferase (MT) mediated reaction can be represented as:
GpppN(pN)n + S-adenosylmethionine (Adomet) ->m7GpppN(pN)n + S-adenosylhomocysteine (Adohcy).
References
T Tsukamoto, Y Shibagaki, Y Niikura, K Mizumoto, "Cloning and characterization of three human cDNAs encoding mRNA
(guanine-7-)-methyltransferase, an mRNA cap methylase.", Biochem Biophys Res Commun, 251, 1998, 27-34.
Reaction
10.2.3.7 Recognition and binding of the mRNA cap by the cap-binding complex
Authors
Editors
Description
The cap binding complex binds to the methylated GMP cap on the nascent mRNA transcript.
Reaction
10.2.4 Elongation and Processing of Capped Transcripts
10.2.4.1 Elongation of Intron-Containing Transcripts and co-transcriptional mRNA splicing
Authors
Editors
Joshi-Tope, G, 0000-00-00.
Description
Co-transcriptional pre-mRNA splicing is not obligatory. Pre-mRNA splicing begins co-transcriptionally and often continues post-transcriptionally.
Human genes contain an average of nine introns per gene, which cannot serve as splicing substrates until both 5' and 3' ends of each intron are
synthesized. Thus the time that it takes for pol II to synthesize each intron defines a minimal time and distance along the gene in which splicing
factors can be recruited. The time that it takes for pol II to reach the end of the gene defines the maximal time in which splicing could occur
co-transcriptionally. Thus, the kinetics of transcription can affect the kinetics of splicing.
10.2.4.1.1 RNA Polymerase II Transcription Elongation
Authors
Conaway, J, Conaway, R, 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
The mechanisms governing the process of elongation during eukaryotic mRNA synthesis are being unraveled by recent studies. These studies
have led to the expected discovery of a diverse collection of transcription factors that directly regulate the activities of RNA Polymerase II and
unexpected discovery of roles for many elongation factors in other basic processes like DNA repair, recombination, etc. The transcription
machinery and structural features of the major RNA polymerases are conserved across species. The genes active during elongation fall under
different classes like, housekeeping, cell-cycle regulated, development and differentiation specific genes etc. The list of genes involved in
elongation has been growing in recent times, and include: -TFIIS,DSIF, NELF, P-Tefb etc. that are involved in drug induced or
sequence-dependent arrest - TFIIF, ELL, elongin, elongator etc. that are involved in increasing the catalytic rate of elongation by altering the Km
and/or the Vmax of Pol II -FACT, Paf1 and other factors that are involved chromatin modification - DNA repair proteins, RNA processing and
export factors, the 19S proteasome and a host of other factors like Spt5-Spt5, Paf1, and NELF complexes, FCP1P etc. (Arndt and Kane, 2003).
Elongation also represents processive phase of transcription in which the activities of several mRNA processing factors are coupled to
transcription through their binding to RNA polymerase (Pol II). One of the key events that enables this interaction is the differential
phosphorylation of Pol II CTD. Phosphorylation pattern of CTD changes during transcription, most significantly at the beginning and during
elongation process. TFIIH-dependent Ser5 phosphorylation is observed primarily at promoter regions while P-Tefb mediated Ser2
phosphorylation is seen mainly in the coding regions, during elongation. Experimental evidence suggests a dynamic association of RNA
processing factors with differently modified forms of the polymerase during the transcription cycle. (Komarnitsky et al., 2000). [Komarnitsky et al
2000, Arndt & Kane 2003, Shilatifard et al 2003]
References
P Komarnitsky, EJ Cho, S Buratowski, "Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during
transcription.", Genes Dev, 14, 2000, 2452-60.
KM Arndt, CM Kane, "Running with RNA polymerase: eukaryotic transcript elongation.", Trends Genet, 19, 2003, 543-50.
A Shilatifard, RC Conaway, JW Conaway, "The RNA polymerase II elongation complex.", Annu Rev Biochem, 72, 2003, 693-715.
10.2.4.1.1.1 Formation of the Early Elongation Complex
Authors
Description
Transcription elongation by RNA polymerase II (RNAPII) is controlled by a number of trans-acting transcription elongation factors as well as by
cis-acting elements. Transcription elongation is a rate-limiting step for proper mRNA production in which the phosphorylation of Pol II CTD is a
crucial biochemical event. The role of CTD phosphorylation in transcription by Pol II is greatly impaired by protein kinase inhibitors such as
5,6-dichloro-1- ribofuranosylbenzimidazole (DRB), which block CTD phosphorylation and induce arrest of elongating Pol II. DRB-sensitive
activation Pol II CTD during elongation has enabled the isolation of two sets of factors -Negative Elongation Factors (NELF) and DRB sensitivity
inducing factor (DSIF). P-Tefb is a DRB-sensitive, cyclin-dependent CTD kinase composed of Cdk9 that carries out Serine-2 phosphorylation of
Pol II CTD during elongation.
The mechanism by which DSIF, NELF and P-TEFb act together in Pol II-regulated elongation is yet to be fully understood. Various biochemical
evidences point to a model in which DSIF and NELF negatively regulate elongation through interactions with polymerase containing a
hypophosphorylated CTD. Subsequent phosphorylation of the Pol II CTD by P-Tefb might promote elongation by inhibiting interactions of DSIF
and NELF with the elongation complex.
References
JW Conaway, A Shilatifard, A Dvir, RC Conaway, "Control of elongation by RNA polymerase II.", Trends Biochem Sci, 25, 2000, 375-80.
Y Yamaguchi, N Inukai, T Narita, T Wada, H Handa, "Evidence that negative elongation factor represses transcription elongation through binding
to a DRB sensitivity-inducing factor/RNA polymerase II complex and RNA.", Mol Cell Biol, 22, 2002, 2918-27.
10.2.4.1.1.1.1 Hypophosphorylation of RNA Pol II CTD by FCP1P protein
Authors
Description
FCP1 dephosphorylates RNAP II in ternary elongation complexes as well as in solution and, therefore, is thought to function in the recycling of
RNAP II during the transcription cycle. Biochemical experiments suggest that human FCP1 targets CTDs that are phosphorylated at serine 2
(CTD-serine 2) and/or CTD-serine 5. It is also observed to stimulate elongation independent of its catalytic activity. Dephosphorylation of Ser2 -
phosphorylated Pol II results in hypophosphorylated form that disengages capping enzymes (CE).
References
SS Mandal, H Cho, S Kim, K Cabane, D Reinberg, "FCP1, a phosphatase specific for the heptapeptide repeat of the largest subunit of RNA
polymerase II, stimulates transcription elongation.", Mol Cell Biol, 22, 2002, 7543-52.
Reaction
10.2.4.1.1.1.2 DSIF complex binds to RNA Pol II (hypophosphorylated)
Authors
Description
DSIF is a heterodimer consisting of hSPT4 (human homolog of yeast Spt4- p14) and hSPT5 (human homolog of yeast Spt5-p160). DSIF
association with Pol II may be enabled by Spt5 binding to Pol II creating a scaffold for NELF binding (Wada et al.,1998). Spt5 subunit of DSIF
can be phosphorylated by P-TEFb.
References
DK Kim, N Inukai, T Yamada, A Furuya, H Sato, Y Yamaguchi, T Wada, H Handa, "Structure-function analysis of human Spt4: evidence that
hSpt4 and hSpt5 exert their roles in transcriptional elongation as parts of the DSIF complex.", Genes Cells, 8, 2003, 371-8.
T Wada, T Takagi, Y Yamaguchi, A Ferdous, T Imai, S Hirose, S Sugimoto, K Yano, GA Hartzog, F Winston, S Buratowski, H Handa, "DSIF, a
novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs.", Genes
Dev, 12, 1998, 343-56.
Reaction
10.2.4.1.1.1.3 Formation of DSIF:NELF:early elongation complex
Authors
Description
NELF complex is a ~ 300 kDa multiprotein complex composed of 5 peptides (A - E): ~66,61,59,58 and 46 kDa. All these peptides are required
for NELF-mediated inhibition of Pol II elongation. NELF complex has been reported to bind to the pre-formed DSIF:RNA Pol II complex that may
act as a scaffold for its binding. NELF-A is suspected to be involved in Wolf-Hirschhorn syndrome.
Binding of DSIF:NELF to RNA Pol II CTD results in abortive termination of early elongation steps by the growing transcripts.
References
Reaction
10.2.4.1.1.2 Formation of RNA Pol II elongation complex
Authors
Description
TFIIS is a transcription factor involved in different phases of transcription, occurring in a major ubiquitous form and other tissue specific forms.
TFIIS stimulates RNA Pol II complex out of elongation arrest.
Other transcription factors like ELL, Elongin family members and TFIIF interact directly with elongating Pol II and increase its elongation rate.
These factors have been observed to act on naked DNA templates by suppressing transient pausing by the enzyme at all or most steps of
nucleotide addition. In Drosophila, ELL is found at a large number of transcriptionally active sites on polytene chromosomes. In general, ELL is
suspected to have more unidentified functions.
Elongin is a heterotrimeric protein complex that stimulates the overall rate of elongation. In addition, Elongin may act as an E3 Ubiquitin ligase.
Ubiquitylation of RNA Pol II occurs rapidly after genotoxic assault by UV light or chemicals, and results in degradation by proteasome. The FACT
complex appears to promote elongation by facilitating passage of polymerase through chromatin.
All these factors contribute to the formation of a processive elongation complex centered around the RNA Pol II complex positioned on the
DNA:RNA hybrid. This enables the RNA Pol II elongation complex to function as a platform that coordinates mRNA processing and export
(Reviewed by Shilatifard et al., 2003).
References
10.2.4.1.1.2.1 Hyperphosphorylation (Ser2) of RNA Pol II CTD by P-TEFb complex
Authors
Description
Cdk-9 is the kinase subunit of P-TEFb that phosphorylates Serine 2 on the heptapeptide repeats of Pol II CTD alleviating the negative action of
DSIF-NELF complex. This reaction is considered to be a rate limiting step for processive elongation. P-TEFb complex, that has a DRB-sensitive
cyclin-dependent kinase activity, is composed of ~43 kDa, Cdk9 kinase (PITALRE), and either Cyclin T1, Cyclin T2a, Cyclin T2b, or Cyclin K.
The exact mechanism by which P-TEFb removes the inhibition of elongation by DSIF-NELF is not yet known. P-TEFb is also capable of
phosphorylating Spt5 subunit of DSIF complex.
A P-TEFb complex (which contains only the Cyclin T1) is implicated in the efficient synthesis of human immunodeficiency virus-1 (HIV-1)
transcripts. Cyclin T1 subunit of the P-TEFb(Cyclin T1:Cdk9) complex interacts with HIV-1 encoded Tat protein that binds to the transactivation
response (TAR) element in the nascent HIV-1 transcript (reviewed in Price,2000).
The mechanism by which DSIF, NELF and P-TEFb or TAK/P-TEFb act together in Pol II-regulated elongation is yet to be fully understood.
Various biochemical evidences point to a model in which DSIF and NELF negatively regulate elongation through interactions with polymerase
containing a hypophosphorylated CTD. Subsequent phosphorylation of the Pol II CTD by P-TEFb might promote elongation by inhibiting
interactions of DSIF and NELF with the elongation complex.
References
T Wada, T Takagi, Y Yamaguchi, D Watanabe, H Handa, "Evidence that P-TEFb alleviates the negative effect of DSIF on RNA polymerase
II-dependent transcription in vitro.", EMBO J, 17, 1999, 7395-403.
DH Price, "P-TEFb, a cyclin-dependent kinase controlling elongation by RNA polymerase II.", Mol Cell Biol, 20, 2000, 2629-34.
Reaction
10.2.4.1.1.2.2 Recruitment of elongation factors to form elongation complex
Description
At the beginning of this reaction, 1 molecule of 'FACT complex', 1 molecule of 'Elongin Complex', 1 molecule of 'Early elongation complex with
hyperphosphorylated Pol II CTD', 1 molecule of 'TFIIH', 1 molecule of 'RNA polymerase II elongation factor ELL', and 1 molecule of 'TFIIS
protein' are present. At the end of this reaction, 1 molecule of 'Elongation complex' is present.
Reaction
10.2.4.1.1.3 Addition of nucleotides leads to transcript elongation
Authors
Description
High-resolution structures of free, catalytically active yeast Pol II and of an elongating form reveal that Pol II elongation complex includes
features like:
- RNA-DNA hybrid, an unwound template ahead of 3'-OH terminus of growing transcript and an exit groove at the base of the CTD, possibly for
dynamic interaction of processing and transcriptional factors.
- a cleft or channel created by Rpb1 and Rpb2 subunits to accommodate DNA template, extending to Mg2+ ion located deep in the enzyme core
-a 50 kDa "clamp" with open confirmation in free polymerase, allowing entry of DNA strands but closed in the processive elongation phase.
The clamp is composed of portions of Rpb1,Rpb2 and Rpb3 , five loops or "switches" that change from unfolded to well-folded structures
stabilizing the elongation complex, and a long "bridging helix" that emanates from Rpb1 subunit, crossing near the Mg2+ ion. The bridging helix
is thought to "bend" to push on the base pair at the 3'-end of RNA-DNA hybrid like a ratchet, translocating Pol II along the DNA (Cramer et
al.,2001; Gnatt et al.,2001).In addition to its dynamic biochemical potential, Pol II possess a repertoire of functions to serve as a critical platform
of recruiting and coordinating the actions of a host of additional enzyme and proteins involved in various pathways.
References
P Cramer, DA Bushnell, RD Kornberg, "Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution.", Science, 292, 2001,
1863-76.
Reaction
10.2.4.1.1.4 Pol II elongation complex moves on the template as transcript elongates

Description
At the beginning of this reaction, 1 molecule of 'Processive elongation complex', and 1 molecule of 'NTP' are present. At the end of this reaction,
1 molecule of 'Elongation complex prior to separation', and 1 molecule of 'NTP' are present.
Reaction
10.2.4.1.1.5 Separation of elongating transcript from template
Description
At the beginning of this reaction, 1 molecule of 'Elongation complex prior to separation' is present. At the end of this reaction, 1 molecule of
'Elongation complex with separated and uncleaved transcript' is present.
Reaction
10.2.4.1.2 Internal Methylation of mRNA
Editors
Joshi-Tope, G, 0000-00-00.
Description
In addition to the methylation of the 5'-cap, there is methylation of internal nucleotides in the mRNA. This methylation can occur in translated and
untranslated regions. One to three methyl groups have been seen per mRNA molecule, but methylation is non-stoichiometric. The most frequent
methylation observed is at the N6 position of adenosine. The function of mRNA internal methylation, if any, is unknown.
References
JA Bokar, ME Shambaugh, D Polayes, AG Matera, FM Rottman, "Purification and cDNA cloning of the AdoMet-binding subunit of the human
mRNA (N6-adenosine)-methyltransferase.", RNA, 3, 1998, 1233-47.
Reaction
10.2.4.1.3 Formation of pre-mRNPs
Description
After the nascent pre-mRNA undergoes the initial capping and methylation reactions, it gets associated with numerous factors, including the
various heterogeneous nuclear ribonucleoproteins (hnRNPS), the nuclear Cap-Binding Complex, and many splicing factors that make the
pre-mRNA a substrate for splicing, 3'-end processing, and in some cases editing.
Reaction
10.2.4.1.4 mRNA Splicing
Description
The process in which excision of introns from the primary transcript of messenger RNA (mRNA) is followed by ligation of the two exon termini
exposed by removal of each intron, is called mRNA splicing. Most of the mRNA is spliced by the major pathway, involving the U1, U2, U4, U5
and U6 snRNPs. A minor fraction, about 1 %, of the mRNAs are spliced via the U12 dependent pathway.
References
Z Zhou, LJ Licklider, SP Gygi, R Reed, "Comprehensive proteomic analysis of the human spliceosome.", Nature, 419, 2002, 182-5.
10.2.4.1.4.1 mRNA Splicing - Major Pathway
Description
The splicing of pre-mRNA occurs within a large, very dynamic complex, designated the 'spliceosome'. The 50-60S spliceosomes are estimated
to be 40-60 nm in diameter, and have molecular weights in the range of 3-5 million kDa. Small nuclear RNAs (snRNAs) U1, U2, U4, U5, and U6,
are some of the best characterized components of spliceosomes, and are known to play key roles not only in spliceosomal assembly, but also in
the two catalytic steps of the splicing reaction. Over 150 proteins have been detected in spliceosomes, and only a subset of these has been
characterized. The characterization, and the determination of the functions of the protein components of the spliceosome, is still work in
progress.
During spliceosome assembly, the snRNAs and the spliceosomal proteins assemble on the pre-mRNA in a stepwise pathway. First the E
complex forms, followed by complexes A and B; the C complex forms next and contains the products of the first step of the splicing reaction.
Complexes called i and D form as a consequence of the second step of the splicing reaction, which contain the excised intron and the spliced
exons, respectively.
10.2.4.1.4.1.1 Formation of the Spliceosomal E complex
Description
Pre-mRNA transcripts become rapidly associated with many RNA-binding proteins, including hnRNP proteins, cap-binding proteins, SR proteins,
etc; in the test tube this binding does not require splice sites or ATP. The E complex, or early complex, is the first detectable functional
intermediate in spliceosome assembly in vitro. It is an ATP-independent complex. When a functional 5' splice site is present, it is bound by the
U1 snRNP. The splicing factor U2AF (65 and 35 kDa subunits) binds to the polypyrimidine tract (Y)n and the AG dinucleotide at the 3' splice site,
respectively. SF1/mBBP binds to the branch site. Binding of many of these factors is cooperative; e.g., SR proteins and U2AF apparently interact
with each other, facilitating their binding to the pre-mRNA. In the presence of ATP, the E complex is converted to the first ATP-dependent
spliceosomal complex, namely the A complex.
References
J Rappsilber, U Ryder, AI Lamond, M Mann, "Large-scale proteomic analysis of the human spliceosome.", Genome Res, 12, 2002, 1231-45.
K Hartmuth, H Urlaub, HP Vornlocher, CL Will, M Gentzel, M Wilm, R LÃ¼hrmann, "Protein composition of human prespliceosomes isolated by
a tobramycin affinity-selection method.", Proc Natl Acad Sci U S A, 99, 2002, 16719-24.
Reaction
10.2.4.1.4.1.2 Formation of the Spliceosomal A Complex
Description
The A complex is the first ATP-dependent complex in spliceosome assembly. U2AF recruits the U2 snRNP to bind to the branch site in the E
complex in an ATP-dependent fashion, to form the A complex. The U2 snRNA base-pairs with the branch site, causing the branch-site
adenosine to bulge out, which later positions it for nucleophilic attack at the 5' splice site. The A complex serves as a substrate for formation of
the B complex.
References
Reaction
10.2.4.1.4.1.3 Formation of the Spliceosomal B Complex
Authors
Krainer, AR, 2003-06-05.

Editors
Joshi-Tope, G, 0000-00-00.
Description
The formation of the B complex is ATP-dependent, and both the 5' and 3' splice sites are essential for B complex assembly. The U4 and U6
snRNPS are extensively base-paired, and this U4:U6 complex associates with the U5 snRNP to form a tri-snRNP particle. This tri-snRNP
particle then binds to the spliceosomal A complex, to form the spliceosomal B complex.
References
Reaction
10.2.4.1.4.1.4 Formation of an intermediate Spliceosomal C complex
Authors
Krainer, AR, 2003-06-05.
Editors
Joshi-Tope, G, 0000-00-00.
Description
The spliceosomal C complex is a very short-lived intermediate; the splicing intermediates are rapidly converted to splicing products. Also, the
spliced products are released very rapidly, and no complex containing both the splicing products has been isolated. Conversion of the
spliceosomal B complex to the spliceosomal C complex requires ATP. The extensive base-pairing between the U4 and U6 snRNAs is disrupted
during the formation of the C complex, which is thought to require helicase-type activity associated with the DEAD box factors.
References
MS Jurica, LJ Licklider, SR Gygi, N Grigorieff, MJ Moore, "Purification and characterization of native spliceosomes suitable for three-dimensional
structural analysis.", RNA, 8, 2002, 426-39.
Reaction
10.2.4.1.4.1.5 Formation of the active Spliceosomal C complex
Description
The active C complex is formed due to a conformational change in the intermediate C complex. After formation of the active C complex, the
splicing reactions occur very rapidly.
References
Reaction
10.2.4.1.4.1.6 Lariat Formation and 5'-Splice Site Cleavage

Authors
Krainer, AR, 2003-06-05.
Description
In the first catalytic step of mRNA splicing, the 2' OH group of the bulged A at the branch site performs a nucleophilic attack on the 5' splice site
phosphodiester bond, resulting in cleavage of the bond between the 5' exon and the 5' end of the intron, and formation of a new bond between
the 5' end of the intron and the branch site A. This results in a lariat-shaped intermediate, with the intron still attached to the 3' exon. The branch
site A has a 2'-5' phosphodiester bond with the G at the beginning of the intron, in addition to the usual 5'-3' and 3'-5'phosphodiester bonds.
References
RA Padgett, MM Konarska, PJ Grabowski, SF Hardy, PA Sharp, "Lariat RNA's as intermediates and products in the splicing of messenger RNA
precursors.", Science, 225, 1984, 898-903.
B Ruskin, AR Krainer, T Maniatis, MR Green, "Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro.", Cell, 38,
1984, 317-31.
Reaction
10.2.4.1.4.1.7 Formation of Exon Junction Complex
Description
At the beginning of this reaction, 1 molecule of 'Magoh-Y14 complex', and 1 molecule of 'Spliceosomal active C complex with lariat containing,
5'-end cleaved pre-mRNP:CBC complex' are present. At the end of this reaction, 1 molecule of 'Exon Junction Complex' is present.
References
H Le Hir, D Gatfield, E Izaurralde, MJ Moore, "The exon-exon junction complex provides a binding platform for factors involved in mRNA export
and nonsense-mediated mRNA decay", EMBO J, 20, 2001, 4987-97.
H Le Hir, E Izaurralde, LE Maquat, MJ Moore, "The spliceosome deposits multiple proteins 20-24 nucleotides upstream of mRNA exon-exon
junctions", EMBO J, 19, 2000, 6860-9.
VL Reichert, Hir H Le, MS Jurica, MJ Moore, "5' exon interactions within the human spliceosome establish a framework for exon junction
complex structure and assembly", Genes Dev, 16, 2002, 2778-91.
Reaction
10.2.4.1.4.1.8 Cleavage at the 3'-Splice Site and Exon Ligation
Description
The second step of the splicing reaction results in cleavage of the transcript at the 3'splice site, and results in ligation of the two exons and
excision of the intron.
Reaction
10.2.4.1.4.2 mRNA Splicing - Minor Pathway
Editors
Joshi-Tope, G, 0000-00-00.
Description
The splicing of a subset of pre-mRNA introns occurs by a second pathway, designated the AT-AC or U12-dependent splicing pathway. AT-AC
introns have highly conserved, non-canonical splice sites that are removed by the AT-AC spliceosome, which contains distinct snRNAs (U11,
U12, U4atac, U6atac) that are structurally and functionally analogous to the major spliceosome. U5 snRNA as well as many of the protein factors
appear to be conserved between the two spliceosomes.
References
WY Tarn, JA Steitz, "Pre-mRNA splicing: the discovery of a new spliceosome doubles the challenge.", Trends Biochem Sci, 22, 1997, 132-7.
Q Wu, AR Krainer, "AT-AC pre-mRNA splicing mechanisms and conservation of minor introns in voltage-gated ion channel genes.", Mol Cell
Biol, 19, 1999, 3225-36.
10.2.4.1.4.2.1 Formation of AT-AC A complex
Authors
Joshi-Tope, G, 2003-10-27.
Description
U12-type AT-AC introns are distinguished from the major U2-type introns by the consensus sequences of their highly conserved splicing signals.
U12 introns have the 5' ss consensus sequence (G/A)TATCCTTT, the branchpoint sequence TTTCCTTAACT and the 3' ss (C/T)AG. Initial
recognition of AT-AC introns involves interaction of U12 snRNP with the branch-point sequence and U11 with the 5' ss. Unlike the major splicing
pathway, U11 and U12 are in a complex and interact with the pre-mRNA simultaneously, binding in an ATP-dependent manner as a di-snRNP
complex and likely bridging the 5' ss and 3' ss region.
Twenty proteins have been identified in the U11/U12 di-snRNP complex including the snRNP Sm proteins BÃ¢â‚¬â„¢, B, D3, D2, D1, E, F, and
G which are identical to the major splicing pathway Sm proteins. A U2 snRNP core protein complex, SF3b is also found in the U11/U12
di-snRNP, including p14, a protein that interacts with the branchpoint adenosine.
SR proteins are required for formation of A complex in AT-AC splicing. The same SR proteins involved in splicing of the major introns are also
active in splicing of AT-AC introns, though, as in the major pathway, there is substrate specificity.
Reaction
10.2.4.1.4.2.2 Formation of AT-AC B Complex
Authors
Joshi-Tope, G, 2003-10-27.
Description
The U4atac/U6atac enters the spliceosome and U6atac snRNA forms base pairing interactions with the 5' ss and also forms base pairing
interactions with U12 and U4atac is partially displaced. U5 snRNP, the only snRNP common to both the major and minor splicing pathways, also
joins the spliceosome to form the B complex and interacts with nucleotides within the 3' end of the exon flanking the 5' ss.
Reaction
10.2.4.1.4.2.3 Formation of AT-AC C complex
Description
At the beginning of this reaction, 1 molecule of 'ATAC B Complex' is present. At the end of this reaction, 1 molecule of 'U4 ATAC snRNP', and 1
molecule of 'ATAC C Complex' are present.

Reaction
10.2.4.1.4.2.4 ATAC spliceosome mediated Lariat formation,5' splice site cleavage
Description
At the beginning of this reaction, 1 molecule of 'ATAC C Complex' is present. At the end of this reaction, 1 molecule of 'ATAC C Complex with
lariat containing 5'-end cleaved mRNA' is present.

Reaction
10.2.4.1.4.2.5 ATAC spliceosome mediated 3' splice site cleavage, exon ligation
Description
At the beginning of this reaction, 1 molecule of 'ATAC C Complex with lariat containing 5'-end cleaved mRNA' is present. At the end of this
reaction, 1 molecule of 'U6 ATAC snRNP', 1 molecule of 'post exon ligation complex', 1 molecule of 'U12 snRNP', 1 molecule of 'U11 snRNP',
and 1 molecule of 'U5 snRNP' are present.

Reaction
10.2.4.2 Elongation of Intronless Transcripts
Authors
Joshi-Tope, G, 2004-06-28.
Description
This section will be annotated in a later release.
10.2.5 Post-Elongation Processing of the Transcript
Authors
Editors
Joshi-Tope, G, 0000-00-00.
Description
Post-transcriptional splicing of introns is affected neither by the elongating properties of RNA polymerase II, nor by the binding of splicing
regulatory factors to the enzyme. It is only affected by the relative abundance of constitutive and/or regulatory splicing factors at the sites where
splicing takes place. Nevertheless it is important to point out that cytological evidence indicates that unspliced or partially spliced pre-mRNAs do
not diffuse in the nucleoplasm far from the transcription sites. Therefore, recruitment of splicing factors to transcription sites would still favor the
post-transcriptional splicing of introns.
10.2.5.1 Post-Elongation Processing of Intron-Containing pre-mRNA
10.2.5.1.1 mRNA 3'-end processing
Editors
Joshi-Tope, G, 0000-00-00.
Description
The 3' ends of eukaryotic mRNAs are generated by posttranscriptional processing of an extended primary transcript. For almost all RNAs, 3'-end
processing consists of two steps: (i) the mRNA is first cleaved at a particular phosphodiester bond downstream of the coding sequence, (ii) the
upstream fragment then receives a poly(A) tail of approximately 250 adenylate residues, whereas the downstream fragment is degraded. The
two partial reactions are coupled so that reaction intermediates are usually undetectable. While 3' processing can be studied as an isolated
event in vitro, it appears to be connected to transcription, splicing, and transcription termination in vivo.
The only known exception to the rule of cleavage followed by polyadenylation are the major histone mRNAs, which are cleaved but not
polyadenylated.
References
J Zhao, L Hyman, C Moore, "Formation of mRNA 3' ends in eukaryotes: mechanism, regulation, and interrelationships with other steps in mRNA
synthesis.", Microbiol Mol Biol Rev, 63, 1999, 405-45.
CL Moore, PA Sharp, "Accurate cleavage and polyadenylation of exogenous RNA substrate.", Cell, 41, 1985, 845-55.
E Wahle, U RÃ¼egsegger, "3'-End processing of pre-mRNA in eukaryotes.", FEMS Microbiol Rev, 23, 1999, 277-95.
10.2.5.1.1.1 Cleavage of mRNA at the 3'-end
Editors
Joshi-Tope, G, 0000-00-00.
Description
Endonucleolytic cleavage separates the pre-mRNA into an upstream fragment destined to become the mature mRNA, and a downstream
fragment that is rapidly degraded. Cleavage depends on two signals in the RNA, a highly conserved hexanucleotide, AAUAAA, 10 to 30
nucleotides upstream of the cleavage site, and a poorly conserved GU- or U-rich downstream element. Additional sequences, often upstream of
AAUAAA, can enhance the efficiency of the reaction. Cleavage occurs most often after a CA dinucleotide. A single gene can have more than
one 3' processing site.
Cleavage is preceded by the assembly of a large processing complex, the composition of which is poorly defined. ATP, but not its hydrolysis, is
required for assembly. Cleavage at the 3'-end of mRNAs depends on a number of protein factors. CPSF, a heterotetramer, binds specifically to
the AAUAAA sequence. The heterotrimer CstF binds the downstream element. CF I, which appears to be composed of two subunits, one of
several related larger polypeptides and a common smaller one, also binds RNA, but with unknown specificity. RNA recognition by these proteins
is cooperative. Cleavage also requires CF II, composed of at least two subunits, and poly(A) polymerase, the enzyme synthesizing the poly(A)
tail in the second step of the reaction. The polypeptide catalyzing the hydrolysis of the phosphodiester bond remains to be identified.
Cleavage produces a 3'-OH on the upstream fragment and a 5'-phosphate on the downstream fragment. At some unknown point after cleavage,
the downstream RNA fragment, CstF, CF I and CF II are thought to be released, whereas CPSF and poly(A) polymerase remain to carry out
polyadenylation.
References
Reaction
10.2.5.1.1.2 mRNA polyadenylation
Editors
Joshi-Tope, G, 0000-00-00.
Description
The upstream fragment generated by 3' cleavage of the pre-mRNA receives a poly(A) tail of approximately 250 AMP residues in a reaction
depending on the AAUAAA sequence 10 to 30 nucleotides upstream of the 3' end. Polyadenylation is carried out by three proteins: Poly(A)
polymerase carries the catalytic activity. The enzyme has no specificity for any particular RNA sequence, and it also has a very low affinity for
the RNA.
Under physiological conditions, the activity of poly(A) polymerase thus depends on two auxiliary factors, both of which bind to specific RNA
sequences and recruit the enzyme by a direct contact. One of these proteins is the heterotetrameric CPSF, which binds the AAUAAA sequence
and is also essential for 3' cleavage. The second is the nuclear poly(A) binding protein (PABPN1), which binds the growing poly(A) tails once this
has reached a length of about ten nucleotides. Stimulation of poly(A) polymerase by both proteins is synergistic and results in processive
elongation of the RNA, i.e. the polymerase adds AMP residues without dissociating from the RNA. The processive reaction is terminated when
the tail has reached a length of about 250 nucleotides.
References
Reaction
10.2.5.2 Post-Elongation Processing of Intronless pre-mRNA
10.2.5.2.1 Processing of Capped Intronless Pre-mRNA
Authors
Marzluff, WF, 2003-08-22.
Editors
Joshi-Tope, G, 0000-00-00.
Description
There are two classes of intronless pre-mRNAs (mRNAs expressed from genes that lack introns). The first class encodes the replication
dependent histone mRNAs. These mRNAs have unique 3Ã¢â‚¬â„¢ ends that do not have a polyA tail. The replication dependent histone
mRNAs in all metazoans, as well as Chlamydomonas and Volvox fall into this class.
The second class of mRNAs end in polyA tails, which are formed by a mechanism similar to that which poly-adenylate pre-mRNAs containing
introns. In the intronless genes there is a different method of replacing the 3Ã¢â‚¬â„¢ splice site that activates polyadenylation.
References
Z Dominski, WF Marzluff, "Formation of the 3' end of histone mRNA.", Gene, 239, 1999, 1-14.
WF Marzluff, P Gongidi, KR Woods, J Jin, LJ Maltais, "The human and mouse replication-dependent histone genes.", Genomics, 80, 2002,
487-98.
R Sanchez, WF Marzluff, "The stem-loop binding protein is required for efficient translation of histone mRNA in vivo and in vitro", Mol Cell Biol,
22, 2002, 7093-104.
JG Gall, "Cajal bodies: the first 100 years.", Annu Rev Cell Dev Biol, 16, 2001, 273-300.
MR Frey, AG Matera, "Coiled bodies contain U7 small nuclear RNA and associate with specific DNA sequences in interphase human cells.",
Proc Natl Acad Sci U S A, 92, 1995, 5915-9.
10.2.5.2.1.1 SLBP Dependent Processing of Replication-Dependent Histone Pre-mRNAs
Authors
Editors
Description
There are two well-documented trans-acting factors required for histone pre-mRNA processing. These are:
1 Stem-loop binding protein (SLBP), also termed hairpin binding protein (HBP). This 32 kDa protein is likely the first protein that binds to the
histone pre-mRNA as it is being transcribed.
The U7 snRNP. This particle contains the U7 snRNA, the smallest of the snRNAs which varies from 57-70 nts long depending on the species.
The 5Ã¢â‚¬â„¢ end of U7 snRNA binds to a sequence 3Ã¢â‚¬â„¢ of the stemloop, termed the histone downstream element (HDE). There are a
number of proteins found in the U7 snRNP. There are 7 Sm proteins, as are present in the spliceosomal snRNP. Five of these proteins are the
same as ones found in the spliceosomal snRNPs and there are 2, Lsm10 and Lsm11 that are unique to U7 snRNP.
A third protein joins the U7 snRNP, ZFP100, a large zinc finger protein. ZFP100 interacts with SLBP bound to the histone pre-mRNA and with
Lsm11 and likely plays a critical role in recruiting U7 snRNP to the histone pre-mRNA.
It should be noted that there must be other trans-acting factors, including the factor that catalyzes the cleavage reaction. The cleavage occurs in
the presence of EDTA as does the cleavage reaction in polyadenylation, it is likely that this reaction is catalyzed by a protein. There may well be
additional proteins associated with U7 snRNP, and since under some conditions in vitro processing occurs in the absence of SLBP, it is possible
that all of the other factors required for processing are associated with the active form of U7 snRNP.
References
A Streit, TW Koning, D Soldati, L Melin, D SchÃ¼mperli, "Variable effects of the conserved RNA hairpin element upon 3' end processing of
histone pre-mRNA in vitro.", Nucleic Acids Res, 21, 1993, 1569-75.
ME Harris, R BÃ¶hni, MH Schneiderman, L Ramamurthy, D SchÃ¼mperli, WF Marzluff, "Regulation of histone mRNA in the unperturbed cell
cycle: evidence suggesting control at two posttranscriptional steps.", Mol Cell Biol, 11, 1991, 2416-24.
10.2.5.2.1.1.1 Binding of SLBP to Replication-Dependent Histone Pre-mRNA
Authors
Editors

Description
The 32 kDa stem-loop binding protein (SLBP), also termed hairpin binding protein (HBP) is likely the first protein that binds to the histone
pre-mRNA as it is being transcribed.
References
F Martin, A Schaller, S Eglite, D SchÃ¼mperli, B MÃ¼ller, "The gene for histone RNA hairpin binding protein is located on human chromosome
4 and encodes a novel type of RNA binding protein.", EMBO J, 16, 1997, 769-78.
Z Dominski, LX Zheng, R Sanchez, WF Marzluff, "Stem-loop binding protein facilitates 3'-end formation by stabilizing U7 snRNP binding to
histone pre-mRNA.", Mol Cell Biol, 19, 1999, 3561-70.
ZF Wang, ML Whitfield, TC Ingledue, Z Dominski, WF Marzluff, "The protein that binds the 3' end of histone mRNA: a novel RNA-binding protein
required for histone pre-mRNA processing.", Genes Dev, 10, 1997, 3028-40.
Reaction
10.2.5.2.1.1.2 Recruitment of U7 snRNP:ZFP100 complex to the SLBP Bound Pre-mRNA
Authors

Description
same as ones found in the spliceosomal snRNPs and there are 2, Lsm10 and Lsm11 that are unique to U7 snRNP. A third protein joins the U7
snRNP, ZFP100, a large zinc finger protein. ZFP100 interacts with SLBP bound to the histone pre-mRNA and with Lsm11 and likely plays a
critical role in recruiting U7 snRNP to the histone pre-mRNA.
References
F Schaufele, GM Gilmartin, W Bannwarth, ML Birnstiel, "Compensatory mutations suggest that base-pairing with a small nuclear RNA is
required to form the 3' end of H3 messenger RNA.", Nature, 323, 1986, 777-81.
Reaction
10.2.5.2.1.1.3 Cleavage of the 3'-end of Replication Dependent Histone Pre-mRNA
Authors
Editors
Description
Processing is initiated once the SLBP (bound to the stem loop) and the U7 snRNP (bound to the HDE) are both loaded onto the pre-mRNA. The
pre-mRNA HDE makes base-pairing contacts with the 5Ã¢â‚¬Â² end of U7 snRNA. Binding of the U7 snRNP to the pre-mRNA is stabilized by
interactions between a U7 snRNP protein, hZFP100 and SLBP. It should be noted that there must be other trans-acting factors, including the
factor that catalyzes the cleavage reaction, which have yet to be defined. The cleavage occurs in the presence of EDTA as does the cleavage
reaction in polyadenylation, it is likely that this reaction is catalyzed by a protein. There may well be additional proteins associated with the U7
snRNP, and since in some conditions in vitro processing occurs in the absence of SLBP, it is possible that all the other factors required for
processing are associated with the active form of the U7 snRNP.
References
Reaction
10.2.5.2.1.2 SLBP independent Processing of Histone Pre-mRNAs
Authors
Editors
Description
This class of mRNAs is expressed from genes that lack introns yet the transcripts end in polyA tails. These tails are formed by a mechanism
similar to that for pre-mRNAs containing introns. It is believed that there is a cis-element that replaces the 3Ã¢â‚¬â„¢ splice site that normally
serves to activate polyadenylation of intron containing pre-mRNAs.
10.2.5.2.1.2.1 Recruitment of U7 snRNP:ZFP100 complex to the Histone Pre-mRNA
Authors
Editors
Description
References
Reaction
10.2.5.2.1.2.2 Cleavage of the 3'-end of the Histone Pre-mRNA
Authors
Editors
Description
Processing is initiated once the U7 snRNP is loaded onto the pre-mRNA. The pre-mRNA HDE makes base-pairing contacts with the 5Ã¢â‚¬Â²
end of U7 snRNA. Binding of the U7 snRNP to the pre-mRNA is stabilized by interactions between a U7 snRNP protein, hZFP100 and other
trans-acting factors, including the factor that catalyzes the cleavage reaction, which have yet to be defined. The cleavage occurs in the presence
of EDTA as does the cleavage reaction in polyadenylation, it is likely that this reaction is catalyzed by a protein. There may well be additional
proteins associated with the U7 snRNP, since the in vitro processing occurs in the absence of SLBP, it is possible that all the other factors
required for processing are associated with the active form of the U7 snRNP.
References
Reaction
10.2.5.2.1.3 Processing of Intronless Pre-mRNAs
Authors
Wahle, E, 2003-06-05.
Editors
Description
The 3' ends of eukaryotic mRNAs are generated by posttranscriptional processing of an extended primary transcript. For almost all RNAs, 3'
processing consists of two steps: The mRNA is first cleaved at a particular phosphodiester bond downstream of the coding sequence. The
upstream fragment then receives a poly(A) tail of approximately 250 adenylate residues whereas the downstream fragment is degraded. The two
partial reactions are coupled so that reaction intermediates are usually undetectable. While 3' processing can be studied as an isolated event in
vitro, it appears to be connected to transcription, splicing and transcription termination in vivo.
References
10.2.5.2.1.3.1 Recognition of AAUAAA sequence by CPSF
Authors
Wahle, E, 2003-06-05.
Editors
Description
Poly(A) polymerase has no specificity for any particular RNA sequence, as well as a very low affinity for the RNA. Under physiological
conditions, the activity of poly(A) polymerase depends on two auxiliary factors, both of which bind to specific RNA sequences and recruit the
poly(A) polymerase by a direct contact. One of these proteins is the heterotetrameric CPSF, which binds the AAUAAA sequence and is also
essential for 3' cleavage.
References
Reaction
10.2.5.2.1.3.2 Recruitment of CstF to the CPSF Bound Pre-mRNA
Authors
Wahle, E, 2003-06-05.
Editors
Description
Endonucleolytic cleavage separates the pre-mRNA into an upstream fragment destined to become the mature mRNA and a downstream
fragment that is rapidly degraded. Polyadenylation and cleavage occur concurrently, with complexes co-assembling, cleavage depends on two
signals in the RNA, a highly conserved hexanucleotide, AAUAAA, 10 to 30 nucleotides upstream of the cleavage site, and a poorly conserved
GU- or U-rich downstream element. Additional sequences, often upstream of AAUAAA, can enhance the efficiency of the reaction. Cleavage
occurs most often after a CA dinucleotide. A single gene can have more than one 3' processing site.
required for assembly. Cleavage depends on a number of protein factor. CPSF, a heterotetramer, binds specifically to the AAUAAA sequence.
The heterotrimer CstF binds the GU- or U-rich downstream element.
References
Reaction
10.2.5.2.1.3.3 Binding of Cleavage factors and Poly(A)Polymerase to the CstF:CPSF:Pre-mRNA Complex
Authors
Wahle, E, 2003-06-05.
Editors
Description
CF I, which appears to be composed of two subunits, one of several related larger polypeptides and a common smaller one, also binds RNA, but
with unknown specificity. RNA recognition by these proteins is cooperative. Cleavage also requires CF II, composed of at least two subunits, and
poly(A) polymerase, the enzyme synthesizing the poly(A) tail in the second step of the reaction.
References
Reaction
10.2.5.2.1.3.4 Cleavage and polyadenylation of Intronless Pre-mRNA
Authors
Wahle, E, 2003-06-05.
Editors
Description
The nuclear poly(A) binding protein (PABPN1), which binds the growing poly(A) tails once it has reached a length of about ten nucleotides.
Stimulation of poly(A) polymerase by both CPSF and PABPN1 is synergistic and results in processive elongation of the RNA (the polymerase
adds AMP residues without dissociating from the RNA). The processive reaction is terminated when the tail has reached a length of about 250
nucleotides.
References
Reaction
10.3 Transport of Mature Transcript to Cytoplasm
Authors
Joshi-Tope, G, 2003-09-02.
Description
Transport of mRNA through the Nuclear Pore Complex (NPC) is a dynamic process involving distinct machinery and receptor subsets. The
separation of the two compartments and the regulation of this transport provide spatial and temporal control over mRNA expression and
ultimately control over translation. It should be noted that mRNA export does not rely on a specific motif in the mRNA molecule, but rather
transport appears to be coupled to processing and regulation. The specific proteins that are bound to the mRNA determine when it will be
transported to the cytoplasm. This limitation insures that transport overwhelmingly favors transport of fully processed mRNA molecules.
10.3.1 Transport of Mature mRNA derived from an Intron-Containing Transcript
Description
Transport of mRNA from the nucleus to the cytoplasm, where it is translated into protein, is highly selective and closely coupled to correct RNA
processing. This coupling is achieved by the nuclear pore complex, which recognizes and transports only completed mRNAs.
10.3.1.1 Recruitment of TAP to the EJC
Authors
Joshi-Tope, G, 2005-02-17.
Editors
Joshi-Tope, G, 0000-00-00.
Description
Aly/Ref recruits TAP to the Exon Junction Complex. This makes the mRNP complex ready for export to the cytoplasm.
Reaction
10.3.1.2 Docking of the TAP:EJC Complex with the NPC
Description
At the beginning of this reaction, 1 molecule of 'TAP:3'-polyadenylated, capped mRNA complex' is present. At the end of this reaction, 1
molecule of 'SRp55', 1 molecule of 'U2AF 65 kDa subunit', 1 molecule of 'SR9 / SRp30', 1 molecule of 'hTra2', 1 molecule of 'hPrp16', 1
molecule of 'SR 11/ p54', 1 molecule of 'hPrp22', 1 molecule of 'SRp40', 1 molecule of 'hPrp17', 1 molecule of 'SF2/ASF/SFRS1', 1 molecule of
'hSLU7', 1 molecule of 'Export Receptor bound mature mRNA Complex', 1 molecule of 'U2AF 35 kDa subunit', 1 molecule of 'SR2 / SC35', 1
molecule of 'hPrp43', 1 molecule of 'SRp20', 1 molecule of 'SR7/ 9G8 protein', 1 molecule of 'hPrp18', and 1 molecule of 'SR4 / SRp75' are
present.

Reaction
10.3.1.3 Transport of the export-competent complex through the NPC
Description
In this reaction, 1 molecule of 'Export Receptor bound mature mRNA Complex' is translocated from nucleoplasm to cytosol.
Reaction
10.3.1.4 Release from the NPC and Disassembly of the mRNP
Description
At the beginning of this reaction, 1 molecule of 'eIF4E', and 1 molecule of 'Export Receptor bound mature mRNA Complex' are present. At the
end of this reaction, 1 molecule of 'THOC4(Aly/Ref)', 1 molecule of 'Mature mRNP Complex', 1 molecule of 'SRm160', and 1 molecule of 'TAP'
are present.
This reaction takes place in the 'cytoplasm'.
Reaction
10.3.2 Transport of Mature mRNAs Derived from Intronless Transcripts
Authors
Editors
Description
Transport of mature mRNAs derived from intronless transcripts require some of the same protein complexes as mRNAs derived from intron
containing complexes, including TAP and Aly/Ref. However a number of the splicing related factors are lacking from the intronless derived
mRNAs, as they required no splicing.
10.3.2.1 Transport of the SLBP Dependant Mature mRNA

Description
Transport of U7 snRNP and stem-loop binding protein (SLBP) processed mRNA.
10.3.2.1.1 Docking of Mature Replication Dependent Histone mRNA with the NPC
Authors
Editors
Description
Histone mRNAs are exported by a mechanism that requires TAP, the key factor requires for transport of polyadenylated mRNAs. How TAP is
recruited to the histone mRNAs is not known, but it is clear that transport can occur in the absence of either the stemloop or of SLBP. The
stemloop and SLBP enhance the rate of transport of histone mRNAs in Xenopus oocytes, but are not essential for transport
References
Reaction
10.3.2.1.2 Transport of the Mature IntronlessTranscript Derived Histone mRNA:SLBP:TAP:Aly/Ref complex through the NPC
Authors
Editors
Description
Once the transport complex is fully assembled the mature mRNA can be translocated from the nucleoplasm to the cytoplasm. The assembled
complex starts at the nucleoplasmic basket, travels through the pore, and ends it journey at the cytoplasmic face of the nuclear pore complex.
Reaction
10.3.2.1.3 Release of the Mature intronless transcript derived Histone mRNA:SLBP:eIF4E Complex
Authors
Description
At some point eIF4E binds the mature mRNA. While TAP and Aly/Ref are released and will be reycled back to the nucleoplasm.
Reaction
10.3.2.2 Transport of the SLBP independent Mature mRNA
Description
Transport of the SLBP independent Mature mRNA through the nuclear pore.
10.3.2.2.1 Docking of Mature Histone mRNA complex:TAP at the NPC
Authors
Editors
Description
mature transcript docks at the NPC, in the course of transport CBC will be lost from the mRNA cap, and remain in the nucleous.
References
Reaction
10.3.2.2.2 Transport of the Mature Intronless Transcript Derived Histone mRNA:TAP:Aly/Ref Complex through the NPC
Authors
Description
The mature SLBP independent intronless histone mRNA is transported through the nucler pore to the cytoplasmic side.
Reaction
10.3.2.2.3 Release of the SLBP independent Histone mRNA from the NPC
Authors

Description
Reaction
10.3.2.3 Transport of Mature mRNA Derived from an Intronless Transcript
Description
processing.
10.3.2.3.1 Docking of the Mature intronless derived transcript derived mRNA, TAP and Aly/Ref at the NPC
Authors
Description
The polyadenylated, capped transcript and TAP dock at the nucleoplasmic side of the NPC. The Cap Binding Complex (CBC) and CPSF
complexes are released back into the nucleoplasm.
Reaction
10.3.2.3.2 Transport of the Mature intronless transcript derived mRNA:TAP:Aly/Ref Complex through the NPC
Authors
Description
The nucleoplasmic 3' polyadenylated, capped intronless mRNA and TAP are transported through the NPC to the cyotplasmic side of the pore.
Reaction
10.3.2.3.3 Release of the Mature intronless derived mRNA, TAP, and Aly/Ref from the NPC
Authors
Description
The cytoplasmic 3' polyadenylated, capped intronless mRNA and TAP are released from the NPC into the cytosol. Cytosolic TAP will be
recycled to the nucleous, while the 3' polyadenylated, capped intronless mRNA is bound by eIF4E and destined for translation.
Reaction
10.4 Translation
Authors
Merrick, WC, Bedwell, D, Gebauer, F, Anand, M, Balar, BA, Gross, S, Ortiz, PA, Ozturk, S, Pittman, YR, Ulloque, R, Hentze, MW, Kinzy, TG,
2005-03-29.
Editors
Tello-Ruiz, MK, Gillespie, ME, Gopinathrao, G, Matthews, L, 0000-00-00.
Reviewers
Hershey, JW, Sonenberg, N, Hinnebusch, AG, 0000-00-00.
Description
Protein synthesis is accomplished through the process of translation of an mRNA sequence into a polypeptide chain. This process can be
divided into three distinct stages: initiation, elongation and termination. During the initiation phase, the two subunits of the ribosome are brought
together to the translation start site on the mRNA where the polypeptide chain is to begin. Extension of the polypeptide chain occurs when a
specific aminoacyl-tRNA, as determined by the template mRNA, binds an elongating ribosome. The protein chain is released from the ribosome
when any one of three stop codons in the relevant reading frame on the mRNA is reached. Individual reactions at each one of these stages are
catalyzed by a number of initiation, elongation and release factors, respectively.
10.4.1 Eukaryotic Translation Initiation
Description
Initiation of translation in the majority of eukaryotic cellular mRNAs depends on the 5'-cap (m7GpppN) and involves ribosomal scanning of the 5'
untranslated region (5'-UTR) for an initiating AUG start codon. Therefore, this mechanism is often called cap-dependent translation initiation.
Proximity to the cap, as well as the nucleotides surrounding an AUG codon, influence the efficiency of the start site recognition during the
scanning process. However, if the recognition site is poor enough, scanning ribosomal subunits will ignore and skip potential starting AUGs, a
phenomenon called leaky scanning. Leaky scanning allows a single mRNA to encode several proteins that differ in their amino-termini. Several
eukaryotic cell and viral mRNAs initiate translation by an alternative mechanism that involves internal initiation rather than ribosomal scanning.
These mRNAs contain complex nucleotide sequences, called internal ribosomal entry sites, where ribosomes bind in a cap-independent manner
and start translation at the closest downstream AUG codon.
10.4.1.1 Cap-dependent Translation Initiation

Description
Translation initiation is a complex process in which the Met-tRNAi initiator, 40S, and 60S ribosomal subunits are assembled by eukaryotic
initiation factors (eIFs) into an 80S ribosome at the start codon of an mRNA. The basic mechanism for this process can be described as a series
of five steps: 1) formation of a pool of free 40S subunits, 2) formation of the ternary complex (Met-tRNAi/eIF2/GTP), and subsequently, the 43S
complex (comprising the 40S subunit, Met-tRNAi/eIF2/GTP, eIF3 and eIF1A), 3) activation of the mRNA upon binding of the cap-binding
complex eIF4F, and factors eIF4A, eIF4B and eIF4H, with subsequent binding to the 43S complex, 4) ribosomal scanning and start codon
recognition, and 5) GTP hydrolysis and joining of the 60S ribosomal subunit.
10.4.1.1.1 Formation of a pool of free 40S subunits
Description
The 80S ribosome dissociates into free 40S (small) and 60S (large) ribosomal subunits. Each ribosomal subunit is constituted by several
individual ribosomal proteins and rRNA.
10.4.1.1.1.1 Release of 40S and 60S subunits from the 80S ribosome
Description
80S monosomes dissociate into 40S and 60S ribosomal subunits. eIF1A promotes this dissociation.
References
JW Hershey, WC Merrick, "Pathway and mechanism of initiation of protein synthesis.", Translational Control of Gene Expression. (Sonenberg,
Hershey and Mathews, eds.), 2000, 33-88.
Reaction
10.4.1.1.1.2 eIF3 and eIF1A bind to the 40S subunit
Description
eIF3 and eIF1A bind to the 40S ribosomal subunit.
References
R Benne, JW Hershey, "The mechanism of action of protein synthesis initiation factors from rabbit reticulocytes.", J Biol Chem, 253, 1978,
3078-87.
H Goumans, A Thomas, A Verhoeven, HO Voorma, R Benne, "The role of eIF-4C in protein synthesis initiation complex formation.", Biochim
Biophys Acta, 608, 1980, 39-46.
DT Peterson, WC Merrick, B Safer, "Binding and release of radiolabeled eukaryotic initiation factors 2 and 3 during 80 S initiation complex
formation.", J Biol Chem, 254, 1979, 2509-16.
H Trachsel, B Erni, MH Schreier, T Staehelin, "Initiation of mammalian protein synthesis. II. The assembly of the initiation complex with purified
initiation factors.", J Mol Biol, 116, 1978, 755-67.
R Majumdar, A Bandyopadhyay, U Maitra, "Mammalian translation initiation factor eIF1 functions with eIF1A and eIF3 in the formation of a stable
40 S preinitiation complex.", J Biol Chem, 278, 2003, 6580-7.
Reaction
10.4.1.1.2 Formation of the ternary complex, and subsequently, the 43S complex
Description
Binding of the methionyl-tRNA initiator to the active eIF2:GTP complex results in the formation of the ternary complex. Subsequently, this
Met-tRNAi:eIF2:GTP (ternary) complex binds to the complex formed by the 40S subunit, eIF3 and eIF1A, to form the 43S complex.
References
3078-87.
B Safer, SL Adams, WF Anderson, WC Merrick, "Binding of MET-TRNAf and GTP to homogeneous initiation factor MP.", J Biol Chem, 250,
1976, 9076-82.
S Gupta, AL Roy, MK Nag, TG Kinzy, S MacMillan, RE Hileman, TE Dever, S Wu, WC Merrick, JW Hershey, "New insights into an old problem:
ternary complex (Met-tRNAf.eIF.GTP) formation in animal cells.", Post-transcriptional control of gene expression. J.E. McCarty and M.F.Tuite
(eds)., 1990, 521-526.
10.4.1.1.2.1 De novo formation of eIF2:GTP
Description
Activation of eIF2 through direct binding of GTP.

References
1976, 9076-82.
Reaction
10.4.1.1.2.2 Met-tRNAi binds to eIF2:GTP to form the ternary complex
Description
The ternary complex forms upon binding of the initiator methionyl-tRNA to the active eIF2:GTP complex.
References
1976, 9076-82.
Reaction
10.4.1.1.2.3 Formation of the 43S pre-initiation complex
Description
The ternary complex (Met-tRNAi:eIF2:GTP) binds to the complex formed by the 40S subunit, eIF3 and eIF1A, to form the 43S complex. eIF1A
promotes binding of the ternary complex to the 40S subunit within 43S. The initiator methionyl-tRNA from the ternary complex is positioned at
the ribosomal P site.
References
3078-87.
(eds)., 1990, 521-526.
Reaction
10.4.1.1.3 Activation of the mRNA upon binding of the cap-binding complex and eIFs, and subsequent binding to 43S
Description
The cap-binding complex is constituted by the initiation factors eIF4A, eIF4G and eIF4E. First, eIF4E must be released from the inactive
eIF4E:4E-BP complex. Then eIF4A interacts with eIF4G, and eIF4E binds to the amino-terminal domain of eIF4G, resulting in the formation of
the cap-binding complex eIF4F. eIF4A together with eIF4B or eIF4H is thought to unwind RNA secondary structures near the 5'-end of the
mRNA. The translation initiation complex is formed when the 43S complex binds the cap-bound mRNA.
References
TV Pestova, SI Borukhov, CU Hellen, "Eukaryotic ribosomes require initiation factors 1 and 1A to locate initiation codons.", Nature, 394, 1999,
854-9.
3078-87.
JA Grifo, SM Tahara, MA Morgan, AJ Shatkin, WC Merrick, "New initiation factor activity required for globin mRNA translation.", J Biol Chem,
258, 1983, 5804-10.
10.4.1.1.3.1 Release of eIF4E from the inactive eIF4E:4E-BP complex
Description
eIF4E gets released from the inactive eIF4E:4EBP complex.
References
A Pause, GJ Belsham, AC Gingras, O DonzÃ©, TA Lin, JC Lawrence, N Sonenberg, "Insulin-dependent stimulation of protein synthesis by
phosphorylation of a regulator of 5'-cap function.", Nature, 371, 1994, 762-7.
Reaction
10.4.1.1.3.2 Formation of the cap-binding eIF4F complex
Description
eIF4A interacts with eIF4G, and eIF4E interacts with the amino-terminal domain of eIF4G to form the cap-binding complex eIF4F.
References
J Yoder-Hill, A Pause, N Sonenberg, WC Merrick, "The p46 subunit of eukaryotic initiation factor (eIF)-4F exchanges with eIF-4A.", J Biol Chem,
268, 1993, 5566-73.
Reaction
10.4.1.1.3.3 eIF4F binds to mRNP
Description
The factor eIF4E within the eIF4F (cap-binding) complex directly binds the 5'-cap on eukaryotic mRNAs. Note that the mRNA is in complex with
cytoplasmic proteins constituting an mRNP complex.
References
N Sonenberg, KM Rupprecht, SM Hecht, AJ Shatkin, "Eukaryotic mRNA cap binding protein: purification by affinity chromatography on
sepharose-coupled m7GDP.", Proc Natl Acad Sci U S A, 76, 1980, 4345-9.
258, 1983, 5804-10.
Reaction
10.4.1.1.3.4 Cap-bound mRNA is activated by helicases
Description
The DEAD-box RNA helicase eIF4A, together with the RNA-binding proteins eIF4B or eIF4H, is thought to unwind RNA secondary structures
near the 5'-end of the mRNA and in the presence of ATP.
References
258, 1983, 5804-10.
Reaction
10.4.1.1.3.5 Translation initiation complex formation
Description
The translation initiation complex forms when the 43S complex binds the mRNA that is associated with eIF4F, eIF4B and eIF4H. eIF4G in the
eIF4F complex can directly contact eIF3 in the 43S complex. eIF1A is necessary for the formation of this complex.
References
854-9.
3078-87.
258, 1983, 5804-10.
10.4.1.1.3.5.1 Formation of translation initiation complexes containing mRNA that does not circularize
Description
References
854-9.
3078-87.
258, 1983, 5804-10.
Reaction
10.4.1.1.3.5.2 Formation of translation initiation complexes yielding circularized Ceruloplasmin mRNA in a 'closed-loop' conformation
Authors
Matthews, L, 2004-12-13.
Editors
Matthews, L, 0000-00-00.
Description
The precise order of events leading to the circularization of poly (A) mRNA during translation initiation is unknown. Here the association of PABP
with the poly (A) mRNA and the association of PABP with eIF4F are represented as occuring simultaneously after formation of the initiation
complex. However, it is also possible that these interactions occur during the formation of the translation initiation complex. The binding of eIF4F
to the cap and binding of PABP to the poly (A) tail, for example, may occur at the same time. In fact, the eIF4G-PABP interaction helps eIF4F to
bind tighter to the cap (Borman et al. 2000.) In addition, eIF4B and eIF4H bind more transiently to the mRNA and may not be part of an initial
complex in which PABP has not yet touched eIF4G.
References
H Imataka, A Gradi, N Sonenberg, "A newly identified N-terminal amino acid sequence of human eIF4G binds poly(A)-binding protein and
functions in poly(A)-dependent translation", EMBO J, 17, 1998, 7480-9.
AM Borman, YM Michel, KM Kean, "Biochemical characterisation of cap-poly(A) synergy in rabbit reticulocyte lysates: the eIF4G-PABP
interaction increases the functional affinity of eIF4E for the capped mRNA 5'-end", Nucleic Acids Res, 28, 2000, 4068-75.
Source reaction
This reaction was inferred from the corresponding reaction "Translation initiation complex formation" in species Homo sapiens.
854-9.
3078-87.
258, 1983, 5804-10.
Reaction
10.4.1.1.4 Ribosomal scanning and start codon recognition
Description
The 80S ribosome bound to the mRNA moves along the mRNA molecule from its initial site to the initiation codon and forms a 48S complex, in
which the initiation codon is base paired to the anticodon of the Met-tRNAi. Proper recognition of the AUG initiation codon depends on base
pairing with the anticodon of the Met-tRNAi and requires eIF1, eIF1A, eIF2 and eIF5.
References
854-9.
3078-87.
TF Donahue, "Genetic approaches to translation initiation in Saccharomyces cerevisiae.", Translational Control of Gene Expression. N.
Sonenberg, J.W. Hershey and M.B. Mathews (eds.), 2000, 487-502.
M Kozak, "Evaluation of the "scanning model" for initiation of protein synthesis in eucaryotes.", Cell, 22, 1981, 7-8.
10.4.1.1.4.1 Ribosomal scanning
Description
The mRNA-bound ribosomal complex moves along the 5'-untranslated region (5'-UTR) of the mRNA from its initial site to the initiation codon to
form a 48S complex, in which the initiation codon (AUG) is base paired to the anticodon of the Met-tRNAi. It is not known whether eIF4A (or
another ATPase, such as DED1) facilitates scanning by melting mRNA secondary structures or by actively propelling the ribosome.
References
TV Pestova, IN Shatsky, CU Hellen, "Functional dissection of eukaryotic initiation factor 4F: the 4A subunit and the central domain of the 4G
subunit are sufficient to mediate internal entry of 43S preinitiation complexes.", Mol Cell Biol, 16, 1997, 6870-8.
3078-87.
RY Chuang, PL Weaver, Z Liu, TH Chang, "Requirement of the DEAD-Box protein ded1p for messenger RNA translation.", Science, 275, 1997,
1468-71.
I Iost, M Dreyfus, P Linder, "Ded1p, a DEAD-box protein required for translation initiation in Saccharomyces cerevisiae, is an RNA helicase.", J
Biol Chem, 274, 1999, 17677-83.
Reaction
10.4.1.1.4.2 Start codon recognition
Description
The AUG initiation codon in the mRNA is recognized by base pairing with the anticodon of the Met-tRNAi. This reaction requires eIF1, eIF1A,
eIF2 and eIF5.
References
854-9.
Reaction
10.4.1.1.5 GTP hydrolysis and joining of the 60S ribosomal subunit
Description
Hydrolysis of eIF2-GTP occurs after the Met-tRNAi has recognized the AUG. This reaction is catalyzed by eIF5 (or eIF5B) and is thought to
cause dissociation of all other initiation factors and allow joining of the large 60S ribosomal subunit. The 60S subunit joins - a reaction catalyzed
by eIF5 or eIF5B - resulting in a translation-competent 80S ribosome. Following 60S subunit joining, eIF5B hydrolyzes its GTP and is released
from the 80S ribosome, which is now ready to start elongating the polypeptide chain.
References
MH Schreier, B Erni, T Staehelin, "Initiation of mammalian protein synthesis. I. Purification and characterization of seven initiation factors.", J Mol
Biol, 116, 1978, 727-53.
3078-87.
A Chakrabarti, U Maitra, "Function of eukaryotic initiation factor 5 in the formation of an 80 S ribosomal polypeptide chain initiation complex.", J
Biol Chem, 266, 1991, 14039-45.
WC Merrick, WM Kemper, WF Anderson, "Purification and characterization of homogeneous initiation factor M2A from rabbit reticulocytes.", J
Biol Chem, 250, 1975, 5556-62.
TV Pestova, IB Lomakin, JH Lee, SK Choi, TE Dever, CU Hellen, "The joining of ribosomal subunits in eukaryotes requires eIF5B.", Nature, 403,
2000, 332-5.
K Asano, J Clayton, A Shalev, AG Hinnebusch, "A multifactor complex of eukaryotic initiation factors, eIF1, eIF2, eIF3, eIF5, and initiator
tRNA(Met) is an important translation initiation intermediate in vivo.", Genes Dev, 14, 2000, 2534-46.
10.4.1.1.5.1 eIF2:GTP is hydrolyzed, eIFs are released

Description
Once the Met-tRNAi has recognized the AUG, eIF2-bound GTP is hydrolyzed. The reaction is catalyzed by eIF5 (or eIF5B) and is thought to
cause dissociation of all other initiation factors and allow joining of the large 60S ribosomal subunit. Release of the initiation factors from 40S
leaves the Met-tRNAi in the ribosomal P-site base-paired to the start codon on the mRNA.
References
Biol, 116, 1978, 727-53.
3078-87.
Biol Chem, 266, 1991, 14039-45.
Biol Chem, 250, 1975, 5556-62.
2000, 332-5.
Reaction
10.4.1.1.5.2 eIF5B:GTP is hydrolyzed and released
Description
Once the 60S subunit joins the translation initiation complex, eIF5B hydrolyzes its GTP and is released from the now 80S monosome. The fully
assembled 80s ribosome is now ready to start elongation of the polypeptide chain.
References
Biol Chem, 250, 1975, 5556-62.
2000, 332-5.
Reaction
10.4.1.1.5.3 The 60S subunit joins the translation initiation complex
Description
Joining of the 60S subunit to form the 80S ribosome is catalyzed by the presence of GTP-bound eIF5B.
References
Biol, 116, 1978, 727-53.
Biol Chem, 250, 1975, 5556-62.
2000, 332-5.
Reaction
10.4.1.1.6 Recycling of eIF2:GDP
Description
The active eIF2:GTP complex may be formed by direct binding of GTP to free eIF2 or by GDP-GTP exchange on the eIF2:GDP:eIF2B complex.
The eIF2:GDP complex binds eIF2B forming an eIF2:GDP:eIF2B intermediate complex. eIF2B is a guanine nucleotide releasing factor required
to cause GDP release so that a new GTP molecule can bind and activate eIF2. Phosphorylated eIF2:GDP sequesters all eIF2B as an inactive
complex, and thus, reuse of eIF2 is inhibited as a consequence of the tight bond it forms with eIF2B, which prevents nucleotide exchange.
Therefore, in the absence of free eIF2B, excess eIF2 remains in its inactive GDP-bound form and protein synthesis slows dramatically.
References
MJ Clemens, VM Pain, ST Wong, EC Henshaw, "Phosphorylation inhibits guanine nucleotide exchange on eukaryotic initiation factor 2.",
Nature, 296, 1982, 93-5.
10.4.1.1.6.1 Formation of eIF2:GDP:eIF2B intermediate
Description
Inactive eIF2:GDP binds eIF2B to form an eIF2:GDP:eIF2B intermediate.
References
JN Dholakia, AJ Wahba, "Mechanism of the nucleotide exchange reaction in eukaryotic polypeptide chain initiation. Characterization of the
guanine nucleotide exchange factor as a GTP-binding protein.", J Biol Chem, 264, 1989, 546-50.
AG Rowlands, R Panniers, EC Henshaw, "The catalytic mechanism of guanine nucleotide exchange factor action and competitive inhibition by
phosphorylated eukaryotic initiation factor 2.", J Biol Chem, 263, 1988, 5526-33.
Reaction
10.4.1.1.6.2 eIF2 activation
Description
eIF2B is a guanine nucleotide releasing factor that is required to cause GDP release so that a new GTP molecule can bind and activate eIF2, so
that it can be reused.
References
Reaction
10.4.1.2 Cap-independent Translation Initiation
Description
Initiation on several viral and cellular mRNAs is cap-independent and is mediated by binding of the ribosome to internal ribosome entry site
(IRES) elements. These elements are often found in characteristically long structured regions on the 5'-UTR of an mRNA that may or may not
have regulatory upstream open reading frames (uORFs). Both of these features on the 5'-end of the mRNA hinder ribosomal scanning, and thus
promote a cap-independent translation initiation mechanism. IRESs act as specific translational enhancers that allow translation initiation to
occur in response to specific stimuli and under the control of different trans-acting factors, as for example when cap-dependent protein synthesis
is shut off during viral infection. Such regulatory elements have been identified in the mRNAs of growth factors, protooncogenes, angiogenesis
factors, and apoptosis regulators, which are translated under a variety of stress conditions, including hypoxia, serum deprivation, irradiation and
apoptosis. Thus, cap-independent translational control might have evolved to regulate cellular responses in acute but transient stress conditions
that would otherwise lead to cell death, while the same mechanism is of major importance for viral mRNAs to bypass the shutting-off of host
protein synthesis after infection. Encephalomyocarditis virus (EMCV) and hepatitis C virus exemplify two distinct mechanisms of IRES-mediated
initiation. In contrast to cap-dependent initiation, the eIF4A and eIF4G subunits of eIF4F bind immediately upstream of the EMCV initiation codon
and promote binding of a 43S complex. Accordingly, EMCV initiation does not involve scanning and does not require eIF1, eIF1A, and the eIF4E
subunit of eIF4F. Nonetheless, initiation on some EMCV-like IRESs requires additional non-canonical initiation factors, which alter IRES
conformation and promote binding of eIF4A/eIF4G. Initiation on the hepatitis C virus IRES is simpler: a 43S complex containing only eIF2 and
eIF3 binds directly to the initiation codon as a result of specific interaction of the IRES and the 40S subunit.
References
M Holcik, N Sonenberg, RG Korneluk, "Internal ribosome initiation of translation and the control of cell death.", Trends Genet, 16, 2000, 469-73.
10.4.2 Eukaryotic Translation Elongation
Authors
Description
The translation elongation cycle adds one amino acid at a time to a growing polypeptide according to the sequence of codons found in the
mRNA. The next available codon on the mRNA is exposed in the aminoacyl-tRNA (aa-tRNA) binding site (A site) on the 30S subunit.
A: Ternary complexes of aa -tRNA:eEF1A:GTP enter the ribosome and enable the anticodon of the tRNA to make a codon/anticodon interaction
with the A-site codon of the mRNA. B: Upon cognate recognition, the eEF1A:GTP is brought into the GTPase activating center of the ribosome,
GTP is hydrolyzed and eEF1A:GDP leaves the ribosome. C: The peptidyl transferase center of ribosome catalyses the formation of a peptide
bond between the incoming amino acid and the peptide found in the peptidyl-tRNA binding site (P site). D: In the pre-translocation state of the
ribosome, the eEF2:GTP enters the ribosome, physically translocating the peptidyl-tRNA out of the A site to P site and leaves the ribosome
eEF2:GDP. This action of eEF2:GTP accounts for the precise movement of the mRNA by 3 nucleotides.Consequently, deacylated tRNA is
shifted to the E site. A ribosome associated ATPase activity is proposed to stimulate the release of deacylated tRNA from the E site subsequent
to translocation (Elskaya et al., 1991). In this post-translocation state, the ribosome is now ready to receive a new ternary complex.
This process is illustrated below with: an amino acyl-tRNA with an amino acid, a peptidyl-tRNA with a growing peptide, a deacylated tRNA with
an -OH, and a ribosome with A,P and E sites to accommodate these three forms of tRNA.
References
WC Merrick, J Nyborg, "The Protein Biosynthesis Elongation Cycle", Translational Control of Gene Expression, 2000, 89-127.
AV El'skaya, GV Ovcharenko, SS Palchevskii, ZM Petrushenko, FJ Triana-Alonso, KH Nierhaus, "Three tRNA binding sites in rabbit liver
ribosomes and role of the intrinsic ATPase in 80S ribosomes from higher eukaryotes", Biochemistry, 36, 1997, 10492-7.
LD Kapp, JR Lorsch, "The molecular mechanics of eukaryotic translation", Annu Rev Biochem, 73, 2004, 657-704.
10.4.2.1 eEF1A complexes with GTP

Authors
Description
The cycle of elongation starts with an empty ribosomal A-site and the peptidyl-tRNA in the P-site. eEF1A is activated by GTP binding and allows
for the subsequent binding of aminoacyl-tRNA (aa-tRNA).This process is illustrated below with a GTP molecule in white and eEF1A protein in
yellow.
Source reaction
This reaction was inferred from the corresponding reaction "reEF1A complexes with GTP" in species Oryctolagus cuniculus.
MD Carvalho, JF Carvalho, WC Merrick, "Biological characterization of various forms of elongation factor 1 from rabbit reticulocytes", Arch
Reaction
10.4.2.2 eEF1A:GTP:aminoacyl tRNA ternary complex formation.
Authors
Description
The binding of eEF1A:GTP to aminoacyl tRNA (aa-tRNA) results in the formation of a ternary complex (eEF1A:GTP:aa-tRNA). Human eEF1A
and rabbit eEF1A are 100% identical, and prokaryotic homologue of eEF1A (EF-Tu) shows 59% identity in the GTP-binding domain.This
process is illustrated below with: a GTP molecule in white and eEF1A protein in yellow.
References
WC Merrick, TE Dever, TG Kinzy, SC Conroy, J Cavallius, CL Owens, "Characterization of protein synthesis factors from rabbit reticulocytes",
Biochim Biophys Acta, 1050, 1990, 235-40.
Source reaction
This reaction was inferred from the corresponding reaction "reEF1A:GTP:Aminoacyl-tRNA ternary complex formation" in species Oryctolagus
cuniculus.
Reaction
10.4.2.3 Peptide chain elongation
Authors
Description
The mechanism of a peptide bond requires the movement of three protons. First the deprotonation of the ammonium ion generates a reactive
amine, allowing a nucleophilic attack on the carbonyl group. This is followed by the loss of a proton from the reaction intermediate, only to be
taken up by the oxygen on the leaving group (from the end of the amino acid chain bound to the tRNA in the P-site). The peptide bond formation
results in the net loss of one water molecule, leaving a deacylated-tRNA in the P-site, and a nascent polypeptide chain one amino acid larger in
the A-site.
For the purpose of illustration, the figures used in the section show one amino acid being added to a peptidyl-tRNA with a growing peptide chain.
References
R Green, JR Lorsch, "The path to perdition is paved with protons", Cell, 110, 2002, 665-8.
10.4.2.3.1 Aminoacyl-tRNA binds to the ribosome at the A-site
Authors
Description
Once the correct codon-anticodon match occurs between the mRNA and aa-tRNA, the decoding event triggers GTP hydrolysis on eEF1A. The
resulting conformational change releases the aa-tRNA to the A-site, and GDP bound form eEF1A is released from the ribosome.
Insight into the mechanics of this system has been obtained from earlier works with rabbit reticulocytes and the E.coli system.
This process is illustrated below with: an amino acyl-tRNA with an amino acid, a peptidyl-tRNA with a growing peptide and a ribosome with A,P
and E sites to accommodate these two forms of tRNA.
References
MV Rodnina, T Pape, R Fricke, W Wintermeyer, "Elongation factor Tu, a GTPase triggered by codon recognition on the ribosome: mechanism
and GTP consumption", Biochem Cell Biol, 73, 1995, 1221-7.
Source reaction
This reaction was inferred from the corresponding reaction "Aminoacyl-tRNA binds to the ribosome at the A-site" in species Oryctolagus
cuniculus.
Reaction
10.4.2.3.2 Hydrolysis of eEF1A:GTP
Authors
Description
resulting conformational change releases the aa-tRNA to the A-site, and GDP bound form of eEF1A is released from the ribosome.
This process is illustrated below with: an amino acyl-tRNA with an amino acid,a peptidyl-tRNA with a growing peptide and a ribosome with A,P
References
Source reaction
This reaction was inferred from the corresponding reaction "Hydrolysis of reEF1A:GTP" in species Oryctolagus cuniculus.
Reaction
10.4.2.3.3 Peptide transfer from P-site tRNA to the A-site tRNA
Description
The A- and P-sites of the ribosome positions the aa-tRNA and peptidyl-tRNA such that a nucleophilic attack can occur between the amine group
of the A-site aa-tRNA and the carbonyl group of the growing peptide chain on the P-site tRNA, resulting in the formation of a peptide bond. The
carboxyl end of the peptide chain is uncoupled from the tRNA molecule in the P-site and forms a new peptide bond with the amino acid that is in
the A-site.
This process is illustrated below with: a peptidyl-tRNA with a growing peptide,a deacylated tRNA with an -OH and a ribosome with A,P and E
sites to accommodate these three forms of tRNA.
References
WC Merrick, "Mechanism and regulation of eukaryotic protein synthesis", Microbiol Rev, 56, 1992, 291-315.
Source reaction
This reaction was inferred from the corresponding reaction "Peptide transfer from P-site tRNA to the A-site tRNA" in species Escherichia coli.
RK Tompkins, "Sequential translation of trinucleotide codons for peptide bond formation, translocation, and termination", Proc Natl Acad Sci U S
A, 66, 1970, 1164-9.
Reaction
10.4.2.3.4 Translocation of ribosome by 3 bases in the 3' direction
Authors
Description
Following peptide bond formation, GTP-bound eEF2 catalyzes the translocation of the deacylated tRNA in the P-site and the peptidyl-tRNA in
the A-site (the pre-translocation state) into the E- and P- sites (the post-translocation state), respectively. Thus, the mRNA advances by three
bases to expose the next codon in the A-site. After translocation, GDP-bound eEF2 leaves the ribosome to allow another round of elongation.
eEF2 is reactivated by the release of GDP and binds GTP for subsequent rounds.
This process is illustrated below with a peptidyl-tRNA with a growing peptide, a deacylated tRNA with an -OH and a ribosome with A,P and E
sites to accommodate these three forms of tRNA is also shown.
References
OW Odom, WD Picking, B Hardesty, "Movement of tRNA but not the nascent peptide during peptide bond formation on ribosomes",
Biochemistry, 29, 1990, 10734-44.
Source reaction
This reaction was inferred from the corresponding reaction "Translocation of ribosome by 3 bases in the 3' direction" in species Escherichia coli.
Biochemistry, 29, 1990, 10734-44.
K Holschuh, D Riesner, HG Gassen, "Steps of mRNA translocation in protein biosynthesis", Nature, 293, 1981, 675-7.
A, 66, 1970, 1164-9.
Reaction
10.4.2.4 Regeneration of eEF1A:GTP by eEF1B activity
Authors
Description
The eEF1B complex binds to eEF1A and regulates its activity by catalyzing the release of GDP. Subsequently, GTP is able to bind eEF1A
allowing the formation of the ternary complex (eEF1A-GTP-aa-tRNA).In metazoans eEF1 protein family is composed of four subunits: eEF1A
and eEF1B alpha, beta, and gamma (formerly EF-1alpha, EF-1beta, EF-1delta, and EF-1gamma, respectively). Both eEF1B alpha and eEF1B
beta function as nucleotide exchange proteins. eEF1B gamma associates with eEF1B alpha and stimulates its exchange activity.
This process is illustrated below with a GTP molecule in white and eEF1A protein in yellow.The three subunits of eEF1B are also shown.
References
JM Perez, G Siegal, J Kriek, K Hard, J Dijk, GW Canters, W Moller, "The solution structure of the guanine nucleotide exchange domain of
human elongation factor 1beta reveals a striking resemblance to that of EF-Ts from Escherichia coli", Structure Fold Des, 7, 1999, 217-26.
Damme HT van, R Amons, R Karssies, CJ Timmers, GM Janssen, W Moller, "Elongation factor 1 beta of artemia: localization of functional sites
and homology to elongation factor 1 delta", Biochim Biophys Acta, 1050, 1990, 241-7.
GM Janssen, W Moller, "Elongation factor 1 beta gamma from Artemia. Purification and properties of its subunits.", Eur J Biochem, 171, 1988,
119-29.
Reaction
10.4.3 Eukaryotic Translation Termination
Authors
Bedwell, DM, 2004-11-09.
Editors
Description
The arrival of any of the three stop codons (UAA, UAG and UGA) into the ribosomal A-site triggers the binding of a release factor (RF) to the
ribosome and subsequent polypeptide chain release. In eukaryotes, the RF is composed of two proteins, eRF1 and eRF3. eRF1 is responsible
for the hydrolysis of the peptidyl-tRNA, while eRF3 provides a GTP-dependent function. The ribosome releases the mRNA and dissociates into
its two complex subunits, which can reassemble on another molecule to begin a new round of protein synthesis. It should be noted that at
present, there is no factor identified in eukaryotes that would be the functional equivalent of the bacterial ribosome release (or recycling) factor,
RRF, that catalyzes dissociation of the ribosome from the mRNA following release of the polypeptide
References
J Salas-Marco, DM Bedwell, "GTP hydrolysis by eRF3 facilitates stop codon decoding during eukaryotic translation termination", Mol Cell Biol,
24, 2004, 7769-78.
S Ichikawa, A Kaji, "Molecular cloning and expression of ribosome releasing factor", J Biol Chem, 264, 1989, 20054-9.
10.4.3.1 GTP bound eRF3:eRF1 complex binds the peptidyl tRNA:mRNA:80S Ribosome complex
Authors
Description
Please note that this reaction was inferred from experiments performed using Saccharomyces cerevisiae.
References
L Frolova, Goff X Le, HH Rasmussen, S Cheperegin, G Drugeon, M Kress, I Arman, AL Haenni, JE Celis, M Philippe, "A highly conserved
eukaryotic protein family possessing properties of polypeptide chain release factor", Nature, 372, 1994, 701-3.
S Hoshino, M Imai, M Mizutani, Y Kikuchi, F Hanaoka, M Ui, T Katada, "Molecular cloning of a novel member of the eukaryotic polypeptide
chain-releasing factors (eRF). Its identification as eRF3 interacting with eRF1.", J Biol Chem, 273, 1998, 22254-9.
Source reaction
This reaction was inferred from the corresponding reaction "GTP bound eRF3:eRF1 complex binds the peptidyl-tRNA:mRNA:Ribosome
complex" in species Saccharomyces cerevisiae.
24, 2004, 7769-78.
Reaction
10.4.3.2 GTP Hydrolysis by eRF3 bound to the eRF1:mRNA:polypeptide:80S Ribosome complex
Authors
Description
References
S Hoshino, M Imai, T Kobayashi, N Uchida, T Katada, "The eukaryotic polypeptide chain releasing factor (eRF3/GSPT) carrying the translation
termination signal to the 3'-Poly(A) tail of mRNA. Direct association of erf3/GSPT with polyadenylate-binding protein.", J Biol Chem, 274, 1999,
16677-80.
Source reaction
This reaction was inferred from the corresponding reaction "GTP Hydrolysis by eRF3 bound to the eRF1:mRNA:polypeptide:80S Ribosome
24, 2004, 7769-78.
Reaction
10.4.3.3 Polypeptide release from the eRF3-GDP:eRF1:mRNA:80S Ribosome complex
Authors
Description
References
16677-80.
Source reaction
This reaction was inferred from the corresponding reaction "Polypeptide release from the eRF3-GDP:eRF1:mRNA:80S Ribosome complex" in
species Saccharomyces cerevisiae.
24, 2004, 7769-78.
Reaction
11 HIV Infection
Authors
Bukrinsky, M, D'Eustachio, P, Gillespie, ME, Gopinathrao, G, Iordanskiy, S, Morrow, MP, Matthews, L, Rice, AP, 0000-00-00.
Description
The global pandemic of Human Immunodeficiency Virus (HIV) infection has resulted in tens of millions of people infected by the virus and
millions more affected. UNAIDS estimates around 40 million HIV/AIDS patients worldwide with 75% of them living in sub-Saharan Africa. The
primary method of HIV infection is by sexual exposure while nonsexual HIV transmission also can occur through transfusion with contaminated
blood products, injection drug use, occupational exposure,accidental needlesticks or mother-to-child transmission. HIV damages the immune
system, leaving the infected person vulnerable to a variety of "opportunistic" infections arising from host immune impairment (Hare, 2004).
HIV-1 and the less common HIV-2 belong to the family of retroviruses. HIV-1 contains a single-stranded RNA genome that is 9 kilobases in
length and contains 9 genes that encode 15 different proteins. These proteins are classified as: structural proteins (Gag, Pol, and Env),
regulatory proteins (Tat and Rev), and accessory proteins (Vpu, Vpr, Vif, and Nef) (Frankel and Young,1998).
HIV infection cycle can be divided into two phases:
1. An Early phase consisting of early events occuring after HIV infection of a susceptible target cell and a
2. Late phase comprising the later events in the HIV-infected cell resulting in the assembly of new infectious virions. The section titled HIV
lifecycle consists of annotations of events in these two phases.
The virus has developed various molecular strategies to suppress the antiviral immune responses (innate, cellular and humoral) of the host.
HIV-1 viral auxiliary proteins (Tat, Rev, Nef, Vif, Vpr and Vpu) play important roles in these host-pathogen interactions (Li et al.,2005). The
section titled Host interactions of HIV factors will highlight these complex post-infection processes and the annotations will be released in near
future.
References
L Li, HS Li, CD Pauza, M Bukrinsky, RY Zhao, "Roles of HIV-1 auxiliary proteins in viral pathogenesis and host-pathogen interactions", Cell Res,
15, 2005, 923-34.
BC Hare, "Clinical Overview of HIV Disease", HIV InSite knowledgebase (http://hivinsite.ucsf.edu/InSite?page=kb-03-01-01), 2004.
AD Frankel, JA Young, "HIV-1: fifteen proteins and an RNA", Annu Rev Biochem, 67, 1998, 1-25.
11.1 HIV Life Cycle
Authors
Bukrinsky, M, D'Eustachio, P, Gillespie, ME, Gopinathrao, G, Iordanskiy, S, Morrow, MP, Matthews, L, Rice, AP, 0000-00-00.
Description
The life cycle of HIV-1 is divided into early and late phases, shown schematically in the figure. In the early phase, an HIV-1 virion binds to
receptors and co-receptors on the human host cell surface (a), viral and host cell membranes fuse and the viral particle is uncoated (b), the viral
genome is reverse transcribed and the viral preintegration complex (PIC) forms (c), the PIC is transported through the nuclear pore into the
nucleoplasm (d), and the viral reverse transcript is integrated into a host cell chromosome (e). In the late phase, viral RNAs are transcribed from
the integrated viral genome and processed to generate viral mRNAs and full-length viral genomic RNAs (f), the viral RNAs are exported through
the nuclear pore into the cytosol (g), viral mRNAs are translated and the resulting viral proteins are post-translationally processed (h), core
particles containing viral genomic RNA and proteins assemble at the host cell membrane and immature viral particles are released by budding.
The released particles mature to become infectious (j), completing the cycle (Frankel and Young 1998; Miller and Bushman 1997).
Most of the crucial concepts used to describe these processes were originally elucidated in studies of retroviruses associated with tumors in
chickens, birds, and other animal model systems, and the rapid elucidation of the basic features of the HIV-1 life cycle was critically dependent
on the intellectual framework provided by these earlier studies. This earlier work has been very well summarized (e.g., Weiss et al. 1984; Coffin
et al. 1997); here for brevity and clarity we focus on experimental studies specific to the HIV-1 life cycle.
References
MD Miller, FD Bushman, "Human immunodeficiency virus type 1 preintegration complexes: studies of organization and composition", J Virol, 71,
1997, 5382-90.
JM Coffin, SH Hughes, HE Varmus, Retroviruses (CSHL Press), 1997, 1-843.
AD Frankel, JA Young, "HIV-1: fifteen proteins and an RNA", Annu Rev Biochem, 67, 1998, 1-25.
11.1.1 Early Phase of HIV Life Cycle
Authors
Iordanskiy, S, Morrow, MP, Gopinathrao, G, D'Eustachio, P, Bukrinsky, M, 2006-05-16.
Editors
Gopinathrao, G, 2006-02-17, Gopinathrao, G, D'Eustachio, P, 2006-05-18.
Reviewers
Aiken, C, Bushman, FD, Hughes, SH, Reeves, J, 2006-10-30.
Description
In the early phase of HIV lifecycle, an active virion binds and enters a target cell mainly by specific interactions of the viral envelope proteins with
host cell surface receptors. The virion core is uncoated to expose a viral nucleoprotein complex containing RNA and viral proteins. HIV RNA
genome is reverse transcribed by the viral Reverse Transcriptase to form a cDNA copy, that gets inserted into host cell DNA. The viral Integrase
enzyme is vital to carry out the integration of the viral cDNA into the host genome. The host DNA repair enzymes probably repair the breaks in
DNA at the sites of integration.
References
WC Greene, BM Peterlin, "Charting HIV's remarkable voyage through the cell: Basic science as a passport to future therapy", Nat Med, 8, 2002,
673-80.
11.1.1.1 Binding and entry of HIV virion
Authors
Morrow, MP, Bukrinsky, M, 2006-03-07.
Editors
Reviewers
Reeves, J, 2006-06-12.
Description
HIV enters cells by fusion at the cell surface, that results in a productive infection. The envelope (Env) protein of HIV mediates entry. Env is
composed of a surface subunit, gp120, and a transmembrane subunit, gp41, which assemble as heterotrimers on the virion surface.The trimeric,
surface gp120 protein (SU) on the virion engages CD4 on the host cell, inducing conformational changes that promote binding to select
chemokine receptors CCR5 and CXCR4.
The sequential interplay between SU, CD4 and chemokine coreceptors prompts a conformational change in the transmembrane gp41. This
coiled coil protein, assembled as a trimer on the virion membrane, springs open to project three peptide fusion domains that 'harpoon' the lipid
bilayer of the target cell. A hairpin structure (also referred to as a "coiled coil bundle") is subsequently formed when the extracellular portion of
gp41 collapses, and this hairpin formation promotes the fusion of virion and target cell membranes by bringing them into close proximity. Virion
and target cell membrane fusion leads to the release of HIV viral cores into the cell interior.
11.1.1.1.1 Binding of gp120 of ENV oligomer to the host CD4
Authors
Editors
Reviewers
Reeves, J, 2006-06-12.
Description
CD4, located on the host cell membrane, is the main cellular receptor for the HIV protein gp120, which aids in mediating viral entry into target
cells. The initial step in this cascade of events is the binding of viral gp120 protein to its host receptor, CD4. The key binding sites in CD4 for
interaction with gp120 are located in the amino-terminal part of the CD4 molecule, distal to the transmembrane domain. The gp120 protein forms
an oligomer (trimer) on the viral membrane with each gp120 protein containing variable domains (known as loops) and conservative domains.
The V3 loop is also often obscured by gp120 glycosylation. Crystallization studies of CD4 suggest that the molecule has two immunoglobulin like
domains important for the CD4/gp120 interaction, with one of the domains (D1) playing a more prominent role. Further studies suggest the Phe
43 and Arg 59 residues of CD4 play a major role in complex formation. Crystallization of gp120 shows that the polypeptide chain is folded into
two major domains (an "inner" and "outer" domain with respect to the N and C termini), with the distal end of the Ã¢â‚¬Å"outerÃ¢â‚¬Â• domain
containing the V3 loop. Studies of CD4 complexed with gp120 show that CD4 is bound to gp120 in a depression which is formed at the interface
between the inner and outer domains. The complex itself is held together through van der Waals forces and hydrogen bonding.
References
PD Kwong, R Wyatt, J Robinson, RW Sweet, J Sodroski, WA Hendrickson, "Structure of an HIV gp120 envelope glycoprotein in complex with
the CD4 receptor and a neutralizing human antibody", Nature, 393, 1998, 648-59.
D Klatzmann, E Champagne, S Chamaret, J Gruest, D Guetard, T Hercend, JC Gluckman, L Montagnier, "T-lymphocyte T4 molecule behaves
as the receptor for human retrovirus LAV", Nature, 312, 1984, 767-8.
JS McDougal, MS Kennedy, JM Sligh, SP Cort, A Mawle, JK Nicholson, "Binding of HTLV-III/LAV to T4+ T cells by a complex of the 110K viral
protein and the T4 molecule", Science, 231, 1986, 382-5.
AG Dalgleish, PC Beverley, PR Clapham, DH Crawford, MF Greaves, RA Weiss, "The CD4 (T4) antigen is an essential component of the
receptor for the AIDS retrovirus", Nature, 312, 1984, 763-7.
SE Ryu, PD Kwong, A Truneh, TG Porter, J Arthos, M Rosenberg, XP Dai, NH Xuong, R Axel, RW Sweet, "Crystal structure of an HIV-binding
recombinant fragment of human CD4", Nature, 348, 1990, 419-26.
JS McDougal, JK Nicholson, GD Cross, SP Cort, MS Kennedy, AC Mawle, "Binding of the human retrovirus HTLV-III/LAV/ARV/HIV to the CD4
(T4) molecule: conformation dependence, epitope mapping, antibody inhibition, and potential for idiotypic mimicry", J Immunol, 137, 1986,
2937-44.
Reaction
11.1.1.1.2 Conformational change in gp120 of Env oligomer
Authors
Editors
Reviewers
Reeves, J, 2006-06-12.
Description
HIV-1 infection of target cells depends on the sequential interaction of the gp120 glycoprotein with the cellular CD4 receptor as well as members
of the chemokine receptor family, such as CCR5. Upon interaction with the cellular CD4 receptor, gp120 undergoes a conformation change
which allows interaction with these chemokine receptors to occur. Studies indicate that upon binding to CD4, this conformational change results
in a repositioning of V1 and V2 loops of gp120, and exposes or forms the "bridging sheet domain" epitopes, which are then available for
co-receptor (chemokine receptor) binding along with other domains of gp120. These epitopes are recognized by 17b, a member of a class of
antibodies that recognize CD4-induced (CD4i) epitopes (Kwong et al., 1998, Rizzuto et al., 1998, Zhang et al., 1999).
References
M Kowalski, J Potz, L Basiripour, T Dorfman, WC Goh, E Terwilliger, A Dayton, C Rosen, J Sodroski, "Functional regions of the envelope
glycoprotein of human immunodeficiency virus type 1", Science, 237, 1987, 1351-5.
CD Rizzuto, R Wyatt, N Hernandez-Ramos, Y Sun, PD Kwong, WA Hendrickson, J Sodroski, "A conserved HIV gp120 glycoprotein structure
involved in chemokine receptor binding", Science, 280, 1998, 1949-53.
W Zhang, G Canziani, C Plugariu, R Wyatt, J Sodroski, R Sweet, P Kwong, W Hendrickson, I Chaiken, "Conformational changes of gp120 in
epitopes near the CCR5 binding site are induced by CD4 and a CD4 miniprotein mimetic", Biochemistry, 38, 1999, 9405-16.
JA McKeating, C Shotton, J Cordell, S Graham, P Balfe, N Sullivan, M Charles, M Page, A Bolmstedt, S Olofsson, "Characterization of
neutralizing monoclonal antibodies to linear and conformation-dependent epitopes within the first and second variable domains of human
immunodeficiency virus type 1 gp120", J Virol, 67, 1993, 4932-44.
LA Lasky, G Nakamura, DH Smith, C Fennie, C Shimasaki, E Patzer, P Berman, T Gregory, DJ Capon, "Delineation of a region of the human
immunodeficiency virus type 1 gp120 glycoprotein critical for interaction with the CD4 receptor", Cell, 50, 1987, 975-85.
N Sullivan, Y Sun, Q Sattentau, M Thali, D Wu, G Denisova, J Gershoni, J Robinson, J Moore, J Sodroski, "CD4-Induced conformational
changes in the human immunodeficiency virus type 1 gp120 glycoprotein: consequences for virus entry and neutralization", J Virol, 72, 1998,
4694-703.
Reaction
11.1.1.1.3 CD4:gp120 binds to chemokine co-receptor CCR5/CXCR4
Authors
Editors
Reviewers
Reeves, J, 2006-06-12.
Description
Once the viral gp120 protein has bound to cellular CD4, its bridging sheet region becomes exposed/formed as a result of conformation changes
in the V1 and V2 loops as well as a conformational change in the gp120 core domain. Once this region is exposed, it is free to bind the HIV
co-receptors CCR5 or CXCR4 (also known as chemokine receptors). Different viruses use different co-receptors (CCR5 or CXCR4) for entry,
and many studies investigated the structural determinants of interaction between gp120 and the co-receptor.
Studies of CCR5 binding by gp120 revealed that active regions in the second extracellular loop (ECL2), the N-terminal extracellular domain
(specifically the NYYTSE motif) and at the junction between the fifth transmembrane domain and third cytoplasmic loop of the receptor are
important for viral attachment and subsequent fusion. The N-terminal region likely interacts with the core of gp120 (bridging sheet and adjacent
regions) and the base of V3, while ECL2 may be important for interacting with the tip of V3. The transmembrane 5 / cytoplasmic loop 3 junction
of CCR5 has been shown to influence the conformation of the receptor which allows for subsequent binding of gp120 (Wang et al.,1999).
Deletion of the V3 loop in gp120 abolished Env interaction with co-receptor without affecting the binding of soluble gp120 to CD4, underscoring
the importance of this loop in chemokine receptor, but not CD4, binding. Furthermore, the V3 loop is a major determinant of coreceptor
specificity, with amino acid at positions 11 and 25 being partly predictive of CCR5 or CXCR4 use. Single amino acid changes in V3 can alter
coreceptor use, however sequences outside of V3 can also contribute to coreceptor specificity.
References
I Mondor, M Moulard, S Ugolini, PJ Klasse, J Hoxie, A Amara, T Delaunay, R Wyatt, J Sodroski, QJ Sattentau, "Interactions among HIV gp120,
CD4, and CXCR4: dependence on CD4 expression level, gp120 viral origin, conservation of the gp120 COOH- and NH2-termini and V1/V2 and
V3 loops, and sensitivity to neutralizing antibodies", Virology, 248, 1998, 394-405.
G Alkhatib, C Combadiere, CC Broder, Y Feng, PE Kennedy, PM Murphy, EA Berger, "CC CKR5: a RANTES, MIP-1alpha, MIP-1beta receptor
as a fusion cofactor for macrophage-tropic HIV-1", Science, 272, 1996, 1955-8.
CD Rizzuto, R Wyatt, N Hernandez-Ramos, Y Sun, PD Kwong, WA Hendrickson, J Sodroski, "A conserved HIV gp120 glycoprotein structure
involved in chemokine receptor binding", Science, 280, 1998, 1949-53.
JC Bandres, QF Wang, J O'Leary, F Baleaux, A Amara, JA Hoxie, S Zolla-Pazner, MK Gorny, "Human immunodeficiency virus (HIV) envelope
binds to CXCR4 independently of CD4, and binding can be enhanced by interaction with soluble CD4 or by HIV envelope deglycosylation", J
Virol, 72, 1998, 2500-4.
Z Wang, B Lee, JL Murray, F Bonneau, Y Sun, V Schweickart, T Zhang, SC Peiper, "CCR5 HIV-1 coreceptor activity. Role of cooperativity
between residues in N-terminal extracellular and intracellular domains.", J Biol Chem, 274, 1999, 28413-9.
H Deng, R Liu, W Ellmeier, S Choe, D Unutmaz, M Burkhart, P Di Marzio, S Marmon, RE Sutton, CM Hill, CB Davis, SC Peiper, TJ Schall, DR
Littman, NR Landau, "Identification of a major co-receptor for primary isolates of HIV-1", Nature, 381, 1996, 661-6.
A Trkola, T Dragic, J Arthos, JM Binley, WC Olson, GP Allaway, C Cheng-Mayer, J Robinson, PJ Maddon, JP Moore, "CD4-dependent,
antibody-sensitive interactions between HIV-1 and its co-receptor CCR-5", Nature, 384, 1996, 184-7.
H Choe, M Farzan, Y Sun, N Sullivan, B Rollins, PD Ponath, L Wu, CR Mackay, G LaRosa, W Newman, N Gerard, C Gerard, J Sodroski, "The
beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates", Cell, 85, 1996, 1135-48.
A Bjorndal, H Deng, M Jansson, JR Fiore, C Colognesi, A Karlsson, J Albert, G Scarlatti, DR Littman, EM Fenyo, "Coreceptor usage of primary
human immunodeficiency virus type 1 isolates varies according to biological phenotype", J Virol, 71, 1997, 7478-87.
CC Huang, M Tang, MY Zhang, S Majeed, E Montabana, RL Stanfield, DS Dimitrov, B Korber, J Sodroski, IA Wilson, R Wyatt, PD Kwong,
"Structure of a V3-containing HIV-1 gp120 core", Science, 310, 2005, 1025-8.
Reaction
11.1.1.1.4 Conformational changes in gp120 exposes gp41
Authors
Editors
Reviewers
Reeves, J, 2006-06-12.
Description
The HIV protein known as gp41 is a transmembrane protein which is considered the major mediator of fusion of extracellular virions to the target
cells in the host. HIV gp120 and gp41 proteins form non-covalently linked oligomers on the surface of virions. The gp41 subunit of the oligomer
is anchored in the viral membrane and contains a non-polar fusion peptide at its N-terminus. Upon CD4 and receptor binding, gp120 undergoes
a second conformation change. The conformation change exposes gp41 which continues to mediate fusion of the viral envelope with the host
plasma membrane. Electron microscopy and circular dichroism measurements of the gp41 protein suggest a rod-like conformation with a high
alpha-helical content. Although some studies suggest that gp41must dissociate from gp120 in order to cause fusion between HIV envelope and
the target cell plasma membrane, evidence on this point is not conclusive.
References
ML Bosch, PL Earl, K Fargnoli, S Picciafuoco, F Giombini, F Wong-Staal, G Franchini, "Identification of the fusion peptide of primate
immunodeficiency viruses", Science, 244, 1989, 694-7.
W Weissenhorn, SA Wharton, LJ Calder, PL Earl, B Moss, E Aliprandis, JJ Skehel, DC Wiley, "The ectodomain of HIV-1 env subunit gp41 forms
a soluble, alpha-helical, rod-like oligomer in the absence of gp120 and the N-terminal fusion peptide", EMBO J, 15, 1996, 1507-14.
CM Carr, PS Kim, "A spring-loaded mechanism for the conformational change of influenza hemagglutinin", Cell, 73, 1993, 823-32.
Reaction
11.1.1.1.5 Fusogenic activation of gp41
Authors
Editors
Reviewers
Reeves, J, 2006-06-12.
Description
Fusion of HIV with target cell plasma membranes is mediated largely by the gp41 glycoprotein. This glycoprotein contains a stretch of strongly
hydrophobic amino acids flanked by a series of polar amino acids at its N terminus. Subsequent to the second conformation change in gp120,
the N-terminal fusion peptide of gp41 adopts a position which brings it into close proximity with the target cell plasma membrane. As gp41 is
found in trimers within the viral membrane, the resulting structure of this conformational change is often referred to as a Ã¢â‚¬Å"prongÃ¢â‚¬Â•,
in which three N-terminal peptides extend towards the target cell plasma membrane. The process of fusion begins at this time, with the
N-terminus of gp41 inserting itself into the membrane of the target cell.
References
H Schaal, M Klein, P Gehrmann, O Adams, A Scheid, "Requirement of N-terminal amino acid residues of gp41 for human immunodeficiency
virus type 1-mediated cell fusion", J Virol, 69, 1995, 3308-14.
J Cao, L Bergeron, E Helseth, M Thali, H Repke, J Sodroski, "Effects of amino acid changes in the extracellular domain of the human
immunodeficiency virus type 1 gp41 envelope glycoprotein", J Virol, 67, 1993, 2747-55.
EO Freed, DJ Myers, R Risser, "Characterization of the fusion domain of the human immunodeficiency virus type 1 envelope glycoprotein gp41",
Reaction
11.1.1.1.6 Insertion of gp41 fusion peptide into the target membrane
Authors
Editors
Reviewers
Reeves, J, 2006-06-12.
Description
Insertion of the N-terminal fusion peptide of the HIV gp41 protein is the first step in the fusion of viral and target cell membranes. Substitutions of
polar amino acids at residues 2, 9, 15 and 26 of the N terminus of this peptide completely eliminated its ability to cause fusion, implicating these
residues in gp41Ã¢â‚¬â„¢s role in insertion and fusion. Studies have also shown that mutations in a stretch of residues from 36-64(568 to 596 of
ENV protein) caused gp41 to become partially or completely defective in mediating membrane fusion, suggesting that conformation of the
peptide is important for proper insertion and fusion to occur.
References
J Cao, L Bergeron, E Helseth, M Thali, H Repke, J Sodroski, "Effects of amino acid changes in the extracellular domain of the human
immunodeficiency virus type 1 gp41 envelope glycoprotein", J Virol, 67, 1993, 2747-55.
S Jiang, K Lin, N Strick, AR Neurath, "Inhibition of HIV-1 infection by a fusion domain binding peptide from the HIV-1 envelope glycoprotein
GP41", Biochem Biophys Res Commun, 195, 1993, 533-8.
C Wild, T Oas, C McDanal, D Bolognesi, T Matthews, "A synthetic peptide inhibitor of human immunodeficiency virus replication: correlation
between solution structure and viral inhibition", Proc Natl Acad Sci U S A, 89, 1992, 10537-41.
EO Freed, DJ Myers, R Risser, "Characterization of the fusion domain of the human immunodeficiency virus type 1 envelope glycoprotein gp41",
Reaction
11.1.1.1.7 N and C terminal heptad repeat helices of gp41 form six-helix bundle
Authors
Editors
Reviewers
Reeves, J, 2006-06-12.
Description
The gp41 glycoprotein contains N- and C-terminal heptad repeats, which form a stable six-helical bundle. This six-helix bundle represents a
fusion-active gp41 core, and its conformation is critical for membrane fusion. Among the interactions necessary for the six helix bundle
conformation is the formation of a salt bridge between the Asp632 residue in the C-terminal heptad repeat and the Lys574 terminal in the
N-terminal coiled-coil. Disruption of this interaction has been found to lead to destabilization of the six helix bundle formation, with a subsequent
severe reduction in viral fusion activity. Also, the N-terminal heptad repeat alone was found to be important in viral fusion, as removal or
truncation of this repeat reduced the fusion activity of the peptide even when the adjacent, full length N-terminal fusion peptide was in place. The
bundle itself is formed during the fusion process, prior to pore formation but after insertion of the gp41 fusion peptide into the target cell
membrane. Upon insertion of the fusion peptide, the three N-terminal helices of gp41 adjacent to the target cell membrane and three C-terminal
helices adjacent to the viral membrane undergo a conformational change which brings them into close proximity with one another, creating a
six-helix bundle and leading to eventual fusion.
References
EL Delwart, G Mosialos, T Gilmore, "Retroviral envelope glycoproteins contain a leucine zipper-like repeat", AIDS Res Hum Retroviruses, 6,
1990, 703-6.
DC Chan, D Fass, JM Berger, PS Kim, "Core structure of gp41 from the HIV envelope glycoprotein", Cell, 89, 1997, 263-73.
RA Furuta, CT Wild, Y Weng, CD Weiss, "Capture of an early fusion-active conformation of HIV-1 gp41", Nat Struct Biol, 5, 1998, 276-9.
W Weissenhorn, SA Wharton, LJ Calder, PL Earl, B Moss, E Aliprandis, JJ Skehel, DC Wiley, "The ectodomain of HIV-1 env subunit gp41 forms
a soluble, alpha-helical, rod-like oligomer in the absence of gp120 and the N-terminal fusion peptide", EMBO J, 15, 1996, 1507-14.
WR Gallaher, JM Ball, RF Garry, MC Griffin, RC Montelaro, "A general model for the transmembrane proteins of HIV and other retroviruses",
AIDS Res Hum Retroviruses, 5, 1989, 431-40.
CH Chen, TJ Matthews, CB McDanal, DP Bolognesi, ML Greenberg, "A molecular clasp in the human immunodeficiency virus (HIV) type 1 TM
protein determines the anti-HIV activity of gp41 derivatives: implication for viral fusion", J Virol, 69, 1995, 3771-7.
S Jiang, H Lu, S Liu, Q Zhao, Y He, AK Debnath, "N-substituted pyrrole derivatives as novel human immunodeficiency virus type 1 entry
inhibitors that interfere with the gp41 six-helix bundle formation and block virus fusion", Antimicrob Agents Chemother, 48, 2004, 4349-59.
SA Gallo, A Puri, R Blumenthal, "HIV-1 gp41 six-helix bundle formation occurs rapidly after the engagement of gp120 by CXCR4 in the HIV-1
Env-mediated fusion process", Biochemistry, 40, 2001, 12231-6.
Y He, S Liu, AK Debnath, S Jiang, "Formation of a Salt Bridge between the N- and C-terminal Heptad Repeats of HIV-1 gp41 Is Critical for
Stabilization of 6-Helix Bundle and Virus Fusion", http://www.retroconference.org/2004/cd/Abstract/295.htm (11th Conference on Retrovirus and
Opportunistic Infections), 2004.
K Sackett, Y Shai, "The HIV-1 gp41 N-terminal heptad repeat plays an essential role in membrane fusion", Biochemistry, 41, 2002, 4678-85.
JW Dubay, SJ Roberts, B Brody, E Hunter, "Mutations in the leucine zipper of the human immunodeficiency virus type 1 transmembrane
glycoprotein affect fusion and infectivity", J Virol, 66, 1992, 4748-56.
Reaction
11.1.1.1.8 Fusion of viral membrane with host cell membrane
Authors
Editors
Reviewers
Reeves, J, 2006-06-12.
Description
With the transition of gp41 into the six-helix bundle, fusion of the viral and target cell membranes begins to take place. The specifics of fusion are
not completely clear, but it is understood that fusion proceeds after insertion of the gp41 fusion peptide, which results in curvature of viral and
target cell membranes. This results in a state of hemi-fusion, where only the outer lipid bilayers of each membrane are fused, whereas
membrane leaflets that are distal with respect to the intermembrane gap remain separate at this stage. Hemi-fusion allows the exchange of lipids
between the contacting leaflets, whereas the exchange of aqueous content between the virus and the cell remains blocked. The next step in
fusion is the merger of the distal leaflets, leading to the formation of a nascent fusion pore, which leads to mixing of viral and cellular contents.
Studies of fusion of Influenza virus suggested that multiple hairpin structures may form a narrow fusion pore which subsequently expands to a
larger opening. In the case of HIV, this larger opening allows for passage of the Matrix-surrounded viral core out of the virus and into the host
cell cytoplasm.
References
F Sinangil, A Loyter, DJ Volsky, "Quantitative measurement of fusion between human immunodeficiency virus and cultured cells using
membrane fluorescence dequenching", FEBS Lett, 239, 1988, 88-92.
GB Melikyan, RM Markosyan, H Hemmati, MK Delmedico, DM Lambert, FS Cohen, "Evidence that the transition of HIV-1 gp41 into a six-helix
bundle, not the bundle configuration, induces membrane fusion", J Cell Biol, 151, 2000, 413-23.
M Ferrer, TM Kapoor, T Strassmaier, W Weissenhorn, JJ Skehel, D Oprian, SL Schreiber, DC Wiley, SC Harrison, "Selection of gp41-mediated
HIV-1 cell entry inhibitors from biased combinatorial libraries of non-natural binding elements", Nat Struct Biol, 6, 1999, 953-60.
E Hunter, "Viral Entry and Receptors", Retroviruses (Ed.) Coffin, Hughes and Varmus, 1997.
B Chackerian, EM Long, PA Luciw, J Overbaugh, "Human immunodeficiency virus type 1 coreceptors participate in postentry stages in the virus
replication cycle and function in simian immunodeficiency virus infection", J Virol, 71, 1997, 3932-9.
U Fischer, S Meyer, M Teufel, C Heckel, R Luhrmann, G Rautmann, "Evidence that HIV-1 Rev directly promotes the nuclear export of
unspliced", EMBO J, 13, 1994, 4105-12.
JM Kilby, JJ Eron, "Novel therapies based on mechanisms of HIV-1 cell entry", N Engl J Med, 348, 2003, 2228-38.
T Kanaseki, K Kawasaki, M Murata, Y Ikeuchi, S Ohnishi, "Structural features of membrane fusion between influenza virus and liposome as
revealed by quick-freezing electron microscopy", J Cell Biol, 137, 1997, 1041-56.
Reaction
11.1.1.2 Uncoating of the HIV Virion
Authors
Iordanskiy, S, Bukrinsky, M, 2006-04-03.
Editors
Reviewers
Aiken, C, 2006-10-30.
Description
HIV-1 uncoating is a poorly understood process. It likely involves a progressive and partial dissembly of matrix and capsid layers. While viral
proteins like MA and Nef are thought to be involved, the primary cause seems to be the cytosolic pH and a simple dilution effect. Successful
uncoating generates the viral reverse transcription complex, which comprises the diploid viral RNA genome, tRNALys primer, RT, IN, MA,
nucleocapsid (NC), viral protein R (Vpr) and various host proteins; the reverse-transcription complex is thus liberated from the plasma
membrane. It is believed that the transiting viral nucleoprotein complex associates with the elements of cytoskeleton like actin microfilaments.
References
M Bukrinsky, "A hard way to the nucleus", Mol Med, 10, 2004, 1-5.
D McDonald, MA Vodicka, G Lucero, TM Svitkina, GG Borisy, M Emerman, TJ Hope, "Visualization of the intracellular behavior of HIV in living
cells", J Cell Biol, 159, 2002, 441-52.
J Lehmann-Che, A Saib, "Early stages of HIV replication: how to hijack cellular functions for a successful infection", AIDS Rev, 6, 2004, 199-207.
WC Greene, BM Peterlin, "Charting HIV's remarkable voyage through the cell: Basic science as a passport to future therapy", Nat Med, 8, 2002,
673-80.
11.1.1.2.1 Disintegration of matrix layer
Authors
Editors
Reviewers
Aiken, C, 2006-10-30.
Description
After fusion of the viral membrane with the target cell membrane, the viral core, which is surrounded by a layer of Matrix (p17) proteins, is
exposed to the cytoplasm. Disintegration of the Matrix layer allows for the conical-shaped viral core to be fully released, and allow for viral capsid
dissociation and eventually reverse transcription. Dissociation of the Matrix layer is not well characterized, but is believed to occur due to
disruption of protein-protein interactions as a result of the conditions of the cytoplasm (including pH), which differ from that of the internal viral
structure.
References
H Zhang, G Dornadula, J Orenstein, RJ Pomerantz, "Morphologic changes in human immunodeficiency virus type 1 virions secondary to
intravirion reverse transcription: evidence indicating that reverse transcription may not take place within the intact viral core", J Hum Virol, 3,
2000, 165-72.
A Fassati, SP Goff, "Characterization of intracellular reverse transcription complexes of human immunodeficiency virus type 1", J Virol, 75, 2001,
3626-35.
Reaction
11.1.1.2.2 Disassembly of viral capsid

Authors
Editors
Reviewers
Aiken, C, 2006-10-30.
Description
The HIV capsid protein (p24) surrounds the viral genome and associated proteins to make up the viral core. Dissolution of the viral capsid allows
for release of the viral RNA and other proteins such as Vpr into the cytoplasm, which will subsequently form the Reverse Transcription Complex.
Dissolution of capsid proteins may be caused by interaction with cellular proteins, e.g. TRIM5, or may occur in a similar fashion to that of matrix
dissolution; as a reaction to a change in pH. Indeed, studies observing capsid assembly and conformation show that this protein-protein
interaction is heavily influenced by even small changes in pH (pH7.0 to 6.8).
References
2000, 165-72.
M Yamashita, M Emerman, "The Cell Cycle Independence of HIV Infections Is Not Determined by Known Karyophilic Viral Elements", PLoS
Pathog, 1, 2005, e18.
3626-35.
LS Ehrlich, T Liu, S Scarlata, B Chu, CA Carter, "HIV-1 capsid protein forms spherical (immature-like) and tubular (mature-like) particles in vitro:
structure switching by pH-induced conformational changes", Biophys J, 81, 2001, 586-94.
Reaction
11.1.1.3 Formation of RTC (Reverse Transcription Complex)
Authors
Editors
Reviewers
Aiken, C, 2006-10-30.
Description
Reverse transcription complex is a transitory structure where reverse transcription takes place. Initially, it is likely identical to the RNA-protein
complex found inside the virion core. Upon maturation, it may shed some HIV proteins (such as MA or Vpr) and incorporate cellular proteins
(such as INI1 or PML).
References
M Bukrinsky, N Sharova, TL McDonald, T Pushkarskaya, WG Tarpley, M Stevenson, "Association of integrase, matrix, and reverse transcriptase
antigens of human immunodeficiency virus type 1 with viral nucleic acids following acute infection", Proc Natl Acad Sci U S A, 90, 1993, 6125-9.
S Iordanskiy, R Berro, M Altieri, F Kashanchi, M Bukrinsky, "Intracytoplasmic maturation of the human immunodeficiency virus type 1 reverse
transcription complexes determines their capacity to integrate into chromatin", Retrovirology, 3, 2006, 4.
2000, 165-72.
3626-35.
Reaction
11.1.1.4 Annealing of 3'-end of unwound transfer RNA primer with genomic RNA
Authors
Gopinathrao, G, D'Eustachio, P, 2006-05-18.
Editors
Reviewers
Hughes, SH, 2006-10-30.
Description
Retroviruses use cellular tRNAs as primers for reverse transcription of the viral genomic RNA (Mak and Kleiman 1997). The primer tRNA is
selectively packaged during assembly of retrovirus particles. In the case of HIV-1, lysine tRNAs are preferentially incorporated during retroviral
packaging, and lysine tRNA 3, the specific isoacceptor form that serves as a primer for reverse transcription, anneals to the PBS (primer binding
site) within the U5 region of the viral genomic RNA. This association appears to be mediated by the viral reverse transcriptase (RT) protein,
possibly its "thumb" and "connection" domains (Jiang et al. 1993; Mak et al. 1994; Mishima and Steitz 1995).
References
Y Mishima, JA Steitz, "Site-specific crosslinking of 4-thiouridine-modified human tRNA(3Lys) to reverse transcriptase from human
immunodeficiency virus type I", EMBO J, 14, 1995, 2679-87.
M Jiang, J Mak, A Ladha, E Cohen, M Klein, B Rovinski, L Kleiman, "Identification of tRNAs incorporated into wild-type and mutant human
immunodeficiency virus type 1", J Virol, 67, 1993, 3246-53.
J Mak, L Kleiman, "Primer tRNAs for reverse transcription", J Virol, 71, 1997, 8087-95.
J Mak, M Jiang, MA Wainberg, ML Hammarskjold, D Rekosh, L Kleiman, "Role of Pr160gag-pol in mediating the selective incorporation of
tRNA(Lys) into human immunodeficiency virus type 1 particles", J Virol, 68, 1994, 2065-72.
Reaction
11.1.1.5 Reverse Transcription of HIV RNA
Authors
Editors
Reviewers
Hughes, SH, 2006-10-30.
Description
The RNA genome of HIV-1, like that of other retroviruses, is reverse-transcribed (Baltimore 1970; Temin and Mizutani 1970) into
double-stranded DNA, which is then integrated into a host cell chromosome and transcribed to yield both viral mRNAs and viral genomic RNAs.
HIV-1 reverse transcription takes place in the cytosol of a newly infected host cell and involves multiple steps of RNA synthesis and degradation
of the RNA strand of RNA:DNA duplexes mediated by the HIV-1 RT protein, as well as two template switches, to yield a DNA duplex colinear
with the viral genomic RNA but with additional Long Terminal Repeat (LTR) sequence motifs at both ends (Telesnitsky and Goff 1997;
Jonckheere et al. 2000).
HIV-1 RT has two catalytic activities essential for transcription of a DNA duplex copy of the viral genomic RNA: a reverse transcriptase activity
and an RNase H activity. The reverse transcriptase is primer dependent and can transcribe both RNA and DNA templates in a 5'-3' direction.
The RNaseH acts on the RNA strand of RNA:DNA duplexes and can catalyze both endo- and exonucleolytic cleavage of such an RNA strand.
RT is a heterodimer of 66 and 51 kD polypeptides, both generated by cleavage of the HIV-1 Pol gene product: p66 contains Pol amino acid
residues 599-1158; p51 contains residues 599-1038. Both active sites of the HIV-1 RT enzyme are contained in the p66 polypeptide, the
polymerase activity in its aminoterminal region, and the RNase in its carboxyterminus. The p51 subunit lacks an RNaseH domain, and while its
polymerase domain is intact, its conformation in the p66:p51 heterodimer occludes the active site (Hughes et al. 1996; Jacobo-Molina et al.
1993; Kohlstaedt et al. 1992; Wang et al. 1994).
The process of reverse transcription is outlined in the figure below: viral genomic RNA and primer tRNA are shown in black, "minus" strand DNA
is shown in red, and "plus" strand DNA is shown in blue.
References
SH Hughes, Z Hostomsky, SF Le Grice, K Lentz, E Arnold, "What is the orientation of DNA polymerases on their templates?", J Virol, 70, 1996,
2679-83.
H Jonckheere, J Anne, E De Clercq, "The HIV-1 reverse transcription (RT) process as target for RT inhibitors", Med Res Rev, 20, 2000, 129-54.
HM Temin, S Mizutani, "RNA-dependent DNA polymerase in virions of Rous sarcoma virus", Nature, 226, 1970, 1211-3.
A Jacobo-Molina, J Ding, RG Nanni, Jr Clark AD, X Lu, C Tantillo, RL Williams, G Kamer, AL Ferris, P Clark, A Hizi, E Arnold, SH Hughes,
"Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows
bent DNA.", Proc Natl Acad Sci U S A, 90, 1993, 6320-4.
D Baltimore, "RNA-dependent DNA polymerase in virions of RNA tumour viruses", Nature, 226, 1970, 1209-11.
A Telesnitsky, SP Goff, "Reverse Transcriptase and the Generation of Retroviral DNA", Retroviruses (Coffin, JM, et al., editors) [Book], 1997,
121-160.
J Wang, SJ Smerdon, J Jager, LA Kohlstaedt, PA Rice, JM Friedman, TA Steitz, "Structural basis of asymmetry in the human immunodeficiency
virus type 1 reverse transcriptase heterodimer", Proc Natl Acad Sci U S A, 91, 1994, 7242-6.
LA Kohlstaedt, J Wang, JM Friedman, PA Rice, TA Steitz, "Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an
inhibitor.", Science, 256, 1992, 1783-90.
11.1.1.5.1 Minus-strand DNA synthesis
Authors
Editors
Reviewers
Hughes, SH, 2006-10-30.
Description
In the first part of reverse transcription, minus-strand synthesis, a DNA strand complementary to the HIV genomic RNA is synthesized, using the
viral RNA as a template and a host cell lysine tRNA molecule as primer. The synthesis proceeds in two discrete steps, separated by a strand
transfer event. As minus strand DNA is synthesized, the viral genomic RNA is degraded, also in several discrete steps. Two specific polypurine
tracts (PPT sequences) in the viral RNA, one within the pol gene (central or cPPT) and one immediately preceding the U3 sequence (3' PPT) are
spared from degradation and serve to prime synthesis of DNA complementary to the minus strand (plus-strand synthesis). During plus-strand
synthesis, Preston and colleagues observed secondary sites of plus-strand initiation at low frequency both in the cell-free system and in cultured
virus (Klarman et al., 1997). Both DNA synthesis and RNA degradation activities are catalyzed by the HIV-1 reverse transcriptase (RT)
heterodimer.
References
GJ Klarmann, H Yu, X Chen, JP Dougherty, BD Preston, "Discontinuous plus-strand DNA synthesis in human immunodeficiency virus type
1-infected cells and in a partially reconstituted cell-free system", J Virol, 71, 1997, 9259-69.
ML Andreola, GA Nevinsky, PJ Barr, L Sarih-Cottin, B Bordier, M Fournier, S Litvak, L Tarrago-Litvak, "Interaction of tRNALys with the p66/p66
form of HIV-1 reverse transcriptase stimulates DNA polymerase and ribonuclease H activities", J Biol Chem, 267, 1992, 19356-62.
JM Whitcomb, SH Hughes, "Retroviral reverse transcription and integration: progress and problems", Annu Rev Cell Biol, 8, 1992, 275-306.
L Tarrago-Litvak, ML Andreola, GA Nevinsky, L Sarih-Cottin, S Litvak, "The reverse transcriptase of HIV-1: from enzymology to therapeutic
intervention", FASEB J, 8, 1994, 497-503.
121-160.
11.1.1.5.1.1 Synthesis of minus strand strong stop DNA (-sssDNA)
Authors
Editors
Reviewers
Hughes, SH, 2006-10-30.
Description
To catalyze DNA synthesis, retroviral reverse transcriptase requires a primer strand to extend and a template strand to copy. For HIV-1, the
primer is the 3'-end of a partially unwound lysine(3) tRNA annealed to the PBS (primer binding site) 179 bases from the 5' end of the retroviral
genomic RNA (Isel et al. 1995). Reverse transcription of the viral genomic RNA proceeds from the bound tRNA primer to the 5' end of the viral
RNA, yielding a minus-strand strong-stop DNA (-sssDNA) complementary to the R and U5 elements of the HIV-1 viral genome, as shown in the
figure below (Telesnitsky and Goff 1997; Jonckheere et al. 2000). The reaction takes place in the host cell cytosol, and is catalyzed by the
reverse transcriptase activity of the HIV-1 RT heterodimer.
NucleoCapsid (NC) protein prevents self-priming by generating or stabilizing a thermodynamically favored RNA-DNA heteroduplex instead of the
kinetically favored TAR hairpin seen in reverse transcription experiments in vitro (Driscoll and Hughes 2000).
References
MD Driscoll, SH Hughes, "Human immunodeficiency virus type 1 nucleocapsid protein can prevent self-priming of minus-strand strong stop DNA
by promoting the annealing of short oligonucleotides to hairpin sequences", J Virol, 74, 2000, 8785-92.
121-160.
C Isel, C Ehresmann, G Keith, B Ehresmann, R Marquet, "Initiation of reverse transcription of HIV-1: secondary structure of the HIV-1
RNA/tRNA(3Lys) (template/primer)", J Mol Biol, 247, 1995, 236-50.
Reaction
11.1.1.5.1.2 RNase H-mediated cleavage of the RNA strand of the -sssDNA:RNA duplex
Authors
Editors
Reviewers
Hughes, SH, 2006-10-30.
Description
As the reverse transcriptase activity of the HIV-1 RT heterodimer catalyzes the synthesis of minus-strand strong stop DNA (-sssDNA), the
RNaseH activity of the same RT heterodimer catalyzes the degradation of the complementary viral genomic RNA sequences. Degradation of
this RNA is required for the efficient transfer of the -sssDNA to the 5' end of the viral genomic RNA. The RNase H active site is positioned within
the HIV-1 RT heterodimer so as to attack the RNA strand of the RNA:DNA duplex at a point 18 bases behind the site of reverse transcription
(Furfine and Reardon 1991; Ghosh et al. 1995; Gopalakrishnan et al. 1992; Wohrl and Moelling 1990). The rate of RNase H cleavage is
substantially lower than the rate of DNA synthesis, however (Kati et al. 1992), and may further depend on RT stalling and structural features of
the viral genomic RNA template. The product of these combined DNA synthesis and RNA degradation events is a DNA strand still duplexed with
extended viral genomic RNA fragments.
References
ES Furfine, JE Reardon, "Reverse transcriptase.RNase H from the human immunodeficiency virus. Relationship of the DNA polymerase and
RNA hydrolysis activities.", J Biol Chem, 266, 1991, 406-12.
BM Wohrl, K Moelling, "Interaction of HIV-1 ribonuclease H with polypurine tract containing RNA-DNA hybrids", Biochemistry, 29, 1990,
10141-7.
M Ghosh, KJ Howard, CE Cameron, SJ Benkovic, SH Hughes, SF Le Grice, "Truncating alpha-helix E' of p66 human immunodeficiency virus
reverse transcriptase modulates RNase H function and impairs DNA strand transfer", J Biol Chem, 270, 1995, 7068-76.
121-160.
V Gopalakrishnan, JA Peliska, SJ Benkovic, "Human immunodeficiency virus type 1 reverse transcriptase: spatial and temporal relationship
between the polymerase and RNase H activities", Proc Natl Acad Sci U S A, 89, 1992, 10763-7.
WM Kati, KA Johnson, LF Jerva, KS Anderson, "Mechanism and fidelity of HIV reverse transcriptase", J Biol Chem, 267, 1992, 25988-97.
Reaction
11.1.1.5.1.3 RNase H-mediated degradation of the RNA strand of the -sssDNA:RNA duplex
Authors
Reviewers
Hughes, SH, 2006-10-30.
Description
The rate of RNase H cleavage is substantially lower than the rate of DNA synthesis (Kati et al. 1992), so the product of the combined DNA
synthesis and RNA degradation events catalyzed by the RT heterodimer mediating minus-strand strong stop DNA (-sssDNA) synthesis is a DNA
segment still duplexed with extended viral genomic RNA fragments. In vitro, other RT heterodimers bind the remaining RNA:DNA
heteroduplexes and their RNase H domains further degrade the viral genomic RNA (Wisniewski et al. 2000a, b).
References
M Wisniewski, M Balakrishnan, C Palaniappan, PJ Fay, RA Bambara, "The sequential mechanism of HIV reverse transcriptase RNase H", J Biol
Chem, 275, 2000, 37664-71.
M Wisniewski, M Balakrishnan, C Palaniappan, PJ Fay, RA Bambara, "Unique progressive cleavage mechanism of HIV reverse transcriptase
RNase H", Proc Natl Acad Sci U S A, 97, 2000, 11978-83.
Reaction
11.1.1.5.1.4 First strand transfer mediated by Repeated (R) sequence
Authors
Editors
Reviewers
Hughes, SH, 2006-10-30.
Description
The minus strand strong stop DNA (-sssDNA) is transferred to the 3' end of the HIV-1 genomic RNA, where the 3' end of the -sssDNA anneals to
the viral genomic R sequence motif (Ghosh et al. 1995; Klaver and Berkhout 1994; Ohi and Clever 2000; Telesnitsky and Goff 1997). Viral NC
(nucleocapsid) protein may play a role in this transfer (Driscoll and Hughes 2000).
References
MD Driscoll, SH Hughes, "Human immunodeficiency virus type 1 nucleocapsid protein can prevent self-priming of minus-strand strong stop DNA
by promoting the annealing of short oligonucleotides to hairpin sequences", J Virol, 74, 2000, 8785-92.
B Klaver, B Berkhout, "Premature strand transfer by the HIV-1 reverse transcriptase during strong-stop DNA synthesis", Nucleic Acids Res, 22,
1994, 137-44.
121-160.
Y Ohi, JL Clever, "Sequences in the 5' and 3' R elements of human immunodeficiency virus type 1 critical for efficient reverse transcription", J
Virol, 74, 2000, 8324-34.
Reaction
11.1.1.5.1.5 Minus strand DNA synthesis resumes
Authors
Editors
Reviewers
Hughes, SH, 2006-10-30.
Description
Synthesis of minus-strand DNA proceeds toward the 5' end of the PBS motif of the template HIV genomic RNA.
References
121-160.
Reaction
11.1.1.5.1.6 RNase H-mediated cleavage of the template strand
Authors
Editors
Reviewers
Hughes, SH, 2006-10-30.
Description
As the reverse transcriptase activity of the HIV-1 RT heterodimer catalyzes the extension of the minus-strand DNA, the RNaseH activity
catalyzes the degradation of the complementary viral genomic RNA sequences. Telesnitsky and Goff (1993) observed that two defective forms
of reverse transcriptase can complement to restore retroviral infectivity. The RNase H active site is positioned within the HIV-1 RT heterodimer
so as to attack the RNA strand of the RNA:DNA duplex at a point 18 bases behind the site of reverse transcription (Furfine and Reardon 1991;
Ghosh et al. 1995; Gopalakrishnan et al. 1992; Wohrl and Moelling 1990). The rate of RNase H cleavage is substantially lower than the rate of
DNA synthesis and the level of its activity in vivo is unclear, however (Kati et al. 1992). The product of these combined DNA synthesis and RNA
degradation events is a DNA strand still duplexed with extended viral genomic RNA fragments.
References
ES Furfine, JE Reardon, "Reverse transcriptase.RNase H from the human immunodeficiency virus. Relationship of the DNA polymerase and
RNA hydrolysis activities.", J Biol Chem, 266, 1991, 406-12.
A Telesnitsky, SP Goff, "Two defective forms of reverse transcriptase can complement to restore retroviral infectivity", EMBO J, 12, 1993,
4433-8.
BM Wohrl, K Moelling, "Interaction of HIV-1 ribonuclease H with polypurine tract containing RNA-DNA hybrids", Biochemistry, 29, 1990,
10141-7.
JG Julias, MJ McWilliams, SG Sarafianos, WG Alvord, E Arnold, SH Hughes, "Effects of mutations in the G tract of the human immunodeficiency
virus type 1 polypurine tract on virus replication and RNase H cleavage", J Virol, 78, 2004, 13315-24.
KA Pullen, AJ Rattray, JJ Champoux, "The sequence features important for plus strand priming by human immunodeficiency virus type 1 reverse
transcriptase", J Biol Chem, 268, 1993, 6221-7.
121-160.
V Gopalakrishnan, JA Peliska, SJ Benkovic, "Human immunodeficiency virus type 1 reverse transcriptase: spatial and temporal relationship
between the polymerase and RNase H activities", Proc Natl Acad Sci U S A, 89, 1992, 10763-7.
Reaction
11.1.1.5.1.7 RNase H-mediated degradation of the template strand
Authors
Reviewers
Hughes, SH, 2006-10-30.
Description
The rate of RNase H cleavage is substantially lower than the rate of DNA synthesis (Kati et al. 1992), so the product of the combined DNA
synthesis and RNA degradation events catalyzed by the RT heterodimer mediating minus-strand DNA synthesis is a DNA segment still duplexed
with extended viral genomic RNA fragments. Other RT heterodimers bind the remaining RNA:DNA heteroduplexes and their RNase H domains
further degrade the viral genomic RNA (Wisniewski et al. 2000a, b). Two PPT (polypurine tract) sequence motifs in the template, one
immediately 5' to the U3 sequence and one located within the pol gene in the center of the viral genome, are spared from degradation
(Charneau et al. 1992; Julias et al. 2004; Pullen et al. 1993).
References
M Wisniewski, M Balakrishnan, C Palaniappan, PJ Fay, RA Bambara, "The sequential mechanism of HIV reverse transcriptase RNase H", J Biol
Chem, 275, 2000, 37664-71.
JG Julias, MJ McWilliams, SG Sarafianos, WG Alvord, E Arnold, SH Hughes, "Effects of mutations in the G tract of the human immunodeficiency
virus type 1 polypurine tract on virus replication and RNase H cleavage", J Virol, 78, 2004, 13315-24.
HE Huber, CC Richardson, "Processing of the primer for plus strand DNA synthesis by human immunodeficiency virus 1 reverse transcriptase",
J Biol Chem, 265, 1990, 10565-73.
M Alizon, P Charneau, F Clavel, "A second origin of DNA plus-strand synthesis is required for optimal Human Immunodeficiency Virus
replication", J Virol, 66, 1992, 2814-2820.
M Wisniewski, M Balakrishnan, C Palaniappan, PJ Fay, RA Bambara, "Unique progressive cleavage mechanism of HIV reverse transcriptase
RNase H", Proc Natl Acad Sci U S A, 97, 2000, 11978-83.
Reaction
11.1.1.5.2 Plus-strand DNA synthesis
Authors
Editors
Reviewers
Hughes, SH, 2006-10-30.
Description
Two specific polypurine tracts (PPT sequences) in the viral RNA, one within the pol gene (central or cPPT) and one immediately preceding the
U3 sequence (3' PPT), are spared from degradation during minus strand DNA synthesis and prime plus-strand synthesis. At least two discrete
steps of DNA replication, removal of the PPT RNAs and the tRNA primer that initiated minus-strand synthesis, and a strand transfer lead to the
synthesis of a linear duplex DNA corresponding to the full length of the HIV genomic RNA with long terminal repeat (LTR) sequences at both
ends. Both DNA synthesis and RNA degradation are catalyzed by domains of the HIV-1 reverse transcriptase (RT) heterodimer. During
plus-strand synthesis, Preston and colleagues observed secondary sites of plus-strand initiation at low frequency both in the cell-free system
and in cultured virus-infected cells (Klarman et al., 1997).
References
GJ Klarmann, H Yu, X Chen, JP Dougherty, BD Preston, "Discontinuous plus-strand DNA synthesis in human immunodeficiency virus type
1-infected cells and in a partially reconstituted cell-free system", J Virol, 71, 1997, 9259-69.
121-160.
11.1.1.5.2.1 3' PPT-primed initiation of plus-strand DNA synthesis
Authors
Editors
Reviewers
Hughes, SH, 2006-10-30.
Description
HIV-1 genomic RNA contains a centrally located PPT (cPPT) within the pol gene that, like 3'PPT, is spared by RNase H during minus-strand
DNA synthesis and persists to prime plus-strand DNA synthesis. This ribonucleotide primes the synthesis of a plus-strand DNA extending
through the U3 and R regions of the HIV sequence and terminating in the PBS region (the tRNA primer-binding site). This DNA segment is
known as plus-strand strong-stop DNA (+sssDNA) (Telesnitsky and Goff 1997; Pullen et al. 1993; Huber and Richardson 1990). cPPT priming is
important for efficient viral replication (Alizon et al. 1992; Rausch and Le Grice 2004). Several features of cPPT priming in vivo remain to be
clarified.
References
121-160.
HE Huber, CC Richardson, "Processing of the primer for plus strand DNA synthesis by human immunodeficiency virus 1 reverse transcriptase",
J Biol Chem, 265, 1990, 10565-73.
M Alizon, P Charneau, F Clavel, "A second origin of DNA plus-strand synthesis is required for optimal Human Immunodeficiency Virus
JW Rausch, SF Le Grice, "'Binding, bending and bonding': polypurine tract-primed initiation of plus-strand DNA synthesis in human
immunodeficiency virus", Int J Biochem Cell Biol, 36, 2004, 1752-66.
Reaction
11.1.1.5.2.2 RNase H-mediated digestion of tRNA, 3'PPT and cPPT RNA primers
Authors
Editors
Reviewers
Hughes, SH, 2006-10-30.
Description
RNase H catalyzes the precise cleavage of the bonds linking the primer tRNA attached to the minus-strand DNA, the 3' PPT RNA primer to the
plus-strand strong-stop DNA, and the cPPT primer to the stretch of plus-strand DNA whose synthesis it primed. In each case, precise cleavage
near the RNA-DNA junction occurs (Pullen et al. 1992). HIV-1 RT is the only reverse transcriptase that cleaves the tRNA:DNA junction so as to
leave a ribo A residue from the tRNA at the 5' end of the minus strand.
While a single RT heterodimer could in principle catalyze DNA synthesis and primer RNA:DNA bond cleavage, evidence from several in vitro
systems suggests that separate RT heterodimers are likely to catalyze these two reactions (Rausch and Le Grice 2004).
References
KA Pullen, LK Ishimoto, JJ Champoux, "Incomplete removal of the RNA primer for minus-strand DNA synthesis by human immunodeficiency
virus type 1 reverse transcriptase", J Virol, 66, 1992, 367-73.
121-160.
Reaction
11.1.1.5.2.3 Second strand transfer by annealing complementary PBS sequences
Authors
Editors
Reviewers
Hughes, SH, 2006-10-30.
Description
With the removal of all viral genomic RNA and tRNA, the PBS sequence at the 3' end of the plus-strand strong-stop DNA (+sssDNA) is free to
pair with the complementary PBS sequence at the 3' end of the minus-strand DNA, to generate a circular structure (Telesnitsky and Goff 1997).
References
121-160.
Reaction
11.1.1.5.2.4 Synthesis of full-length duplex viral DNA with a discontinuous plus strand
Authors
Editors
Reviewers
Hughes, SH, 2006-10-30.
Description
After the second jump, elongation of the plus and minus strands continues. The elongation process requires strand displacement, which RT can
mediate, at least in vitro (Huber et al. 1989; Hottiger et al. 1994; Rausch and Le Grice 2004). The final product is a blunt-ended linear duplex
DNA with a discontinuity in its "plus" strand at the site of the cPPT sequence motif.
References
HE Huber, JM McCoy, JS Seehra, CC Richardson, "Human immunodeficiency virus 1 reverse transcriptase. Template binding, processivity,
strand displacement synthesis, and template switching.", J Biol Chem, 264, 1989, 4669-78.
M Hottiger, VN Podust, RL Thimmig, C McHenry, U Hubscher, "Strand displacement activity of the human immunodeficiency virus type 1
reverse transcriptase heterodimer and its individual subunits", J Biol Chem, 269, 1994, 986-91.
121-160.
Reaction
11.1.1.6 Removal of plus-strand flap and gap closure complete synthesis of linear duplex viral DNA
Authors
Reviewers
Hughes, SH, 2006-10-30.
Description
The fate of the discontinuous viral DNA duplex synthesized in the cytosol of an infected cell by HIV-1 reverse transcriptase is not entirely clear.
Studies of some viral systems suggest that this discontinuous structure is required for passage of the viral duplex DNA into the nucleus while
there are evidence contrary to this observation. Studies in vitro indicate that human nuclear flap endonuclease and DNA ligase can remove the
flap and seal the plus-strand discontinuity in HIV-1 DNA (Miller et al. 1995; Rausch and Le Grice 2004; Rumbaugh et al. 1998), although role of
flap is not yet clear.
References
JA Rumbaugh, GM Fuentes, RA Bambara, "Processing of an HIV replication intermediate by the human DNA replication enzyme FEN1", J Biol
Chem, 273, 1998, 28740-5.
MD Miller, B Wang, FD Bushman, "Human immunodeficiency virus type 1 preintegration complexes containing discontinuous plus strands are
competent to integrate in vitro", J Virol, 69, 1995, 3938-44.
Reaction
11.1.1.7 Integration of provirus
Authors
Editors
Reviewers
Bushman, FD, 2006-10-30.
Description
For retroviral DNA to direct production of progeny virions it must become covalently integrated into the host cell chromosome (reviewed in Coffin
et al. 1997; Hansen et al. 1998). Analyses of mutants have identified the viral integrase coding region (part of the retroviral pol gene) as essential
for the integration process (Donehower 1988; Donehower and Varmus 1984; Panganiban and Temin 1984; Quinn and Grandgenett 1988;
Schwartzberg et al. 1984). Also essential are regions at the ends of the viral long terminal repeats (LTRs) that serve as recognition sites for
integrase protein (Colicelli and Goff 1985, 1988; Panganiban and Temin 1983).
The viral genomic RNA is reverse transcribed to form a linear double-stranded DNA molecule, the precursor to the integrated provirus (Brown et
al. 1987, 1989; Fujiwara and Mizuuchi 1988). The provirus is colinear with unintegrated linear viral DNA (Dhar et al. 1980; Hughes et al. 1978)
but differs from the reverse transcription product in that it is missing two bases from each end (Hughes et al. 1981). Flanking the integrated HIV
provirus are direct repeats of the cellular DNA that are 5 base pairs in length (Vincent et al. 1990). This duplication of cellular sequences flanking
the viral DNA is generated as a consequence of the integration mechanism (Coffin et al., 1997).
Linear viral DNA is found in a complex with proteins in the cytoplasm of infected cells. These complexes (termed "preintegration complexes",
PICs) can be isolated and have been shown to mediate integration of viral DNA into target DNA in vitro (Bowerman et al. 1989; Brown et al.
1987; Ellison et al. 1990; Farnet and Haseltine 1990, 1991).
The development of in vitro assays with purified integrase has allowed its enzymatic functions to be elucidated. The provirus is formed by two
reactions catalyzed by the viral integrase: terminal cleavage and strand transfer. Studies with purified integrase have shown that it is sufficient
for both 3' end cleavage (Bushman and Craigie 1991; Craigie et al. 1990; Katzman et al. 1989; Sherman and Fyfe 1990) and joining of the viral
DNA to the cellular chromosome or naked target DNA (Bushman et al. 1990; Craigie et al. 1990; Katz et al. 1990). HIV integrase catalyze the
removal of two bases from the 3' end of each viral DNA strand, leaving recessed 3' hydroxyl groups (Brown et al. 1989; Fujiwara and Mizuuchi
1988; Roth et al. 1989; Sherman and Fyfe 1990). This terminal cleavage reaction is required for proper integration. It may allow the virus to
create a standard end from viral DNA termini that can be heterogeneous due to the terminal transferase activity of reverse transcriptase (Miller et
al. 1997; Patel and Preston 1994). In addition, the terminal cleavage step is coupled to the formation of a stable integrase-DNA complex (Ellison
and Brown 1994; Vink et al. 1994). Following terminal cleavage, a recessed hydroxyl is exposed that immediately follows a CA dinucleotide.
More internal LTR sites are also important for integration (Balakrishnan and Jonsson 1997; Bushman and Craigie 1990; Leavitt et al. 1992). After
end processing, integrase catalyzes the covalent attachment of hydroxyl groups at the viral DNA termini to protruding 5' phosphoryl ends of the
host cell DNA (Brown et al. 1987; Brown et al. 1989; Fujiwara and Mizuuchi 1988). The DNA cleavage and joining reactions involved in
integration are shown in the figure below. Both the viral DNA 3' end cleavage and strand transfer reactions are mediated by single-step
transesterification chemistry as shown by stereochemical analysis of reaction products (Engelman et al. 1991). Biochemical analysis of purified
integrase revealed that it requires a divalent metal - either Mg2+ or Mn2+ - to carry out reactions with model substrates, that probably mediate
the reaction chemistry (Bushman and Craigie 1991; Craigie et al. 1990; Katzman et al. 1989; Sherman and Fyfe 1990; Gao et al. 2004).
References
PH Patel, BD Preston, "Marked infidelity of human immunodeficiency virus type 1 reverse transcriptase at RNA and DNA template ends", Proc
Natl Acad Sci U S A, 91, 1994, 549-53.
PL Schwartzberg, J Colicelli, SP Goff, "Construction and analysis of deletion mutations in the pol gene of Moloney murine leukemia virus: a new
viral function required for productive infection", Cell, 37, 1984, 1043-52.
B Bowerman, PO Brown, JM Bishop, HE Varmus, "A nucleoprotein complex mediates the integration of retroviral DNA", Genes Dev, 3, 1989,
469-78.
MJ Roth, PL Schwartzberg, SP Goff, "Structure of the termini of DNA intermediates in the integration of retroviral DNA: dependence on IN
function and terminal DNA sequence", Cell, 58, 1989, 47-54.
KA Vincent, D York-Higgins, M Quiroga, PO Brown, "Host sequences flanking the HIV provirus", Nucleic Acids Res, 18, 1990, 6045-7.
PA Sherman, JA Fyfe, "Human immunodeficiency virus integration protein expressed in Escherichia coli possesses selective DNA cleaving
activity", Proc Natl Acad Sci U S A, 87, 1990, 5119-23.
LA Donehower, "Analysis of mutant Moloney murine leukemia viruses containing linker insertion mutations in the 3' region of pol", J Virol, 62,
1988, 3958-64.
RA Katz, G Merkel, J Kulkosky, J Leis, AM Skalka, "The avian retroviral IN protein is both necessary and sufficient for integrative recombination
in vitro", Cell, 63, 1990, 87-95.
PO Brown, B Bowerman, HE Varmus, JM Bishop, "Correct integration of retroviral DNA in vitro", Cell, 49, 1987, 347-56.
FD Bushman, R Craigie, "Sequence requirements for integration of Moloney murine leukemia virus DNA in vitro", J Virol, 64, 1990, 5645-8.
MS Hansen, S Carteau, C Hoffmann, L Li, FD Bushman, "Retroviral cDNA integration: mechanism, applications and inhibition", Genet Eng (N
Y), 20, 1998, 41-61.
V Ellison, PO Brown, "A stable complex between integrase and viral DNA ends mediates human immunodeficiency virus integration in vitro",
K Mizuuchi, "Retroviral DNA integration: structure of an integration intermediate", Cell, 54, 1988, 497-504.
M Katzman, RA Katz, AM Skalka, J Leis, "The avian retroviral integration protein cleaves the terminal sequences of linear viral DNA at the in
vivo sites of integration", J Virol, 63, 1989, 5319-27.
PO Brown, B Bowerman, HE Varmus, JM Bishop, "Retroviral integration: structure of the initial covalent product and its precursor, and a role for
the viral IN protein", Proc Natl Acad Sci U S A, 86, 1989, 2525-9.
WA Haseltine, "Integration of human immunodeficiency virus type 1 DNA in vitro", Proc Natl Acad Sci U S A, 87, 1990, 4164-8.
FD Bushman, R Craigie, "Activities of human immunodeficiency virus (HIV) integration protein in vitro: specific cleavage and integration of HIV
DNA", Proc Natl Acad Sci U S A, 88, 1991, 1339-43.
M Balakrishnan, CB Jonsson, "Functional identification of nucleotides conferring substrate specificity to retroviral integrase reactions", J Virol,
71, 1997, 1025-35.
AT Panganiban, HM Temin, "The retrovirus pol gene encodes a product required for DNA integration: identification of a retrovirus int locus", Proc
Natl Acad Sci U S A, 81, 1984, 7885-9.
J Colicelli, SP Goff, "Sequence and spacing requirements of a retrovirus integration site", J Mol Biol, 199, 1988, 47-59.
C Vink, RA Lutzke, RH Plasterk, "Formation of a stable complex between the human immunodeficiency virus integrase protein and viral DNA",
SH Hughes, A Mutschler, JM Bishop, HE Varmus, "A Rous sarcoma virus provirus is flanked by short direct repeats of a cellular DNA sequence
present in only one copy prior to integration", Proc Natl Acad Sci U S A, 78, 1981, 4299-303.
LA Donehower, HE Varmus, "A mutant murine leukemia virus with a single missense codon in pol is defective in a function affecting integration",
K Gao, S Wong, FD Bushman, "Metal binding by the D,DX35E motif of human immunodeficiency virus type 1 integrase: selective rescue of Cys
substitutions by Mn2+ in vitro", J Virol, 78, 2004, 6715-22.
FD Bushman, T Fujiwara, R Craigie, "Retroviral DNA integration directed by HIV integration protein in vitro", Science, 249, 1990, 1555-8.
V Ellison, H Abrams, T Roe, J Lifson, PO Brown, "Human immunodeficiency virus integration in a cell-free system", J Virol, 64, 1990, 2711-5.
1997, 5382-90.
AD Leavitt, RB Rose, HE Varmus, "Both substrate and target oligonucleotide sequences affect in vitro integration mediated by human
immunodeficiency virus type 1 integrase protein produced in Saccharomyces cerevisiae", J Virol, 66, 1992, 2359-68.
CM Farnet, WA Haseltine, "Determination of viral proteins present in the human immunodeficiency virus type 1 preintegration complex", J Virol,
65, 1991, 1910-5.
MK Lewinski, FD Bushman, "Retroviral DNA integration--mechanism and consequences", Adv Genet, 55, 2005, 147-81.
SH Hughes, PR Shank, DH Spector, HJ Kung, JM Bishop, HE Varmus, PK Vogt, ML Breitman, "Proviruses of avian sarcoma virus are terminally
redundant, co-extensive with unintegrated linear DNA and integrated at many sites", Cell, 15, 1978, 1397-410.
R Dhar, WL McClements, LW Enquist, GF Vande Woude, "Nucleotide sequences of integrated Moloney sarcoma provirus long terminal repeats
and their host and viral junctions", Proc Natl Acad Sci U S A, 77, 1980, 3937-41.
AT Panganiban, HM Temin, "The terminal nucleotides of retrovirus DNA are required for integration but not virus production", Nature, 306, 1983,
155-60.
TP Quinn, DP Grandgenett, "Genetic evidence that the avian retrovirus DNA endonuclease domain of pol is necessary for viral integration", J
Virol, 62, 1988, 2307-12.
R Craigie, FD Bushman, "The IN protein of Moloney murine leukemia virus processes the viral DNA ends and accomplishes their integration in
vitro", Cell, 62, 1990, 829-37.
A Engelman, K Mizuuchi, R Craigie, "HIV-1 DNA integration: mechanism of viral DNA cleavage and DNA strand transfer", Cell, 67, 1991,
1211-21.
J Colicelli, SP Goff, "Mutants and pseudorevertants of Moloney murine leukemia virus with alterations at the integration site", Cell, 42, 1985,
573-80.
11.1.1.7.1 Formation of Pre-Integration Complex (PIC)
Authors
Editors
Reviewers
Bushman, FD, 2006-10-30.
Description
Concomitant with the completion of reverse transcription, the pre-integration complex is formed by shedding of some viral proteins from the viral
core, and binding of cellular proteins, thereby yielding complexes capable of integration. The terminal cleavage reaction takes place in the
cytoplasm, where two nucleotides are removed from each viral DNA 3' end. This serves to remove heterogeneous extra bases from the viral
DNA ends occasionally added by reverse transcription, thereby yielding a homogeneous substrate for downstream steps, and also serves to
stablilize the PIC. The DNA in PICs is considerably compacted relative to its length when fully extended, probably due to binding of proteins in
addition to the viral integrase. These proteins are not fully clarified, due to the difficulty of biochemical analysis of small amounts of material, but
candidates include the viral NC and MA proteins, and the cellular HMGA, BAF, and PSIP1/LEDGF/p75 proteins. Purified integrase is capable of
carrying out the terminal cleavage and initial strand transfer reactions.
References
JM Coffin, SH Hughes, HE Varmus, "Integration", Retroviruses (Book), 5, 1997, 161-204.
PO Brown, B Bowerman, HE Varmus, JM Bishop, "Correct integration of retroviral DNA in vitro", Cell, 49, 1987, 347-56.
H Chen, A Engelman, "The barrier-to-autointegration protein is a host factor for HIV type 1 integration", Proc Natl Acad Sci U S A, 95, 1998,
15270-4.
M Lewinski, A Ciuffi, S Barr, J Leipzig, S Hannenhalli, C Hoffmann, FD Bushman, "Genome-wide analysis of retroviral DNA integration", Nat Rev
Microbiol, 3, 2005, 848-58.
M Llano, M Vanegas, O Fregoso, D Saenz, S Chung, M Peretz, EM Poeschla, "LEDGF/p75 determines cellular trafficking of diverse lentiviral but
not murine oncoretroviral integrase proteins and is a component of functional lentiviral preintegration complexes", J Virol, 78, 2004, 9524-37.
1997, 5382-90.
MS Lee, R Craigie, "A previously unidentified host protein protects retroviral DNA from autointegration", Proc Natl Acad Sci U S A, 95, 1998,
1528-33.
CM Farnet, FD Bushman, "HIV-1 cDNA integration: requirement of HMG I(Y) protein for function of preintegration complexes in vitro", Cell, 88,
1997, 483-92.
Reaction
11.1.1.7.2 Integrase binds viral DNA ends
Authors
Editors
Reviewers
Bushman, FD, 2006-10-30.
Description
Upon completion of reverse transcription, the viral integrase protein (IN) becomes bound to the ends of the viral DNA. This is inferred by the fact
that this is the site of integrase action, and several biochemical studies have documented integrase interactions with the terminal DNA.
References
CM Farnet, FD Bushman, "HIV cDNA integration: molecular biology and inhibitor development", AIDS, 10, 1996, S3-11.
FD Bushman, "Retroviral cDNA integration: mechanism, applications and inhibition.(Ed) Setlow JK", Genetic Engineering Principles and
Methods, 20, 1998, 41-62.
FD Bushman, R Craigie, "Integration of human immunodeficiency virus DNA: adduct interference analysis of required DNA sites", Proc Natl
Acad Sci U S A, 89, 1992, 3458-62.
M Li, M Mizuuchi, Jr Burke TR, R Craigie, "Retroviral DNA integration: reaction pathway and critical intermediates", EMBO J, 25, 2006,
1295-304.
FD Bushman, T Fujiwara, R Craigie, "Retroviral DNA integration directed by HIV integration protein in vitro", Science, 249, 1990, 1555-8.
Reaction
11.1.1.7.3 Terminal (3' end) cleavage of viral DNA
Authors
Editors
Reviewers
Bushman, FD, 2006-10-30.
Description
Prior to integration, two nucleotides are removed from each 3' end of the linear viral DNA, thereby exposing recessed 3' hydroxyls. This reaction
may serve to remove heterogenous extra bases from the viral DNA end, and to stabilize the IN-DNA complex. The chemistry of cleavage is a
simple hydrolysis by single-step transesterification.
References
Methods, 20, 1998, 41-62.
PO Brown, B Bowerman, HE Varmus, JM Bishop, "Retroviral integration: structure of the initial covalent product and its precursor, and a role for
the viral IN protein", Proc Natl Acad Sci U S A, 86, 1989, 2525-9.
1295-304.
1997, 5382-90.
1211-21.
Reaction
11.1.1.7.4 Import of PIC to the Host Nucleus

Authors
Editors
Reviewers
Bushman, FD, 2006-10-30.
Description
HIV can infect non-dividing cells, implying that the PIC must be able to traverse the nuclear membrane. In contrast, simple retroviruses such as
MLV can only infect cells once they have passed through mitosis, potentially because they require breakdown of the nucleus to access
chromosomal integration sites. The mechanism of nuclear localization is controversial. A variety of proposals have been made for nuclear
localization sequences (NLS) in the PIC, but most of those have now been shown to be dispensible for HIV integration. According to a new idea
from Yamashita and Emerman, it may be that the PIC is imported into the nucleus by a default pathway, while MLV PICs are retained in the
cytoplasm because capsid protein is stably associated with PICs.
References
M Yamashita, M Emerman, "The Cell Cycle Independence of HIV Infections Is Not Determined by Known Karyophilic Viral Elements", PLoS
Pathog, 1, 2005, e18.
M Yamashita, M Emerman, "Retroviral infection of non-dividing cells: old and new perspectives", Virology, 344, 2006, 88-93.
T Roe, TC Reynolds, G Yu, PO Brown, "Integration of murine leukemia virus DNA depends on mitosis", EMBO J, 12, 1993, 2099-108.
M Yamashita, M Emerman, "Capsid is a dominant determinant of retrovirus infectivity in nondividing cells", J Virol, 78, 2004, 5670-8.
Reaction
11.1.1.7.5 Integration of viral DNA into host genomic DNA
Authors
Editors
Reviewers
Bushman, FD, 2006-10-30.
Description
Following nuclear entry, the viral preintegration complex (PIC) must select a site for integration in a host cell chromosome, and then carry out the
chemical steps of the reaction.
At the chromosomal level, HIV has been found to favor active transcription units for integration. Subsequent studies established that the cellular
PSIP1/LEDGF/p75 protein is important in this reaction. PSIP1/LEDGF/p75 binds tightly to HIV integrase, and also to chromatin. Knocking down
PSIP1/LEDGF/p75 in cells resulted in several perturbations of integration targeting in vivo, including reduced integration in transcription units.
Thus PSIP1/LEDGF/p75 has been hypothesized to act as a tethering factor that dictates at least in part the placement of HIV integration sites.
The integration target DNA is also expected to be coated with nucleosomes. Tests of integration into mononucleosomes in vitro have shown that
wrapping integration target DNA actually boosts integration activity. Kinked positions on the DNA gyre are particularly favored for integration.
Integration does not take place at a unique sequence in the integration target DNA (i.e. it is not like a restriction enzyme). However, favored and
disfavored primary sequences can be detected when many integration sites are aligned. Synthesis and testing of favored HIV integration sites
showed that they were favored for integration by PICs in vitro.
After a target DNA is bound, the integration reactions take place via a single-step transesterification.
Integration of both ends of the viral DNA, followed by melting of the target DNA segments between the points of joining, yields single stranded
gaps at each host-virus DNA junction, and a two base overhang derived from the viral DNA. The manner by which this intermediate is
subsequently repaired to yield the fully integrated provirus is unclear. For many parasitic DNA replication reactions, the parasite carries out
reaction steps only up to a point that the host cannot easily reverse, forcing the host to complete the job (Bushman 2001; Craig et al. 2002). For
retroviral integration, it is reasonable to infer that host DNA repair enzymes complete provirus formation. DNA gap repair enzymes are known to
be involved in a variety of DNA repair pathways, so their recruitment to gaps at host-virus DNA junctions is readily envisioned. Consistent with
this, known gap repair enzymes have been shown to act on model host-virus DNA junctions in vitro (Yoder and Bushman, 2000).
References
Microbiol, 3, 2005, 848-58.
1295-304.
1997, 5382-90.
11.1.1.7.5.1 Target DNA binding
Authors
Editors
Reviewers
Bushman, FD, 2006-10-30.
Description
How the PIC finds favored sites on target DNA has not been fully clarified. Active genes are favored for integration, and favored sequences at
the site of integration also influence the reaction. Studies of cells depeleted in PSIP1/LEDGF/p75 suggest that this protein acts as a tethering
factor binding HIV PICs near integration target DNA. Access of PICs to sites on chromosomes may be significant, since centromeric alphoid
repeats are disfavored for integration, perhaps due to wrapping in compact centromeric heterochromatin. Nucleosomes bound to the integration
template also affect target site selection and integration complex binding.
References
A Ciuffi, M Llano, E Poeschla, C Hoffmann, J Leipzig, P Shinn, JR Ecker, FD Bushman, "A role for LEDGF/p75 in targeting HIV DNA
integration", Nat Med, 11, 2005, 1287-9.
AR Schroder, P Shinn, H Chen, C Berry, JR Ecker, FD Bushman, "HIV-1 integration in the human genome favors active genes and local
hotspots", Cell, 110, 2002, 521-9.
RS Mitchell, BF Beitzel, AR Schroder, P Shinn, H Chen, CC Berry, JR Ecker, FD Bushman, "Retroviral DNA integration: ASLV, HIV, and MLV
show distinct target site preferences", PLoS Biol, 2, 2004, E234.
S Carteau, C Hoffmann, FD Bushman, "Chromosome structure and human immunodeficiency virus type 1 cDNA integration: centromeric alphoid
repeats are a disfavored target", J Virol, 72, 1998, 4005-14.
A Engelman, "The ups and downs of gene expression and retroviral DNA integration", Proc Natl Acad Sci U S A, 102, 2005, 1275-6.
AG Holman, JM Coffin, "Symmetrical base preferences surrounding HIV-1, avian sarcoma/leukosis virus, and murine leukemia virus integration
sites", Proc Natl Acad Sci U S A, 102, 2005, 6103-7.
Microbiol, 3, 2005, 848-58.
P Cherepanov, AL Ambrosio, S Rahman, T Ellenberger, A Engelman, "Structural basis for the recognition between HIV-1 integrase and
transcriptional coactivator p75", Proc Natl Acad Sci U S A, 102, 2005, 17308-13.
1211-21.
Reaction
11.1.1.7.5.2 Transesterification to connect viral DNA 3' ends to host DNA 5' ends
Authors
Editors
Reviewers
Bushman, FD, 2006-10-30.
Description
The first chemical step of integration involves a single step transesterification, in which the recessed 3' hydroxyl of the viral DNA becomes
covalently joined to a protruding 5' end in the target DNA. This step at the same time cleaves the target DNA.
References
Methods, 20, 1998, 41-62.
PM Pryciak, HP Muller, HE Varmus, "Simian virus 40 minichromosomes as targets for retroviral integration in vivo", Proc Natl Acad Sci U S A,
89, 1992, 9237-41.
S Carteau, C Hoffmann, FD Bushman, "Chromosome structure and human immunodeficiency virus type 1 cDNA integration: centromeric alphoid
repeats are a disfavored target", J Virol, 72, 1998, 4005-14.
HP Muller, PM Pryciak, HE Varmus, "Retroviral integration machinery as a probe for DNA structure and associated proteins", Cold Spring Harb
Symp Quant Biol, 58, 1993, 533-41.
P Cherepanov, G Maertens, P Proost, B Devreese, J Van Beeumen, Y Engelborghs, E De Clercq, Z Debyser, "HIV-1 integrase forms stable
tetramers and associates with LEDGF/p75 protein in human cells", J Biol Chem, 278, 2003, 372-81.
A Engelman, "The ups and downs of gene expression and retroviral DNA integration", Proc Natl Acad Sci U S A, 102, 2005, 1275-6.
P Cherepanov, AL Ambrosio, S Rahman, T Ellenberger, A Engelman, "Structural basis for the recognition between HIV-1 integrase and
transcriptional coactivator p75", Proc Natl Acad Sci U S A, 102, 2005, 17308-13.
PM Pryciak, HE Varmus, "Nucleosomes, DNA-binding proteins, and DNA sequence modulate retroviral integration target site selection", Cell, 69,
1992, 769-80.
M Llano, M Vanegas, O Fregoso, D Saenz, S Chung, M Peretz, EM Poeschla, "LEDGF/p75 determines cellular trafficking of diverse lentiviral but
not murine oncoretroviral integrase proteins and is a component of functional lentiviral preintegration complexes", J Virol, 78, 2004, 9524-37.
PM Pryciak, A Sil, HE Varmus, "Retroviral integration into minichromosomes in vitro", EMBO J, 11, 1992, 291-303.
Reaction
11.1.1.7.5.3 Gap repair completes provirus integration
Authors
Editors
Reviewers
Bushman, FD, 2006-10-30.
Description
The mechanism by which the integration reaction is completed has not been fully clarified. Unfolding of the integration intermediate resulting
from the IN-catalyzed transesterification produces a branched DNA molecule. Denaturation of the host DNA between the points of joining
produces DNA gaps at each host-virus DNA junction. How these gaps are repaired is unclear. Well studied host cell gap repair enzymes can
carry out this repair step on model virus-host DNA junctions in vitro, providing candidate enzymes. However, efforts to show importance in vivo
are complicated by the fact that the functions are either redundant or lethal when mutated.
Because the strand transfer complex formed at the completion of integration is quite stable, there may be a requirement for a disassembly step
to remove integrase and potentially other proteins to allow access of the gap repair machinery.
In order to complete the last stages of integration, the viral proteins must be removed, and the gaps at the host virus DNA junctions repaired.
The sequence in which the dissembly of PIC occus is not yet understood.
References
Microbiol, 3, 2005, 848-58.
1211-21.
Reaction
11.1.1.7.6 1-LTR circle formation
Authors
Editors

Reviewers
Bushman, FD, 2006-10-30.
Description
The 1-LTR circle can be formed by either of two pathways. The first involves a failure to complete reverse transcription; the second, annotated
here, follows the completion of reverse transcription and is mediated by cellular enzymes. In this pathway, the action of host cell homologous
recombination enzymes on the long terminal repeat (LTR) termini of the viral DNA results in formation of a single LTR. This reaction probably
takes place after partial or complete disassembly of the PIC to expose the viral DNA. Repair of this intermediate as in the late stages of
homologous recombination pathways results in formation of the 1-LTR circle. Mutations in the Mre11/Rad50/NBS pathway influence the
formation of 1-LTR circles.
References
Microbiol, 3, 2005, 848-58.
Reaction
11.1.1.7.7 2-LTR circle formation

Authors
Editors
Reviewers
Bushman, FD, 2006-10-30.
Description
The formation of 2-LTR circles requires the action of the cellular non-homologous DNA end-joining pathway. Specifically the cellular Ku, XRCC4
and ligase IV proteins are needed. Evidence for this is provided by the observation that cells mutant in these functions do not support detectable
formation of 2-LTR circles, though integration and formation of 1-LTR circles are mostly normal. The reaction takes place in the nucleus, and
formation of 2-LTR circles has been used as a surrogate assay for nuclear transport. It has also been suggested that the NHEJ system affects
the toxicity of retroviral infection.
References
JM Kilzer, T Stracker, B Beitzel, K Meek, M Weitzman, FD Bushman, "Roles of host cell factors in circularization of retroviral dna", Virology, 314,
2003, 460-7.
Microbiol, 3, 2005, 848-58.
11.1.1.7.7.1 Association of Ku heterodimer with viral DNA ends

Authors
Editors
Reviewers
Bushman, FD, 2006-10-30.
Description
The Ku protein can be found bound to active PICs in the cytoplasm. However, ligation of the viral DNA ends to form 2-LTR circles takes place in
the nucleus.
References
L Li, JM Olvera, KE Yoder, RS Mitchell, SL Butler, M Lieber, SL Martin, FD Bushman, "Role of the non-homologous DNA end joining pathway in
the early steps of retroviral infection", EMBO J, 20, 2001, 3272-81.
Reaction
11.1.1.7.7.2 Association of XRCC4:DNA ligase IV complex with viral DNA ends
Authors
Editors

Reviewers
Bushman, FD, 2006-10-30.
Description
XRCC4 and DNA ligase 4 are recruited to the complex containing viral DNA.
References
Reaction
11.1.1.7.7.3 2-LTR formation due to circularization of viral DNA
Authors
Editors
Reviewers
Bushman, FD, 2006-10-30.
Description
Viral DNA that does not become integrated can undergo another fate, which is to have the two viral DNA ends joined together to form a 2-LTR
circle. This reaction requires Ku, XRCC4 and ligase 4.
References
Reaction
11.1.1.7.8 Autointegration results in viral DNA circles
Authors
Editors
Reviewers
Bushman, FD, 2006-10-30.
Description
In this pathway, the viral integration machinery uses a site within the viral DNA as an integration target. This results in a covalent rearrangment
of the viral DNA. The resulting DNA forms are not substrates for integration.
It has been suggested that the cellular BAF protein binds to viral DNA and diminishes autointegration by coating and condensing the viral DNA,
thereby making it a less efficient integration target.
References
MS Lee, R Craigie, "Protection of retroviral DNA from autointegration: involvement of a cellular factor", Proc Natl Acad Sci U S A, 91, 1994,
9823-7.
MS Lee, R Craigie, "A previously unidentified host protein protects retroviral DNA from autointegration", Proc Natl Acad Sci U S A, 95, 1998,
1528-33.
11.1.1.7.8.1 Suicidal integration leading to inverted circle formation
Authors
Editors
Reviewers
Bushman, FD, 2006-10-30.

Description
Following the integrase-mediated strand transfer reaction of autointegration, the integration complex must be disassembled and the gapped
intermediate repaired, just as in normal integration.
References
CM Farnet, WA Haseltine, "Circularization of human immunodeficiency virus type 1 DNA in vitro", J Virol, 65, 1991, 6942-52.
Methods, 20, 1998, 41-62.
Reaction
11.1.1.7.8.2 Suicidal integration leading to smaller circles of viral DNA
Authors
Editors
Reviewers
Bushman, FD, 2006-10-30.
Description
Following the integrase-mediated strand transfer reaction of autointegration, the integration complex must be disassembled and the gapped
intermediate repaired, just as in normal integration.
References
CM Farnet, WA Haseltine, "Circularization of human immunodeficiency virus type 1 DNA in vitro", J Virol, 65, 1991, 6942-52.
Methods, 20, 1998, 41-62.
Reaction
11.1.2 Late Phase of HIV Life Cycle
Reviewers
Peterlin, BM, 2005-01-05.
Description
The late phase of the HIV-1 life cycle includes the regulated expression of the HIV gene products and the assembly of viral particles. The
assembly of viral particles will be covered in a later release of Reactome. HIV-1 gene expression is regulated by both cellular and viral proteins.
Although the initial activation of the HIV-1 transcription is facilitated by cellular transcription factors including NF-kappa B (Nabel and Baltimore,
1987), this activation results in the production of primarily short transcripts (Kao et al., 1987). Expression of high levels of the full length HIV-1
transcript requires the function of the HIV-1 Tat protein which promotes elongation of the HIV-1 transcript (reviewed in Karn, 1999; Taube et al.
1999; Liou et al., 2004; Barboric and Peterlin 2005). The HIV-1 Rev protein is required post-transcriptionally for the expression of the late genes.
Rev functions by promoting the nuclear export of unspliced and partially spliced transcripts that encode the major structural proteins Gag, Pol
and Env, and the majority of the accessory proteins (Malim et al., 1989; reviewed in Pollard and Malim 1998 .
References
LY Liou, CH Herrmann, "HIV-1 infection and regulation of Tat function in macrophages", Int J Biochem Cell Biol, 36, 2004, 1767-75.
M Barboric, BM Peterlin, "A new paradigm in eukaryotic biology: HIV Tat and the control of transcriptional elongation", PLoS Biol, 3, 2005, e76.
J Karn, "Tackling Tat", J Mol Biol, 293, 1999, 235-54.
MH Malim, J Hauber, SY Le, JV Maizel, BR Cullen, "The HIV-1 rev trans-activator acts through a structured target sequence to", Nature, 338,
1989, 254-7.
R Taube, K Fujinaga, J Wimmer, M Barboric, BM Peterlin, "Tat transactivation: a model for the regulation of eukaryotic transcriptional
elongation", Virology, 264, 1999, 245-53.
G Nabel, D Baltimore, "An inducible transcription factor activates expression of human immunodeficiency virus in T cells", Nature, 326, 1987,
711-3.
SY Kao, AF Calman, PA Luciw, BM Peterlin, "Anti-termination of transcription within the long terminal repeat of HIV-1 by tat gene product",
Nature, 330, 1987, 489-93.
VW Pollard, MH Malim, "The HIV-1 Rev protein", Annu Rev Microbiol, 52, 1998, 491-532.
11.1.2.1 Transcription of the HIV genome
Authors
Matthews, L, Rice, AP, 2005-07-27.
Editors
Matthews, L, 2005-07-26.
Reviewers
Peterlin, BM, 2005-01-05.
Description
Expression of the integrated HIV-1 provirus is dependent on the host cell Pol II transcription machinery, but is regulated in critical ways by HIV-1
Tat and Rev proteins. The long terminal repeats (LTR) located at either end of the proviral DNA contain regulatory sequences that recruit cellular
transcription factors. The U3 region of the 5' LTR contains numerous cis-acting elements that regulate Pol II-mediated transcription initiation. The
full-length transcript, which encodes nine genes, functions as an mRNA and is packaged as genomic RNA. Smaller (subgenomic) viral mRNAs
are generated by alternative splicing. The activities of Tat and Rev create two phases of gene expression (see Karn 1999; Cullen 1991). The Tat
protein is an RNA specific trans-activator of LTR-mediated transcription. Association of Tat with TAR, a RNA stem-loop within the RNA leader
sequence, is required for efficient elongation of the HIV-1 transcript. In the early phase of viral transcription, a multiply-spliced set of mRNAs is
generated, producing the transcripts of the regulatory proteins, Tat, Rev, and Nef. In the late phase, Rev regulates nuclear export of HIV-1
mRNAs, repressing expression of the early regulatory mRNAs and promoting expression of viral structural proteins. Nuclear export of the
unspliced and partially spliced late HIV-1 transcripts that encode the structural proteins requires the association of Rev with a cis-acting RNA
sequence in the transcripts (Rev Response Element, RRE).
References
BR Cullen, "Retroviruses as model systems for the study of nuclear RNA export", Virology, 249, 1998, 203-10.
BR Cullen, "Human immunodeficiency virus as a prototypic complex retrovirus", J Virol, 65, 1991, 1053-6.
Source pathway
This pathway was inferred from the corresponding pathway "RNA Polymerase II Transcription" in species Homo sapiens.
11.1.2.1.1 HIV-1 Transcription Pre-Initiation
Authors
Editors
Matthews, L, 2005-07-26.
Description
Source pathway
This pathway was inferred from the corresponding pathway "RNA Polymerase II Transcription Pre-Initiation" in species Homo sapiens.
11.1.2.1.1.1 Recognition and Binding of Core HIV-1 Promoter Elements by TFIID
Authors
Description
Source reaction
This reaction was inferred from the corresponding reaction "Recognition and Binding of Core Promoter Elements by TFIID" in species Homo
sapiens.
Reaction
11.1.2.1.1.2 Binding of TFIIA and TFIIB to the HIV-1 promoter:TFIID complex
Authors
Editors
Matthews, L, 2005-07-26.
Description
Source reaction
This reaction was inferred from the corresponding reaction "Binding of TFIIA and TFIIB to the pol II promoter:TFIID complex" in species Homo
sapiens.
Reaction
11.1.2.1.1.3 Recruitment of RNA Polymerase II Holoenzyme by TFIIF to the HIV-1promoter:TFIID:TFIIA:TFIIB complex
Authors
Editors
Matthews, L, 2005-07-26.
Description
Source reaction
This reaction was inferred from the corresponding reaction "Recruitment of RNA Polymerase II Holoenzyme by TFIIF to the pol II
promoter:TFIID:TFIIA:TFIIB complex" in species Homo sapiens.
Reaction
11.1.2.1.1.4 Binding of TFIIE to the growing HIV-1 preinitiation complex
Authors
Editors
Matthews, L, 2005-07-26.
Description
CTD by TFIIH.
Source reaction
This reaction was inferred from the corresponding reaction "Binding of TFIIE to the growing preinitiation complex" in species Homo sapiens.
Reaction
11.1.2.1.1.5 Formation of the HIV-1 closed pre-initiation complex
Authors
Editors
Matthews, L, 2005-07-26.
Description
Source reaction
This reaction was inferred from the corresponding reaction "Formation of the closed pre-initiation complex" in species Homo sapiens.
Reaction
11.1.2.1.2 HIV-1 Promoter Opening: First Transition
Authors
Editors
Matthews, L, 2005-07-26.
Description
start site.
Source reaction
This reaction was inferred from the corresponding reaction "RNA Polymerase II Promoter Opening: First Transition" in species Homo sapiens.
1992, 450-3.
7468-80.
145-56.
Reaction
11.1.2.1.3 HIV-1 Transcription Initiation
Authors
Editors
Matthews, L, 2005-07-26.
Description
initiation complex.
Source pathway
This pathway was inferred from the corresponding pathway "RNA Polymerase II Transcription Initiation" in species Homo sapiens.
1479-90.
14990-7.
7468-80.
22, 2002, 762-73.
11.1.2.1.3.1 NTP binds active site of RNA Polymerase II in HIV-1 open pre-initiation complex
Authors
Editors
Matthews, L, 2005-07-26.
Description
At the beginning of this reaction, 1 molecule of 'HIV-1 open pre-initiation complex', and 2 molecules of 'NTP' are present. At the end of this
reaction, 1 molecule of 'HIV-1 initiation complex' is present.

Source reaction
This reaction was inferred from the corresponding reaction "NTP Binds Active Site of RNA Polymerase II" in species Homo sapiens.
Reaction
11.1.2.1.3.2 Nucleophillic attack by 3'-hydroxyl oxygen of nascent HIV-1 transcript on the Alpha phosphate of NTP
Authors
Editors
Matthews, L, 2005-07-26.
Description
At the beginning of this reaction, 1 molecule of 'HIV-1 initiation complex' is present. At the end of this reaction, 1 molecule of 'HIV-1 initiation
Source reaction
This reaction was inferred from the corresponding reaction "Nucleophillic Attack by 3'-hydroxyl Oxygen of nascent transcript on the Alpha
Phosphate of NTP" in species Homo sapiens.
Reaction
11.1.2.1.3.3 Newly formed phosphodiester bond stabilized and PPi released
Authors
Editors
Matthews, L, 2005-07-26.
Description
At the beginning of this reaction, 1 molecule of 'HIV-1 initiation complex with phosphodiester-PPi intermediate' is present. At the end of this
reaction, 1 molecule of 'HIV-1 transcription complex', and 1 molecule of 'pyrophosphate' are present.
Source reaction
This reaction was inferred from the corresponding reaction "Newly Formed Phosphodiester Bond Stabilized and PPi Released" in species Homo
sapiens.
Reaction
11.1.2.1.4 RNA Polymerase II HIV-1 Promoter Escape
Authors
Editors
Matthews, L, 2005-07-26.
Description
Source pathway
This pathway was inferred from the corresponding pathway "RNA Polymerase II Promoter Escape" in species Homo sapiens.
7468-80.
11.1.2.1.4.1 Addition of the third nucleotide on the nascent HIV-1 transcript
Authors
Editors
Matthews, L, 2005-07-26.
Description
positions -9 to +3.
Source reaction
This reaction was inferred from the corresponding reaction "Addition of the third nucleotide on the nascent transcript" in species Homo sapiens.
7468-80.
22, 2002, 762-73.
Reaction
11.1.2.1.4.2 Addition of the fourth nucleotide on the nascent HIV-1 transcript: Second Transition
Authors
Editors
Matthews, L, 2005-07-26.
Description
positions -9 to +4.
Source reaction
This reaction was inferred from the corresponding reaction "Addition of the fourth nucleotide on the Nascent Transcript: Second Transition" in
species Homo sapiens.
7468-80.
22, 2002, 762-73.
Reaction
11.1.2.1.4.3 Addition of nucleotides 5 through 9 on the growing HIV-1 transcript
Authors
Editors
Matthews, L, 2005-07-26.
Description
Source reaction
This reaction was inferred from the corresponding reaction "Addition of Nucleotides 5 through 9 on the growing Transcript" in species Homo
sapiens.
1479-90.
7468-80.
Acad Sci U S A, 94, 1997, 9006-10.
Reaction
11.1.2.1.4.4 Addition of nucleotides 10 and 11 on the growing HIV-1 transcript: Third Transition
Authors
Editors
Matthews, L, 2005-07-26.
Description
Source reaction
This reaction was inferred from the corresponding reaction "Addition of nucleotides 10 and 11 on the growing transcript: Third Transition" in
1479-90.
7468-80.
Reaction
11.1.2.1.4.5 Addition of nucleotides between position +11 and +30 on HIV-1 transcript
Authors
Editors
Matthews, L, 2005-07-26.
Description
Source reaction
This reaction was inferred from the corresponding reaction "Addition of nucleotides between position +11 and +30" in species Homo sapiens.
1479-90.
Cell Biol, 21, 2001, 5815-25.
Acids Res, 29, 2001, 2706-14.
Reaction
11.1.2.1.5 RNA Pol II CTD phosphorylation and interaction with CE
Authors
Editors
Matthews, L, 2005-07-26.
Description
To facilitate co-transcriptional capping, and thereby restrict the cap structure to RNAs made by RNA polymerase II, the capping enzymes bind
directly to the RNA polymerase II. The C-terminal domain of the largest Pol II subunit contains several phosphorylation sites on its heptapeptide
repeats. The capping enzyme guanylyltransferase and the methyltransferase bind specifically to CTD phosphorylated at Serine 5 within the
CTD. Kinase subunit of TFIIH, Cdk7, catalyzes this phosphorylation event that occurs near the promoter. In addition, it has been shown that
binding of capping enzyme to the Serine-5 phosphorylated CTD stimulates guanylyltransferase activity in vitro.
Source pathway
This pathway was inferred from the corresponding pathway "RNA Pol II CTD phosphorylation and interaction with CE" in species Homo sapiens.
11.1.2.1.5.1 Extrusion of 5'-end of 30 nt long HIV-1 transcript through the pore in Pol II complex
Authors
Editors
Matthews, L, 2005-07-26.
Description
At the beginning of this reaction, 1 molecule of 'HIV-1 transcription complex containing transcript to +30' is present. At the end of this reaction, 1
molecule of 'HIV-1 transcription complex containing extruded transcript to +30' is present.
Source reaction
This reaction was inferred from the corresponding reaction "Extrusion of 5'-end of 30 nt long transcript through the pore in Pol II complex" in
Reaction
11.1.2.1.5.2 Phosphorylation (Ser5) of RNA pol II CTD
Authors
Editors
Matthews, L, 2005-07-26.
Description
Phosphorylation of serine 5 residue at the CTD of pol II largest subunit is an important step signaling the end of initiation and escape into
processive elongation processes. Cdk7 protein subunit of TFIIH phosphorylates RNA Pol II CTD serine 5 residues on its heptad repeats.
Source reaction
This reaction was inferred from the corresponding reaction "Phosphorylation (Ser5) of RNA pol II CTD" in species Homo sapiens.
Reaction
11.1.2.1.5.3 RNA Polymerase II CTD (phosphorylated) binds to CE
Authors
Editors
Matthews, L, 2005-07-26.
Description
At the beginning of this reaction, 1 molecule of 'mRNA capping enzyme', and 1 molecule of 'HIV-1 transcription complex with (ser5)
phosphorylated CTD containing extruded transcript to +30' are present. At the end of this reaction, 1 molecule of 'RNA Pol II with phosphorylated
CTD: CE complex' is present.
Source reaction
This reaction was inferred from the corresponding reaction "RNA Polymerase II CTD (phosphorylated) binds to CE" in species Homo sapiens.
Reaction
11.1.2.1.5.4 Activation of GT
Authors
Editors
Matthews, L, 2005-07-26.
Description
At the beginning of this reaction, 1 molecule of 'RNA Pol II with phosphorylated CTD: CE complex' is present. At the end of this reaction, 1
molecule of 'RNA Pol II with phosphorylated CTD: CE complex with activated GT' is present.
Source reaction
This reaction was inferred from the corresponding reaction "Activation of GT" in species Homo sapiens.
Reaction
11.1.2.1.5.5 SPT5 subunit of Pol II binds the RNA triphosphatase (RTP)
Authors
Editors
Matthews, L, 2005-07-26.
Description
The capping enzyme interacts with the Spt5 subunit of transcription elongation factor DSIF. This interaction may couple the capping reaction
with promoter escape or elongation, thereby acting as a Ã¢â‚¬Å"checkpointÃ¢â‚¬Â• to assure that capping has occurred before the polymerase
proceeds to make the rest of the transcript.
Source reaction
This reaction was inferred from the corresponding reaction "SPT5 subunit of Pol II binds the RNA triphosphatase (RTP)" in species Homo
sapiens.
Reaction
11.1.2.1.6 HIV-1 Transcription Elongation
Authors
Editors
Matthews, L, 2005-07-26.
Description
In the absence of the HIV-1 protein Tat, transcription of the proviral DNA is inefficient and results in the production of truncated transcripts (Kao
et al., 1987). While initiation of transcription from the HIV-1 LTR and formation of the early elongation complex occurs normally, transcription
elongation is incomplete with non-processive polymerases disengaging from the proviral DNA template prematurely (reviewed in Karn 1999).
The mechanism of Tat-mediated elongation is described below.
References
AP Rice, CH Herrmann, "Regulation of TAK/P-TEFb in CD4+ T lymphocytes and macrophages", Curr HIV Res, 1, 2003, 395-404.
Nature, 330, 1987, 489-93.
Source pathway
This pathway was inferred from the corresponding pathway "RNA Polymerase II Transcription Elongation" in species Homo sapiens.
11.1.2.1.6.1 Formation of the HIV-1 Early Elongation Complex
Authors

Editors
Matthews, L, 2005-07-26.
Description
This HIV-1 event was inferred from the corresponding human RNA Poll II transcription event. The details relevant to HIV-1 are described below.
Formation of the early elongation complex involves hypophosphorylation of RNA Pol II CTD by FCP1P protein, association of the DSIF complex
with RNA Pol II, and formation of DSIF:NELF:HIV-1 early elongation complex as described below (Mandal et al 2002; Kim et al 2003; Yamaguchi
et al 2002).
References
Source pathway
This pathway was inferred from the corresponding pathway "Formation of the Early Elongation Complex" in species Homo sapiens.
11.1.2.1.6.1.1 Hypophosphorylation of RNA Pol II CTD by FCP1P protein
Authors
Editors
Matthews, L, 2005-07-26.
Description
This HIV-1 event was inferred from the corresponding human RNA Pol II transcription event. FCP1 dephosphorylates RNAP II in ternary
elongation complexes as well as in solution and, therefore, is thought to function in the recycling of RNAP II during the transcription cycle.
Biochemical experiments suggest that human FCP1 targets CTDs that are phosphorylated at serine 2 (CTD-serine 2) and/or CTD-serine 5. It is
also observed to stimulate elongation independent of its catalytic activity. Dephosphorylation of Ser2 - phosphorylated Pol II results in
hypophosphorylated form that disengages capping enzymes (CE).
References
Source reaction
This reaction was inferred from the corresponding reaction "Hypophosphorylation of RNA Pol II CTD by FCP1P protein" in species Homo
sapiens.
Reaction
11.1.2.1.6.1.2 DSIF complex binds to RNA Pol II (hypophosphorylated)
Authors
Editors
Matthews, L, 2005-07-26.
Description
This HIV-1 event was inferred from the corresponding human RNA Pol II transcription event. DSIF is a heterodimer consisting of hSPT4 (human
homolog of yeast Spt4- p14) and hSPT5 (human homolog of yeast Spt5-p160) (Wada et al. 1998). DSIF association with Pol II may be enabled
by Spt5 binding to Pol II creating a scaffold for NELF binding. Spt5 subunit of DSIF can be phosphorylated by P-TEFb (Ivanov et al. 2000).
References
D Ivanov, YT Kwak, J Guo, RB Gaynor, "Domains in the SPT5 protein that modulate its transcriptional regulatory properties", Mol Cell Biol, 20,
2000, 2970-83.
Dev, 12, 1998, 343-56.
Source reaction
This reaction was inferred from the corresponding reaction "DSIF complex binds to RNA Pol II (hypophosphorylated)" in species Homo sapiens.
Dev, 12, 1998, 343-56.
Reaction
11.1.2.1.6.1.3 Formation of DSIF:NELF:HIV-1 early elongation complex
Authors

Editors
Matthews, L, 2005-07-26.
Description
This HIV-1 event was inferred from the corresponding human RNA Pol II transcription event. NELF complex is a ~ 300 kDa multiprotein complex
composed of 5 peptides (A - E): ~66,61,59,58 and 46 kDa (Yamaguchi et al 1999). All these peptides are required for NELF-mediated inhibition
of Pol II elongation. NELF complex has been reported to bind to the pre-formed DSIF:RNA Pol II complex that may act as a scaffold for its
binding. NELF-A is suspected to be involved in Wolf-Hirschhorn syndrome. Binding of DSIF:NELF to RNA Pol II CTD results in abortive
termination of early elongation steps by the growing transcripts.
References
Y Yamaguchi, T Takagi, T Wada, K Yano, A Furuya, S Sugimoto, J Hasegawa, H Handa, "NELF, a multisubunit complex containing RD,
cooperates with DSIF to repress RNA polymerase II elongation", Cell, 97, 1999, 41-51.
Source reaction
This reaction was inferred from the corresponding reaction "Formation of DSIF:NELF:early elongation complex" in species Homo sapiens.
Reaction
11.1.2.1.6.2 Tat-mediated elongation of the HIV-1 transcript
Description
The Tat protein is a viral transactivator protein that regulates HIV-1 gene expression by controlling RNA Pol II-mediated elongation (reviewed in
Karn 1999; Taube et al. 1999; Liou et al. 2004; Barboric and Peterlin 2005). Tat appears to be required in order to overcome the arrest of RNA
Pol II by the negative transcriptional elongation factors DSIF and NELF (Wada et al. 1998; Yamaguchi et al. 1999; Yamaguchi et al 2002;
Fujinaga et al. 2004). While Pol II can associate with the proviral LTR and initiate transcription in the absence of Tat, these polymerase
complexes are non-processive and dissociate from the template prematurely producing very short transcripts (Kao et al. 1987). Tat associates
with the RNA element, TAR, which forms a stem loop structure in the leader RNA sequence (Dingwall et al. 1989). Tat also associates with the
cellular kinase complex P-TEFb(Cyclin T1:Cdk9) and recruits it to the TAR stem loop structure (Herrmann, 1995) (Wei et al. 1998). This
association between Tat, TAR and P-TEFb(Cyclin T1:Cdk9) is believed to bring the catalytic subunit of this kinase complex (Cdk9) in close
proximity to Pol II where it hyperphosphorylates the CTD of RNA Pol II (Zhou et al. 2000). The RD subunits of NELF and the SPT5 subunit of
DSIF, which associate through RD with the bottom stem of TAR, are also phosphorylated by P-TEFb(Cyclin T1:Cdk9) (Yamaguchi et al. 2002;
Fujinaga et al. 2004; Ivanov et al. 2000). Phosphorylation of RD results in its dissociation from TAR. Thus, Tat appears to facilitate transcriptional
elongation of the HIV-1 transcript by hyperphosphorylating the RNA Poll II CTD and by removing the negative transcription elongation factors
from TAR. In addition, there is evidence that the association of Tat with P-TEFb(Cyclin T1:Cdk9) alters the substrate specificity of P-TEFb
enhancing phosphorylation of ser5 residues in the CTD of RNA Pol II (Zhou et al. 2000).
References
CH Herrmann, AP Rice, "Lentivirus Tat proteins specifically associate with a cellular protein kinase, TAK, that hyperphosphorylates the
carboxyl-terminal domain of the large subunit of RNA polymerase II: candidate for a Tat cofactor", J Virol, 69, 1995, 1612-20.
P Wei, ME Garber, SM Fang, WH Fischer, KA Jones, "A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its
high-affinity, loop-specific binding to TAR RNA", Cell, 92, 1998, 451-62.
2000, 2970-83.
LY Liou, CH Herrmann, "HIV-1 infection and regulation of Tat function in macrophages", Int J Biochem Cell Biol, 36, 2004, 1767-75.
K Fujinaga, D Irwin, Y Huang, R Taube, T Kurosu, BM Peterlin, "Dynamics of human immunodeficiency virus transcription: P-TEFb
phosphorylates RD and dissociates negative effectors from the transactivation response element", Mol Cell Biol, 24, 2004, 787-95.
Dev, 12, 1998, 343-56.
M Barboric, BM Peterlin, "A new paradigm in eukaryotic biology: HIV Tat and the control of transcriptional elongation", PLoS Biol, 3, 2005, e76.
C Dingwall, I Ernberg, MJ Gait, SM Green, S Heaphy, J Karn, AD Lowe, M Singh, MA Skinner, R Valerio, "Human immunodeficiency virus 1 tat
protein binds trans-activation-responsive region (TAR) RNA in vitro", Proc Natl Acad Sci U S A, 86, 1989, 6925-9.
M Zhou, MA Halanski, MF Radonovich, F Kashanchi, J Peng, DH Price, JN Brady, "Tat modifies the activity of CDK9 to phosphorylate serine 5
of the RNA polymerase II carboxyl-terminal domain during human immunodeficiency virus type 1 transcription", Mol Cell Biol, 20, 2000, 5077-86.
R Taube, K Fujinaga, J Wimmer, M Barboric, BM Peterlin, "Tat transactivation: a model for the regulation of eukaryotic transcriptional
elongation", Virology, 264, 1999, 245-53.
Nature, 330, 1987, 489-93.
11.1.2.1.6.2.1 Formation of HIV-1 elongation complex containing HIV-1 Tat
Authors
Editors
Matthews, L, 2005-07-26.
Description
This HIV-1 event was inferred from the corresponding human RNA Poll II transcription event in Reactome. The details relevant to HIV-1 are
described below. For a more detailed description of the general mechanism, see the link to the corresponding RNA Pol II transcription event
below. The formation of the HIV-1 elongation complex involves Tat mediated recruitment of P-TEFb(Cyclin T1:Cdk9) to the TAR sequence (Wei
et al, 1998) and P-TEFb(Cyclin T1:Cdk9) mediated phosphorylation of the RNA Pol II CTD as well as the negative transcriptional elongation
factors DSIF and NELF (Herrmann, 1995; Ivanov et al. 2000; Fujinaga et al. 2004; Zhou et al., 2004).
References
2000, 2970-83.
M Zhou, L Deng, V Lacoste, HU Park, A Pumfery, F Kashanchi, JN Brady, A Kumar, "Coordination of transcription factor phosphorylation and
histone methylation by the P-TEFb kinase during human immunodeficiency virus type 1 transcription", J Virol, 78, 2004, 13522-33.
Source pathway
This pathway was inferred from the corresponding pathway "Formation of RNA Pol II elongation complex " in species Homo sapiens.
11.1.2.1.6.2.1.1 Association of Tat with P-TEFb(Cyclin T1:Cdk9)
Description
Tat associates with the Cyclin T1 subunit of P-TEFb (Cyclin T1:Cdk9) through a region of cysteine-rich and core sequences referred to as the
ARM domain within Tat (Wei et al., 1998; see also Herrmann 1995). This interaction is believed to involve metal ions stabilized by cysteine
residues in both proteins (Bieniasz et al., 1998; Garber et al., 1998).
References
PD Bieniasz, TA Grdina, HP Bogerd, BR Cullen, "Recruitment of a protein complex containing Tat and cyclin T1 to TAR governs the species
specificity of HIV-1 Tat", EMBO J, 17, 1998, 7056-65.
ME Garber, P Wei, VN KewalRamani, TP Mayall, CH Herrmann, DR Littman, KA Jones, "The interaction between HIV-1 Tat and human cyclin
T1 requires zinc and a critical cysteine residue that is not conserved in the murine CycT1 protein", Genes Dev, 12, 1998, 3512-27.
Reaction
11.1.2.1.6.2.1.2 Hyperphosphorylation (Ser2) of RNA Pol II CTD by the P-TEFb(Cyclin T1:Cdk9) complex
Authors
Editors
Matthews, L, 2005-07-26.
Description
The association between Tat, TAR and P-TEFb is believed to bring the catalytic subunit of P-TEFb(Cyclin T1:Cdk9) in close proximity to Pol II
where it hyperphosphorylates the CTD of Pol II (Herrmann et al., 1995; Zhou et al. 2000). In the presence of Tat, P-TEFb(Cyclin T1:CDK9) has
been shown to phosphorylate serine 5 in addition to serine 2 suggesting that modification of the substrate specificity of CDK9 may play a role in
the ability of Tat to promote transcriptional elongation (Zhou et al. 2000).
References
M Zhou, MA Halanski, MF Radonovich, F Kashanchi, J Peng, DH Price, JN Brady, "Tat modifies the activity of CDK9 to phosphorylate serine 5
of the RNA polymerase II carboxyl-terminal domain during human immunodeficiency virus type 1 transcription", Mol Cell Biol, 20, 2000, 5077-86.
Source reaction
This reaction was inferred from the corresponding reaction "Hyperphosphorylation (Ser2) of RNA Pol II CTD by P-TEFb complex" in species
Homo sapiens.
Reaction
11.1.2.1.6.2.1.3 Phosphorylation of NEFL by the P-TEFb(Cyclin T1:Cdk9) complex
Authors

Description
Phosphorylation of the RD subunit of NEFL by P-TEFb(Cyclin T1:Cdk9) results in the dissociation of NEFL from TAR as well as the conversion
of NEFL to an elongation factor (Fujinaga et al., 2004)
References
Reaction
11.1.2.1.6.2.1.4 Phosphorylation of DSIF by the P-TEFb(Cyclin T1:Cdk9) complex
Authors
Editors
Matthews, L, 2006-01-10.
Description
Phosphorylation of the Spt5 subunit of DSIF by P-TEFb(Cyclin T1:Cdk9) results in the conversion of DSIF to an elongation factor (Ivanov al.
2000).
References
2000, 2970-83.
Reaction
11.1.2.1.6.2.1.5 Recruitment of elongation factors to form HIV-1 elongation complex
Authors
Editors
Matthews, L, 2005-07-26.
Description
At the beginning of this reaction, 1 molecule of 'FACT complex', 1 molecule of 'Elongin Complex', 1 molecule of 'TFIIH', 1 molecule of 'RNA
polymerase II elongation factor ELL', 1 molecule of 'Tat-containing early elongation complex with hyperphosphorylated Pol II CTD (
phospho-NELF phospho DSIF)', and 1 molecule of 'TFIIS protein' are present. At the end of this reaction, 1 molecule of 'HIV-1 elongation
complex containing Tat' is present.
Source reaction
This reaction was inferred from the corresponding reaction "Recruitment of elongation factors to form elongation complex" in species Homo
sapiens.
Reaction
11.1.2.1.6.2.2 Addition of nucleotides leads to HIV-1 transcript elongation
Authors

Editors
Matthews, L, 2005-07-26.
Description
This HIV-1 event was inferred from the corresponding human RNA Pol II transcription event. High-resolution structures of free, catalytically
active yeast Pol II and of an elongating form reveal that Pol II elongation complex includes features like:
References
1863-76.
Source reaction
This reaction was inferred from the corresponding reaction "Addition of nucleotides leads to transcript elongation" in species Homo sapiens.
1863-76.
Reaction
11.1.2.1.6.2.3 Pol II elongation complex moves on the HIV-1 template as transcript elongates
Authors
Editors
Matthews, L, 2005-07-26.
Description
This event was inferred from the corresponding human Poll II transcription elongation event.
Source reaction
This reaction was inferred from the corresponding reaction "Pol II elongation complex moves on the template as transcript elongates" in species
Homo sapiens.
Reaction
11.1.2.1.6.2.4 Separation of elongating HIV-1 transcript from template
Authors
Editors
Matthews, L, 2005-07-26.
Description
This event was inferred from the corresponding human Poll II transcription elongation event.
Source reaction
This reaction was inferred from the corresponding reaction "Separation of elongating transcript from template" in species Homo sapiens.
Reaction
11.1.2.1.6.3 Abortive elongation of HIV-1 transcript in the absence of Tat

Description
This event was inferred from the corresponding Reactome human Poll II transcription elongation event. The details specific to HIV-1 transcription
elongation are described below. In the absence of the HIV-1 Tat protein, the RNA Pol II complexes associated with the HIV-1 template are
non-processive. RNA Pol II is arrested after promoter clearance by the negative transcriptional elongation factors DSIF and NELF as occurs
during early elongation of endogenous templates (Wada et al, 1998; Yamaguchi et al. 1999). This arrest cannot be overcome by P-TEFb
mediated phosphorylation in the absence of Tat however, and elongation aborts resulting in the accumulation of short transcripts (Kao et al.,
1987).
References
Dev, 12, 1998, 343-56.
Nature, 330, 1987, 489-93.
11.1.2.1.6.3.1 Limited elongation of the HIV-1 transcript
Authors
Editors
Matthews, L, 2005-07-26.
Description
In the absence of Tat, transcriptional elongation beyond position +59 does not occur (Kao et al., 1987).
References
Nature, 330, 1987, 489-93.
Source reaction
This reaction was inferred from the corresponding reaction "Addition of nucleotides leads to transcript elongation" in species Homo sapiens.
1863-76.
Reaction
11.1.2.1.6.3.2 Separation of abortive HIV-1 transcript from template
Authors
Editors
Matthews, L, 2005-07-26.
Description
At the beginning of this reaction, 1 molecule of 'DSIF:NELF:early elongation complex after limited nucleotide addition' is present. At the end of
this reaction, 1 molecule of 'Early elongation complex with separated aborted transcript' is present.
Source reaction
This reaction was inferred from the corresponding reaction "Separation of elongating transcript from template" in species Homo sapiens.
Reaction
11.1.2.1.7 HIV-1 Transcription Termination
Authors
Editors
Matthews, L, 2005-07-26.
Description
Termination of HIV-1 transcription is believed to be mechanistically the same as termination of endogenous human messages.
Source pathway
This pathway was inferred from the corresponding pathway "RNA Polymerase II Transcription Termination" in species Homo sapiens.
11.1.2.2 Rev-mediated nuclear export of HIV-1 RNA
Authors
Editors
Matthews, L, 2006-06-07.
Reviewers
Kumar, A, 2007-01-31.
Description
The HIV-1 genome contains 9 genes encoded by a single transcript. In order for the virus to replicate, unspliced, singly-spliced and fully spliced
viral mRNA must be exported from the nucleus. The HIV-1 mRNA splice sites are inefficient resulting it the accumulation of a pool of
incompletely spliced RNAs (Staffa and Cochrane, 1994). In the early stages of the viral life cycle, or in the absence of the viral Rev protein,
completely spliced viral mRNA which encode the regulatory proteins Tat, Nef and Rev are exported from the nucleus while the incompletely
spliced structural protein encoding transcripts are held within the nucleus by cellular proteins that normally function in preventing the nuclear
export of cellular pre-mRNA. Export of both unspliced and partially spliced mRNA is mediated by the viral protein Rev which is recruited, along
with cellular cofactors, to the Rev Response Element (RRE) within the HIV-1 mRNA sequence (Malim et al., 1990; Fischer et al., 1994). The
cellular hRIP protein is essential for correct Rev-mediated export of viral RNAs to the cytoplasm (Sanchez-Velar et al., 2004; Yu et al., 2005).
References
CC Fritz, MR Green, "HIV Rev uses a conserved cellular protein export pathway for the nucleocytoplasmic transport of viral RNAs", Curr Biol, 6,
1996, 848-54.
1989, 254-7.
MH Malim, LS Tiley, DF McCarn, JR Rusche, J Hauber, BR Cullen, "HIV-1 structural gene expression requires binding of the Rev trans-activator
to its RNA target sequence", Cell, 60, 1990, 675-83.
BR Cullen, "Nuclear mRNA export: insights from virology", Trends Biochem Sci, 28, 2003, 419-24.
N Sanchez-Velar, EB Udofia, Z Yu, ML Zapp, "hRIP, a cellular cofactor for Rev function, promotes release of HIV RNAs from the perinuclear
region", Genes Dev, 18, 2004, 23-34.
A Staffa, A Cochrane, "The tat/rev intron of human immunodeficiency virus type 1 is inefficiently spliced because of suboptimal signals in the 3'
splice site", J Virol, 68, 1994, 3071-9.
Z Yu, N Sanchez-Velar, IE Catrina, EL Kittler, EB Udofia, ML Zapp, "The cellular HIV-1 Rev cofactor hRIP is required for viral replication", Proc
Natl Acad Sci U S A, 102, 2005, 4027-32.
unspliced", EMBO J, 13, 1994, 4105-12.
11.1.2.2.1 Rev molecules assemble onto the RRE RNA sequence through their ARM sequence
Authors
Editors
Matthews, L, 2005-07-26.
Reviewers
Kumar, A, 2007-01-31.
Description
Nuclear export of the unspliced and partially spliced HIV-1 transcripts requires the association of the HIV-1 Rev protein with a cis-acting RNA
sequence known as the Rev Response Element (RRE) located within the env gene. The RRE forms a stem loop structure that associates with
an arginine-rich RNA binding motif (ARM) within Rev.
References
ML Zapp, MR Green, "Sequence-specific RNA binding by the HIV-1 Rev protein", Nature, 342, 1989, 714-6.
Reaction
11.1.2.2.2 Multimerization of Rev
Authors

Editors
Matthews, L, 2005-07-26.
Reviewers
Kumar, A, 2007-01-31.
Description
In order for Rev to function, multiple molecules must bind sequentiallly to the RRE (Malim and Cullen 1991).
References
MH Malim, BR Cullen, "HIV-1 structural gene expression requires the binding of multiple Rev monomers to the viral RRE: implications for HIV-1
latency", Cell, 65, 1991, 241-8.
Reaction
11.1.2.2.3 Rev multimer-bound HIV-1 mRNA associates with Crm1
Authors
Editors
Matthews, L, 2007-01-31.
Reviewers
Kumar, A, 2007-01-31.
Description
CRM1 associates directly with Rev through the Rev nuclear export signal (NES) domain and acts as the nuclear export receptor for the
Rev-RRE ribonucleoprotein complex.
References
P Askjaer, TH Jensen, J Nilsson, L Englmeier, J Kjems, "The specificity of the CRM1-Rev nuclear export signal interaction is mediated by
RanGTP", J Biol Chem, 273, 1998, 33414-22.
Reaction
11.1.2.2.4 Rev multimer-bound HIV-1 mRNA:CRM1 complex associates with Ran:GTP
Authors
Editors
Matthews, L, 2006-07-07.
Reviewers
Kumar, A, 2007-01-31.
Description
RanGTP binds to a preformed Rev-CRM1 complex.
References
Reaction
11.1.2.2.5 Rev multimer-bound HIV-1 mRNA:Crm1:Ran:GTP complex associates with the NPC
Authors
Editors
Matthews, L, 2006-07-13.
Reviewers
Kumar, A, 2007-01-31.
Description
The Rev multimer-bound HIV-1 mRNA:Crm1:Ran:GTP complex associates with the NPC.
Reaction
11.1.2.2.6 Translocation of nuclear RNA transport complex to cytoplasm

Authors
Editors
Matthews, L, 2006-07-13.
Reviewers
Kumar, A, 2007-01-31.
Description
Crm1 is a nucleocytoplasmic transport factor that is believed to interact with nucleoporins facilitating docking of the RRE-Rev-CRM1-RanGTP
complex to the nuclear pore and the translocation of the complex across the nuclear pore complex (see Cullen 1998) Crm1 has been found in
complex with two such nucleoporins, CAN/Nup214 and Nup88 which have been shown to be components of the human nuclear pore complex
(Fornerod et al., 1997).
References
BE Meyer, MH Malim, "The HIV-1 Rev trans-activator shuttles between the nucleus and the cytoplasm", Genes Dev, 8, 1994, 1538-47.
M Fornerod, J van Deursen, S van Baal, A Reynolds, D Davis, KG Murti, J Fransen, G Grosveld, "The human homologue of yeast CRM1 is in a
dynamic subcomplex with CAN/Nup214 and a novel nuclear pore component Nup88", EMBO J, 16, 1997, 807-16.
R Yi, HP Bogerd, BR Cullen, "Recruitment of the Crm1 nuclear export factor is sufficient to induce cytoplasmic expression of incompletely
spliced human immunodeficiency virus mRNAs", J Virol, 76, 2002, 2036-42.
M Fornerod, M Ohno, M Yoshida, IW Mattaj, "CRM1 is an export receptor for leucine-rich nuclear export signals", Cell, 90, 1997, 1051-60.
Reaction
11.1.2.2.7 Association of RanBP1 with Ran-GTP:CRM1:Rev:mRNA complex

Authors
Editors
Matthews, L, 2006-05-23.
Reviewers
Kumar, A, 2007-01-31.
Description
Upon translocation to the cytoplasm, RanBP1 associates with Ran-GTP in the Rev-CRM1-Ran-GTP complex.
Reaction
11.1.2.2.8 Release of the HIV-1 mRNA and Crm1 from Rev in the cytoplasm
Authors
Editors
Matthews, L, 2006-07-13.
Reviewers
Kumar, A, 2007-01-31.
Description
The association of RanBp1 with RanGTP:CRM1:Rev promotes disassembly of the complex and release of the Rev:RNA cargo.
Reaction
11.1.2.2.9 Hydrolysis of Ran:GTP to Ran:GDP
Authors
Editors
Matthews, L, 2006-07-13.
Reviewers
Kumar, A, 2007-01-31.
Description
Ran-GAP, a Ran-specific GTPase-activating protein converts Ran-GTP to Ran-GDP, producing a Ran-GTP gradient across the nuclear
membrane.
References
FR Bischoff, H Krebber, T Kempf, I Hermes, H Ponstingl, "Human RanGTPase-activating protein RanGAP1 is a homologue of yeast Rna1p
involved in mRNA processing and transport", Proc Natl Acad Sci U S A, 92, 1995, 1749-53.
Reaction
11.1.2.3 Assembly of HIV virion
Description
This pathway module includes annotations of events leading to synthesis and organization of GAG, GAGPOL and ENV proteins. This section will
annotated in future.
11.1.2.4 Budding and maturation of HIV-1 virion
Description
This part of the Late Phase will be annotated in a future release of Reactome.
11.2 Host Interactions of HIV factors
Reviewers
Benarous, R, Zhao, RY, Peterlin, BM, 2006-10-30.
Description
Like all viruses, HIV-1 must co-opt the host cell macromolecular transport and processing machinery. HIV-1 Vpr and Rev proteins play key roles
in this co-optation. Efficient HIV-1 replication likewise requires evasion of APOBEC3G-mediated mutagenesis of reverse transcripts, a process
mediated by the viral Vif protein.
11.2.1 Interactions of Vpr with host cellular proteins
Authors
Matthews, L, 2006-05-15.
Editors
Matthews, L, 2006-05-15.
Reviewers
Zhao, RY, 2006-07-11.
Description
Vpr has been implicated in multiple processes during HIV-1 replication, including nuclear import of the pre-integration complex (PIC)(Heinzinger
et al., 1994), apoptosis (Stewart et al., 1997) and induction of cell cycle G2/M arrest (He et al., 1995; Re et al., 1995; Zhao et al., 1996).
Interactions between Vpr and host nucleoporins (importin ÃŽÂ±) appear to facilitate the nuclear import of the PIC (Popov et al., 1998; Vodicka et
al., 1998) while interactions between Vpr the adenine nucleotide transporter (ANT) protein at the inner mitochondrial membrane may contribute
to release of apoptosis factors by promoting permeabilization of the mitochondrial outer membrane (Jacotot et al., 2000).
Vpr induces cell cycle G2/M arrest by promoting hyperphosphorylation of Cdk1/Cdc2 (Re et al., 1995; Zhao et al., 1996). However, it is unclear
which protein(s) Vpr interacts with to cause this effect. For recent reviews, see, (Li et al., 2005; Zhao, Bukrinsky, and Elder, 2005). Progression
of cells from G2 phase of the cell cycle to mitosis is a tightly regulated cellular process that requires activation of the Cdk1/Cdc2 kinase, which
determines onset of mitosis in all eukaryotic cells. The activity of Cdk1/Cdc2 is regulated in part by the phosphorylation status of tyrosine 15
(Tyr15) on Cdk1/Cdc2, which is phosphorylated by Wee1 kinase during late G2 and is rapidly dephosphorylated by the Cdc25 tyrosine
phosphatase to trigger entry into mitosis. These Cdk1/Cdc2 regulators are the downstream targets of two well-characterized G2/M checkpoint
pathways which prevent cells from entering mitosis when cellular DNA is damaged or when DNA replication is inhibited. It is clear that Vpr
induces cell cycle G2/M arrest by promoting Tyr15 phosphorylation of Cdk1/Cdc2 both in human and fission yeast cells (Elder et al., 2000; Re et
al., 1995; Zhao et al., 1996), which modulates host cell cycle machinery to benefit viral survival or replication. Although some aspects of
Vpr-induced G2/M arrest resembles induction of host cellular checkpoints, increasing evidence suggests that Vpr induces cell cycle G2 arrest
through a mechanism that is to some extent different from the classic G2/M checkpoints. One the unique features distinguishing Vpr-induced G2
arrest from the classic checkpoints is the role of phosphatase 2A (PP2A) in Vpr-induced G2 arrest (Elder, Benko, and Zhao, 2002; Elder et al.,
2001; Masuda et al., 2000). Interestingly, PP2A is targeted by a number of other viral proteins including SV40 small T antigen, polyomavirus T
antigen, HTLV Tax and adenovirus E4orf4. Thus an in-depth understanding of the molecular mechanisms underlying Vpr-induced G2 arrest will
provide additional insights into the basic biology of cell cycle G2/M regulation and into the biological significance of this effect during
host-pathogen interactions.
References
S Popov, M Rexach, L Ratner, G Blobel, M Bukrinsky, "Viral protein R regulates docking of the HIV-1 preintegration complex to the nuclear pore
complex", J Biol Chem, 273, 1998, 13347-52.
E Jacotot, L Ravagnan, M Loeffler, KF Ferri, HL Vieira, N Zamzami, P Costantini, S Druillennec, J Hoebeke, JP Briand, T Irinopoulou, E Daugas,
SA Susin, D Cointe, ZH Xie, JC Reed, BP Roques, G Kroemer, "The HIV-1 viral protein R induces apoptosis via a direct effect on the
mitochondrial permeability transition pore", J Exp Med, 191, 2000, 33-46.
M Masuda, Y Nagai, N Oshima, K Tanaka, H Murakami, H Igarashi, H Okayama, "Genetic studies with the fission yeast Schizosaccharomyces
pombe suggest involvement of wee1, ppa2, and rad24 in induction of cell cycle arrest by human immunodeficiency virus type 1 Vpr", J Virol, 74,
2000, 2636-46.
RT Elder, M Yu, M Chen, X Zhu, M Yanagida, Y Zhao, "HIV-1 Vpr induces cell cycle G2 arrest in fission yeast (Schizosaccharomyces pombe)
through a pathway involving regulatory and catalytic subunits of PP2A and acting on both Wee1 and Cdc25", Virology, 287, 2001, 359-70.
RY Zhao, M Bukrinsky, RT Elder, "HIV-1 viral protein R (Vpr) & host cellular responses", Indian J Med Res, 121, 2005, 270-86.
Y Zhao, J Cao, MR O'Gorman, M Yu, R Yogev, "Effect of human immunodeficiency virus type 1 protein R (vpr) gene expression on basic cellular
function of fission yeast Schizosaccharomyces pombe", J Virol, 70, 1996, 5821-6.
F Re, D Braaten, EK Franke, J Luban, "Human immunodeficiency virus type 1 Vpr arrests the cell cycle in G2 by inhibiting the activation of
p34cdc2-cyclin B", J Virol, 69, 1995, 6859-64.
J He, S Choe, R Walker, P Di Marzio, DO Morgan, NR Landau, "Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the
G2 phase of the cell cycle by inhibiting p34cdc2 activity", J Virol, 69, 1995, 6705-11.
MA Vodicka, DM Koepp, PA Silver, M Emerman, "HIV-1 Vpr interacts with the nuclear transport pathway to promote macrophage infection",
Genes Dev, 12, 1998, 175-85.
15, 2005, 923-34.
RT Elder, Z Benko, Y Zhao, "HIV-1 VPR modulates cell cycle G2/M transition through an alternative cellular mechanism other than the classic
mitotic checkpoints", Front Biosci, 7, 2002, d349-57.
RT Elder, M Yu, M Chen, S Edelson, Y Zhao, "Cell cycle G2 arrest induced by HIV-1 Vpr in fission yeast (Schizosaccharomyces pombe) is
independent of cell death and early genes in the DNA damage checkpoint", Virus Res, 68, 2000, 161-73.
SA Stewart, B Poon, JB Jowett, IS Chen, "Human immunodeficiency virus type 1 Vpr induces apoptosis following cell cycle arrest", J Virol, 71,
1997, 5579-92.
NK Heinzinger, MI Bukinsky, SA Haggerty, AM Ragland, V Kewalramani, MA Lee, HE Gendelman, L Ratner, M Stevenson, M Emerman, "The
Vpr protein of human immunodeficiency virus type 1 influences nuclear localization of viral nucleic acids in nondividing host cells", Proc Natl
Acad Sci U S A, 91, 1994, 7311-5.
11.2.1.1 Vpr-mediated induction of apoptosis by mitochondrial outer membrane permeabilization
Authors
Matthews, L, 2006-05-15.
Editors
Matthews, L, 2006-06-02.
Reviewers
Zhao, RY, 2006-07-11.
Description
In one model of Vpr mediated induction of apoptosis, Vpr acts directly on the mitochondrial permeability transition pore complex through its
interaction with adenine nucleotide translocator (ANT). This interaction promotes the permeabiliztion of the mitochondrial membranes resulting in
the release of cytochrome c and apoptosis-inducing factors.
References
11.2.1.1.1 Translocation of Vpr to the mitochondria
Authors
Matthews, L, 2006-05-15.
Editors
Matthews, L, 2006-06-02.
Reviewers
Zhao, RY, 2006-07-11.
Description
Vpr translocates to the mitochondria.
References
Reaction
11.2.1.1.2 Association of Vpr with ANT1
Authors
Matthews, L, 2006-05-15.
Editors
Matthews, L, 2006-06-07.
Reviewers
Zhao, RY, 2006-07-11.
Description
Vpr interacts with the PTPC component ANT1. This interaction induces mitochondrial membrane permeabilization and release of cytochrome c
and apoptotic factors.
References
Reaction
11.2.1.2 Vpr-mediated nuclear import of PICs
Authors
Matthews, L, 2006-05-15.
Editors
Matthews, L, 2006-06-07.
Reviewers
Zhao, RY, 2006-07-11.
Description
Vpr appears to function in anchoring the PIC to the nuclear envelope. This anchoring likely involves interactions between Vpr and host
nucleoporins.
11.2.1.2.1 Vpr binds nucleoporins
Authors
Matthews, L, 2006-05-15.
Editors
Matthews, L, 2006-05-15.
Reviewers
Zhao, RY, 2006-07-11.
References
E Le Rouzic, A Mousnier, C Rustum, F Stutz, E Hallberg, C Dargemont, S Benichou, "Docking of HIV-1 Vpr to the nuclear envelope is mediated
by the interaction with the nucleoporin hCG1", J Biol Chem, 277, 2002, 45091-8.
Reaction
11.2.1.2.2 Interaction of Vpr with importin alpha
Authors
Matthews, L, 2006-05-15.
Editors
Matthews, L, 2006-05-23.
Reviewers
Zhao, RY, 2006-07-11.
Description
Vpr interacts with importin-alpha through alphaH1 and alphaH2. The interaction via alphaH1 is indispensable for the nuclear entry of Vpr
(Kamata et al., 2005) .
References
S Popov, M Rexach, L Ratner, G Blobel, M Bukrinsky, "Viral protein R regulates docking of the HIV-1 preintegration complex to the nuclear pore
complex", J Biol Chem, 273, 1998, 13347-52.
MA Vodicka, DM Koepp, PA Silver, M Emerman, "HIV-1 Vpr interacts with the nuclear transport pathway to promote macrophage infection",
Genes Dev, 12, 1998, 175-85.
M Kamata, Y Nitahara-Kasahara, Y Miyamoto, Y Yoneda, Y Aida, "Importin-alpha promotes passage through the nuclear pore complex of
human immunodeficiency virus type 1 Vpr", J Virol, 79, 2005, 3557-64.
Reaction
11.2.2 Interactions of Rev with host cellular proteins
Authors
Editors
Matthews, L, 2006-03-24.
Reviewers
Kumar, A, 2007-01-31.
Description
In order to facilitate the transport of incompletely spliced HIV-1 transcripts, Rev shuttles between the cytoplasm and nucleus using host cell
transport mechanisms (reviewed in Li et al. 2005). Nuclear import appears to be achieved by the association of Rev with importinÃ¢â‚¬"beta and
B23 and docking at the nuclear pore through interactions between importin-beta and nucleoporins. The dissociation of Rev with the import
machinery and the subsequent export of Rev-associated HIV-1 mRNA complex requires Ran-GTP. Ran GTP associates with importin-beta,
displacing its cargo. Crm1 associates with the Rev:RNA complex and Ran:GTP and is believed to interact with nucleoporins facilitating docking
of the RRE-Rev-CRM1-RanGTP complex to the nuclear pore and the translocation of the complex across the nuclear pore complex. In the
cytoplasm, RanBP1 associates with Ran-GTP causing the Crm1-Rev-Ran-GTP complex to disassemble. The Ran GAP protein promotes the
hydrolysis of RanGTP to Ran GDP. The activities of Ran GAP in the cytoplasm and Ran-GEF, which converts RAN-GDP to Ran-GTP in the
nucleus, produce a gradient of Ran-GTP/GDP required for this shuttling of Rev and other cellular transport proteins.
References
11.2.2.1 Nuclear import of Rev protein
Authors
Matthews, L, 2006-06-08.
Editors
Matthews, L, 2007-01-31.
Reviewers
Kumar, A, 2007-01-31.
Description
Nuclear import of Rev involves the cellular proteins including importin-beta and B23 and is mediated by an arginine-rich nuclear localization
signal (NLS) within the RNA binding domain of the Rev protein. The NLS of Rev associates with importin- beta as well as B23 which has been
shown to function in the nuclear import of ribosomal proteins. The Rev-importin ÃŽÂ²-B23 complex associates with the nuclear pore through
interactions between importin ÃŽÂ² and nucleoporin. Upon entry into the nucleus, Ran-GTP associates with importin ÃŽÂ² resulting in in the
disassembly of the importin ÃŽÂ²-Rev-B23 complex and the release of Rev cargo.
11.2.2.1.1 Association of multimerized Rev with beta-importin
Authors
Matthews, L, 2006-06-08.
Editors
Matthews, L, 2007-01-31.
Reviewers
Kumar, A, 2007-01-31.
Description
The association of Rev with importin-beta is mediated by the Rev nuclear localisation signal.
References
BR Henderson, P Percipalle, "Interactions between HIV Rev and nuclear import and export factors: the Rev nuclear localisation signal mediates
specific binding to human importin-beta", J Mol Biol, 274, 1997, 693-707.
Reaction
11.2.2.1.2 Rev associates with B23
Authors
Matthews, L, 2006-06-08.
Editors
Matthews, L, 2007-01-31.
Reviewers
Kumar, A, 2007-01-31.
Description
B23 may function as a shuttle for the import of HIV Rev from the cytoplasm into the nucleus or nucleolus permitting additional rounds of export of
viral RNAs.
References
A Szebeni, B Mehrotra, A Baumann, SA Adam, PT Wingfield, MO Olson, "Nucleolar protein B23 stimulates nuclear import of the HIV-1 Rev
protein and NLS-conjugated albumin", Biochemistry, 36, 1997, 3941-9.
C Fankhauser, E Izaurralde, Y Adachi, P Wingfield, UK Laemmli, "Specific complex of human immunodeficiency virus type 1 rev and nucleolar
B23 proteins: dissociation by the Rev response element", Mol Cell Biol, 11, 1991, 2567-75.
Reaction
11.2.2.1.3 Rev:importin beta:B23 recruited to the nuclear pore
Authors
Matthews, L, 2006-06-08.
Editors
Matthews, L, 2007-01-31.
Reviewers
Kumar, A, 2007-01-31.
Description
The Rev-importin ÃŽÂ²-B23 complex is recruited to the nuclear pore by an interaction between importin ÃŽÂ² and nucleoporin.
Source reaction
This reaction was inferred from the corresponding reaction "Rev associates with B23" in species Homo sapiens.
C Fankhauser, E Izaurralde, Y Adachi, P Wingfield, UK Laemmli, "Specific complex of human immunodeficiency virus type 1 rev and nucleolar
B23 proteins: dissociation by the Rev response element", Mol Cell Biol, 11, 1991, 2567-75.
This reaction was inferred from the corresponding reaction "Translocation of Rev:importin-beta:B23 to the nucleus" in species Homo sapiens.
Reaction
11.2.2.1.4 Translocation of Rev:importin-beta:B23 to the nucleus

Authors
Matthews, L, 2006-06-08.
Editors
Matthews, L, 2007-01-31.
Reviewers
Kumar, A, 2007-01-31.
Description
Following the association of Rev with importin-beta, the Rev:B23:importin-beta complex is imported into the nucleus.
References
Source reaction
This reaction was inferred from the corresponding reaction "Disassembly of the Rev-importin beta-B23:Ran-GTP complex" in species Homo
sapiens.
Reaction
11.2.2.1.5 Association of Ran-GTP with importin-beta

Authors
Matthews, L, 2006-06-08.
Editors
Matthews, L, 2007-01-31.
Reviewers
Kumar, A, 2007-01-31.
Description
Inside the nucleus, Ran-GTP associates with importin-beta.
References
Reaction
11.2.2.1.6 Disassembly of the Rev-importin beta-B23:Ran-GTP complex
Authors
Matthews, L, 2006-06-08.
Editors
Matthews, L, 2007-01-31.
Reviewers
Kumar, A, 2007-01-31.
Description
The association of importin-beta with Ran-GTP causes the disassembly of the Rev-importin ÃŽÂ²-B23 complex releasing the Rev in the nucleus.
References
Reaction
11.2.2.2 Rev-mediated nuclear export of HIV-1 RNA
Authors
Editors
Matthews, L, 2006-06-07.
Reviewers
Kumar, A, 2007-01-31.
Description
The HIV-1 genome contains 9 genes encoded by a single transcript. In order for the virus to replicate, unspliced, singly-spliced and fully spliced
viral mRNA must be exported from the nucleus. The HIV-1 mRNA splice sites are inefficient resulting it the accumulation of a pool of
incompletely spliced RNAs (Staffa and Cochrane, 1994). In the early stages of the viral life cycle, or in the absence of the viral Rev protein,
completely spliced viral mRNA which encode the regulatory proteins Tat, Nef and Rev are exported from the nucleus while the incompletely
spliced structural protein encoding transcripts are held within the nucleus by cellular proteins that normally function in preventing the nuclear
export of cellular pre-mRNA. Export of both unspliced and partially spliced mRNA is mediated by the viral protein Rev which is recruited, along
with cellular cofactors, to the Rev Response Element (RRE) within the HIV-1 mRNA sequence (Malim et al., 1990; Fischer et al., 1994). The
cellular hRIP protein is essential for correct Rev-mediated export of viral RNAs to the cytoplasm (Sanchez-Velar et al., 2004; Yu et al., 2005).
References
CC Fritz, MR Green, "HIV Rev uses a conserved cellular protein export pathway for the nucleocytoplasmic transport of viral RNAs", Curr Biol, 6,
1996, 848-54.
1989, 254-7.
BR Cullen, "Nuclear mRNA export: insights from virology", Trends Biochem Sci, 28, 2003, 419-24.
N Sanchez-Velar, EB Udofia, Z Yu, ML Zapp, "hRIP, a cellular cofactor for Rev function, promotes release of HIV RNAs from the perinuclear
region", Genes Dev, 18, 2004, 23-34.
A Staffa, A Cochrane, "The tat/rev intron of human immunodeficiency virus type 1 is inefficiently spliced because of suboptimal signals in the 3'
splice site", J Virol, 68, 1994, 3071-9.
Z Yu, N Sanchez-Velar, IE Catrina, EL Kittler, EB Udofia, ML Zapp, "The cellular HIV-1 Rev cofactor hRIP is required for viral replication", Proc
Natl Acad Sci U S A, 102, 2005, 4027-32.
unspliced", EMBO J, 13, 1994, 4105-12.
11.2.2.2.1 Rev molecules assemble onto the RRE RNA sequence through their ARM sequence
Authors
Editors
Matthews, L, 2005-07-26.
Reviewers
Kumar, A, 2007-01-31.
Description
Nuclear export of the unspliced and partially spliced HIV-1 transcripts requires the association of the HIV-1 Rev protein with a cis-acting RNA
sequence known as the Rev Response Element (RRE) located within the env gene. The RRE forms a stem loop structure that associates with
an arginine-rich RNA binding motif (ARM) within Rev.
References
ML Zapp, MR Green, "Sequence-specific RNA binding by the HIV-1 Rev protein", Nature, 342, 1989, 714-6.
Reaction
11.2.2.2.2 Multimerization of Rev
Authors
Editors
Matthews, L, 2005-07-26.
Reviewers
Kumar, A, 2007-01-31.
Description
In order for Rev to function, multiple molecules must bind sequentiallly to the RRE (Malim and Cullen 1991).
References
MH Malim, BR Cullen, "HIV-1 structural gene expression requires the binding of multiple Rev monomers to the viral RRE: implications for HIV-1
latency", Cell, 65, 1991, 241-8.
Reaction
11.2.2.2.3 Rev multimer-bound HIV-1 mRNA associates with Crm1
Authors
Editors
Matthews, L, 2007-01-31.
Reviewers
Kumar, A, 2007-01-31.
Description
CRM1 associates directly with Rev through the Rev nuclear export signal (NES) domain and acts as the nuclear export receptor for the
Rev-RRE ribonucleoprotein complex.
References
Reaction
11.2.2.2.4 Rev multimer-bound HIV-1 mRNA:CRM1 complex associates with Ran:GTP
Authors
Editors
Matthews, L, 2006-07-07.
Reviewers
Kumar, A, 2007-01-31.
Description
RanGTP binds to a preformed Rev-CRM1 complex.
References
Reaction
11.2.2.2.5 Rev multimer-bound HIV-1 mRNA:Crm1:Ran:GTP complex associates with the NPC
Authors

Editors
Matthews, L, 2006-07-13.
Reviewers
Kumar, A, 2007-01-31.
Description
The Rev multimer-bound HIV-1 mRNA:Crm1:Ran:GTP complex associates with the NPC.
Reaction
11.2.2.2.6 Translocation of nuclear RNA transport complex to cytoplasm
Authors
Editors
Matthews, L, 2006-07-13.
Reviewers
Kumar, A, 2007-01-31.
Description
Crm1 is a nucleocytoplasmic transport factor that is believed to interact with nucleoporins facilitating docking of the RRE-Rev-CRM1-RanGTP
complex to the nuclear pore and the translocation of the complex across the nuclear pore complex (see Cullen 1998) Crm1 has been found in
complex with two such nucleoporins, CAN/Nup214 and Nup88 which have been shown to be components of the human nuclear pore complex
(Fornerod et al., 1997).
References
BE Meyer, MH Malim, "The HIV-1 Rev trans-activator shuttles between the nucleus and the cytoplasm", Genes Dev, 8, 1994, 1538-47.
M Fornerod, J van Deursen, S van Baal, A Reynolds, D Davis, KG Murti, J Fransen, G Grosveld, "The human homologue of yeast CRM1 is in a
dynamic subcomplex with CAN/Nup214 and a novel nuclear pore component Nup88", EMBO J, 16, 1997, 807-16.
R Yi, HP Bogerd, BR Cullen, "Recruitment of the Crm1 nuclear export factor is sufficient to induce cytoplasmic expression of incompletely
spliced human immunodeficiency virus mRNAs", J Virol, 76, 2002, 2036-42.
M Fornerod, M Ohno, M Yoshida, IW Mattaj, "CRM1 is an export receptor for leucine-rich nuclear export signals", Cell, 90, 1997, 1051-60.
Reaction
11.2.2.2.7 Association of RanBP1 with Ran-GTP:CRM1:Rev:mRNA complex
Authors
Editors
Matthews, L, 2006-05-23.
Reviewers
Kumar, A, 2007-01-31.
Description
Upon translocation to the cytoplasm, RanBP1 associates with Ran-GTP in the Rev-CRM1-Ran-GTP complex.
Reaction
11.2.2.2.8 Release of the HIV-1 mRNA and Crm1 from Rev in the cytoplasm
Authors
Editors
Matthews, L, 2006-07-13.
Reviewers
Kumar, A, 2007-01-31.
Description
The association of RanBp1 with RanGTP:CRM1:Rev promotes disassembly of the complex and release of the Rev:RNA cargo.
Reaction
11.2.2.2.9 Hydrolysis of Ran:GTP to Ran:GDP
Authors
Editors
Matthews, L, 2006-07-13.
Reviewers
Kumar, A, 2007-01-31.
Description
Ran-GAP, a Ran-specific GTPase-activating protein converts Ran-GTP to Ran-GDP, producing a Ran-GTP gradient across the nuclear
membrane.
References
FR Bischoff, H Krebber, T Kempf, I Hermes, H Ponstingl, "Human RanGTPase-activating protein RanGAP1 is a homologue of yeast Rna1p
involved in mRNA processing and transport", Proc Natl Acad Sci U S A, 92, 1995, 1749-53.
Reaction
11.2.3 APOBEC3G mediated resistance to HIV-1 infection
Authors
Matthews, L, 2006-06-07.
Editors
Matthews, L, 2007-01-30.
Reviewers
Mulder, L, 2007-01-30, Simon, V, 2007-01-30.
Description
Representatives of the apolipoprotein B mRNA editing enzyme catalytic polypeptide 3 (APOBEC3) family provide innate resistance to
exogeneous and endogenous retroviruses (see Cullen 2006 for a recent review). Humans and other primates encode a cluster of seven different
cytidine deaminases with APOBEC3G, APOBEC3F and APOBEC3B having some anti HIV-1 activity. Our understanding is most complete for
APOBEC3G which has been described first and the reactions described herein will focus on this representative enzyme.
APOBEC3G is a cytoplasmic protein which strongly restricts replication of Vif deficient HIV-1 (Sheehy 2002). It is expressed in cell populations
that are susceptible to HIV infection (e.g., T-lymphocytes and macrophages). In the producer cell, APOBEC3G is incorporated into budding
HIV-1 particles through an interaction with HIV-1 gag nucleocapsid (NC) protein in a RNA-dependent fashion.
Within the newly infected cell (= target cell), virus-associated APOBEC3G regulates the infectivity of HIV-1 by deaminating cytidine to uracil in
the minus-strand viral DNA intermediate during reverse transcription. Deamination results in the induction of G-to-A hypermutations in the
plus-strand viral DNA which subsequently can either be integrated as a non-functional provirus or degraded before integration.
References
HL Wiegand, BP Doehle, HP Bogerd, BR Cullen, "A second human antiretroviral factor, APOBEC3F, is suppressed by the HIV-1 and HIV-2 Vif
proteins", EMBO J, 23, 2004, 2451-8.
BR Cullen, "Role and mechanism of action of the APOBEC3 family of antiretroviral resistance factors", J Virol, 80, 2006, 1067-76.
Q Yu, R Konig, S Pillai, K Chiles, M Kearney, S Palmer, D Richman, JM Coffin, NR Landau, "Single-strand specificity of APOBEC3G accounts
for minus-strand deamination of the HIV genome", Nat Struct Mol Biol, 11, 2004, 435-42.
AM Sheehy, NC Gaddis, JD Choi, MH Malim, "Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein",
Nature, 418, 2002, 646-50.
11.2.3.1 Association of APOBEC3G with Gag

Authors
Matthews, L, 2006-06-07.
Editors
Matthews, L, 2007-01-30.
Reviewers
Mulder, L, 2007-01-30, Simon, V, 2007-01-30.
Description
APOBEC3G is incorporated into virus particles through its association with components of the viral RNA packaging machinery. It binds to the
nucleocapsid portion of Gag (NC), a region of the polyprotein that associates with genomic RNA and functions in RNA encapsidation.
References
V Zennou, D Perez-Caballero, H Gottlinger, PD Bieniasz, "APOBEC3G incorporation into human immunodeficiency virus type 1 particles", J
Virol, 78, 2004, 12058-61.
A Schafer, HP Bogerd, BR Cullen, "Specific packaging of APOBEC3G into HIV-1 virions is mediated by the nucleocapsid domain of the gag
polyprotein precursor", Virology, 328, 2004, 163-8.
Reaction
11.2.3.2 Association of APOBEC3G with single-stranded region of forming HIV-1 minus strand
Authors
Matthews, L, 2006-06-07.
Editors
Matthews, L, 2007-01-30.
Reviewers
Mulder, L, 2007-01-30, Simon, V, 2007-01-30.
Description
In the target cell, HIV-1-associated APOBEC3G binds to the HIV-1 reverse transcript minus strand and catalyzes the deamination of cytidines in
a specific dinucleotide context (e.g., dCC). In contrast, APOBEC3F and APOBEC3B display a preference for dTC.
References
Reaction
11.2.3.3 Deamination of C residues during synthesis of HIV-1 reverse transcript minus-strand
Authors
Matthews, L, 2006-06-07.
Editors
Matthews, L, 2007-01-30.
Reviewers
Mulder, L, 2007-01-30, Simon, V, 2007-01-30.
Description
During reverse transcription, APOBEC3G-mediated minus-strand deamination occurs within a CC dinucleotide context over the entire length of
the HIV-1 genome (Yu et al., 2004).
The polypurine tract is essential for plus strand synthesis and is located at the 3Ã¢â‚¬â„¢ end of the retroviral genome. HIV-1 encodes an
additional central polypurine tract located in the middle of the genome which also serves as primer for plus strand synthesis.
Deamination of the minus strand continues throughout its synthesis with the frequency of deamination events increasing from the 5Ã¢â‚¬â„¢ to
3Ã¢â‚¬â„¢ regions. A 400bp region downstream of the central polypurine tract seems to be protected from deamination (Wurtzer et al., 2006)
References
H Zhang, B Yang, RJ Pomerantz, C Zhang, SC Arunachalam, L Gao, "The cytidine deaminase CEM15 induces hypermutation in newly
synthesized HIV-1 DNA", Nature, 424, 2003, 94-8.
S Wurtzer, A Goubard, F Mammano, S Saragosti, D Lecossier, AJ Hance, F Clavel, "Functional central polypurine tract provides downstream
protection of the human immunodeficiency virus type 1 genome from editing by APOBEC3G and APOBEC3B", J Virol, 80, 2006, 3679-83.
Reaction
11.2.4 Vif-mediated degradation of APOBEC3G
Authors
Matthews, L, 2006-05-15.
Editors
Matthews, L, 2007-01-30.
Reviewers
Mulder, L, 2007-01-30, Simon, V, 2007-01-30.
Description
The HIV-1 accessory protein Vif (Viral infectivity factor) is required for the efficient infection of primary cell populations (e.g., lymphocytes and
macrophages) and Ã¢â‚¬Å"non-permissiveÃ¢â‚¬Â• cell lines. Vif neutralises the host DNA editing enzyme, APOBEC3G, in the producer cell.
Indeed, in the absence of a functional Vif, APOBEC3G is selectively incorporated into the budding virions and in the next cycle of infection leads
to the deamination of deoxycytidines (dC) within the minus-strand cDNA during reverse transcription (Sheehy et al 2003; Li et al., 2005 ; Stopak
et al. 2003).
Deamination changes cytidine to uracil and thus results in G to A transitions and stop codons in the provirus. The aberrant cDNAs produced in
the infected cell can either be integrated in form of non-functional proviruses or degraded. Vif counteracts the antiviral activity of APOBEC3G by
associating directly with it and promoting its polyubiquitination and degradation by the 26S proteasome.
Vif binds APOBEC3G and recruits it into an E3 ubiquitin-enzyme complex composed by the cytoplasmic proteins Cullin5, Rbx, ElonginC and
ElonginB (Yu et al., 2003) . Thus, in the presence of Vif, APOBEC3G incorporation into the virion is minimal.
References
M Kobayashi, A Takaori-Kondo, Y Miyauchi, K Iwai, T Uchiyama, "Ubiquitination of APOBEC3G by an HIV-1 Vif-Cullin5-Elongin B-Elongin C
complex is essential for Vif function", J Biol Chem, 280, 2005, 18573-8.
AM Sheehy, NC Gaddis, MH Malim, "The antiretroviral enzyme APOBEC3G is degraded by the proteasome in response to HIV-1 Vif", Nat Med,
9, 2003, 1404-7.
X Yu, Y Yu, B Liu, K Luo, W Kong, P Mao, XF Yu, "Induction of APOBEC3G ubiquitination and degradation by an HIV-1 Vif-Cul5-SCF complex",
Science, 302, 2003, 1056-60.
15, 2005, 923-34.
K Stopak, C de Noronha, W Yonemoto, WC Greene, "HIV-1 Vif blocks the antiviral activity of APOBEC3G by impairing both its translation and
intracellular stability", Mol Cell, 12, 2003, 591-601.
AM Sheehy, NC Gaddis, JD Choi, MH Malim, "Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein",
Nature, 418, 2002, 646-50.
11.2.4.1 Association of Vif with APOBEC3G
Authors
Matthews, L, 2006-05-15.
Editors
Matthews, L, 2007-01-30.
Reviewers
Mulder, L, 2007-01-30, Simon, V, 2007-01-30.
Description
The HIV-1 Vif protein associates with the DNA editing enzyme APOBEC3G Marin et al) . The binding site has not yet been mapped but
emerging evidence suggest that the N-terminal lregion of Vif is essential for APOBEC3G recognition (Tian et al) .
Substitution of a single amino acid in the human APOBEC3G (Asp128Lys) abolishes binding and renders it resistant to HIV-1 Vif (Schrofelbauer
et al; Bogerd et al.).
References
HP Bogerd, BP Doehle, HL Wiegand, BR Cullen, "A single amino acid difference in the host APOBEC3G protein controls the primate species
specificity of HIV type 1 virion infectivity factor", Proc Natl Acad Sci U S A, 101, 2004, 3770-4.
B Schrofelbauer, D Chen, NR Landau, "A single amino acid of APOBEC3G controls its species-specific interaction with virion infectivity factor
(Vif)", Proc Natl Acad Sci U S A, 101, 2004, 3927-32.
M Marin, KM Rose, SL Kozak, D Kabat, "HIV-1 Vif protein binds the editing enzyme APOBEC3G and induces its degradation", Nat Med, 9,
2003, 1398-403.
C Tian, X Yu, W Zhang, T Wang, R Xu, XF Yu, "Differential requirement for conserved tryptophans in human immunodeficiency virus type 1 Vif
for the selective suppression of APOBEC3G and APOBEC3F", J Virol, 80, 2006, 3112-5.
Reaction
11.2.4.2 Association of APOBEC3G:Vif with the Cul5-SCF complex
Authors
Matthews, L, 2006-05-15.
Editors
Matthews, L, 2007-01-30.
Reviewers
Mulder, L, 2007-01-30, Simon, V, 2007-01-30.
Description
The interaction between Vif and the E3 ubiquitin ligase complex (Cullin5, Elongin B and Elongin C, and Rbx1) takes place through direct binding
of the SOCS box motif in the viral protein Vif to the host protein Elongin C. Moreover, a conserved HCCH motif in Vif allows binding to Cullin 5.
References
Science, 302, 2003, 1056-60.
Z Xiao, E Ehrlich, Y Yu, K Luo, T Wang, C Tian, XF Yu, "Assembly of HIV-1 Vif-Cul5 E3 ubiquitin ligase through a novel zinc-binding
domain-stabilized hydrophobic interface in Vif", Virology, 349, 2006, 290-9.
A Mehle, J Goncalves, M Santa-Marta, M McPike, D Gabuzda, "Phosphorylation of a novel SOCS-box regulates assembly of the HIV-1 Vif-Cul5
complex that promotes APOBEC3G degradation", Genes Dev, 18, 2004, 2861-6.
Reaction
11.2.4.3 Multi-ubiquitination of APOBEC3G
Authors
Matthews, L, 2006-05-15.
Editors
Matthews, L, 2007-01-30.
Reviewers
Mulder, L, 2007-01-30, Simon, V, 2007-01-30.
Description
APOBEC3G is multi-ubiquitinated by the Vif-Cul5-SCF complex.
References
Reaction
11.2.4.4 Proteosome-mediated degradation of APOBEC3G
Authors
Matthews, L, 2006-05-15.
Editors
Matthews, L, 2007-01-30.
Reviewers
Mulder, L, 2007-01-30, Simon, V, 2007-01-30.
Description
Following multi-ubiquitination by the Vif-Cul5-SCF complex, APOBEC3G is degraded by the 26S proteasome.
References
AM Sheehy, NC Gaddis, MH Malim, "The antiretroviral enzyme APOBEC3G is degraded by the proteasome in response to HIV-1 Vif", Nat Med,
9, 2003, 1404-7.
Science, 302, 2003, 1056-60.
Reaction
11.2.5 Interactions of Tat with host cellular proteins
Authors
Editors
Matthews, L, 2006-03-24.
Description
The elongation of HIV-1 mRNA depends upon the interaction of Tat with the host P-TEFb complex (Hermann and Rice, 1995; Wei et al., 1998).
References
11.2.5.1 Association of Tat with P-TEFb(Cyclin T1:Cdk9)
Description
Tat associates with the Cyclin T1 subunit of P-TEFb (Cyclin T1:Cdk9) through a region of cysteine-rich and core sequences referred to as the
ARM domain within Tat (Wei et al., 1998; see also Herrmann 1995). This interaction is believed to involve metal ions stabilized by cysteine
residues in both proteins (Bieniasz et al., 1998; Garber et al., 1998).
References
PD Bieniasz, TA Grdina, HP Bogerd, BR Cullen, "Recruitment of a protein complex containing Tat and cyclin T1 to TAR governs the species
specificity of HIV-1 Tat", EMBO J, 17, 1998, 7056-65.
ME Garber, P Wei, VN KewalRamani, TP Mayall, CH Herrmann, DR Littman, KA Jones, "The interaction between HIV-1 Tat and human cyclin
T1 requires zinc and a critical cysteine residue that is not conserved in the murine CycT1 protein", Genes Dev, 12, 1998, 3512-27.
Reaction
11.2.6 The role of Nef in HIV-1 replication and disease pathogenesis
Authors
Reviewers
Skowronski, J, 2007-08-07.
Description
The HIV-1 Nef protein is a 27-kDa myristoylated protein that is abundantly produced during the early phase of viral replication cycle. It is highly
conserved in all primate lentiviruses, suggesting that its function is essential for survival of these pathogens. The protein name "Nef" was derived
from early reports of its negative effect on viral replication, thus 'negative factor' or Nef. Subsequently it has been demonstrated that Nef plays an
important role in several steps of HIV replication. In addition, it appears to be a critical pathogenic factor, as Nef-deficient SIV and HIV are
significantly less pathogenic than the wild-type viruses, whereas Nef-transgenic mice show many features characteristic to HIV disease.
The role of Nef in HIV-1 replication and disease pathogenesis is determined by at least four independent activities of this protein. Nef affects the
cell surface expression of several cellular proteins, interferes with cellular signal transduction pathways, enhances virion infectivity and viral
replication, and regulates cholesterol trafficking in HIV-infected cells.
References
C Cheng-Mayer, P Iannello, K Shaw, PA Luciw, JA Levy, "Differential effects of nef on HIV replication: implications for viral pathogenesis in the
host", Science, 246, 1989, 1629-32.
MD Daniel, F Kirchhoff, SC Czajak, PK Sehgal, RC Desrosiers, "Protective effects of a live attenuated SIV vaccine with a deletion in the nef
gene", Science, 258, 1992, 1938-41.
Z Hanna, DG Kay, N Rebai, A Guimond, S Jothy, P Jolicoeur, "Nef harbors a major determinant of pathogenicity for an AIDS-like disease
induced by HIV-1 in transgenic mice", Cell, 95, 1998, 163-75.
R Hofmann-Lehmann, J Vlasak, AL Williams, AL Chenine, HM McClure, DC Anderson, S O'Neil, RM Ruprecht, "Live attenuated, nef-deleted SIV
is pathogenic in most adult macaques after prolonged observation", AIDS, 17, 2003, 157-66.
15, 2005, 923-34.
Z Hanna, DG Kay, M Cool, S Jothy, N Rebai, P Jolicoeur, "Transgenic mice expressing human immunodeficiency virus type 1 in immune cells
develop a severe AIDS-like disease", J Virol, 72, 1998, 121-32.
N Ahmad, S Venkatesan, "Nef protein of HIV-1 is a transcriptional repressor of HIV-1 LTR", Science, 241, 1988, 1481-5.
WB Dyer, AF Geczy, SJ Kent, LB McIntyre, SA Blasdall, JC Learmont, JS Sullivan, "Lymphoproliferative immune function in the Sydney Blood
Bank Cohort, infected with natural nef/long terminal repeat mutants, and in other long-term survivors of transfusion-acquired HIV-1 infection",
AIDS, 11, 1997, 1565-74.
11.2.6.1 Nef and signal transduction
Authors
Reviewers
Description
Nef interferes with cellular signal transduction pathways in a number of ways. Nef is associated with lipid rafts through its amino-terminal
myristoylation and a proline-rich SH3-binding domain. These cholesterol-rich membrane microdomains appear to concentrate potent signaling
mediators. Nef was found to complex with and activate serine/threonine protein kinase PAK-2, which may contribute to activation of infected
cells. In vitro, HIV-infected T cells produce enhanced levels of interleukin-2 during activation. When expressed in macrophages, Nef intersects
the CD40L signaling pathway inducing secretion of chemokines and other factors that attract resting T cells and promote their infection by HIV.
References
S Swingler, B Brichacek, JM Jacque, C Ulich, J Zhou, M Stevenson, "HIV-1 Nef intersects the macrophage CD40L signalling pathway to
promote resting-cell infection", Nature, 424, 2003, 213-9.
JK Wang, E Kiyokawa, E Verdin, D Trono, "The Nef protein of HIV-1 associates with rafts and primes T cells for activation", Proc Natl Acad Sci
U S A, 97, 2000, 394-9.
H Schmidtmayerova, HS Nottet, G Nuovo, T Raabe, CR Flanagan, L Dubrovsky, HE Gendelman, A Cerami, M Bukrinsky, B Sherry, "Human
immunodeficiency virus type 1 infection alters chemokine beta peptide expression in human monocytes: implications for recruitment of
leukocytes into brain and lymph nodes", Proc Natl Acad Sci U S A, 93, 1996, 700-4.
11.2.6.1.1 Nef mediated activation of the T-cell receptor
Authors
Reviewers
Description
Nef drives the formation of lipid raft complexes.

References
K Agopian, BL Wei, JV Garcia, D Gabuzda, "CD4 and MHC-I downregulation are conserved in primary HIV-1 Nef alleles from brain and
lymphoid tissues, but Pak2 activation is highly variable", Virology, 358, 2007, 119-35.
VK Arora, RP Molina, JL Foster, JL Blakemore, J Chernoff, BL Fredericksen, JV Garcia, "Lentivirus Nef specifically activates Pak2", J Virol, 74,
2000, 11081-7.
GH Renkema, A Manninen, K Saksela, "Human immunodeficiency virus type 1 Nef selectively associates with a catalytically active
subpopulation of p21-activated kinase 2 (PAK2) independently of PAK2 binding to Nck or beta-PIX", J Virol, 75, 2001, 2154-60.
JK Wang, E Kiyokawa, E Verdin, D Trono, "The Nef protein of HIV-1 associates with rafts and primes T cells for activation", Proc Natl Acad Sci
U S A, 97, 2000, 394-9.
BL Wei, VK Arora, A Raney, LS Kuo, GH Xiao, E O'Neill, JR Testa, JL Foster, JV Garcia, "Activation of p21-activated kinase 2 by human
immunodeficiency virus type 1 Nef induces merlin phosphorylation", J Virol, 79, 2005, 14976-80.
GH Renkema, A Manninen, DA Mann, M Harris, K Saksela, "Identification of the Nef-associated kinase as p21-activated kinase 2", Curr Biol, 9,
1999, 1407-10.
A Raney, LS Kuo, LL Baugh, JL Foster, JV Garcia, "Reconstitution and molecular analysis of an active human immunodeficiency virus type 1
Nef/p21-activated kinase 2 complex", J Virol, 79, 2005, 12732-41.
Reaction
11.2.6.1.2 Nef Binds and activates the Src-family tyrosine kinase Hck
Authors
Reviewers
Description
The protein Hck is a member of the Src family of non-receptor tyrosine kinases which is preferentially expressed in haematopoietic cells of the
myeloid and B-lymphoid lineages. Src kinases are inhibited by tyrosine-phosphorylation at a carboxy-terminal site. The SH2 domains of these
enzymes play an essential role in this regulation by binding to the tyrosine-phosphorylated tail. The SH2 domain of Hck regulates enzymatic
activity indirectly; intramolecular interactions between the SH3 and catalytic domains appear to stabilize an inactive form of the kinase. The
HIV-1 Nef protein, which is a high-affinity ligand for the Hck SH3 domain, binds to either the downregulated or activated form of Hck causing a
large increase in Hck catalytic activity. The intact SH3-binding motif in Nef is crucial for Hck activation.
References
K Saksela, G Cheng, D Baltimore, "Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for
the enhanced growth of Nef+ viruses but not for down-regulation of CD4", EMBO J, 14, 1995, 484-91.
I Moarefi, M LaFevre-Bernt, F Sicheri, M Huse, CH Lee, J Kuriyan, WT Miller, "Activation of the Src-family tyrosine kinase Hck by SH3 domain
displacement", Nature, 385, 1997, 650-3.
S Grzesiek, A Bax, GM Clore, AM Gronenborn, JS Hu, J Kaufman, I Palmer, SJ Stahl, PT Wingfield, "The solution structure of HIV-1 Nef reveals
an unexpected fold and permits delineation of the binding surface for the SH3 domain of Hck tyrosine protein kinase", Nat Struct Biol, 3, 1996,
340-5.
Reaction
11.2.6.1.3 Nef Binds and activates the Src-family tyrosine kinase Fyn
Authors
Reviewers
Description
Nef has been shown to bind specifically to a subset of the Src family of kinases. Nef/Fyn interaction centers on a proline-rich motif (Pro-x-x-Pro),
which is implicated in SH3 binding. This domain is partially disordered in the absence of the binding partner; when bound this motif fully adopts a
left-handed polyproline type II helix conformation upon complex formation with the Fyn SH3 domain. Within this structure the arginine residue
(Arg77) of Nef interacts with Asp 100 of the RT loop within the Fyn SH3 domain, and triggers a hydrogen-bond rearrangement which allows the
loop to adapt to complement the Nef surface. The Arg96 residue of the Fyn SH3 domain is specifically accommodated in the same hydrophobic
pocket of Nef. The Nef-Fyn complex forms in vivo and may have a crucial role in the T cell perturbating action of Nef by altering T cell receptor
signaling.
References
S Arold, P Franken, MP Strub, F Hoh, S Benichou, R Benarous, C Dumas, "The crystal structure of HIV-1 Nef protein bound to the Fyn kinase
SH3 domain suggests a role for this complex in altered T cell receptor signaling", Structure, 5, 1997, 1361-72.
Reaction
11.2.6.1.4 Nef Binds and activates the Src-family tyrosine kinase Lck
Authors
Reviewers
Description
The Nef protein of the primate lentiviruses, including human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), is
a myristylated protein associated with increased viral replication and enhanced pathogenicity. Both the potentiation of T-lymphocyte activation
and the enhanced serine-phosphorylation of HIV-1 capsid by Nef correlate with increased viral replication. The Nef proteins from HIV-1 and SIV
bind to Lck. The SH3 and SH2 domains of Lck are sufficient for coprecipitation with non-tyrosine-phosphorylated Nef proteins. The conserved
core region of HIV-1 Nef is essential for the interaction with Lck and is also important for enhanced HIV-1 replication in T-lymphocytes. The SIV
and HIV-1 Nef proteins are differentially tyrosine-phosphorylated. The kinase-active Lck tyrosine-phosphorylates SIVmac239 Nef but does not
phosphorylate HIV-1 Nef.
References
H Cheng, JP Hoxie, WP Parks, "The conserved core of human immunodeficiency virus type 1 Nef is essential for association with Lck and for
enhanced viral replication in T-lymphocytes", Virology, 264, 1999, 5-15.
Reaction
11.2.6.1.5 Nef binds a ternary complex comprising DOCK2 guanine nucleotide exchange factor for small Rho-family GTPase Rac, its
cofactor ELMO1, and Rac, and activates Rac through this interaction
Reviewers
Description
The infectious cycle of primate lentiviruses is intimately linked to interactions between cells of the immune system. Nef, a potent virulence factor,
alters cellular environments to increase lentiviral replication in the host, functioning as an adaptor protein. Nef activates Rac in T cell lines and in
primary T cells following infection with HIV-1 in the absence of antigenic stimuli. Nef activates Rac by binding the DOCK2-ELMO1 complex, and
this interaction is linked to the abilities of Nef to inhibit chemotaxis and promote T cell activation. Nef targets a critical switch that regulates Rac
GTPases downstream of chemokine- and antigen-initiated signaling pathways. This interaction enables Nef to influence multiple aspects of T
cell function and thus provides an important mechanism by which Nef impacts pathogenesis by primate lentiviruses.
References
A Janardhan, T Swigut, B Hill, MP Myers, J Skowronski, "HIV-1 Nef binds the DOCK2-ELMO1 complex to activate rac and inhibit lymphocyte
chemotaxis", PLoS Biol, 2, 2004, E6.
Reaction
11.2.6.2 Nef-mediates down modulation of cell surface receptors by recruiting them to clathrin adapters
Authors
Reviewers
Description
The maximal virulence of HIV-1 requires Nef, a virally encoded peripheral membrane protein. Nef binds to the adaptor protein (AP) complexes of
coated vesicles, inducing an expansion of the endosomal compartment and altering the surface expression of cellular proteins including CD4
and class I major histocompatibility complex.
Nef affects the cell surface expression of several cellular proteins. It down-regulates CD4, CD8, CD28, and major histocompatibility complex
class I and class II proteins, but upregulates the invariant chain of MHC II (CD74). To modulate cell surface receptor expression, Nef utilizes
several strategies, linked to distinct regions within the Nef protein.
Since all these receptors are essential for proper functions of the immune system, modulation of their surface expression by Nef has profound
effects on anti-HIV immune responses. Down-regulation of MHC I protects HIV-infected cells from host CTL response, whereas
down-modulation of CD28 and CD4 probably limits the adhesion of a Nef-expressing T cell to the antigen-presenting cell, thus promoting the
movement of HIV-infected cells into circulation and the spread of the virus.
References
JV Garcia, AD Miller, "Serine phosphorylation-independent downregulation of cell-surface CD4 by nef", Nature, 350, 1991, 508-11.
T Swigut, N Shohdy, J Skowronski, "Mechanism for down-regulation of CD28 by Nef", EMBO J, 20, 2001, 1593-604.
K Janvier, H Craig, D Hitchin, R Madrid, N Sol-Foulon, L Renault, J Cherfils, D Cassel, S Benichou, J Guatelli, "HIV-1 Nef stabilizes the
association of adaptor protein complexes with membranes", J Biol Chem, 278, 2003, 8725-32.
ME Greenberg, S Bronson, M Lock, M Neumann, GN Pavlakis, J Skowronski, "Co-localization of HIV-1 Nef with the AP-2 adaptor protein
complex correlates with Nef-induced CD4 down-regulation", EMBO J, 16, 1997, 6964-76.
M Greenberg, L DeTulleo, I Rapoport, J Skowronski, T Kirchhausen, "A dileucine motif in HIV-1 Nef is essential for sorting into clathrin-coated
pits and for downregulation of CD4", Curr Biol, 8, 1998, 1239-42.
HM Craig, MW Pandori, JC Guatelli, "Interaction of HIV-1 Nef with the cellular dileucine-based sorting pathway is required for CD4
down-regulation and optimal viral infectivity", Proc Natl Acad Sci U S A, 95, 1998, 11229-34.
V Stove, I Van de Walle, E Naessens, E Coene, C Stove, J Plum, B Verhasselt, "Human immunodeficiency virus Nef induces rapid
internalization of the T-cell coreceptor CD8alphabeta", J Virol, 79, 2005, 11422-33.
V Piguet, YL Chen, A Mangasarian, M Foti, JL Carpentier, D Trono, "Mechanism of Nef-induced CD4 endocytosis: Nef connects CD4 with the
mu chain of adaptor complexes", EMBO J, 17, 1998, 2472-81.
L Erdtmann, K Janvier, G Raposo, HM Craig, P Benaroch, C Berlioz-Torrent, JC Guatelli, R Benarous, S Benichou, "Two independent regions of
HIV-1 Nef are required for connection with the endocytic pathway through binding to the mu 1 chain of AP1 complex", Traffic, 1, 2000, 871-83.
S Le Gall, L Erdtmann, S Benichou, C Berlioz-Torrent, L Liu, R Benarous, JM Heard, O Schwartz, "Nef interacts with the mu subunit of clathrin
adaptor complexes and reveals a cryptic sorting signal in MHC I molecules", Immunity, 8, 1998, 483-95.
PA Bresnahan, W Yonemoto, S Ferrell, D Williams-Herman, R Geleziunas, WC Greene, "A dileucine motif in HIV-1 Nef acts as an internalization
signal for CD4 downregulation and binds the AP-1 clathrin adaptor", Curr Biol, 8, 1998, 1235-8.
M Geyer, OT Fackler, BM Peterlin, "Structure--function relationships in HIV-1 Nef", EMBO Rep, 2, 2001, 580-5.
O Schwartz, V Marechal, S Le Gall, F Lemonnier, JM Heard, "Endocytosis of major histocompatibility complex class I molecules is induced by
the HIV-1 Nef protein", Nat Med, 2, 1996, 338-42.
M Schindler, S Wurfl, P Benaroch, TC Greenough, R Daniels, P Easterbrook, M Brenner, J Munch, F Kirchhoff, "Down-modulation of mature
major histocompatibility complex class II and up-regulation of invariant chain cell surface expression are well-conserved functions of human and
simian immunodeficiency virus nef alleles", J Virol, 77, 2003, 10548-56.
11.2.6.2.1 Nef mediated downregulation of CD28 cell surface expression
Authors
Reviewers
Description
Down-regulation of CD28 receptors involves a dileucine-based motif in the second disordered loop of Nef, which connects Nef to adaptor protein
(AP) complex, which is a part of cellular endocytosis machinery. Nef induces accelerated endocytosis of CD28 via clathrin-coated pits followed
by lysosomal degradation.
References
K Janvier, H Craig, D Hitchin, R Madrid, N Sol-Foulon, L Renault, J Cherfils, D Cassel, S Benichou, J Guatelli, "HIV-1 Nef stabilizes the
association of adaptor protein complexes with membranes", J Biol Chem, 278, 2003, 8725-32.
11.2.6.2.1.1 Formation of Nef CD28 cytoplasmic tail complex
Authors
Reviewers
Description
Down-regulation of CD28 receptors involves a dileucine-based motif in the second disordered loop of Nef, which connects Nef to adaptor protein
(AP) complex, which is a part of cellular endocytosis machinery.
References
Reaction
11.2.6.2.1.2 Formation of Nef:Cd28:Clathrin-coated Pit Adapter Protein complex
Authors
Reviewers
Description
Nef induces accelerated endocytosis of CD28 via clathrin-coated pits.
References
Reaction
11.2.6.2.1.3 Internalization of Nef:CD28:Clathrin-Coated Pit Adapter Protein Complex
Authors
Reviewers
Description
Once Nef has induced endocytosis of CD28, CD28 containing vesicles are targeted for lysosomal degradation.
References
Reaction
11.2.6.2.2 Nef mediated downregulation of MHC class I complex cell surface expression
Authors
Reviewers
Description
Down-regulation of MHC class I involves Nef-mediated connection in the endosomes between MHC-I's cytoplasmic tail and the phosphofurin
acidic cluster sorting protein-1 (PACS-1)-dependent protein-sorting pathway. Down-regulation of MHC I protects HIV-infected cells from host
CTL response.
References
P Stumptner-Cuvelette, S Morchoisne, M Dugast, S Le Gall, G Raposo, O Schwartz, P Benaroch, "HIV-1 Nef impairs MHC class II antigen
presentation and surface expression", Proc Natl Acad Sci U S A, 98, 2001, 12144-9.
M Williams, JF Roeth, MR Kasper, RI Fleis, CG Przybycin, KL Collins, "Direct binding of human immunodeficiency virus type 1 Nef to the major
histocompatibility complex class I (MHC-I) cytoplasmic tail disrupts MHC-I trafficking", J Virol, 76, 2002, 12173-84.
JF Roeth, M Williams, MR Kasper, TM Filzen, KL Collins, "HIV-1 Nef disrupts MHC-I trafficking by recruiting AP-1 to the MHC-I cytoplasmic tail",
J Cell Biol, 167, 2004, 903-13.
S Le Gall, L Erdtmann, S Benichou, C Berlioz-Torrent, L Liu, R Benarous, JM Heard, O Schwartz, "Nef interacts with the mu subunit of clathrin
adaptor complexes and reveals a cryptic sorting signal in MHC I molecules", Immunity, 8, 1998, 483-95.
AD Blagoveshchenskaya, L Thomas, SF Feliciangeli, CH Hung, G Thomas, "HIV-1 Nef downregulates MHC-I by a PACS-1- and PI3K-regulated
ARF6 endocytic pathway", Cell, 111, 2002, 853-66.
O Schwartz, V Marechal, S Le Gall, F Lemonnier, JM Heard, "Endocytosis of major histocompatibility complex class I molecules is induced by
the HIV-1 Nef protein", Nat Med, 2, 1996, 338-42.
V Piguet, L Wan, C Borel, A Mangasarian, N Demaurex, G Thomas, D Trono, "HIV-1 Nef protein binds to the cellular protein PACS-1 to
downregulate class I major histocompatibility complexes", Nat Cell Biol, 2, 2000, 163-7.
11.2.6.2.2.1 Formation of MHC I:Nef Complex
Authors
Reviewers
Description
Nef disrupts the transport of major histocompatibility complex class I molecules by first binding to the the cytoplasmic side of the transmembrane
complex.
References
J Cell Biol, 167, 2004, 903-13.
Reaction
11.2.6.2.2.2 Formation of MHC I:Nef:AP-1:PACS-1 Complex
Authors
Reviewers
Description
The complex formed by Nef and the major histocompatibility complex class I molecules creates binding sites for PACS-1 and the AP-1 complex.
References
J Cell Biol, 167, 2004, 903-13.
Reaction
11.2.6.2.2.3 Transport of MHC I:Nef:AP-1:PACS-1 Complex

Authors
Reviewers
Description
The complex of Nef, major histocompatibility complex class I molecules, PACS-1 and AP-1 is transported from the trans-Golgi network to an
endosome, where the MHC I complex will be degraded.
References
J Cell Biol, 167, 2004, 903-13.
Reaction
11.2.6.2.2.4 Degradation of MHC I Complex
Authors
Reviewers
Description
Once the complex of Nef, major histocompatibility complex class I molecules, PACS-1 and AP-1 arrives at the endosome, the MHC I complex is
targeted for degradation.
References
J Cell Biol, 167, 2004, 903-13.
Reaction
11.2.6.2.3 Nef Mediated CD4 Down-regulation
Authors
Reviewers
Description
The presence of Nef accelerates endocytosis and lysosomal degradation of the transmembrane glycoprotein CD4. CD4 has its own
internalization motif, though this motif is normally concealed by CD4 interaction with Lck, a tyrosine kinase. Nef is known to disrupt this
interaction and then facilitate a cascade of protein interactions that ultimately result in the degradation of internalized CD4 protein. The final set
of protein interactions that direct Nef to the beta-subunit of the COPI coatomers are at this time unclear.
A benefit for the virus from CD4 down-modulation is abolition of interaction between the receptor and the Env protein of the budding virus, which
likely increases HIV release from infected cell as well as infectivity of viral particles.
References
A Preusser, L Briese, AS Baur, D Willbold, "Direct in vitro binding of full-length human immunodeficiency virus type 1 Nef protein to CD4
cytoplasmic domain", J Virol, 75, 2001, 3960-4.
S Salghetti, R Mariani, J Skowronski, "Human immunodeficiency virus type 1 Nef and p56lck protein-tyrosine kinase interact with a common
element in CD4 cytoplasmic tail", Proc Natl Acad Sci U S A, 92, 1995, 349-53.
S Grzesiek, SJ Stahl, PT Wingfield, A Bax, "The CD4 determinant for downregulation by HIV-1 Nef directly binds to Nef. Mapping of the Nef
binding surface by NMR.", Biochemistry, 35, 1996, 10256-61.
C Aiken, J Konner, NR Landau, D Trono, ME Lenburg, "Nef induces CD4 endocytosis: requirement for a critical dileucine motif in the
membrane-proximal CD4 cytoplasmic domain", Cell, 76, 1994, 853-64.
11.2.6.2.3.1 Nef mediated disruption of CD4:Lck Complex
Authors
Reviewers
Description
Nef disrupts the CD4 Lck complex
References
Reaction
11.2.6.2.3.2 Formation of CD4:Nef:AP-2 Complex:v-ATPase Complex
Authors
Reviewers
Description
AP-2 is recruited to the newly formed Nef:CD4 complex
References
Reaction
11.2.6.2.3.3 Internalization of the CD4:Nef:AP-2 Complex:v-ATPase Complex
Authors
Reviewers
Description
The Nef:CD4:AP-2 complex is internalized
References
Reaction
11.2.6.2.3.4 Formation of a Nef:ARF1:CD4 complex
Authors
Reviewers
Description
The HIV Nef protein downregulates CD4 through sequential connection with clathrin-coated pits and the COP1 coatomer, resulting in
accelerated endocytosis and lysosomal targeting. The small GTPase ARF1 controls the Nef-induced, COP-mediated late-endosomal targeting of
CD4. Nef binds ARF1 directly and can recruit the GTPase onto endosomal membranes, leading to the eventual degradation of CD4 (Faure et al.
2004).
References
J Faure, R Stalder, C Borel, K Sobo, V Piguet, N Demaurex, J Gruenberg, D Trono, "ARF1 regulates Nef-induced CD4 degradation", Curr Biol,
14, 2004, 1056-64.
Reaction
11.2.6.2.3.5 Degradation of CD4
Authors

Reviewers
Description
CD4 is degraded
References
Reaction
11.2.6.2.4 Nef Mediated CD8 Down-regulation
Authors
Reviewers
Description
Human immunodeficiency virus (HIV) Nef is a membrane-associated protein decreasing surface expression of CD4, CD28, and major
histocompatibility complex class I on infected cells. Nef also strongly down-modulates surface expression of the beta-chain of the CD8alphabeta
receptor by accelerated endocytosis, while CD8 alpha-chain expression is less affected. Mutational analysis of the cytoplasmic tail of the CD8
beta-chain indicates that an FMK amino acid motif is critical for the Nef-induced endocytosis. Although independent of CD4, endocytosis of the
CD8 beta-chain is abrogated by the same mutations in Nef that affect CD4 down-regulation, suggesting common molecular interactions. The
ability to down-regulate the human CD8 beta-chain was conserved in HIV-1, HIV-2, and simian immunodeficiency virus SIVmac239 Nef and
required an intact AP-2 complex.
References
M Geyer, H Yu, R Mandic, T Linnemann, YH Zheng, OT Fackler, BM Peterlin, "Subunit H of the V-ATPase binds to the medium chain of adaptor
protein complex 2 and connects Nef to the endocytic machinery", J Biol Chem, 277, 2002, 28521-9.
11.2.6.2.4.1 Formation of CD8:Nef:AP-2 Complex:v-ATPase Complex
Authors
Reviewers
Description
The presence of Nef accelerates endocytosis and lysosomal degradation of the transmembrane glycoprotein CD8. Nef facilitates a cascade of
protein interactions that ultimately result in the degradation of internalized CD8 protein. The final set of protein interactions that direct Nef to the
beta-subunit of the COPI coatomers are at this time unclear.
A number of sites within Nef are proposed to be required for CD8 down-regulation, the myristoylation signal and N-terminal anchor regions, the
C-terminal flexible loop, and amino acid positions 57 to 58. Consistent with all reported Nef functions, the myristoylation signal was found to be
essential for CD8 down-modulation. The flexible loop contains a dileucine-based internalization motif, which is flanked by acidic clusters and is
involved in enhanced internalization of the Nef-CD4 complex.
References
Reaction
11.2.6.2.4.2 Internalization of the CD8:Nef:AP-2 Complex:v-ATPase Complex
Authors
Reviewers
Description
The CD8alphabeta receptor is internalized via endocytosis.
References
Reaction
11.2.6.2.4.3 Degradation of CD8
Authors
Reviewers
Description
Once the CD8alphabeta receptor has been internalized via endocytosis, the vesicles are targeted for lysosomal degradation.
References
Reaction
11.2.7 Vpu mediated degradation of CD4
Authors
Matthews, L, 2006-05-15.
Editors
Matthews, L, 2006-05-15.
Reviewers
Benarous, R, 2006-09-21.
Description
The HIV-1 Vpu protein promotes the degradation of the CD4 receptor by recruiting an SCF like ubiquitination complex that promotes CD4
degradation. Vpu links h-ÃŽÂ²TrCP to CD4 at the ER membrane through interactions with h-ÃŽÂ²TrCP and the cytoplasmic tail of CD4. The
SKP1 component of the SCF complex is then recruited to the VpuÃ¢â‚¬"h-ÃŽÂ²TrCP:CD4 promoting ubiquitination and subsequent
proteasome-mediated degradation of CD4 (reviewed in Li et al., 2005). Vpu has also been shown to also increases progeny virus secretion from
infected cells. Although the precise role of Vpu in this process is not yet known, it may affect ion conductive membrane pore formation and/or
interference with TASK-1, an acid-sensitive K+ channel that inhibits virion release in some cells (see references in Li et al., 2005).
References
J Binette, EA Cohen, "Recent advances in the understanding of HIV-1 Vpu accessory protein functions", Curr Drug Targets Immune Endocr
Metabol Disord, 4, 2004, 297-307.
15, 2005, 923-34.
11.2.7.1 Association of Vpu with CD4
Authors
Matthews, L, 2006-05-15.
Editors
Matthews, L, 2006-05-15.
Reviewers
Benarous, R, 2006-09-21.
Description
Vpu is expressed in the ER and associates with a membrane-proximal region in the cytoplasmic tail of CD4.
References
F Margottin, SP Bour, H Durand, L Selig, S Benichou, V Richard, D Thomas, K Strebel, R Benarous, "A novel human WD protein, h-beta TrCp,
that interacts with HIV-1 Vpu connects CD4 to the ER degradation pathway through an F-box motif", Mol Cell, 1, 1998, 565-74.
Reaction
11.2.7.2 Vpu:CD4 associates with beta-TrCP
Authors
Matthews, L, 2006-05-15.
Editors
Matthews, L, 2006-05-15.
Reviewers
Benarous, R, 2006-09-21.
Description
Vpu links beta-TrCP to CD4 at the ER membrane through interactions with beta-TrCP and the cytoplasmic tail of CD4.
References
Reaction
11.2.7.3 The Vpu:CD4:beta-TrCP complex recruits SKP1
Authors
Matthews, L, 2006-05-15.
Editors
Matthews, L, 2006-05-15.
Reviewers
Benarous, R, 2006-09-21.
Description
The SKP1 component of the SCF complex is recruited to the Vpu:beta-TrCP:CD4 complex.
References
Reaction
11.2.7.4 Ubiquitination of CD4 by Vpu:CD4:beta-TrCP:SKP1 complex
Authors
Matthews, L, 2006-05-15.
Editors
Matthews, L, 2006-05-15.
Reviewers
Benarous, R, 2006-09-21.
Description
CD4 is ubiquitinated in the CD4:VpuÃ¢â‚¬"h-ÃŽÂ²TrCP:Skp1 complex.
References
Reaction
11.2.7.5 Degradation of ubiquitinated CD4
Authors
Matthews, L, 2006-05-15.
Editors
Matthews, L, 2006-05-15.
Reviewers
Benarous, R, 2006-09-21.
Description
Ubiquitinated CD4 is then subject to proteasome-mediated degradation.
References
Reaction
12 Hemostasis
Authors
D'Eustachio, P, Pace, N.P., Farndale, R, de Bono, B, 2004-01-21.
Editors
Joshi-Tope, G, 0000-00-00.
Reviewers
Brummel, K, Rush, MG, Stafford, DW, 0000-00-00.
Description
Two principal mechanisms limit blood loss after vascular injury. Initially, platelets are activated, adhere to the site of the injury, and aggregate
into a plug that limits blood loss. Proteins and small molecules released from activated platelets stimulate the plug formation process, and
fibrinogen from the plasma forms bridges between activated platelets. These events allow the initiation of the clotting cascade, the second
mechanism to limit blood loss. Negatively charged phospholipids exposed on cell surfaces at the site of injury and on activated platelets interact
with tissue factor, setting off a cascade of reactions leading to generation of fibrin and the formation of an insoluble fibrin clot that strengthens the
platelet plug.
12.1 Formation of Platelet plug
Authors
de Bono, B, 2004-08-12.
Editors
de Bono, B, 0000-00-00.
Reviewers
Pace, N.P., 0000-00-00.

Description
Hemostasis is a physiological response that culminates in the arrest of bleeding from an injured vessel. Acute vessel injury results in its
constriction to reduce the loss of blood. Under normal conditions vascular endothelium supports vasodilation, inhibits platelet adhesion and
activation, suppresses coagulation, enhances fibrin cleavage and is anti-inflammatory in character. Under acute vascular trauma vasoconstrictor
mechanisms predominate and the endothelium becomes prothrombotic, procoagulatory and proinflammatory in nature. This is achieved by a
reduction of endothelial dilating agents: adenosine, NO and prostacyclin; and the direct action of ADP, serotonin and thromboxane on vascular
smooth muscle cells to elicit their contraction (Becker, Heindl et al. 2000).
The chief trigger for the change in endothelial function that leads to the formation of haemostatic thrombus is the loss of the endothelial cell
barrier between blood and ECM components (Ruggeri 2002). Circulating platelets identify and discriminate areas of endothelial lesions; here,
they adhere to the exposed sub endothelium. Their interaction with the various thrombogenic substrates and locally generated or released
agonists results in platelet activation. This process is described as possessing two stages, firstly, adhesion - the initial tethering to a surface, and
secondly aggregation - the platelet-platelet cohesion (Savage, Cattaneo et al. 2001).
12.1.1 Platelet Adhesion to exposed collagen
Authors
de Bono, B, 2004-08-13.
Description
Initiation is the first step in the formation of the platelet plug. This occurs whenever circulating platelets are arrested and subsequently activated
by exposed collagen and vWF. Several collagen binding proteins are expressed on platelets, including integrin Alpha2 Beta1, GP VI, and GP IV.
Integrin Alpha2 Beta1, also known on leukocytes as VLA-2, is the major collagen receptor. It requires Mg2+ for interacting with collagen. Binding
occurs via the alpha2 subunit I domain to a collagen motif with the sequence Gly-Phe-Hyp-Gly-Glu-Arg (Emsley 2000). Binding of collagen to
Alpha2 Beta1 also generates the intracellular signals that contribute to platelet activation. These facilitate the engagement of the lower-affinity
collagen receptor, GP VI (Keely 1996). The second platelet receptor for collagen is GP VI. This is part of the immunoglobulin superfamily and is
the most potent receptor that generates intracellular signals. Its signaling ability is strictly dependent on its association with the Fc receptor
epsilon chain, which contains Immunoreceptor Tyrosine-based Activation Motifs (ITAM). GP VI is the key player in collagen-induced platelet
activation. vWF protein is a polymeric structure of variable size. It is secreted in two directions by the endothelium - basolaterally and into the
bloodstream. Shear-induced aggregation is achieved when vWF binds via its A1 domain to GPIb (part of GPIb - IX - V), and via its A3 domain
mediating collagen binding to the subendothelium. The interaction between vWF and GPIb is regulated by shear force, an increase in the shear
stress results in a corresponding increase in the affinity of vWF for GPIb.
12.1.1.1 Collagen adhesion via alpha 2 beta 1 glycoprotein

12.1.1.1.1 Adhesion via alpha 2 beta 1 glycoprotein
Authors
Geiger, B, Horwitz, R, 2008-05-07.
Reviewers
Yamada, K, Humphries, MJ, Hynes, R, 2008-05-07.
Description
At the beginning of this reaction, 1 molecule of 'Magnesium', 1 molecule of 'Collagen I', and 1 molecule of 'Alpha 2 Beta 1 Integrin' are present.
At the end of this reaction, 1 molecule of 'Alpha 2 Beta 1 Integrin : Collagen IV: Mg++ complex' is present.
References
J Emsley, CG Knight, RW Farndale, MJ Barnes, RC Liddington, "Structural basis of collagen recognition by integrin alpha2beta1", Cell, 101,
2000, 47-56.
Reaction
12.1.1.2 Collagen adhesion via Gp IV
12.1.1.2.1 Platelet glycoprotein IV [plasma membrane] GP IV : Collagen IV complex formation
Description
At the beginning of this reaction, 1 molecule of 'Platelet glycoprotein IV', and 1 molecule of 'Collagen I' are present. At the end of this reaction, 1
molecule of 'GP IV : Collagen IV complex' is present.
References
MM Huang, JB Bolen, JW Barnwell, SJ Shattil, JS Brugge, "Membrane glycoprotein IV (CD36) is physically associated with the Fyn, Lyn, and
Yes protein-tyrosine kinases in human platelets", Proc Natl Acad Sci U S A, 88, 1991, 7844-8.
Reaction
12.1.1.3 vWF interaction with collagen

12.1.1.3.1 vWF binds to collagen
Description
At the beginning of this reaction, 1 molecule of 'Collagen I', and 1 molecule of 'Von Willebrand factor precursor' are present. At the end of this
reaction, 1 molecule of 'Collagen IV : vWF complex' is present.
References
VT Turitto, HJ Weiss, TS Zimmerman, II Sussman, "Factor VIII/von Willebrand factor in subendothelium mediates platelet adhesion", Blood, 65,
1985, 823-31.
Reaction
12.1.1.3.2 GP Ib-IX-V binds to [vWF:Collagen] complex
Description
At the beginning of this reaction, 1 molecule of 'Collagen IV : vWF complex', and 1 molecule of 'GP Ib-IX-V complex' are present. At the end of
this reaction, 1 molecule of 'GP Ib-IX-V : vWF : Collagen IV complex' is present.
References
MH Kroll, TS Harris, JL Moake, RI Handin, AI Schafer, "von Willebrand factor binding to platelet GpIb initiates signals for platelet activation", J
Clin Invest, 88, 1991, 1568-73.
Reaction
12.1.2 Platelet Activation
Authors
de Bono, B, 2004-08-13.
Description
Platelet activation begins with the initial binding of adhesive ligands and of the excitatory platelet agonists (released or generated at the sites of
vascular trauma) to cognate receptors on the platelet membrane (Ruggeri 2002). Intracellular signaling reactions will then enhance the adhesive
and procoagulant properties of tethered platelets or of platelets circulating in the proximity. From the subendothelial adhesive substrates,
collagen and possibly vWF are the main inducers of platelet activation. GP VI is the most potent collagen receptor initiating signal generation, an
ability derived from its interaction with the FcRI gamma chain. This results in the phosphorylation of the gamma-chain by the non-receptor
tyrosine kinases of the Src family. The phosphotyrosine motif is recognized by the SH2 domains of Syk, a tyrosine kinase. This association
activates the Syk enzyme, leading to activation (by tyrosine phosphorylation) of PLC gamma2. Thrombin is an important platelet agonist
generated on the membrane of stimulated platelets. Thrombin acts via cell surface Protease Activated Receptors (PARs). PARs are G-protein
coupled receptors activated by a proteolytic cleavage in an extracellular loop (Vu 1991). Four PARs are identified, of which PARs 1 ,3 and 4 are
substrates for thrombin. PAR 1 is the predominant thrombin receptor, PAR 3 is minimally expressed and PAR 4 is less responsive to thrombin.
Platelets do not store PAR1, due to limited protein synthesis, they are capable of responding to thrombin only once.
12.1.2.1 Platelet activation triggers
12.1.2.1.1 Collagen-mediated activation cascade
12.1.2.1.1.1 Binding of GP VI:Fc Epsilon R1 gamma receptor complex with collagen
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
GPVI is the major signaling receptor for collagen on the platelet surface. It has two Ig domains, a mucin-rich stalk and a cytosolic sequence of 51
amino acids in human. It is coupled to a disulfide linked Fc receptor gamma-chain homodimer in the membrane via a salt-bridge between
charged amino acids within the transmembrane sequences and through specific sequences with the cytosolic tails . This chain is essential for
expression of GPVI on platelets.
References
M Tsuji, Y Ezumi, M Arai, H Takayama, "A novel association of Fc receptor gamma-chain with glycoprotein VI and their co-expression as a
collagen receptor in human platelets", J Biol Chem, 272, 1997, 23528-31.
Reaction
12.1.2.1.1.2 Src-mediated phoshorylation of FcR1 gamma

Description
At the beginning of this reaction, 1 molecule of 'GP VI:Fc Epsilon R1 gamma:Collagen IV complex', and 1 molecule of 'ATP' are present. At the
end of this reaction, 1 molecule of 'ADP', and 1 molecule of 'GP VI:phosphorylated Fc Epsilon R1 gamma:Collagen IV complex' are present.
This reaction is mediated by the 'protein-tyrosine kinase activity' of 'GP VI: Fc Epsilon R1 gamma: Collagen IV: SRC'.
References
J Asselin, JM Gibbins, M Achison, YH Lee, LF Morton, RW Farndale, MJ Barnes, SP Watson, "A collagen-like peptide stimulates tyrosine
phosphorylation of syk and phospholipase C gamma2 in platelets independent of the integrin alpha2beta1", Blood, 89, 1997, 1235-42.
Reaction
12.1.2.1.1.3 Syk-mediated phosphorylation of Phospholipase C gamma 2
Description
At the beginning of this reaction, 1 molecule of 'Phospholipase C gamma 2' is present. At the end of this reaction, 1 molecule of 'Phosphorylated
phospholipase C gamma 2' is present.
This reaction is mediated by the 'protein-tyrosine kinase activity' of 'GP VI:phosphorylated Fc Epsilon R1 gamma:Collagen IV: Syk complex'.
References
JM Gibbins, M Okuma, R Farndale, M Barnes, SP Watson, "Glycoprotein VI is the collagen receptor in platelets which underlies tyrosine
phosphorylation of the Fc receptor gamma-chain", FEBS Lett, 413, 1997, 255-9.
Reaction
12.1.2.1.1.4 Binding of SRC tyrosine kinase
Description
At the beginning of this reaction, 1 molecule of 'SRC', and 1 molecule of 'GP VI:Fc Epsilon R1 gamma:Collagen IV complex' are present. At the
end of this reaction, 1 molecule of 'GP VI: Fc Epsilon R1 gamma: Collagen IV: SRC' is present.
Reaction
12.1.2.1.1.5 Binding of Syk tyrosine kinase
Description
At the beginning of this reaction, 1 molecule of 'Tyrosine-protein kinase SYK ', and 1 molecule of 'GP VI:phosphorylated Fc Epsilon R1
gamma:Collagen IV complex' are present. At the end of this reaction, 1 molecule of 'GP VI:phosphorylated Fc Epsilon R1 gamma:Collagen IV:
Syk complex' is present.
Reaction
12.1.2.1.2 Thrombin-activated activation cascade
12.1.2.1.2.1 Thrombin-mediated activation of PARs
References
SR Coughlin, "Thrombin signalling and protease-activated receptors", Nature, 407, 2000, 258-64.
12.1.2.1.2.1.1 Thrombin-mediated activation of PAR1
Description
At the beginning of this reaction, 1 molecule of 'Proteinase activated receptor 1 precursor' is present. At the end of this reaction, 1 molecule of
'Activated PAR1', and 1 molecule of 'Cleaved portion of PAR1' are present.
This reaction is mediated by the 'thrombin activity' of 'activated thrombin (factor IIa)'.
References
TK Vu, DT Hung, VI Wheaton, SR Coughlin, "Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of
receptor activation", Cell, 64, 1991, 1057-68.
Reaction
Description
'Cleaved portion of PAR3', and 1 molecule of 'Activated PAR3' are present.
References
H Ishihara, AJ Connolly, D Zeng, ML Kahn, YW Zheng, C Timmons, T Tram, SR Coughlin, "Protease-activated receptor 3 is a second thrombin
receptor in humans", Nature, 386, 1997, 502-6.
Reaction
Description
'Activated PAR4', and 1 molecule of 'Cleaved portion of PAR4' are present.
References
WF Xu, H Andersen, TE Whitmore, SR Presnell, DP Yee, A Ching, T Gilbert, EW Davie, DC Foster, "Cloning and characterization of human
protease-activated receptor 4", Proc Natl Acad Sci U S A, 95, 1998, 6642-6.
Reaction
12.1.2.1.2.2 G-protein cascades
12.1.2.1.2.2.1 G alpha 12 cascade
12.1.2.1.2.2.1.1 GTP loading of G alpha 12
Description
At the beginning of this reaction, 1 molecule of 'GTP', and 1 molecule of 'Alpha 12 G protein : GDP] : Beta G protein : Gamma G protein]
complex' are present. At the end of this reaction, 1 molecule of 'GDP', and 1 molecule of 'Alpha 12 G protein : GTP] : Beta G protein : Gamma G
protein] complex' are present.
This reaction is mediated by the 'G-protein coupled receptor activity' of 'Activated PAR'.
References
M Bauer, M Retzer, JI Wilde, P Maschberger, M Essler, M Aepfelbacher, SP Watson, W Siess, "Dichotomous regulation of myosin
phosphorylation and shape change by Rho-kinase and calcium in intact human platelets", Blood, 94, 1999, 1665-72.
Reaction
12.1.2.1.2.2.1.2 Activation of Rho guanine nucleotide exchange factor 1 (p115-RhoGEF)
Description
At the beginning of this reaction, 1 molecule of 'Rho guanine nucleotide exchange factor 1 (p115-RhoGEF)', and 1 molecule of 'Alpha 12 G
protein : GTP] : Beta G protein : Gamma G protein] complex' are present. At the end of this reaction, 1 molecule of 'Activated 115Rho GEF', and
1 molecule of 'Beta and gamma subunit G-protein complex' are present.
References
S Fukuhara, H Chikumi, JS Gutkind, "RGS-containing RhoGEFs: the missing link between transforming G proteins and Rho?", Oncogene, 20,
2001, 1661-8.
Reaction
12.1.2.1.2.2.1.3 Activation of RAC1
Description
At the beginning of this reaction, 1 molecule of 'GTP', and 1 molecule of 'Deactivated RAC1' are present. At the end of this reaction, 1 molecule
of 'Activated RAC1', and 1 molecule of 'GDP' are present.
This reaction is mediated by the 'guanyl-nucleotide exchange factor activity' of 'Activated 115Rho GEF'.
References
AC Azim, K Barkalow, J Chou, JH Hartwig, "Activation of the small GTPases, rac and cdc42, after ligation of the platelet PAR-1 receptor", Blood,
95, 2000, 959-64.
Reaction
12.1.2.1.2.2.1.4 Activation of PI3K
Description
At the beginning of this reaction, 1 molecule of 'PI3K', and 1 molecule of 'Activated RAC1' are present. At the end of this reaction, 1 molecule of
'Activated PI3K' is present.
References
TJ Kovacsovics, C Bachelot, A Toker, CJ Vlahos, B Duckworth, LC Cantley, JH Hartwig, "Phosphoinositide 3-kinase inhibition spares actin
assembly in activating platelets but reverses platelet aggregation", J Biol Chem, 270, 1995, 11358-66.
Reaction
12.1.2.1.2.2.2 G alpha Q cascade
12.1.2.1.2.2.2.1 GTP loading of G alpha Q
Description
At the beginning of this reaction, 1 molecule of 'Alpha Q G protein : GDP] : Beta G protein : Gamma G protein] complex', and 1 molecule of 'GTP'
are present. At the end of this reaction, 1 molecule of 'Alpha Q G protein : GTP] : Beta G protein : Gamma G protein] complex', and 1 molecule
of 'GDP' are present.
This reaction is mediated by the 'G-protein coupled receptor activity' of 'Activated PAR'.
References
A Shenker, P Goldsmith, CG Unson, AM Spiegel, "The G protein coupled to the thromboxane A2 receptor in human platelets is a member of the
novel Gq family", J Biol Chem, 266, 1991, 9309-13.
Reaction
12.1.2.1.2.2.2.2 Activation of PLC beta 1
Description
At the beginning of this reaction, 1 molecule of 'Alpha Q G protein : GTP] : Beta G protein : Gamma G protein] complex', and 1 molecule of
'Phospholipase C beta 1' are present. At the end of this reaction, 1 molecule of 'Activated phospholipase C beta 1', and 1 molecule of 'Beta and
gamma subunit G-protein complex' are present.
References
LF Brass, DR Manning, K Cichowski, CS Abrams, "Signaling through G proteins in platelets: to the integrins and beyond", Thromb Haemost, 78,
1997, 581-9.
Reaction
12.1.2.2 Phospholipase-mediated signalling
12.1.2.2.1 PLC-mediated hydrolysis
12.1.2.2.1.1 PLC beta 1-mediated hydrolysis
Description
At the beginning of this reaction, 1 molecule of '1-Phosphatidyl-D-myo-inositol 4,5-bisphosphate' is present. At the end of this reaction, 1
molecule of '1D-myo-Inositol 1,4,5-trisphosphate', and 1 molecule of '1,2-Diacylglycerol' are present.
This reaction is mediated by the 'phospholipase C activity' of 'Activated phospholipase C beta 1'.
References
Y Banno, Y Yada, Y Nozawa, "Purification and characterization of membrane-bound phospholipase C specific for phosphoinositides from human
platelets", J Biol Chem, 263, 1988, 11459-65.
Reaction
12.1.2.2.1.2 PLC gamma 2-mediated hydrolysis
Description
At the beginning of this reaction, 1 molecule of '1-Phosphatidyl-D-myo-inositol 4,5-bisphosphate' is present. At the end of this reaction, 1
molecule of '1D-myo-Inositol 1,4,5-trisphosphate', and 1 molecule of '1,2-Diacylglycerol' are present.
This reaction is mediated by the 'phospholipase C activity' of 'Phosphorylated phospholipase C gamma 2'.
References
Y Banno, Y Yada, Y Nozawa, "Purification and characterization of membrane-bound phospholipase C specific for phosphoinositides from human
platelets", J Biol Chem, 263, 1988, 11459-65.
Reaction
12.1.2.2.2 Effects of IP3
12.1.2.2.2.1 Effects on Ca++ levels
12.1.2.2.2.1.1 Binding of IP3 to IP3 receptor
Description
At the beginning of this reaction, 1 molecule of '1D-myo-Inositol 1,4,5-trisphosphate', and 1 molecule of 'IP3 receptor' are present. At the end of
this reaction, 1 molecule of 'IP3 receptor bound to IP3 and Ca++' is present.
References
MJ Berridge, P Lipp, MD Bootman, "The versatility and universality of calcium signalling", Nat Rev Mol Cell Biol, 1, 2000, 11-21.
Reaction
12.1.2.3 Elevation of cytosolic Ca++ levels
12.1.2.3.1 Entry of Ca++ from platelet dense tubular system
Description
In this reaction, 1 molecule of 'Calcium' is translocated from platelet dense tubular network lumen to cytosol.
This reaction takes place in the 'platelet dense tubular network membrane' and is mediated by the 'calcium channel activity' of 'IP3 receptor
bound to IP3 and Ca++'.
References
MP Mahaut-Smith, SO Sage, TJ Rink, "Receptor-activated single channels in intact human platelets", J Biol Chem, 265, 1990, 10479-83.
Reaction
12.1.2.3.2 Entry of Ca++ from plasma
Description
In this reaction, 1 molecule of 'Calcium' is translocated from extracellular region to cytosol.
This reaction takes place on the 'plasma membrane' and is mediated by the 'calcium channel activity' of 'P2X1 purinoreceptor bound to ATP'.
References
A Zschauer, Breemen C van, FR Buhler, MT Nelson, "Calcium channels in thrombin-activated human platelet membrane", Nature, 334, 1988,
703-5.
Reaction
12.1.2.3.3 Response to elevated platelet cytosolic Ca++
Authors
de Bono, B, 2004-08-13.
Description
Activation of phospholipase C enzymes results in the generation of second messengers of the phosphatidylinositol pathway. The events
resulting from this pathway are a rise in intracellular Ca and activation of Protein Kinase C(PKC). Phospholipase C cleaves the phosphodiester
bond in PIP2 to form 1,2 DiAcylGlycerol (DAG) and 1,4,5-inositol trisphosphate (IP3). IP3 opens Ca2+ channels in the platelet dense tubular
system, raising intracellular Ca2+ levels. DAG is second messenger that regulates a family of Ser/Thr kinases consisting of PKC isozymes
(Nishizuka 1995). DAG achieves activation of PKC isozymes by increasing their affinity for phospholipid. Most PKC enzymes are also
Ca-dependent, so the activation is in synergy with the rise in intracellular Ca2+. Platelets contain several PKC isoforms that can be activated by
DAG and/or Ca2+ (Chang 1997).
References
TR Walker, SP Watson, "Synergy between Ca2+ and protein kinase C is the major factor in determining the level of secretion from human
platelets", Biochem J, 289, 1993, 277-82.
12.1.2.3.3.1 Activation of PKC
Description
At the beginning of this reaction, 1 molecule of 'Calcium', 1 molecule of 'Calcium-sensitive protein kinase C', and 1 molecule of
'1,2-Diacylglycerol' are present. At the end of this reaction, 1 molecule of 'Activated Ca++ and DAG dependent PKC' is present.
References
E Oancea, T Meyer, "Protein kinase C as a molecular machine for decoding calcium and diacylglycerol signals", Cell, 95, 1998, 307-18.
Reaction
12.1.2.3.3.2 Disinhibition of SNARE formation
12.1.2.3.3.2.1 Phosphorylation of Platelet Sec-1
Description
At the beginning of this reaction, 1 molecule of 'ATP', and 1 molecule of 'Platelet SEC1 protein' are present. At the end of this reaction, 1
molecule of 'Phosphorylated platelet SEC1 protein', and 1 molecule of 'ADP' are present.
This reaction is mediated by the 'calcium-dependent protein kinase C activity' of 'Activated Ca++ and DAG dependent PKC'.
References
GL Reed, AK Houng, ML Fitzgerald, "Human platelets contain SNARE proteins and a Sec1p homologue that interacts with syntaxin 4 and is
phosphorylated after thrombin activation: implications for platelet secretion", Blood, 93, 1999, 2617-26.
Reaction
12.1.2.3.3.2.2 Phosphorylation of Syntaxin-4
Description
At the beginning of this reaction, 1 molecule of 'ATP', and 1 molecule of 'Syntaxin 4' are present. At the end of this reaction, 1 molecule of
'Phosphorylated syntaxin 4', and 1 molecule of 'ADP' are present.
This reaction is mediated by the 'calcium-dependent protein kinase C activity' of 'Activated Ca++ and DAG dependent PKC'.
References
SH Chung, J Polgar, GL Reed, "Protein kinase C phosphorylation of syntaxin 4 in thrombin-activated human platelets", J Biol Chem, 275, 2000,
25286-91.
Reaction
12.1.2.3.3.3 Platelet degranulation
12.1.2.3.3.3.1 Exocytosis of Dense granule
References
RJ Skaer, PD Peters, JP Emmines, "The localization of calcium and phosphorus in human platelets", J Cell Sci, 15, 1974, 679-82.
12.1.2.3.3.3.1.1 Exocytosis of ADP
Description
In this reaction, 1 molecule of 'ADP' is translocated from platelet dense granule lumen to extracellular region.
Reaction
12.1.2.3.3.3.1.2 Exocytosis of ATP
Description
In this reaction, 1 molecule of 'ATP' is translocated from platelet dense granule lumen to extracellular region.
Reaction
12.1.2.3.3.3.1.3 Exocytosis of Ca++
Description
In this reaction, 1 molecule of 'Calcium' is translocated from platelet dense granule lumen to extracellular region.
References
JH Martin, FL Carson, GJ Race, "Calcium-containing platelet granules", J Cell Biol, 60, 1974, 775-7.
Reaction
12.1.2.3.3.3.1.4 Exocytosis of GDP
Description
In this reaction, 1 molecule of 'GDP' is translocated from platelet dense granule lumen to extracellular region.
References
MH Fukami, "Isolation of dense granules from human platelets", Methods Enzymol, 215, 1992, 36-42.
Reaction
12.1.2.3.3.3.1.5 Exocytosis of GTP
Description
In this reaction, 1 molecule of 'GTP' is translocated from platelet dense granule lumen to extracellular region.
Reaction
12.1.2.3.3.3.1.6 Exocytosis of Mg++
Description
In this reaction, 1 molecule of 'Magnesium' is translocated from platelet dense granule lumen to extracellular region.
Reaction
12.1.2.3.3.3.1.7 Exocytosis of orthophosphate
Description
In this reaction, 1 molecule of 'Orthophosphate' is translocated from platelet dense granule lumen to extracellular region.
Reaction
12.1.2.3.3.3.1.8 Exocytosis of pyrophosphate
Description
In this reaction, 1 molecule of 'pyrophosphate' is translocated from platelet dense granule lumen to extracellular region.
Reaction
12.1.2.3.3.3.1.9 Exocytosis of serotonin
Description
In this reaction, 1 molecule of '3-(2-Aminoethyl)-1H-indol-5-ol' is translocated from platelet dense granule lumen to extracellular region.
References
JL Costa, TS Reese, DL Murphy, "Serotonin storage in platelets: estimation of storage-packet size", Science, 183, 1974, 537-8.
Reaction
12.1.2.3.3.3.1.10 Surface deployment of CD63
Description
In this reaction, 1 molecule of 'CD63 antigen' is translocated from platelet dense granule membrane to plasma membrane.
Reaction
12.1.2.3.3.3.1.11 Surface deployment of LAMP2
Description
In this reaction, 1 molecule of 'Lysosome-associated membrane glycoprotein 2 precursor' is translocated from platelet dense granule membrane
to plasma membrane.
References
SJ Israels, EM McMillan, C Robertson, S Singhory, A McNicol, "The lysosomal granule membrane protein, LAMP-2, is also present in platelet
dense granule membranes", Thromb Haemost, 75, 1996, 623-9.
Reaction
12.1.2.3.3.3.2 Exocytosis of Alpha granule
References
P Harrison, EM Cramer, "Platelet alpha-granules", Blood Rev, 7, 1993, 52-62.
12.1.2.3.3.3.2.1 Surface deployment of alpha IIb beta 3 integrin

Description
In this reaction, 1 molecule of 'Alpha IIb Beta 3 Integrin' is translocated from platelet alpha granule membrane to plasma membrane.
References
K Niiya, E Hodson, R Bader, V Byers-Ward, JA Koziol, EF Plow, ZM Ruggeri, "Increased surface expression of the membrane glycoprotein
IIb/IIIa complex induced by platelet activation. Relationship to the binding of fibrinogen and platelet aggregation.", Blood, 70, 1987, 475-83.
Reaction
12.1.2.3.3.3.2.2 Exocytosis of albumin
Description
In this reaction, 1 molecule of 'Serum albumin precursor' is translocated from platelet alpha granule lumen to extracellular region.
References
GO Gogstad, I Hagen, R Korsmo, NO Solum, "Characterization of the proteins of isolated human platelet alpha-granules. Evidence for a
separate alpha-granule-pool of the glycoproteins IIb and IIIa.", Biochim Biophys Acta, 670, 1981, 150-62.
Reaction
12.1.2.3.3.3.2.3 Exocytosis of beta-thromboglobulin
Description
In this reaction, 1 molecule of 'Beta-thromboglobulin' is translocated from platelet alpha granule lumen to extracellular region.
References
S Moore, DS Pepper, JD Cash, "The isolation and characterisation of a platelet-specific beta-globulin (beta-thromboglobulin) and the detection of
antiurokinase and antiplasmin released from thrombin-aggregated washed human platelets", Biochim Biophys Acta, 379, 1975, 360-9.
Reaction
12.1.2.3.3.3.2.4 Exocytosis of factor V
Description
In this reaction, 1 molecule of 'factor V' is translocated from platelet alpha granule lumen to extracellular region.
References
CM Chesney, D Pifer, RW Colman, "Subcellular localization and secretion of factor V from human platelets", Proc Natl Acad Sci U S A, 78,
1981, 5180-4.
Reaction
12.1.2.3.3.3.2.5 Exocytosis of factor VIII
Description
In this reaction, 1 molecule of 'Coagulation factor VIII precursor' is translocated from platelet alpha granule lumen to extracellular region.
References
MB Zucker, MJ Broekman, KL Kaplan, "Factor VIII-related antigen in human blood platelets: localization and release by thrombin and collagen",
J Lab Clin Med, 94, 1979, 675-82.
Reaction
12.1.2.3.3.3.2.6 Exocytosis of fibrinogen
Description
In this reaction, 1 molecule of 'fibrinogen' is translocated from platelet alpha granule lumen to extracellular region.
References
JP Keenan, NO Solum, "Quantitative studies on the release of platelet fibrinogen by thrombin", Br J Haematol, 23, 1972, 461-6.
Reaction
12.1.2.3.3.3.2.7 Exocytosis of fibronectin
Description
In this reaction, 1 molecule of 'Fibronectin precursor' is translocated from platelet alpha granule lumen to extracellular region.
References
MB Zucker, MW Mosesson, MJ Broekman, KL Kaplan, "Release of platelet fibronectin (cold-insoluble globulin) from alpha granules induced by
thrombin or collagen; lack of requirement for plasma fibronectin in ADP-induced platelet aggregation", Blood, 54, 1979, 8-12.
Reaction
12.1.2.3.3.3.2.8 Exocytosis of low-affinity platelet factor IV
Description
In this reaction, 1 molecule of 'Low-affinity platelet factor IV' is translocated from platelet alpha granule lumen to extracellular region.
Reaction
12.1.2.3.3.3.2.9 Exocytosis of multimerin

Description
In this reaction, 1 molecule of 'Multimerin 1' is translocated from platelet alpha granule lumen to extracellular region.
References
CP Hayward, DF Bainton, JW Smith, P Horsewood, RH Stead, TJ Podor, TE Warkentin, JG Kelton, "Multimerin is found in the alpha-granules of
resting platelets and is synthesized by a megakaryocytic cell line", J Clin Invest, 91, 1993, 2630-9.
Reaction
12.1.2.3.3.3.2.10 Exocytosis of neutrophil-activating peptide 2
Description
In this reaction, 1 molecule of 'Neutrophil-activating peptide 2' is translocated from platelet alpha granule lumen to extracellular region.
Reaction
12.1.2.3.3.3.2.11 Exocytosis of osteonectin
Description
In this reaction, 1 molecule of 'Osteonectin' is translocated from platelet alpha granule lumen to extracellular region.
References
J Breton-Gorius, P Clezardin, J Guichard, N Debili, L Malaval, W Vainchenker, EM Cramer, PD Delmas, "Localization of platelet osteonectin at
the internal face of the alpha-granule membranes in platelets and megakaryocytes", Blood, 79, 1992, 936-41.
Reaction
12.1.2.3.3.3.2.12 Surface deployment of P-selectin
Description
In this reaction, 1 molecule of 'P-selectin precursor' is translocated from platelet alpha granule membrane to plasma membrane.
References
SJ Israels, JM Gerrard, YV Jacques, A McNicol, B Cham, M Nishibori, DF Bainton, "Platelet dense granule membranes contain both
granulophysin and P-selectin (GMP-140)", Blood, 80, 1992, 143-52.
Reaction
12.1.2.3.3.3.2.13 Exocytosis of plasminogen
Description
In this reaction, 1 molecule of 'Plasminogen precursor ' is translocated from platelet alpha granule lumen to extracellular region.
Reaction
12.1.2.3.3.3.2.14 Exocytosis of plasminogen activator inhibitor
Description
In this reaction, 1 molecule of 'Plasminogen activator inhibitor-1 precursor' is translocated from platelet alpha granule lumen to extracellular
region.
Reaction
12.1.2.3.3.3.2.15 Exocytosis of Platelet factor 4
Description
In this reaction, 1 molecule of 'Platelet factor 4 precursor' is translocated from platelet alpha granule lumen to extracellular region.
References
S Niewiarowski, DP Thomas, "Platelet factor 4 and adenosine diphosphate release during human platelet aggregation", Nature, 222, 1969,
1269-70.
Reaction
12.1.2.3.3.3.2.16 Exocytosis of TGF beta
Description
In this reaction, 1 molecule of 'TGF beta ' is translocated from platelet alpha granule lumen to extracellular region.
References
RK Assoian, MB Sporn, "Type beta transforming growth factor in human platelets: release during platelet degranulation and action on vascular
smooth muscle cells", J Cell Biol, 102, 1986, 1217-23.
Reaction
12.1.2.3.3.3.2.17 Exocytosis of thrombospondin
Description
In this reaction, 1 molecule of 'Thrombospondin' is translocated from platelet alpha granule lumen to extracellular region.
References
KM McLaren, "Immunohistochemical localisation of thrombospondin in human megakaryocytes and platelets", J Clin Pathol, 36, 1983, 197-9.
Reaction
12.1.2.3.3.3.2.18 Exocytosis of vWF
Description
In this reaction, 1 molecule of 'Von Willebrand factor precursor' is translocated from platelet alpha granule lumen to extracellular region.
Reaction
12.1.2.3.3.3.2.19 Surface deployment of GpIV
Description
In this reaction, 1 molecule of 'Platelet glycoprotein IV' is translocated from platelet alpha granule membrane to plasma membrane.
References
G Berger, JP Caen, MC Berndt, EM Cramer, "Ultrastructural demonstration of CD36 in the alpha-granule membrane of human platelets and
megakaryocytes", Blood, 82, 1993, 3034-44.
Reaction
12.1.2.3.3.3.2.20 Surface deployment of CD9

Description
In this reaction, 1 molecule of 'CD9 antigen' is translocated from platelet alpha granule membrane to plasma membrane.
References
M Inngjerdingen, K Waterhouse, NO Solum, "Studies on the dual effects on platelets of a monoclonal antibody to CD9, and on the properties of
platelet CD9", Thromb Res, 95, 1999, 215-27.
Reaction
12.1.2.3.3.3.2.21 Surface deployment of GP Ib-IX-V complex
Description
In this reaction, 1 molecule of 'GP Ib-IX-V complex' is translocated from platelet alpha granule membrane to plasma membrane.
References
G Berger, JM Masse, EM Cramer, "Alpha-granule membrane mirrors the platelet plasma membrane and contains the glycoproteins Ib, IX, and
V", Blood, 87, 1996, 1385-95.
Reaction
12.1.2.3.3.3.2.22 Surface deployment of PECAM-1
Description
In this reaction, 1 molecule of 'Platelet endothelial cell adhesion molecule precursor' is translocated from platelet alpha granule membrane to
plasma membrane.
References
EM Cramer, G Berger, MC Berndt, "Platelet alpha-granule and plasma membrane share two new components: CD9 and PECAM-1", Blood, 84,
1994, 1722-30.
Reaction
12.1.2.3.3.3.2.23 Exocytosis of protease nexin II APP
Description
In this reaction, 1 molecule of 'Amyloid beta A4 protein precursor' is translocated from platelet alpha granule lumen to extracellular region.
References
Nostrand W Van, AH Schmaier, JS Farrow, DD Cunningham, "Protease nexin-II (amyloid beta-protein precursor): a platelet alpha-granule
protein", Science, 248, 1990, 745-8.
Reaction
12.1.2.3.3.3.2.24 Exocytosis of histidine rich glycoprotein
Description
In this reaction, 1 molecule of 'Histidine-rich glycoprotein precursor' is translocated from platelet alpha granule lumen to extracellular region.
References
LL Leung, PC Harpel, RL Nachman, EM Rabellino, "Histidine-rich glycoprotein is present in human platelets and is released following thrombin
stimulation", Blood, 62, 1983, 1016-21.
Reaction
12.1.2.3.3.3.2.25 Exocytosis of alpha 2 macroglobulin
Description
In this reaction, 1 molecule of 'Alpha-2-macroglobulin precursor' is translocated from platelet alpha granule lumen to extracellular region.
References
RL Nachman, PC Harpel, "Platelet alpha2-macroglobulin and alpha1-antitrypsin", J Biol Chem, 251, 1976, 4512-21.
Reaction
12.1.2.3.3.3.2.26 Exocytosis of alpha 1 antitrypsin
Description
In this reaction, 1 molecule of 'Alpha-1-antitrypsin' is translocated from platelet alpha granule lumen to extracellular region.
References
RL Nachman, PC Harpel, "Platelet alpha2-macroglobulin and alpha1-antitrypsin", J Biol Chem, 251, 1976, 4512-21.
Reaction
12.1.2.3.3.3.2.27 Exocytosis of complement factor D
Description
In this reaction, 1 molecule of 'Complement factor D precursor ' is translocated from platelet alpha granule lumen to extracellular region.
References
DM Kenney, AE 3rd Davis, "Association of alternative complement pathway components with human blood platelets: secretion and localization
of factor D and beta 1H Globulin", Clin Immunol Immunopathol, 21, 1981, 351-63.
Reaction
12.1.2.3.3.3.2.28 Exocytosis of kininogen
Description
In this reaction, 1 molecule of 'Kininogen precursor' is translocated from platelet alpha granule lumen to extracellular region.
Reaction
12.1.2.3.3.3.2.29 Exocytosis of hepatocyte growth factor
Description
In this reaction, 1 molecule of 'Hepatocyte growth factor' is translocated from platelet alpha granule lumen to extracellular region.
References
T Nakamura, T Nishizawa, M Hagiya, T Seki, M Shimonishi, A Sugimura, K Tashiro, S Shimizu, "Molecular cloning and expression of human
hepatocyte growth factor", Nature, 342, 1989, 440-3.
Reaction
12.1.2.3.3.3.2.30 Exocytosis of C1 inhibitor

Description
In this reaction, 1 molecule of 'Plasma protease C1 inhibitor precursor' is translocated from platelet alpha granule lumen to extracellular region.
References
AH Schmaier, PM Smith, RW Colman, "Platelet C1- inhibitor. A secreted alpha-granule protein.", J Clin Invest, 75, 1985, 242-50.
Reaction
12.1.2.3.3.3.2.31 Exocytosis of PDGF
Description
In this reaction, 1 molecule of 'Platelet-derived growth factor' is translocated from platelet dense granule lumen to extracellular region.
References
HN Antoniades, CD Scher, CD Stiles, "Purification of human platelet-derived growth factor", Proc Natl Acad Sci U S A, 76, 1979, 1809-13.
Reaction
12.1.2.3.3.3.2.32 Exocytosis of EGF

Description
In this reaction, 1 molecule of 'Pro-epidermal growth factor precursor' is translocated from platelet alpha granule lumen to extracellular region.
References
Y Oka, DN Orth, "Human plasma epidermal growth factor/beta-urogastrone is associated with blood platelets", J Clin Invest, 72, 1983, 249-59.
Reaction
12.1.2.3.3.3.2.33 Exocytosis of Insulin-like growth factor
Description
In this reaction, 1 molecule of 'Insulin-like growth factor' is translocated from platelet alpha granule lumen to extracellular region.
References
KP Karey, DA Sirbasku, "Human platelet-derived mitogens. II. Subcellular localization of insulinlike growth factor I to the alpha-granule and
release in response to thrombin.", Blood, 74, 1989, 1093-100.
Reaction
12.1.2.3.3.3.2.34 Exocytosis of VEGF

Description
In this reaction, 1 molecule of 'Vascular endothelial growth factor' is translocated from platelet alpha granule lumen to extracellular region.
References
U Wartiovaara, P Salven, H Mikkola, R Lassila, J Kaukonen, V Joukov, A Orpana, A Ristimaki, M Heikinheimo, H Joensuu, K Alitalo, A Palotie,
"Peripheral blood platelets express VEGF-C and VEGF which are released during platelet activation", Thromb Haemost, 80, 1998, 171-5.
Reaction
12.1.2.3.3.3.2.35 Exocytosis of alpha 2 antiplasmin
Description
In this reaction, 1 molecule of 'Alpha-2-antiplasmin' is translocated from platelet alpha granule lumen to extracellular region.
References
EF Plow, "The presence and release of alpha 2-antiplasmin from human platelets", Blood, 58, 1981, 1069-74.
Reaction
12.1.2.3.3.3.2.36 Exocytosis of Alpha Actinins
Authors
de Bono, B, Pace, N.P., Farndale, R, 2004-09-25.
Editors
Garapati, Phani Vijay, 2008-05-13.
Reviewers
Description
In this reaction, 1 molecule of each of the 'Alpha-actinin' alpha-actinin 1, alpha-actinin 2 and alpha-actini 4 is translocated from platelet alpha
granule lumen to extracellular region.
References
JA Coppinger, G Cagney, S Toomey, T Kislinger, O Belton, JP McRedmond, DJ Cahill, A Emili, DJ Fitzgerald, PB Maguire, "Characterization of
the proteins released from activated platelets leads to localization of novel platelet proteins in human atherosclerotic lesions", Blood, 103, 2004,
2096-104.
Reaction
12.1.2.3.3.3.2.37 Exocytosis of SRGN
Authors

Editors
Reviewers
References
2096-104.
Reaction
12.1.2.3.3.3.2.38 Exocytosis of Clusterin
Authors
Editors
Reviewers
Description
In this reaction, 1 molecule of 'Clusterin' is translocated from platelet alpha granule lumen to extracellular region.
References
2096-104.
Reaction
12.1.2.3.3.3.2.39 Exocytosis of Coagulation factor XIIIA
Authors
Editors
Reviewers
Description
In this reaction, 1 molecule of 'Coagulation factor XIIIA' is translocated from platelet alpha granule lumen to extracellular region.
References
2096-104.
Reaction
12.1.2.3.3.3.2.40 Exocytosis of Metalloproteinase inhibitor 1
Authors
Editors
Reviewers
Description
In this reaction, 1 molecule of 'Metalloproteinase inhibitor 1' is translocated from platelet alpha granule lumen to extracellular region.
References
2096-104.
Reaction
12.1.2.3.3.3.2.41 Exocytosis of Protein S
Authors
Editors
Reviewers
Description
In this reaction, 1 molecule of 'Protein S' is translocated from platelet alpha granule lumen to extracellular region.
References
2096-104.
Reaction
12.1.2.3.3.3.2.42 Exocytosis of Thymosin beta-4
Authors

Editors
Reviewers
Description
In this reaction, 1 molecule of 'Thymosin beta-4' is translocated from platelet alpha granule lumen to extracellular region.
Source reaction
This reaction was inferred from the corresponding reaction "Exocytosis of Thymosin beta-4" in species Mus musculus.
2096-104.
Reaction
12.1.2.3.3.3.2.43 Exocytosis of Fructose-bisphosphate aldolase
Authors

Editors
Reviewers
Description
In this reaction, 1 molecule of 'Fructose-bisphosphate aldolase' is translocated from platelet alpha granule lumen to extracellular region.
Source reaction
This reaction was inferred from the corresponding reaction "Exocytosis of Fructose-bisphosphate aldolase" in species Mus musculus.
2096-104.
Reaction
12.1.3 Plug Formation
Authors
de Bono, B, 2004-08-13.
Description
The tethering of platelets to the site of vascular injury is the first step in the formation of a platelet thrombus. Firm adhesion of these tethered
platelets, as well as the additional recruitment of others onto their surface leads to the formation of large platelet aggregates. The formation of a
thrombus is strictly dependent on the formation of interplatelet bonds.
12.1.3.1 Adhesion of alpha IIb beta 3 integrin to fibrin network
Authors
Geiger, B, Horwitz, R, 2008-05-07.
Reviewers
Description
At the beginning of this reaction, 1 molecule of 'fibrin multimer', and 1 molecule of 'Alpha IIb Beta 3 Integrin' are present. At the end of this
reaction, 1 molecule of 'Integrin alpha IIb beta 3:Fibrin complex' is present.
References
SJ Shattil, "Signaling through platelet integrin alpha IIb beta 3: inside-out, outside-in, and sideways", Thromb Haemost, 82, 1999, 318-25.
Reaction
12.2 Formation of Fibrin Clot (Clotting Cascade)
Authors
Description
The formation of a fibrin clot at the site of an injury to the wall of a normal blood vessel is an essential part of the process to stop blood loss after
vascular injury. The reactions that lead to fibrin clot formation are commonly described as a cascade, in which the product of each step is an
enzyme or cofactor needed for following reactions to proceed efficiently. The entire clotting cascade can be divided into three portions, the
extrinsic pathway, the intrinsic pathway, and the common pathway. The extrinsic pathway begins with the release of tissue factor at the site of
vascular injury and leads to the activation of factor X. The intrinsic pathway provides an alternative mechanism for activation of factor X, starting
from the activation of factor XII. The common pathway consists of the steps linking the activation of factor X to the formation of a multimeric,
cross-linked fibrin clot. Each of these pathways includes not only a cascade of events that generate the catalytic activities needed for clot
formation, but also numerous positive and negative regulatory events.
References
KG Mann, S Butenas, "The dynamics of thrombin formation", Arterioscler Thromb Vasc Biol, 23, 2003, 17-25.
EW Davie, K Fujikawa, W Kisiel, "The coagulation cascade: initiation, maintenance, and regulation", Biochemistry, 30, 1991, 10363-70.
12.2.1 Extrinsic Pathway
Authors
Description
Factor VII, the protease that initiates the normal blood clotting cascade, circulates in the blood in both its proenzyme (factor VII) and its activated
(factor VIIa) forms. No clotting occurs, however, because neither form of the protein has any catalytic activity when free in solution. Blood clotting
is normally initiated when tissue factor (TF), an intrinsic plasma membrane protein, is exposed to the blood by injury to the wall of a blood vessel.
TF is then able to bind factor VIIa from plasma, and possibly also factor VII, to form complexes capable of catalyzing the conversion of factor X,
from plasma, into its activated form, factor Xa. Factor Xa catalyzes the conversion of additional factor VII molecules to their activated form,
increasing the amount of tissue factor:factor VIIa complex available at the site of injury, accelerating the generation of factor Xa, and allowing the
activation of factor IXa as well. This process is self-limiting because as levels of factor Xa increase, tissue factor:factor VIIa complexes become
trapped in the form of catalytically inactive heterotetramers with factor Xa and the protein TFPI (tissue pathway factor inhibitor). At this point the
intinsic pathway, as an independent source of activated factor X, is thought to become critical for the continuation of clot formation (Broze 1995;
Mann et al. 2003).
The nature of the initial tissue factor:factor VII complexes formed is controversial. One model, building on the observation that the complex of
factor VII and TF has low but measurable proteolytic activity on factor X, suggests that this complex begins the activation of factor X, and that as
factor VIIa accumulates, tissue factor:factor VIIa complexes also form, accelerating the process (Nemerson 1988). A second model, building on
the observation that normal plasma contains low levels of activated factor VII constitutively, suggests that complexes with factor VIIa form
immediately at the onset of clotting (Rapaport and Rao 1995). The two models are not mutually exclusive, and in any event, the central roles of
tissue factor and factor VIIa in generating an initial supply of factors IXa and Xa, and the self-limiting nature of the process due to the action of
TFPI, are all well-established.
These events are outlined in the drawing: black arrows connect the substrates (inputs) and products (outputs) of individual reactions, and blue
lines connect output activated enzymes to the other reactions that they catalyze.
References
KG Mann, S Butenas, "The dynamics of thrombin formation", Arterioscler Thromb Vasc Biol, 23, 2003, 17-25.
Y Nemerson, "Tissue factor and hemostasis", Blood, 71, 1988, 1-8.
SI Rapaport, LV Rao, "The tissue factor pathway: how it has become a "prima ballerina"", Thromb Haemost, 74, 1995, 7-17.
GJ Jr Broze, "Tissue factor pathway inhibitor and the revised theory of coagulation", Annu Rev Med, 46, 1995, 103-12.
12.2.1.1 sequestered tissue factor -> tissue factor
Authors
Description
Tissue factor sequestered in the wall of a blood vessel is exposed to circulating blood when the endothelial lining of the vessel is injured.
References
JN Wilcox, KM Smith, SM Schwartz, D Gordon, "Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque", Proc
Natl Acad Sci U S A, 86, 1989, 2839-43.
Reaction
12.2.1.2 tissue factor (TF) + factor VII (F7) -> TF:F7 complex
Authors
Description
Tissue factor exposed at the endothelial cell surface forms a complex with factor VII from plasma.
References
R Bach, R Gentry, Y Nemerson, "Factor VII binding to tissue factor in reconstituted phospholipid vesicles: induction of cooperativity by
phosphatidylserine", Biochemistry, 25, 1986, 4007-20.
Reaction
12.2.1.3 factor X -> factor Xa + factor X activation peptide (TF:F7 catalyst)
Authors
Description
Factor VII, bound to tissue factor at the endothelial cell surface, catalyzes the activation of factor X from plasma with moderate efficiency. The
amino terminal part of the heavy chain of factor X, the factor X activation peptide, is released. (This peptide has no known function.)
References
GJ Jr Broze, "Binding of human factor VII and VIIa to monocytes", J Clin Invest, 70, 1982, 526-35.
R Bach, R Gentry, Y Nemerson, "Factor VII binding to tissue factor in reconstituted phospholipid vesicles: induction of cooperativity by
phosphatidylserine", Biochemistry, 25, 1986, 4007-20.
Y Nemerson, "Tissue factor and hemostasis", Blood, 71, 1988, 1-8.
Reaction
12.2.1.4 factor VII -> factor VIIa
Authors
Description
Factor Xa catalyzes the activation of factor VII from plasma.
References
S Butenas, KG Mann, "Kinetics of human factor VII activation", Biochemistry, 35, 1996, 1904-10.
Reaction
12.2.1.5 tissue factor (TF) + activated factor VII (F7a) -> TF:F7a complex
Authors
Description
Tissue factor exposed at the endothelial cell surface forms a complex with F7a (activated factor VII) from the plasma
References
DW Banner, A D'Arcy, C Chene, FK Winkler, A Guha, WH Konigsberg, Y Nemerson, D Kirchhofer, "The crystal structure of the complex of blood
coagulation factor VIIa with soluble tissue factor", Nature, 380, 1996, 41-6.
Reaction
12.2.1.6 factor X -> factor Xa + factor X activation peptide (TF:F7a catalyst)
Authors
Description
Factor VIIa, bound to tissue factor at the endothelial cell surface (the "extrinsic tenase complex"), catalyzes the formation of activated factor X
with high efficiency. The amino terminal part of the heavy chain of factor X, the factor X activation peptide, is released. (This peptide has no
known function.)
References
GJ Jr Broze, "Binding of human factor VII and VIIa to monocytes", J Clin Invest, 70, 1982, 526-35.
Y Komiyama, AH Pedersen, W Kisiel, "Proteolytic activation of human factors IX and X by recombinant human factor VIIa: effects of calcium,
phospholipids, and tissue factor", Biochemistry, 29, 1990, 9418-25.
SI Rapaport, LV Rao, "The tissue factor pathway: how it has become a "prima ballerina"", Thromb Haemost, 74, 1995, 7-17.
Reaction
12.2.1.7 factor IX -> factor IXa + factor IX activation peptide (TF:F7a catalyst)
Authors
Description
Factor VIIa, bound to tissue factor at the endothelial cell surface, catalyzes the formation of activated factor IX with high efficiency. The amino
terminal part of the heavy chain of factor IX, the factor IX activation peptide, is released. (This peptide has no known function.)
References
JH Lawson, KG Mann, "Cooperative activation of human factor IX by the human extrinsic pathway of blood coagulation", J Biol Chem, 266,
1991, 11317-27.
Y Komiyama, AH Pedersen, W Kisiel, "Proteolytic activation of human factors IX and X by recombinant human factor VIIa: effects of calcium,
phospholipids, and tissue factor", Biochemistry, 29, 1990, 9418-25.
Reaction
12.2.1.8 TFPI + TF:F7a + factor Xa -> TFPI:TF:F7a:factor Xa
Authors
Description
TFPI binds to the factor VIIa:TF complex and to factor Xa at the endothelial surface, forming a stable heterotetrameric complex in which factor
VIIa is catalytically inactive.
References
GJ Jr Broze, TJ Girard, WF Novotny, "Regulation of coagulation by a multivalent Kunitz-type inhibitor", Biochemistry, 29, 1990, 7539-46.
Reaction
12.2.2 Intrinsic Pathway
Authors
Editors
Reviewers
Rush, MG, 2008-01-11.

Description
The intrinsic pathway of blood clotting connects interactions among kininogen (high molecular weight kininogen, HK), prekallikrein (PK), and
factor XII to the activation of clotting factor X by a series of reactions that is independent of the extrinsic pathway and that is not subject to
inhibition by TFPI. It is thus essential for the prolongation of the clotting cascade: while the reactions of the extrinsic pathway appear to be
sufficient to initiate clot formation, those of the intrinsic pathway are required to maintain it (Broze 1995; Davie et al. 1991; Monroe et al. 2002).
The intrinsic pathway can be divided into three parts: 1) reactions involving interactions of kininogen, prekallikrein, and factor XII, leading to the
activation of factor XII, 2) reactions involving factor XI, factor IX, factor VIII, and von Willebrand factor (vWF) leading to the activation of factors
VIII and IX, and 3) reactions that inactivate factor XIIa and kallikrein.
Kininogen, prekallikrein, and factor XII were first identified as proteins needed for the rapid formation of clots when whole blood is exposed to
negatively charged surfaces in vitro. Studies in vitro have identified several possible sets of interactions, in which small quantities of one or more
of these proteins 'autoactivate' and then catalyze the formation of larger quantities of activated factors. Recent work, however, suggests that
these factors form complexes on endothelial cell surfaces mediated by C1q binding protein (C1q bp), that the first activation event is the
cleavage of prekallikrein by prolylcarboxypeptidase, and that the resulting kallikrein catalyzes the activation of factor XII (Schmaier 2004).
The second group of events, occurs in vivo on the surfaces of activated platelets (although most biochemical characterization of the reactions
was originally done with purified proteins in solution). Factor XI binds to the platelet glycoprotein (GP) Ib:IX:V complex, where it can be activated
by cleavage either by thrombin (generated by reactions of the common pathway) or by activated factor XII (generated in the first part of the
intrinsic pathway). Activated factor XI in turn catalyzes the activation of factor IX. Simultaneously, factor VIII, complexed with vWF, is cleaved by
thrombin, activating it and causing its release from vWF. Activated factors VIII and IX form a complex on the platelet surface that very efficiently
converts factor X to activated factor X. (Activated factors X and V then form a complex that efficiently activates thrombin.)
While these two groups of events can be viewed as forming a single functional pathway (e.g., Davie et al. 1991), human clinical genetic data
cast doubt on this view. Individuals deficient in kininogen, prekallikrein, or factor XII proteins exhibit normal blood clot formation in vivo. In
contrast, deficiencies of factor XI can be associated with failure of blood clotting under some conditions, and deficiencies of vWF, factor VIII, or
factor IX cause severe abnormalities - von Willebrand disease, hemophilia A, and hemophilia B, respectively. These data suggest that while the
second group of events is essential for normal clot formation in vivo, the first group has a different function (e.g., Schmaier 2004).
Finally, reactions neutralize proteins activated in the first part of the intrinsic pathway. Kallikrein forms stable complexes with either C1 inhibitor
(C1Inh) or with alpha2-macroglobulin, and factor XIIa forms stable complexes with C1Inh. The relevance of these neutralization events to the
regulation of blood clotting is unclear, however. The physiological abnormalities observed in individuals who lack C1Inh appear to be due entirely
to abnormalities of complement activation; blood clotting appears to proceed normally. This observation is consistent with the hypothesis, above,
that factor XIIa plays a limited role in normal blood clotting under physiological conditions.
These events are outlined in the drawing: black arrows connect the substrates (inputs) and products (outputs) of individual reactions; blue lines
connect activated enzymes to the reactions they catalyze.
References
AH Schmaier, "The physiologic basis of assembly and activation of the plasma kallikrein/kinin system", Thromb Haemost, 91, 2004, 1-3.
GJ Jr Broze, "Tissue factor pathway inhibitor and the revised theory of coagulation", Annu Rev Med, 46, 1995, 103-12.
DM Monroe, M Hoffman, HR Roberts, "Platelets and thrombin generation", Arterioscler Thromb Vasc Biol, 22, 2002, 1381-9.
12.2.2.1 kininogen + C1q binding protein tetramer -> kininogen:C1q binding protein tetramer
Authors
Editors
Description
Kininogen (high molecular weight kininogen; HK) associates with C1q binding protein on the cell surface in a reaction dependent on Zn++
(Joseph et al. 1996). In the body, the Zn++ needed to drive this reaction may be provided locally by Zn++ release from activated platelets (Mahdi
et al. 2002). The C1q binding protein is inferred to form tetramers based on the properties of purified recombinant protein in vitro (Ghebrehiwet et
al. 1994); the stoichiometry of the cell surface complex has not been determined directly.
References
K Joseph, B Ghebrehiwet, EI Peerschke, KB Reid, AP Kaplan, "Identification of the zinc-dependent endothelial cell binding protein for high
molecular weight kininogen and factor XII: identity with the receptor that binds to the globular "heads" of C1q (gC1q-R)", Proc Natl
Acad Sci U S A, 93, 1996, 8552-7.
DM Kerbiriou, JH Griffin, "Human high molecular weight kininogen. Studies of structure-function relationships and of proteolysis of the molecule
occurring during contact activation of plasma.", J Biol Chem, 254, 1979, 12020-7.
F Mahdi, Z Shariat-Madar, CD Figueroa, AH Schmaier, "Factor XII interacts with the multiprotein assembly of urokinase plasminogen activator
receptor, gC1qR, and cytokeratin 1 on endothelial cell membranes", Blood, 99, 2002, 3585-96.
B Ghebrehiwet, BL Lim, EI Peerschke, AC Willis, KB Reid, "Isolation, cDNA cloning, and overexpression of a 33-kD cell surface glycoprotein that
binds to the globular "heads" of C1q", J Exp Med, 179, 1994, 1809-21.
Reaction
12.2.2.2 prekallikrein + kininogen:C1q binding protein tetramer -> prekallikrein:kininogen:C1q binding protein tetramer
Authors
Description
Prekallikrein (PK) associates specifically with kininogen (HK) on cell surfaces. In vivo, this reaction may occur primarily on the surfaces of
endothelial cells in response to platelet activation (Lin et al. 1997; Motta et al. 1998; Mahdi et al. 2003).
References
G Motta, R Rojkjaer, AA Hasan, DB Cines, AH Schmaier, "High molecular weight kininogen regulates prekallikrein assembly and activation on
endothelial cells: a novel mechanism for contact activation", Blood, 91, 1998, 516-28.
F Mahdi, Z Shariat-Madar, AH Schmaier, "The relative priority of prekallikrein and factors XI/XIa assembly on cultured endothelial cells", J Biol
Chem, 278, 2003, 43983-90.
DW Chung, K Fujikawa, BA McMullen, EW Davie, "Human plasma prekallikrein, a zymogen to a serine protease that contains four tandem
repeats", Biochemistry, 25, 1986, 2410-7.
Y Lin, RB Harris, W Yan, KR McCrae, H Zhang, RW Colman, "High molecular weight kininogen peptides inhibit the formation of kallikrein on
endothelial cell surfaces and subsequent urokinase-dependent plasmin formation", Blood, 90, 1997, 690-7.
Reaction
12.2.2.3 prekallikrein:kininogen:C1q binding protein tetramer -> kallikrein:kininogen:C1q binding protein tetramer
Authors
Editors
Description
Prekallikrein in a complex with kininogen and C1q binding protein on the plasma membrane is cleaved to generate active kallikrein, which
remains bound to the complex. In the body, this reaction appears to occur on the surfaces of endothelial cells and may require the presence of
activated platelets. Recent work indicates that the protease that cleaves prekallikrein under these conditions is prolylcarboxypeptidase. Although
this enzyme was originally isolated from lysosomes (Odya et al. 1978; Tan et al. 1993), it is associated with plasma membranes of cultured
human endothelial cells in vitro (Moreira et al. 2002; Shariat-Madar et al. 2002), and the purified recombinant enzyme efficiently cleaves
prekallikrein (Shariat-Madar et al. 2004). In contrast factor XII, despite its activity on prekallikrein in vitro, appears not to be responsible for
prekallikrein activation on the cell surface (Rojkjaer et al. 1998).
References
CR Moreira, AH Schmaier, F Mahdi, G Motta, HB Nader, Z Shariat-Madar, "Identification of prolylcarboxypeptidase as the cell matrix-associated
prekallikrein activator", FEBS Lett, 523, 2002, 167-70.
F Tan, PW Morris, RA Skidgel, EG Erdos, "Sequencing and cloning of human prolylcarboxypeptidase (angiotensinase C). Similarity to both
serine carboxypeptidase and prolylendopeptidase families.", J Biol Chem, 268, 1993, 16631-8.
CE Odya, DV Marinkovic, KJ Hammon, TA Stewart, EG Erdos, "Purification and properties of prolylcarboxypeptidase (angiotensinase C) from
human kidney", J Biol Chem, 253, 1978, 5927-31.
Z Shariat-Madar, F Mahdi, AH Schmaier, "Identification and characterization of prolylcarboxypeptidase as an endothelial cell prekallikrein
activator", J Biol Chem, 277, 2002, 17962-9.
R Rojkjaer, AA Hasan, G Motta, I Schousboe, AH Schmaier, "Factor XII does not initiate prekallikrein activation on endothelial cells", Thromb
Haemost, 80, 1998, 74-81.
Z Shariat-Madar, F Mahdi, AH Schmaier, "Recombinant prolylcarboxypeptidase activates plasma prekallikrein", Blood, 103, 2004, 4554-61.
Reaction
12.2.2.4 kallikrein:kininogen:C1q binding protein tetramer -> kallikrein + activated kininogen:C1q binding protein tetramer + bradykinin
Authors
Editors
Description
The cleavage of kininogen (HK, high molecular weight kininogen) yields activated kininogen and the vasoactive peptide bradykinin (Kerbirou and
Griffin 1979; Lottspeich et al. 1985; Kellerman et al. 1986). In vivo, this reaction is catalyzed by activated kallikrein, takes places within the
kallikrein:kininogen:C1q binding protein tetramer complex on the endothelial cell surface, and results in the release of kallikrein and bradykinin
(Motta et al. 1998). The hormonal functions of bradykinin will be annotated in a future version of Reactome.
References
G Motta, R Rojkjaer, AA Hasan, DB Cines, AH Schmaier, "High molecular weight kininogen regulates prekallikrein assembly and activation on
endothelial cells: a novel mechanism for contact activation", Blood, 91, 1998, 516-28.
DM Kerbiriou, JH Griffin, "Human high molecular weight kininogen. Studies of structure-function relationships and of proteolysis of the molecule
occurring during contact activation of plasma.", J Biol Chem, 254, 1979, 12020-7.
F Lottspeich, J Kellermann, A Henschen, B Foertsch, W Muller-Esterl, "The amino acid sequence of the light chain of human
high-molecular-mass kininogen", Eur J Biochem, 152, 1985, 307-14.
J Kellermann, F Lottspeich, A Henschen, W Muller-Esterl, "Completion of the primary structure of human high-molecular-mass kininogen. The
amino acid sequence of the entire heavy chain and evidence for its evolution by gene triplication.", Eur J Biochem, 154, 1986, 471-8.
Reaction
12.2.2.5 factor XII -> factor XIIa
Authors
Editors
Description
Cleavage of a single peptide bond converts factor XII to activated factor XII (factor XIIa) (Fujikawa and McMullen 1983; McMullen and Fujikawa
1985). Identification of the catalytic activity or activities responsible for this cleavage has not been straightforward. Studies in vitro have
demonstrated the autoactivation of factor XII as well as activation by kallikrein. Both reactions require the presence of negatively charged
surfaces and are accelerated in the presence of kininogen (high molecular weight kininogen, HK) (Griffin and Cochrane 1976; Meier et al. 1977;
Silverberg et al. 1980). Recent work suggests that factor XII activation in vivo may occur primarily on endothelial cell surfaces and that, as in
vitro, association with kininogen may accelerate the reaction (Mahdi et al. 2002; Schmaier 2004), although alternative pathways and alternative
mechanisms for associating factor XII with the cell surface have not been excluded (Joseph et al. 2001).
References
BA McMullen, K Fujikawa, "Amino acid sequence of the heavy chain of human alpha-factor XIIa (activated Hageman factor)", J Biol Chem, 260,
1985, 5328-41.
K Fujikawa, BA McMullen, "Amino acid sequence of human beta-factor XIIa", J Biol Chem, 258, 1983, 10924-33.
HL Meier, JV Pierce, RW Colman, AP Kaplan, "Activation and function of human Hageman factor. The role of high molecular weight kininogen
and prekallikrein.", J Clin Invest, 60, 1977, 18-31.
K Joseph, Y Shibayama, B Ghebrehiwet, AP Kaplan, "Factor XII-dependent contact activation on endothelial cells and binding proteins gC1qR
and cytokeratin 1", Thromb Haemost, 85, 2001, 119-24.
F Mahdi, Z Shariat-Madar, CD Figueroa, AH Schmaier, "Factor XII interacts with the multiprotein assembly of urokinase plasminogen activator
receptor, gC1qR, and cytokeratin 1 on endothelial cell membranes", Blood, 99, 2002, 3585-96.
M Silverberg, JT Dunn, L Garen, AP Kaplan, "Autoactivation of human Hageman factor. Demonstration utilizing a synthetic substrate.", J Biol
Chem, 255, 1980, 7281-6.
JH Griffin, CG Cochrane, "Mechanisms for the involvement of high molecular weight kininogen in surface-dependent reactions of Hageman
factor", Proc Natl Acad Sci U S A, 73, 1976, 2554-8.
Reaction
12.2.2.6 factor XI + platelet glycoprotein (GP) Ib:IX:V complex -> factor XI:platelet glycoprotein (GP) Ib:IX:V complex
Authors
Editors
Description
Plasma factor XI binds to the platelet glycoprotein Ib:IX:V complex (Baglia et al. 2002; Greengard et al. 1986). In the body, this reaction occurs
specifically on the surfaces of activated platelets, but not on endothelial cells (Baird and Walsh 2002). The stoichiometry of the platelet
glycoprotein Ib:IX:V complex has not been established directly, but is inferred from the relative abundances of its components in platelet
membranes (Modderman et al. 1992; Shrimpton et al. 2002).
References
JS Greengard, MJ Heeb, E Ersdal, PN Walsh, JH Griffin, "Binding of coagulation factor XI to washed human platelets", Biochemistry, 25, 1986,
3884-90.
CN Shrimpton, G Borthakur, S Larrucea, MA Cruz, JF Dong, JA Lopez, "Localization of the adhesion receptor glycoprotein Ib-IX-V complex to
lipid rafts is required for platelet adhesion and activation", J Exp Med, 196, 2002, 1057-66.
FA Baglia, KO Badellino, CQ Li, JA Lopez, PN Walsh, "Factor XI binding to the platelet glycoprotein Ib-IX-V complex promotes factor XI
activation by thrombin", J Biol Chem, 277, 2002, 1662-8.
TR Baird, PN Walsh, "The interaction of factor XIa with activated platelets but not endothelial cells promotes the activation of factor IX in the
consolidation phase of blood coagulation", J Biol Chem, 277, 2002, 38462-7.
PW Modderman, LG Admiraal, A Sonnenberg, AE von dem Borne, "Glycoproteins V and Ib-IX form a noncovalent complex in the platelet
membrane", J Biol Chem, 267, 1992, 364-9.
Reaction
12.2.2.7 factor XI:platelet glycoprotein (GP) Ib:IX:V complex -> factor XIa:platelet glycoprotein (GP) Ib:IX:V complex (XIIa catalyst)
Authors
Editors
Description
Factor XI, bound to the cell surface, is converted to activated factor XI (factor XIa). Chemically, this reaction involves the cleavage of a single
peptide bond in each subunit of the factor XI homodimer; intra- and inter-chain disulfide bonds hold the resulting four polypeptides together
(Bouma and Griffin 1977; Kurachi and Davie 1977; McMullen et al. 1991). In the body, this reaction occurs on the surfaces of activated platelets
(Greengard et al. 1986; Baglia et al. 2002; Baird and Walsh 2002); when this reaction occurs as a step in the intrinsic ("contact") pathway of
blood coagulation, it is catalyzed by activated factor XIIa (Kurachi and Davie 1977, Baglia and Walsh 2000) which in turn is generated through
the interactions of factor XII, kallikrein, and kininogen on endothelial cell surfaces (Schmaier 2004).
References
K Kurachi, EW Davie, "Activation of human factor XI (plasma thromboplastin antecedent) by factor XIIa (activated Hageman factor)",
Biochemistry, 16, 1977, 5831-9.
FA Baglia, PN Walsh, "Thrombin-mediated feedback activation of factor XI on the activated platelet surface is preferred over contact activation
by factor XIIa or factor XIa", J Biol Chem, 275, 2000, 20514-9.
BA McMullen, K Fujikawa, EW Davie, "Location of the disulfide bonds in human coagulation factor XI: the presence of tandem apple domains",
Biochemistry, 30, 1991, 2056-60.
JS Greengard, MJ Heeb, E Ersdal, PN Walsh, JH Griffin, "Binding of coagulation factor XI to washed human platelets", Biochemistry, 25, 1986,
3884-90.
BN Bouma, JH Griffin, "Human blood coagulation factor XI. Purification, properties, and mechanism of activation by activated factor XII.", J Biol
Chem, 252, 1977, 6432-7.
TR Baird, PN Walsh, "The interaction of factor XIa with activated platelets but not endothelial cells promotes the activation of factor IX in the
consolidation phase of blood coagulation", J Biol Chem, 277, 2002, 38462-7.
Reaction
12.2.2.8 factor XI:platelet glycoprotein (GP) Ib:IX:V complex -> factor XIa:platelet glycoprotein (GP) Ib:IX:V complex (thrombin catalyst)
Authors
Editors
Description
Factor XI, bound to the cell surface, is converted to activated factor XI (factor XIa). In the body, this reaction occurs on the surfaces of activated
platelets (Baglia et al. 2002). Small quantities of factor XI can be activated in a reaction catalyzed by factor XIIa, to initiate formation of a fibrin
clot. However, the efficient activation of larger quantities of factor XI, needed to propagate the blood clotting process, appears to be mediated by
thrombin (Baglia and Walsh 2000; Gailani and Broze 1993; Naito and Fujikawa 1991; Oliver et al. 1999; Monroe et al. 2002).
References
D Gailani, GJ Jr Broze, "Factor XII-independent activation of factor XI in plasma: effects of sulfatides on tissue factor-induced coagulation",
Blood, 82, 1993, 813-9.
FA Baglia, PN Walsh, "Thrombin-mediated feedback activation of factor XI on the activated platelet surface is preferred over contact activation
by factor XIIa or factor XIa", J Biol Chem, 275, 2000, 20514-9.
JA Oliver, DM Monroe, HR Roberts, M Hoffman, "Thrombin activates factor XI on activated platelets in the absence of factor XII", Arterioscler
Thromb Vasc Biol, 19, 1999, 170-7.
K Naito, K Fujikawa, "Activation of human blood coagulation factor XI independent of factor XII. Factor XI is activated by thrombin and factor XIa
in the presence of negatively charged surfaces.", J Biol Chem, 266, 1991, 7353-8.
DM Monroe, M Hoffman, HR Roberts, "Platelets and thrombin generation", Arterioscler Thromb Vasc Biol, 22, 2002, 1381-9.
Reaction
12.2.2.9 factor IX -> factor IXa + factor IX activation peptide (factor XIa catalyst)
Authors
Editors
Description
Factor XIa, bound to platelet glycoprotein (GP) Ib:IX:V on the platelet cell surface, catalyzes the formation of activated factor IX with high
efficiency in a reaction that requires Ca++. The amino terminal part of the heavy chain of factor IX, the factor IX activation peptide, is released.
(This peptide has no known function.)
References
B Osterud, BN Bouma, JH Griffin, "Human blood coagulation factor IX. Purification, properties, and mechanism of activation by activated factor
XI.", J Biol Chem, 253, 1978, 5946-51.
D Gailani, D Ho, MF Sun, Q Cheng, PN Walsh, "Model for a factor IX activation complex on blood platelets: dimeric conformation of factor XIa is
essential", Blood, 97, 2001, 3117-22.
Reaction
12.2.2.10 factor VIII + von Willebrand factor multimer -> factor VIII:von Willibrand factor multimer
Authors
Editors
Description
Factor VIII binds to von Willebrand factor to form a complex. This complex stabilizes factor VIII, which otherwise has a very short half-life in the
blood.
Factor VIII (Vehar et al. 1984) is a heterodimer containing a heavy and a light polypeptide chain, generated by the proteolytic cleavage of a
single large precursor polypeptide. Several forms of the heavy chain are found in vivo, all functionally the same but differing in the amount of the
B domain removed by proteolysis. The single form annotated here is the shortest one (Eaton et al. 1986; Hill-Eubanks et al. 1989).
In vitro, von Willebrand factor (Titani et al. 1986) can form complexes with factor VIII with a 1:1 stoichiometry. The complexes that form in vivo,
however, involve large multimers of von Willebrand factor and varied, but always low, proportions of factor VIII (Vlot et al. 1995). A stoichiometry
of one molecule of factor VIII associated with 50 of von Willebrand factor is typical in vivo, and is used here to annotate the factor VIII:von
Willebrand factor complex.
References
P Lollar, DC Hill-Eubanks, CG Parker, "Association of the factor VIII light chain with von Willebrand factor", J Biol Chem, 263, 1988, 10451-5.
AJ Vlot, SJ Koppelman, MH van den Berg, BN Bouma, JJ Sixma, "The affinity and stoichiometry of binding of human factor VIII to von
Willebrand factor", Blood, 85, 1995, 3150-7.
GA Vehar, B Keyt, D Eaton, H Rodriguez, DP O'Brien, F Rotblat, H Oppermann, R Keck, WI Wood, RN Harkins, EG Tuddenham, RM Lawn, DJ
Capon, "Structure of human factor VIII", Nature, 312, 1984, 337-42.
K Titani, S Kumar, K Takio, LH Ericsson, RD Wade, K Ashida, KA Walsh, MW Chopek, JE Sadler, K Fujikawa, "Amino acid sequence of human
von Willebrand factor", Biochemistry, 25, 1986, 3171-84.
D Eaton, H Rodriguez, GA Vehar, "Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and
activated protein C with activation and inactivation of factor VIII coagulant activity.", Biochemistry, 25, 1986, 505-12.
DC Hill-Eubanks, CG Parker, P Lollar, "Differential proteolytic activation of factor VIII-von Willebrand factor complex by thrombin", Proc Natl
Acad Sci U S A, 86, 1989, 6508-12.
Reaction
12.2.2.11 factor VIII:von Willibrand factor multimer -> factor VIIIa + factor VIIIa B A3 acidic polypeptide + von Willibrand factor multimer
Authors
Editors
Description
Factor VIII complexed to von Willibrand factor in the blood is cleaved into several smaller polypeptides that remain associated. The acidic
polypeptide on the aminoterminal side of the A3 domain of the light chain is released, however, and as this polypeptide mediates the association
of factor VIII with von Willibrand factor, the activated factor VIII is released. While several proteases are capable of catalyzing these cleavages in
vitro, only thrombin is active on factor VIII:von Willibrand factor complexes under physiological conditions (Eaton et al. 1986; Hill-Eubanks et al.
1989; Lollar et al. 1988; Pieters et al. 1989)
References
P Lollar, DC Hill-Eubanks, CG Parker, "Association of the factor VIII light chain with von Willebrand factor", J Biol Chem, 263, 1988, 10451-5.
J Pieters, T Lindhout, HC Hemker, "In situ-generated thrombin is the only enzyme that effectively activates factor VIII and factor V in
thromboplastin-activated plasma", Blood, 74, 1989, 1021-4.
D Eaton, H Rodriguez, GA Vehar, "Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and
activated protein C with activation and inactivation of factor VIII coagulant activity.", Biochemistry, 25, 1986, 505-12.
DC Hill-Eubanks, CG Parker, P Lollar, "Differential proteolytic activation of factor VIII-von Willebrand factor complex by thrombin", Proc Natl
Acad Sci U S A, 86, 1989, 6508-12.
Reaction
12.2.2.12 factor VIIIa + factor IXa -> factor VIIIa:factor IXa

Authors
Editors
Description
The activated forms of factors VIII and IX associate on a cell surface to form a complex that very efficiently catalyzes the activation of factor X,
the so-called "intrinsic tenase complex". In vitro, negatively charged phospholipids can provide an appropriate surface. In the body, the surface is
provided by the plasma membranes of activated platelets (Gilbert and Arena 1996).
References
GE Gilbert, AA Arena, "Activation of the factor VIIIa-factor IXa enzyme complex of blood coagulation by membranes containing
phosphatidyl-L-serine", J Biol Chem, 271, 1996, 11120-5.
Reaction
12.2.2.13 factor X -> factor Xa + factor X activation peptide (VIIIa:IXa catalyst)
Authors
Editors
Description
Factor IXa, in a complex with factor VIIIa on the surfaces of activated platelets (the "intrinsic tenase complex"), catalyzes the formation of
activated factor X with high efficiency. The amino terminal part of the heavy chain of factor X, the factor X activation peptide, is released. (This
peptide has no known function.)
References
R Rawala-Sheikh, SS Ahmad, B Ashby, PN Walsh, "Kinetics of coagulation factor X activation by platelet-bound factor IXa", Biochemistry, 29,
1990, 2606-11.
Reaction
12.2.2.14 kallikrein + C1Inh -> kallikrein:C1Inh
Authors
Editors
Description
Activated kallikrein binds to C1Inh (plasma protease C1 inhibitor) (Bock et al. 1986), forming a stable and enzymatically inactive complex. This
reaction appears to be the major means by which kallikrein is inactivated (kallikrein can also be inactivated by binding to alpha2-macroglobulin)
(Harpel et al. 1985; Ratnoff et al. 1969).
References
SC Bock, K Skriver, E Nielsen, HC Thogersen, B Wiman, VH Donaldson, RL Eddy, J Marrinan, E Radziejewska, R Huber, TB Shows, S
Magnusson, "Human C1 inhibitor: primary structure, cDNA cloning, and chromosomal localization", Biochemistry, 25, 1986, 4292-301.
PC Harpel, MF Lewin, AP Kaplan, "Distribution of plasma kallikrein between C-1 inactivator and alpha 2-macroglobulin in plasma utilizing a new
assay for alpha 2-macroglobulin-kallikrein complexes", J Biol Chem, 260, 1985, 4257-63.
OD Ratnoff, J Pensky, D Ogston, GB Naff, "The inhibition of plasmin, plasma kallikrein, plasma permeability factor, and the C'1r subcomponent
of the first component of complement by serum C'1 esterase inhibitor", J Exp Med, 129, 1969, 315-31.
Reaction
12.2.2.15 kallikrein + alpha2-macroglobulin -> kallikrein:alpha2-macrogloulin
Authors
Description
Activated kallikrein binds to alpha2-macroglobulin (Sottrup-Jensen et al. 1984), forming a stable and enzymatically inactive complex. Under
normal conditions in vivo, this reaction appears to be responsible for the inactivation of about 1/6 of activated kallikrein (with C1Inh responsible
for the inactivation of about 5/6) (Harpel et al. 1985).
References
L Sottrup-Jensen, TM Stepanik, T Kristensen, DM Wierzbicki, CM Jones, PB Lonblad, S Magnusson, TE Petersen, "Primary structure of human
alpha 2-macroglobulin. V. The complete structure.", J Biol Chem, 259, 1984, 8318-27.
PC Harpel, MF Lewin, AP Kaplan, "Distribution of plasma kallikrein between C-1 inactivator and alpha 2-macroglobulin in plasma utilizing a new
assay for alpha 2-macroglobulin-kallikrein complexes", J Biol Chem, 260, 1985, 4257-63.
Reaction
12.2.2.16 factor XIIa + C1Inh -> factor XIIa:C1Inh

Authors
Editors
Description
Activated factor XII (factor XIIa) binds to C1Inh (C1 inhibitor - Bock et al. 1986) to form a stable, inactive complex (Schneider et al. 1973). While
several protease inhibitors can form stable complexes with XIIa in vitro, only C1Inh does so to a significant extent under normal conditions in
vivo (Pixley et al. 1985).
References
SC Bock, K Skriver, E Nielsen, HC Thogersen, B Wiman, VH Donaldson, RL Eddy, J Marrinan, E Radziejewska, R Huber, TB Shows, S
Magnusson, "Human C1 inhibitor: primary structure, cDNA cloning, and chromosomal localization", Biochemistry, 25, 1986, 4292-301.
AD Schreiber, AP Kaplan, KF Austen, "Inhibition by C1INH of Hagemann factor fragment activation of coagulation, fibrinolysis, and kinin
generation", J Clin Invest, 52, 1973, 1402-9.
RA Pixley, M Schapira, RW Colman, "The regulation of human factor XIIa by plasma proteinase inhibitors", J Biol Chem, 260, 1985, 1723-9.
Reaction
12.2.3 Common Pathway
Authors
Description
The common pathway consists of the cascade of activation events leading from the formation of activated factor X to the formation of active
thrombin, the cleavage of fibrinogen by thrombin, and the formation of cleaved fibrin into a stable multimeric, cross-linked complex. Thrombin
also efficiently catalyzes the activation of several factors required earlier in the clotting cascade, thus acting in effect as a positive regulator of
clotting. At the same time, thrombin activates protein C, which in turn catalyzes the inactivation of several of these upstream factors, thereby
limiting the clotting process. Thrombin can also be trapped in a stable, inactive complex with antithrombin-3, a circulating blood protein. The
quantitative interplay among these positive and negative modulators is critical to the normal regulation of clotting, facilitating the rapid formation
of a protective clot at the site of injury, while limiting and physically confining the process.
References
12.2.3.1 prothrombin -> activated thrombin (factor IIa) + thrombin activation peptide (Xa catalyst)
Authors
Description
Membrane-bound factor Xa catalyzes the activation of small amounts of thrombin. The amino terminal portion of prothrombin is released as an
activation peptide, which can be cleaved further by activated thrombin. Neither the full-length activation peptide nor its cleavage products have
known functions.
References
T Orfeo, N Brufatto, ME Nesheim, H Xu, S Butenas, KG Mann, "The factor V activation paradox", J Biol Chem, 279, 2004, 19580-91.
Reaction
12.2.3.2 factor V -> factor Va + factor V activation peptide
Authors
Description
Activated thrombin (factor IIa) catalyzes the conversion of factor V to factor Va (activated factor V). The activation peptide released in this
reaction has no known function.
References
Reaction
12.2.3.3 factor Va + factor Xa -> Va:Xa complex (prothrombinase)
Authors
Description
Factors Va and Xa associate on a membrane surface to form a complex in which the activity of factor Xa on prothrombin is greatly increased
(Mann et al. 1988). The presence of negatively charged phospholipid in the membrane greatly facilitates this process, a feature that may
contribute to its localization, as such phospholipids are normally on the cytosolic face of the plasma membrane (Devaux 1992), but could be
exposed to the extracellular space following platelet activation or mechanical injury to endothelial cells.
References
KG Mann, RJ Jenny, S Krishnaswamy, "Cofactor proteins in the assembly and expression of blood clotting enzyme complexes", Annu Rev
Biochem, 57, 1988, 915-56.
PF Devaux, "Protein involvement in transmembrane lipid asymmetry", Annu Rev Biophys Biomol Struct, 21, 1992, 417-39.
Reaction
12.2.3.4 prothrombin -> activated thrombin (factor IIa) + thrombin activation peptide (prothrombinase catalyst)
Authors
Description
The membrane-bound Va:Xa (prothrombinase) complex rapidly activates large amounts of thrombin.
References
KG Mann, RJ Jenny, S Krishnaswamy, "Cofactor proteins in the assembly and expression of blood clotting enzyme complexes", Annu Rev
Biochem, 57, 1988, 915-56.
Reaction
12.2.3.5 fibrinogen -> fibrin monomer + 2 fibrinopeptide A + 2 fibrinopeptide B
Authors
Description
The alpha and beta chains of fibrinogen hexamer are cleaved by thrombin to generate fibrin monomer. The amino terminal regions of the
cleaved alpha and beta chains are released (fibrinopeptides A and B respectively).
References
F Ni, Y Konishi, RB Frazier, HA Scheraga, ST Lord, "High-resolution NMR studies of fibrinogen-like peptides in solution: interaction of thrombin
with residues 1-23 of the A alpha chain of human fibrinogen", Biochemistry, 28, 1989, 3082-94.
Reaction
12.2.3.6 n fibrin monomers -> fibrin multimer
Authors
Description
Fibrin monomers rapidly and spontaneously associate into large multimers, binding to one another via sites created by fibrinopeptide release
(Laudano and Doolittelle 1980). The process of multimerization, and the range of multimer structures that can form in vivo and in vitro, have
been studied in detail (Doolittle 1984). Here, multimer size has arbitrarily been set to three fibrin monomers.
References
AP Laudano, RF Doolittle, "Studies on synthetic peptides that bind to fibrinogen and prevent fibrin polymerization. Structural requirements,
number of binding sites, and species differences.", Biochemistry, 19, 1980, 1013-9.
RF Doolittle, "Fibrinogen and fibrin", Annu Rev Biochem, 53, 1984, 195-229.
Reaction
12.2.3.7 factor XIII -> factor XIII cleaved tetramer + 2 factor XIII A activation peptides
Authors
Description
Activated thrombin cleaves the A chains of factor XIII tetramers in a reaction stimulated by the presence of fibrin multimers. The amino terminal
portions of the A chains are released as activation peptides, which have no known function. The resulting factor XIII tetramer remains
catalytically inactive.
References
SD Lewis, TJ Janus, L Lorand, JA Shafer, "Regulation of formation of factor XIIIa by its fibrin substrates", Biochemistry, 24, 1985, 6772-7.
Reaction
12.2.3.8 factor XIII cleaved tetramer + 2 Ca++ -> factor XIIIa + 2 factor XIII B chain
Authors
Description
Once the A chains of the Factor XIII tetramer have been cleaved by thrombin, the complex dissociates and the resulting A chain dimer binds
Ca++ (one per peptide monomer) to form activated factor XIII (factor XIIIa).
References
SD Lewis, TJ Janus, L Lorand, JA Shafer, "Regulation of formation of factor XIIIa by its fibrin substrates", Biochemistry, 24, 1985, 6772-7.
Reaction
12.2.3.9 fibrin multimer -> fibrin multimer, crosslinked + NH4+
Authors
Description
Fibrin multimers are stabilized by the formation of multiple covalent crosslinks between the side chains of specific lysine and glutamine residues
in fibrinogen alpha and gamma chains, catalyzed by factor XIIIa.
References
EG Tuddenham, DN Cooper, The Molecular Genetics of Haemostasis and Its Disorders, 1994, 274-5.
Reaction
12.2.3.10 antithrombin III + heparin -> antithrombin III:heparin
Authors
Description
Antithrombin III binds to membrane-associated heparin, e.g., on the surface of a normal endothelial cell. This binding event increases the affinity
of antithrombin III for thrombin approximately 1000-fold.
References
A Mushunje, A Zhou, RW Carrell, JA Huntington, "Heparin-induced substrate behavior of antithrombin Cambridge II", Blood, 102, 2003, 4028-34.
Reaction
12.2.3.11 activated thrombin (factor IIa) + antithrombin III:heparin -> thrombin:antithrombin III:heparin
Authors
Description
Activated thrombin binds to the antithrombin III:heparin complex on the cell surface.
References
Reaction
12.2.3.12 thrombin:antithrombin III:heparin -> thrombin:cleaved antithrombin III:heparin
Authors
Description
Antithrombin III in the complex is cleaved by thrombin, thereupon undergoing a conformational change that stabilizes the thrombin:antithrombin
III complex, trapping and inactivating the thrombin moiety.
References
Reaction
12.2.3.13 thrombin:cleaved antithrombin III:heparin -> thrombin:cleaved antithrombin III + heparin
Authors
Description
The same conformational change that traps thrombin in its complex with cleaved antithrombin III also decreases the affinity of the latter for
heparin, and the complex of cleaved antithrombin III and thrombin dissociates from the cell-bound heparin molecule.
References
Reaction
12.2.3.14 activated thrombin (factor IIa) + thrombomodulin -> activated thrombin:thrombomodulin
Authors
Description
Activated thrombin (factor IIa) binds to thrombomodulin at the external face of the plasma membrane, forming a thrombin:thrombomodulin
complex. In this complexed form, the activity of thrombin towards protein C is greatly increased, and as thrombomodulin is particularly abundant
on the surfaces of endothelial cells, this association plays a major role in restricting clot formation.
References
CT Esmon, "The roles of protein C and thrombomodulin in the regulation of blood coagulation", J Biol Chem, 264, 1989, 4743-6.
Reaction
12.2.3.15 protein C -> activated protein C + protein C heavy chain activation peptide
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
Thrombin complexed with thrombomodulin at the endothelial cell surface cleaves the heavy chain of protein C, generating activated protein C
and an activation peptide. The activation peptide has no known function.
References
W Kisiel, "Human plasma protein C: isolation, characterization, and mechanism of activation by alpha-thrombin", J Clin Invest, 64, 1979, 761-9.
Reaction
12.2.3.16 factor Va -> factor Vi
Authors
Description
Activated protein C cleaves peptide bonds in activated factor V (factor Va), converting it to an inactive form (factor Vi). The exact site(s) of
cleavage are unknown. Protein S, on the endothelial cell surface, positively regulates this reaction. Although the mechanism of this regulation is
unclear, the regulation is physiologically important, as people with reduced amounts of protein S, like people with reduced amounts of protein C,
are susceptible to thromboembolism.
References
Reaction
12.3 Dissolution of Fibrin Clot
Editors
Reviewers
Rush, MG, 2008-01-11.
Description
The crosslinked fibrin multimers in a clot are broken down to soluble polypeptides by plasmin, a serine protease. Plasmin can be generated from
its inactive precursor plasminogen and recruited to the site of a fibrin clot in two ways, by interaction with tissue plasminogen activator at the
surface of a fibrin clot, and by interaction with urokinase plasminogen activator at a cell surface. The first mechanism appears to be the major
one responsible for the dissolution of clots within blood vessels. The second, although capable of mediating clot dissolution, may normally play a
major role in tissue remodeling, cell migration, and inflammation (Chapman 1997; Lijnen 2001). These other functions of urokinase plasminogen
activator will be annotated in future versions of Reactome.
Clot dissolution is regulated in two ways. First, efficient plasmin activation and fibrinolysis occur only in complexes formed at the clot surface or
on a cell membrane - proteins free in the blood are inefficient catalysts and are rapidly inactivated. Second, both plasminogen activators and
plasmin itself are inactivated by specific serpins, proteins that bind to serine proteases to form stable, enzymatically inactive complexes (Kohler
and Grant 2000).
References
HA Chapman, "Plasminogen activators, integrins, and the coordinated regulation of cell adhesion and migration", Curr Opin Cell Biol, 9, 1997,
714-24.
HP Kohler, PJ Grant, "Plasminogen-activator inhibitor type 1 and coronary artery disease", N Engl J Med, 342, 2000, 1792-801.
HR Lijnen, "Elements of the fibrinolytic system", Ann N Y Acad Sci, 936, 2001, 226-36.
12.3.1 histidine-rich glycoprotein + plasminogen <-> histidine-rich glycoprotein:plasminogen
Authors
Editors
Description
Plasminogen reversibly binds histidine-rich glycoprotein (HRG). The resulting complex interacts poorly with fibrin, suggesting that HRG might
have an anti-fibrinolytic (clot-stabilizing) effect in vivo (Lijnen et al. 1980). Consistent with this suggestion, individuals with chronically reduced
plasma HRG concentrations are susceptible to thrombosis (Shigekiyo et al. 1998).
References
T Koide, D Foster, S Yoshitake, EW Davie, "Amino acid sequence of human histidine-rich glycoprotein derived from the nucleotide sequence of
its cDNA", Biochemistry, 25, 1986, 2220-5.
HR Lijnen, M Hoylaerts, "Isolation and characterization of a human plasma protein with affinity for the lysine binding sites in plasminogen. Role in
the regulation of fibrinolysis and identification as histidine-rich glycoprotein.", J Biol Chem, 255, 1980, 10214-22.
T Shigekiyo, H Yoshida, K Matsumoto, H Azuma, S Wakabayashi, S Saito, K Fujikawa, T Koide, "HRG Tokushima: molecular and cellular
characterization of histidine-rich glycoprotein (HRG) deficiency", Blood, 91, 1998, 128-33.
Reaction
12.3.2 histidine-rich glycoprotein:plasminogen <-> histidine-rich glycoprotein + plasminogen
Authors
Editors
Description
Plasminogen reversibly binds histidine-rich glycoprotein (HRG). The resulting complex interacts poorly with fibrin, suggesting that HRG might
have an anti-fibrinolytic (clot-stabilizing) effect in vivo (Lijnen et al. 1980). Consistent with this suggestion, individuals with chronically reduced
plasma HRG concentrations are susceptible to thrombosis (Shigekiyo et al. 1998).
References
T Koide, D Foster, S Yoshitake, EW Davie, "Amino acid sequence of human histidine-rich glycoprotein derived from the nucleotide sequence of
its cDNA", Biochemistry, 25, 1986, 2220-5.
HR Lijnen, M Hoylaerts, "Isolation and characterization of a human plasma protein with affinity for the lysine binding sites in plasminogen. Role in
the regulation of fibrinolysis and identification as histidine-rich glycoprotein.", J Biol Chem, 255, 1980, 10214-22.
T Shigekiyo, H Yoshida, K Matsumoto, H Azuma, S Wakabayashi, S Saito, K Fujikawa, T Koide, "HRG Tokushima: molecular and cellular
characterization of histidine-rich glycoprotein (HRG) deficiency", Blood, 91, 1998, 128-33.
Reaction
12.3.3 crosslinked fibrin multimer + tissue plasminogen activator (one-chain) -> crosslinked fibrin
multimer:tissue plasminogen activator (one-chain)
Authors
Editors
Description
The first step in the dissolution of a fibrin clot is the association of the one-chain form of tissue plasminogen activator with fibrin.
References
M Hoylaerts, DC Rijken, HR Lijnen, "Kinetics of the activation of plasminogen by human tissue plasminogen activator. Role of fibrin.", J Biol
Chem, 257, 1982, 2912-9.
DL Higgins, GA Vehar, "Interaction of one-chain and two-chain tissue plasminogen activator with intact and plasmin-degraded fibrin",
Biochemistry, 26, 1987, 7786-91.
G Pohl, M Kallstrom, N Bergsdorf, P Wallen, H Jornvall, "Tissue plasminogen activator: peptide analyses confirm an indirectly derived amino
acid sequence, identify the active site serine residue, establish glycosylation sites, and localize variant differences", Biochemistry, 23, 1984,
3701-7.
Reaction
12.3.4 crosslinked fibrin multimer:tissue plasminogen activator (one-chain) + plasminogen -> crosslinked
fibrin multimer:tissue plasminogen activator (one-chain):plasminogen
Authors
Editors
Description
Plasminogen associates with tissue plasminogen activator bound to fibrin.
References
Chem, 257, 1982, 2912-9.
TE Petersen, MR Martzen, A Ichinose, EW Davie, "Characterization of the gene for human plasminogen, a key proenzyme in the fibrinolytic
system", J Biol Chem, 265, 1990, 6104-11.
Reaction
12.3.5 crosslinked fibrin multimer:tissue plasminogen activator (one-chain):plasminogen -> crosslinked

fibrin multimer:tissue plasminogen activator (one-chain) + plasmin
Authors
Editors
Description
Plasminogen bound to fibrin is cleaved and activated by tissue plasminogen activator also bound to the fibrin. The association of both
plasminogen and tissue plasminogen activator with a fibrin clot juxtaposes the two molecules, facilitating their interaction (Hoylaerts et al. 1982).
Early studies suggested that tissue plasminogen activator itself might require activation (conversion to its two-chain form) before it could catalyze
this reaction (e.g., Higgins and Vehar 1987). More recent work (Boose et al. 1989) indicates that the single-chain form of the molecule is
catalytically active, although cleavage increases its activity and may thus serve to accelerate the later stages of fibrinolysis.
References
Chem, 257, 1982, 2912-9.
Biochemistry, 26, 1987, 7786-91.
JA Boose, E Kuismanen, R Gerard, J Sambrook, MJ Gething, "The single-chain form of tissue-type plasminogen activator has catalytic activity:
studies with a mutant enzyme that lacks the cleavage site", Biochemistry, 28, 1989, 635-43.
Reaction
12.3.6 fibrin multimer, crosslinked -> fibrin digestion products (plasmin)
Authors
Editors
Description
Plasmin, generated at the surfaces of the fibrin clot by tissue plasminogen activator or at the surfaces of cells by urokinase plasminogen
activator, catalyzes the hydrolysis of fibrin to soluble fragments (Chapman 1997).
References
HA Chapman, "Plasminogen activators, integrins, and the coordinated regulation of cell adhesion and migration", Curr Opin Cell Biol, 9, 1997,
714-24.
Reaction
12.3.7 crosslinked fibrin multimer:tissue plasminogen activator (one-chain) -> crosslinked fibrin
multimer:tissue plasminogen activator (two-chain)
Authors
Editors
Description
Once plasmin has been activated, in the initial stage of the fibrinolysis process, it can catalyze the conversion of fibrin-bound tissue plasminogen
activator (one-chain) to its more active two-chain form, increasing the rate at which additional plasminogen molecules can be activated.
References
Biochemistry, 26, 1987, 7786-91.
JA Boose, E Kuismanen, R Gerard, J Sambrook, MJ Gething, "The single-chain form of tissue-type plasminogen activator has catalytic activity:
studies with a mutant enzyme that lacks the cleavage site", Biochemistry, 28, 1989, 635-43.
Reaction
12.3.8 crosslinked fibrin multimer:tissue plasminogen activator (two-chain) + plasminogen -> crosslinked
fibrin multimer:tissue plasminogen activator (two-chain):plasminogen
Description
At the beginning of this reaction, 1 molecule of 'plasminogen', and 1 molecule of 'fibrin multimer, crosslinked:tissue plasminogen activator
(two-chain)' are present. At the end of this reaction, 1 molecule of 'fibrin multimer, crosslinked:tissue plasminogen activator
(two-chain):plasminogen' is present.
This reaction takes place in the 'extracellular region'.

References
Biochemistry, 26, 1987, 7786-91.
Reaction
12.3.9 crosslinked fibrin multimer:tissue plasminogen activator (two-chain):plasminogen -> crosslinked

fibrin multimer:tissue plasminogen activator (two-chain) + plasmin
Description
At the beginning of this reaction, 1 molecule of 'fibrin multimer, crosslinked:tissue plasminogen activator (two-chain):plasminogen' is present. At
the end of this reaction, 1 molecule of 'plasmin', and 1 molecule of 'fibrin multimer, crosslinked:tissue plasminogen activator (two-chain)' are
present.
This reaction takes place in the 'extracellular region' and is mediated by the 'plasminogen activator activity' of 'fibrin multimer, crosslinked:tissue
plasminogen activator (two-chain):plasminogen'.
References
Biochemistry, 26, 1987, 7786-91.
Reaction
12.3.10 fibrin multimer, crosslinked:tissue plasminogen activator (two-chain) + plasminogen activator

inhibitor 1 -> fibrin multimer, crosslinked:tissue plasminogen activator (two-chain):plasminogen activator
inhibitor 1
Authors
Editors
Description
Plasminogen activator inhibitor 1, a serpin, binds to fibrin-associated tissue plasminogen activator. The resulting stable complex remains
associated with fibrin but cannot activate plasminogen (Wagner et al. 1989). The importance of this step in the regulation of clot dissolution in
vivo is indicated by the occurence of thrombosis in individuals with abnormally little tissue plasminogen activator or abnormally much
plasminogen activator inhibitor (Juhan-Vague et al. 1987).
References
I Juhan-Vague, J Valadier, MC Alessi, MF Aillaud, J Ansaldi, C Philip-Joet, P Holvoet, A Serradimigni, "Deficient t-PA release and elevated PA
inhibitor levels in patients with spontaneous or recurrent deep venous thrombosis", Thromb Haemost, 57, 1987, 67-72.
OF Wagner, C de Vries, C Hohmann, H Veerman, H Pannekoek, "Interaction between plasminogen activator inhibitor type 1 (PAI-1) bound to
fibrin and either tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). Binding of t-PA/PAI-1 complexes to
fibrin mediated by both the finger and the kringle-2 domain of t-PA.", J Clin Invest, 84, 1989, 647-55.
Reaction
12.3.11 fibrin multimer, crosslinked:tissue plasminogen activator (one-chain) + plasminogen activator

inhibitor 1 -> fibrin multimer, crosslinked:tissue plasminogen activator (one-chain):plasminogen activator
inhibitor 1
Authors
Editors
Description
Plasminogen activator inhibitor 1, a serpin, binds to fibrin-associated tissue plasminogen activator. The resulting stable complex remains
associated with fibrin but cannot activate plasminogen (Wagner et al. 1989). The importance of this step in the regulation of clot dissolution in
vivo is indicated by the occurence of thrombosis in individuals with abnormally little tissue plasminogen activator or abnormally much
plasminogen activator inhibitor (Juhan-Vague et al. 1987).
References
I Juhan-Vague, J Valadier, MC Alessi, MF Aillaud, J Ansaldi, C Philip-Joet, P Holvoet, A Serradimigni, "Deficient t-PA release and elevated PA
inhibitor levels in patients with spontaneous or recurrent deep venous thrombosis", Thromb Haemost, 57, 1987, 67-72.
OF Wagner, C de Vries, C Hohmann, H Veerman, H Pannekoek, "Interaction between plasminogen activator inhibitor type 1 (PAI-1) bound to
fibrin and either tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). Binding of t-PA/PAI-1 complexes to
fibrin mediated by both the finger and the kringle-2 domain of t-PA.", J Clin Invest, 84, 1989, 647-55.
Reaction
12.3.12 alpha-2-antiplasmin + plasmin -> alpha-2-antiplasmin:plasmin
Authors
Editors
Description
Plasmin binds the serpin alpha-2-antiplasmin, forming a stable and catalytically inactive complex. While several serpin proteins bind and
inactivate plasmin in vitro, alpha-2-antiplasmin appears to be the only one with substantial plasmin-neutralizing activity in vivo (Moroi and Aoki
1976; Lijnen et al. 1987).
References
M Moroi, N Aoki, "Isolation and characterization of alpha2-plasmin inhibitor from human plasma. A novel proteinase inhibitor which inhibits
activator-induced clot lysis.", J Biol Chem, 251, 1976, 5956-65.
HR Lijnen, WE Holmes, B van Hoef, B Wiman, H Rodriguez, "Amino-acid sequence of human alpha 2-antiplasmin", Eur J Biochem, 166, 1987,
565-74.
Reaction
12.3.13 urokinase plasminogen activator + urokinase plasminogen activator receptor (uPAR) -> urokinase
plasminogen activator:uPAR
Authors
Editors
Description
The uncleaved (one-chain) form of urokinase plasminogen activator associates with urokinase plasminogen activator receptor (uPAR), forming a
complex at the cell surface (Cubellis et al. 1986). The complex is anchored to the outer face of the plasma membrane by a
glycophosphatidylinositol moiety at the carboxy terminus of uPAR (Behrendt et al. 1990; Ploug et al. 1991).
References
N Behrendt, E Ronne, M Ploug, T Petri, D Lober, LS Nielsen, WD Schleuning, F Blasi, E Appella, K Dano, "The human receptor for urokinase
plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants.", J Biol Chem, 265, 1990, 6453-60.
MV Cubellis, ML Nolli, G Cassani, F Blasi, "Binding of single-chain prourokinase to the urokinase receptor of human U937 cells", J Biol Chem,
261, 1986, 15819-22.
M Ploug, E Ronne, N Behrendt, AL Jensen, F Blasi, K Dano, "Cellular receptor for urokinase plasminogen activator. Carboxyl-terminal
processing and membrane anchoring by glycosyl-phosphatidylinositol.", J Biol Chem, 266, 1991, 1926-33.
Reaction
12.3.14 plasminogen + histidine-rich glycoprotein -> plasminogen:histidine-rich glycoprotein
Authors
Editors
Description
Extracellular plasminogen binds with high affinity to histidine-rich glycoprotein on the plasma membrane. Binding requires Zn++ in
concentrations higher than those found in normal plasma, but that can be generated, e.g., by platelet activation (Jones et al. 2004).
References
AL Jones, MD Hulett, JG Altin, P Hogg, CR Parish, "Plasminogen is tethered with high affinity to the cell surface by the plasma protein,
histidine-rich glycoprotein", J Biol Chem, 279, 2004, 38267-76.
Reaction
12.3.15 plasminogen:histidine-rich glycoprotein -> plasmin + histidine-rich glycoprotein (uPA [one-chain]

catalyst)
Authors
Editors
Description
Plasminogen, tethered to the cell surface by its association with histidine-rich glycoprotein, is cleaved and activated to plasmin by the action of
urokinase plasminogen activator bound to uPAR, its cell-surface receptor. The association of both substrate and enzyme with the cell surface is
necessary for the reaction to proceed efficiently (Ellis et al. 1991). While the one-chain form of urokinase plasminogen activator is lower than that
of the two-chain form, it is still sufficient to initiate the process of plasmin activation (Ellis et al. 1989; Lijnen et al. 1986).
References
V Ellis, N Behrendt, K Dano, "Plasminogen activation by receptor-bound urokinase. A kinetic study with both cell-associated and isolated
receptor.", J Biol Chem, 266, 1991, 12752-8.
HR Lijnen, C Zamarron, M Blaber, ME Winkler, D Collen, "Activation of plasminogen by pro-urokinase. I. Mechanism.", J Biol Chem, 261, 1986,
1253-8.
V Ellis, MF Scully, VV Kakkar, "Plasminogen activation initiated by single-chain urokinase-type plasminogen activator. Potentiation by U937
monocytes.", J Biol Chem, 264, 1989, 2185-8.
Reaction
12.3.16 urokinase plasminogen activator (one-chain):uPAR -> urokinase plasminogen activator

(two-chain):uPAR
Authors
Editors
Description
The small amount of plasmin generated by the activity of the one-chain form of urokinase plasminogen activator in turn cleaves urokinase
plasminogen activator, converting it to its substantially more active two-chain form (Cubellis et al. 1986; Lijnen et al. 1991).
References
HR Lijnen, C Zamarron, M Blaber, ME Winkler, D Collen, "Activation of plasminogen by pro-urokinase. I. Mechanism.", J Biol Chem, 261, 1986,
1253-8.
MV Cubellis, ML Nolli, G Cassani, F Blasi, "Binding of single-chain prourokinase to the urokinase receptor of human U937 cells", J Biol Chem,
261, 1986, 15819-22.
Reaction
12.3.17 plasminogen:histidine-rich glycoprotein -> plasmin + histidine-rich glycoprotein (uPA [two-chain]

catalyst)
Authors
Editors
Description
Plasminogen, tethered to the cell surface by its association with histidine-rich glycoprotein, is rapidly cleaved and activated to plasmin by the
action of urokinase plasminogen activator(two-chain form) bound to uPAR, its cell-surface receptor. The association of both substrate and
enzyme with the cell surface is necessary for the reaction to proceed efficiently (Ellis et al. 1989, 1991).
References
V Ellis, N Behrendt, K Dano, "Plasminogen activation by receptor-bound urokinase. A kinetic study with both cell-associated and isolated
receptor.", J Biol Chem, 266, 1991, 12752-8.
V Ellis, MF Scully, VV Kakkar, "Plasminogen activation initiated by single-chain urokinase-type plasminogen activator. Potentiation by U937
monocytes.", J Biol Chem, 264, 1989, 2185-8.
Reaction
12.3.18 urokinase plasminogen activator (two-chain):uPAR + plasminogen activator inhibitor 1 (PAI-1) ->
PAI-1:urokinase plasminogen activator (two-chain):uPAR
Authors
Editors
Description
Activated (two-chain) urokinase plasminogen activator binds plasminogen activator inhibitor 1, a serpin, to form a stable, inactive complex that
remains associated with uPAR on the plasma membrane (Cubellis et al. 1989).
References
MV Cubellis, P Andreasen, P Ragno, M Mayer, K Dano, F Blasi, "Accessibility of receptor-bound urokinase to type-1 plasminogen activator
inhibitor", Proc Natl Acad Sci U S A, 86, 1989, 4828-32.
Reaction
12.3.19 urokinase plasminogen activator (two-chain):uPAR + plasminogen activator inhibitor 2 (PAI-2) ->
PAI-2:urokinase plasminogen activator (two-chain):uPAR
Authors
Editors
Description
Activated (two-chain) urokinase plasminogen activator binds plasminogen activator inhibitor 2, a serpin, to form a stable, inactive complex that
remains associated with uPAR on the plasma membrane (Estreicher et al. 1990; Kruithof et al. 1986).
References
EK Kruithof, JD Vassalli, WD Schleuning, RJ Mattaliano, F Bachmann, "Purification and characterization of a plasminogen activator inhibitor
from the histiocytic lymphoma cell line U-937", J Biol Chem, 261, 1986, 11207-13.
A Estreicher, J Muhlhauser, JL Carpentier, L Orci, JD Vassalli, "The receptor for urokinase type plasminogen activator polarizes expression of
the protease to the leading edge of migrating monocytes and promotes degradation of enzyme inhibitor complexes", J Cell Biol, 111, 1990,
783-92.
Reaction
12.4 Cell surface interactions at the vascular wall
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
Leukocyte extravasation is a rigorously controlled process that guides white cell movement from the vascular lumen to sites of tissue
inflammation. The powerful adhesive interactions that are required for leukocytes to withstand local flow at the vessel wall is a multistep process
mediated by different adhesion molecules. Platelets adhered to injured vessel walls form strong adhesive substrates for leukocytes. For
instance, the initial tethering and rolling of leukocytes over the site of injury are mediated by reversible binding of selectins to their cognate
cell-surface glycoconjugates.
Endothelial cells are tightly connected through various proteins, which regulate the organization of the junctional complex and bind to
cytoskeletal proteins or cytoplasmic interaction partners that allow the transfer of intracellular signals. An important role for these junctional
proteins in governing the transendothelial migration of leukocytes under normal or inflammatory conditions has been established.
This pathway describes some of the key interactions that assist in the process of platelet and leukocyte interaction with the endothelium, in
response to injury.
References
P da Costa Martins, N van den Berk, LH Ulfman, L Koenderman, PL Hordijk, JJ Zwaginga, "Platelet-monocyte complexes support monocyte
adhesion to endothelium by enhancing secondary tethering and cluster formation", Arterioscler Thromb Vasc Biol, 24, 2004, 193-9.
C Weber, L Fraemohs, E Dejana, "The role of junctional adhesion molecules in vascular inflammation", Nat Rev Immunol, 7, 2007, 467-77.
SP Jackson, N Mistry, Y Yuan, "Platelets and the injured vessel wall-- "rolling into action": focus on glycoprotein Ib/V/IX and the platelet
cytoskeleton", Trends Cardiovasc Med, 10, 2000, 192-7.
A Schober, C Weber, "Mechanisms of monocyte recruitment in vascular repair after injury", Antioxid Redox Signal, 7, 2005, 1249-57.
BF Becker, B Heindl, C Kupatt, S Zahler, "Endothelial function and hemostasis", Z Kardiol, 89, 2000, 160-7.
B Furie, BC Furie, "The molecular basis of platelet and endothelial cell interaction with neutrophils and monocytes: role of P-selectin and the
P-selectin ligand, PSGL-1", Thromb Haemost, 74, 1995, 224-7.
12.4.1 Binding of GP VI:Fc Epsilon R1 gamma receptor complex with collagen
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
References
Reaction
12.4.2 MAC1 binds JAM-C
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
Recruitment of monocytic cells to the vessel wall by platelets is mediated via CD11b/CD18 (Mac-1) and platelet JAM-C. In the case of dendritic
cells, this interaction leads to their activation and platelet phagocytosis. This process may be of importance for progression of atherosclerotic
lesions.
References
HF Langer, K Daub, G Braun, T Schonberger, AE May, M Schaller, GM Stein, K Stellos, A Bueltmann, D Siegel-Axel, HP Wendel, H Aebert, M
Roecken, P Seizer, S Santoso, S Wesselborg, P Brossart, M Gawaz, "Platelets recruit human dendritic cells via Mac-1/JAM-C interaction and
modulate dendritic cell function in vitro", Arterioscler Thromb Vasc Biol, 27, 2007, 1463-70.
Reaction
12.4.3 JAM-B binds JAM-C
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
JAM-B and -C bind each other and are strongly expressed by endothelial cells of high endothelial venules, the predominant site of leukocyte
extravasation. JAM-B and -C also bind to the leukocyte integrins VLA-4 and Mac-1 respectively.
References
RJ Ludwig, TM Zollner, S Santoso, K Hardt, J Gille, H Baatz, PS Johann, J Pfeffer, HH Radeke, MP Schon, R Kaufmann, WH Boehncke, M
Podda, "Junctional adhesion molecules (JAM)-B and -C contribute to leukocyte extravasation to the skin and mediate cutaneous inflammation",
J Invest Dermatol, 125, 2005, 969-76.
Reaction
12.4.4 VLA-4 binds JAM-B
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Description
Several key IgSF cell adhesion molecules engage integrin and in so doing impact on the multi-step paradigm of leukocyte emigration. The
interaction between JAM-B and VLA-4 is facilitated by prior engagement of JAM-B with JAM-C.
References
SA Cunningham, JM Rodriguez, MP Arrate, TM Tran, TA Brock, "JAM2 interacts with alpha4beta1. Facilitation by JAM3.", J Biol Chem, 277,
2002, 27589-92.
Reaction
12.4.5 JAM-A homodimerises
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
JAM-A is the most widely expressed member of the family, and has been shown to be expressed on endothelial and epithelial cells, on platelets,
and on a number of leukocyte subsets. In endothelial cells, JAM-A locates to the tight junctions, where it appears to engage in homophilic
binding to JAM-A on adjacent cells, an interaction that is considered to play a critical role in angiogenesis.
References
A Woodfin, CA Reichel, A Khandoga, M Corada, MB Voisin, C Scheiermann, DO Haskard, E Dejana, F Krombach, S Nourshargh, "JAM-A
mediates neutrophil transmigration in a stimulus-specific manner in vivo: evidence for sequential roles for JAM-A and PECAM-1 in neutrophil
transmigration", Blood, 110, 2007, 1848-56.
H Huang, F Cruz, G Bazzoni, "Junctional adhesion molecule-A regulates cell migration and resistance to shear stress", J Cell Physiol, 209,
2006, 122-30.
PF Bradfield, S Nourshargh, M Aurrand-Lions, BA Imhof, "JAM family and related proteins in leukocyte migration (Vestweber series)",
Arterioscler Thromb Vasc Biol, 27, 2007, 2104-12.
Reaction
12.4.6 LFA1 binds JAM-A

Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
JAM-A plays a key role in leukocyte transmigration and inflammatory extravasation. Transmigration of human leukocytes has been shown to
involve heterophilic interactions of JAM-A with its integrin receptor LFA-1.
References
G Ostermann, L Fraemohs, T Baltus, A Schober, M Lietz, A Zernecke, EA Liehn, C Weber, "Involvement of JAM-A in mononuclear cell
recruitment on inflamed or atherosclerotic endothelium: inhibition by soluble JAM-A", Arterioscler Thromb Vasc Biol, 25, 2005, 729-35.
L Fraemohs, RR Koenen, G Ostermann, B Heinemann, C Weber, "The functional interaction of the beta 2 integrin lymphocyte
function-associated antigen-1 with junctional adhesion molecule-A is mediated by the I domain", J Immunol, 173, 2004, 6259-64.
G Ostermann, KS Weber, A Zernecke, A Schroder, C Weber, "JAM-1 is a ligand of the beta(2) integrin LFA-1 involved in transendothelial
migration of leukocytes", Nat Immunol, 3, 2002, 151-8.
Reaction
12.4.7 Integrin alphaX beta2 binds JAM-C
Authors
Ouwehand, W.H., 2007-11-12.

Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
Although JAM-C is better known for its interaction with MAC-1, an interaction with CD11c/CD18 (known as alpha X beta 2), has also been
described.
References
S Santoso, UJ Sachs, H Kroll, M Linder, A Ruf, KT Preissner, T Chavakis, "The junctional adhesion molecule 3 (JAM-3) on human platelets is a
counterreceptor for the leukocyte integrin Mac-1", J Exp Med, 196, 2002, 679-91.
PF Bradfield, C Scheiermann, S Nourshargh, C Ody, FW Luscinskas, GE Rainger, GB Nash, M Miljkovic-Licina, M Aurrand-Lions, BA Imhof,
"JAM-C regulates unidirectional monocyte transendothelial migration in inflammation", Blood, 110, 2007, 2545-55.
Reaction
12.4.8 MERTK receptor binds ligands (Gas6 or Protein S)
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
MerTK appears to be required for ingestion of apoptotic cells by professional phagocytes such as monocytes/macrophages, retinal pigment
epithelial cells and dendritic cells. Mer appears to be able to induce the cytoskeletal remodelling that is required for engulfment during
phagocytosis. For instance, a deletion in the MERTK gene was identified as the underlying cause for retinal dystrophy which involves an
impairment in the ingestion of shed photoreceptor cell fragments by retinal pigment epithelial cells.
The biological ligands for MerTK are two highly similar vitamin K-dependent proteins, Gas6 and protein S (PS), a negative regulator of blood
coagulation. Both proteins are composed an N-terminal region containing multiple post-translationally modified gamma-carboxyglutamic acid
residues (Gla). The Gla region possesses the ability to interact in a conformationally specific manner with negatively charged membrane
phospholipids, which is thought to mediate the binding of both Gas6 and PS to apoptotic cells. In this way, they are thought to act as recognition
bridges between apoptotic cells and the phagocyte cell that ingest them.
References
S Hafizi, B Dahlback, "Gas6 and protein S. Vitamin K-dependent ligands for the Axl receptor tyrosine kinase subfamily.", FEBS J, 273, 2006,
5231-44.
S Hafizi, B Dahlback, "Signalling and functional diversity within the Axl subfamily of receptor tyrosine kinases", Cytokine Growth Factor Rev, 17,
2006, 295-304.
HM Seitz, TD Camenisch, G Lemke, HS Earp, GK Matsushima, "Macrophages and dendritic cells use different Axl/Mertk/Tyro3 receptors in
clearance of apoptotic cells", J Immunol, 178, 2007, 5635-42.
Reaction
12.4.9 CD84 homodimerises
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
CD84 is a homophilic receptor expressed on T cells, B cells, dendritic cells, monocytes, macrophages, eosinophils, mast cells, granulocytes, and
platelets. CD84 expression increases following activation of T cells, B cells, and dendritic cells. CD84 homophilic engagement is known to
induce platelet stimulation.
References
Q Yan, VN Malashkevich, A Fedorov, E Fedorov, E Cao, JW Lary, JL Cole, SG Nathenson, SC Almo, "Structure of CD84 provides insight into
SLAM family function", Proc Natl Acad Sci U S A, 104, 2007, 10583-8.
M Martin, X Romero, MA de la Fuente, V Tovar, N Zapater, E Esplugues, P Pizcueta, J Bosch, P Engel, "CD84 functions as a homophilic
adhesion molecule and enhances IFN-gamma secretion: adhesion is mediated by Ig-like domain 1", J Immunol, 167, 2001, 3668-76.
Reaction
12.4.10 P-selectin binds P-selectin ligand
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
PSGL-1 is expressed as a homodimer of two 120-kDa subunits that binds all three selectins, with the highest affinity for P-selectin, and is known
to be constitutively expressed on the surface of platelets and most types of leukocytes. Besides playing a critical role in the inflammatory
response by mediating leukocyte-leukocyte and leukocyte-endothelium interactions, PSGL-1 also participates in the hemostatic process by
mediating leukocyte-platelet interactions.
References
P da Costa Martins, JJ Garcia-Vallejo, JV van Thienen, M Fernandez-Borja, JM van Gils, C Beckers, AJ Horrevoets, PL Hordijk, JJ Zwaginga,
"P-selectin glycoprotein ligand-1 is expressed on endothelial cells and mediates monocyte adhesion to activated endothelium", Arterioscler
Reaction
12.4.11 JAM-B homodimerises
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
Apart from its well-established interaction with VLA-4, JAM-B is also known to homodimerize.
References
Reaction
12.4.12 JAM-C homodimerises
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
JAM-C has been detected in epithelial-cell desmosomes. JAM-C homodimers are prominently located in endothelial-cell tight junctions.
References
G Mandicourt, S Iden, K Ebnet, M Aurrand-Lions, BA Imhof, "JAM-C regulates tight junctions and integrin-mediated cell adhesion and migration",
J Biol Chem, 282, 2007, 1830-7.
Reaction
12.4.13 CD177 binds PECAM-1
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.

Description
CD177 is a 58- to 64-kDa glycosylphosphatidylinositol-anchored glycoprotein expressed exclusively by neutrophils, neutrophilic metamyelocytes,
and myelocytes, but not by any other blood cells. It has been shown that neutrophil-specific CD177 is a heterophilic binding partner of PECAM-1,
constituting a novel pathway that promotes neutrophil transmigration.
References
UJ Sachs, CL Andrei-Selmer, A Maniar, T Weiss, C Paddock, VV Orlova, EY Choi, PJ Newman, KT Preissner, T Chavakis, S Santoso, "The
neutrophil-specific antigen CD177 is a counter-receptor for platelet endothelial cell adhesion molecule-1 (CD31)", J Biol Chem, 282, 2007,
23603-12.
A Kalinowska, J Losy, "PECAM-1, a key player in neuroinflammation", Eur J Neurol, 13, 2006, 1284-90.
Reaction
12.4.14 CD47 binds SIRP
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
Integrin-associated protein (IAP or CD47) is a receptor for thrombospondin family members, a ligand for the transmembrane signaling protein
SIRP-alpha and -gamma, and a component of a supramolecular complex containing specific integrins, heterotrimeric G proteins and cholesterol.
References
AN Barclay, MH Brown, "The SIRP family of receptors and immune regulation", Nat Rev Immunol, 6, 2006, 457-64.
Reaction
12.4.15 CD48 binds CD244
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
CD2, CD48, CD84, CD244 and CD58 have a similar extracellular domain arichitecture consisting of two IgSF domains. CD244 is closely related
to CD84 in having a long cytoplasmic tail with tyrosine-based motifs (TxYxxI/V) resembling immunoreceptor tyrosine-based inhibitory motifs
(ITIMs). CD2 has a cytoplasmic domain with proline-rich regions which recruit an Src homology 3 (SH3)- containing protein called
CD2-associated protein (CD2AP). CD48 is glycosyl-phosphatidyl-inositol (GPI)-anchored to the membrane.
CD244 is known to be activated by binding to CD48 in humans.
References
SG Tangye, JH Phillips, LL Lanier, "The CD2-subset of the Ig superfamily of cell surface molecules: receptor-ligand pairs expressed by NK cells
and other immune cells", Semin Immunol, 12, 2000, 149-57.
B Messmer, P Eissmann, S Stark, C Watzl, "CD48 stimulation by 2B4 (CD244)-expressing targets activates human NK cells", J Immunol, 176,
2006, 4646-50.
Reaction
12.4.16 CD58 binds CD2
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
The crystal structure of the human CD2-CD58 complex also shows that most of the residues at the interface between these two proteins are
charged and form several inter-protein salt bridges.
References
AL Wilkins, W Yang, JJ Yang, "Structural biology of the cell adhesion protein CD2: from molecular recognition to protein folding and design",
Curr Protein Pept Sci, 4, 2003, 367-73.
MV Bayas, A Kearney, A Avramovic, PA van der Merwe, DE Leckband, "Impact of salt bridges on the equilibrium binding and adhesion of
human CD2 and CD58", J Biol Chem, 282, 2007, 5589-96.
Reaction
12.4.17 Integrin alpha 5 beta 1 binds fibronectin
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
Tenacious binding of free fibronectin to cells leads to enhanced fibronectin matrix assembly and the formation of a polymerized fibronectin
"cocoon" around the cells. This process is enhanced in the presence of CEACAM molecules.
References
C Brakebusch, R Fassler, "beta 1 integrin function in vivo: adhesion, migration and more", Cancer Metastasis Rev, 24, 2005, 403-11.
C Ordonez, AB Zhai, P Camacho-Leal, L Demarte, MM Fan, CP Stanners, "GPI-anchored CEA family glycoproteins CEA and CEACAM6
mediate their biological effects through enhanced integrin alpha5beta1-fibronectin interaction", J Cell Physiol, 210, 2007, 757-65.
Z Zhang, AO Morla, K Vuori, JS Bauer, RL Juliano, E Ruoslahti, "The alpha v beta 1 integrin functions as a fibronectin receptor but does not
support fibronectin matrix assembly and cell migration on fibronectin", J Cell Biol, 122, 1993, 235-42.
Reaction
12.4.18 CXADR binds to AMICA1
Authors
Ouwehand, W.H., 2007-11-12.

Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
JAM members, such as JAML, bind coxsackie and adenovirus receptor (CXADR) on epithelial and endothelial cells.
References
A Fuchs, M Colonna, "The role of NK cell recognition of nectin and nectin-like proteins in tumor immunosurveillance", Semin Cancer Biol, 16,
2006, 359-66.
K Zen, Y Liu, IC McCall, T Wu, W Lee, BA Babbin, A Nusrat, CA Parkos, "Neutrophil migration across tight junctions is mediated by adhesive
interactions between epithelial coxsackie and adenovirus receptor and a junctional adhesion molecule-like protein on neutrophils", Mol Biol Cell,
16, 2005, 2694-703.
Reaction
12.4.19 protein C -> activated protein C + protein C heavy chain activation peptide
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
References
Reaction
12.4.20 OLR1 binds to oxidized LDL
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
The lectin-like oxidized low density lipoprotein receptor- 1 (Lox-1) mediates the recognition and internalization of oxidatively modified low density
lipoprotein. This interaction results in a number of pro-atherogenic cellular responses that probably play a significant role in the pathology of
atherosclerosis.
References
Q Xie, S Matsunaga, S Niimi, S Ogawa, K Tokuyasu, Y Sakakibara, S Machida, "Human lectin-like oxidized low-density lipoprotein receptor-1
functions as a dimer in living cells", DNA Cell Biol, 23, 2004, 111-7.
T Sawamura, N Kume, T Aoyama, H Moriwaki, H Hoshikawa, Y Aiba, T Tanaka, S Miwa, Y Katsura, T Kita, T Masaki, "An endothelial receptor
for oxidized low-density lipoprotein", Nature, 386, 1997, 73-7.
Reaction
12.4.21 Platelet-derived TREM-1 ligand binds to TREM-1
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
The triggering receptor expressed on myeloid cells 1 (TREM-1) plays an important role in the innate immune response related to severe
infections and sepsis. Although the identity and occurrence of the natural TREM-1 ligands are so far unknown, the presence of a ligand for
TREM-1 on human platelets has been established.
References
P Haselmayer, L Grosse-Hovest, P von Landenberg, H Schild, MP Radsak, "TREM-1 ligand expression on platelets enhances neutrophil
activation", Blood, 110, 2007, 1029-35.
Reaction
12.4.22 Tie2 Signaling
Authors
Garapati, Phani Vijay, de Bono, B, 2008-03-05.

Reviewers
Trowsdale, J, 2008-02-26.
Description
The Tie2/Tek receptor tyrosine kinase plays a pivotal role in vascular and hematopoietic development and is expressed exclusively on
endothelial lineage. Tie2 interacts with a group of ligands belonging to angiopoietin family and undergoes activation.
These ligands show opposing actions, angiopoietin 1 and angiopoietin 4 stimulate the Tie2 phosphorylation and angiopoietin 2 inhibits it. Upon
tyrosine phosphorylation Tie2 acts as a scaffold for various signaling proteins involved in different signal transduction cascades that can effect
survival of endothelium and angiogenic sprout formation.
References
N Jones, DJ Dumont, "The Tek/Tie2 receptor signals through a novel Dok-related docking protein, Dok-R", Oncogene, 17, 1998, 1097-108.
S Loughna, TN Sato, "Angiopoietin and Tie signaling pathways in vascular development", Matrix Biol, 20, 2001, 319-25.
N Jones, DJ Dumont, "Tek/Tie2 signaling: new and old partners", Cancer Metastasis Rev, 19, 2000, 13-7.
NL Ward, DJ Dumont, "The angiopoietins and Tie2/Tek: adding to the complexity of cardiovascular development", Semin Cell Dev Biol, 13,
2002, 19-27.
12.4.22.1 Interaction of Tie2 with Ang1
Authors
Reviewers
Description
Tie receptors and their angiopoietin ligands play a critical role in angiogenesis or blood vessel formation. They are considered to control
numerous signaling pathways that are involved in diverse cellular processes, such as cell migration, proliferation, survival and reorganization of
the actin cytoskeleton.
Tie (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains) represents a class of receptor tyrosine kinases
(RTKs) that are predominately expressed by vascular endothelial cells. The angiopoietins are a family of growth factors that are largely specific
for endothelium and they bind to Tie2/Tek RTKs.
Tie2 signaling initially involves the activation of Tie2 by the interaction of angiopoietin 1. Angiopoietin interacts with the Tie2 receptor with its
fibrinogen like domain (FLD). This interaction leads to the dimerization of both the receptor and the ligand, and later initiate the
trans-phosphorylation of Tie2.
References
S Davis, TH Aldrich, PF Jones, A Acheson, DL Compton, V Jain, TE Ryan, J Bruno, C Radziejewski, PC Maisonpierre, GD Yancopoulos,
"Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning", Cell, 87, 1996, 1161-9.
Y Xu, Q Yu, "Angiopoietin-1, unlike angiopoietin-2, is incorporated into the extracellular matrix via its linker peptide region", J Biol Chem, 276,
2001, 34990-8.
2002, 19-27.
Reaction
12.4.22.2 Dimerization of Tie2/Ang1 complex
Authors
Reviewers
Description
Receptor tyrosine kinase activation and signaling are typically initiated via dimerization of the receptors through homo-oligomeric ligand binding.
Angiopoietin1 may form homotrimers, but in most cases it assembles into higher-order multimers. This oligomerization is mediated by the N-ter
coiled coil domain (CCD).
The binding of Ang1 oligomers to Tie2 promotes the dimerization of Tie2, which is further assisted by the interaction between the kinase
domains of the receptors.
References
KG Peters, CD Kontos, PC Lin, AL Wong, P Rao, L Huang, MW Dewhirst, S Sankar, "Functional significance of Tie2 signaling in the adult
vasculature", Recent Prog Horm Res, 59, 2004, 51-71.
LM Shewchuk, AM Hassell, B Ellis, WD Holmes, R Davis, EL Horne, SH Kadwell, DD McKee, JT Moore, "Structure of the Tie2 RTK domain:
self-inhibition by the nucleotide binding loop, activation loop, and C-terminal tail", Structure, 8, 2000, 1105-13.
Reaction
12.4.22.3 Trans-phosphorylation of Tie2
Authors
Reviewers
Description
The dimerization of Tie2 leads to autophosphorylation and activation of its kinase domain. There are multiple tyrosine phosphorylation sites in
the Tie2 kinase domain. The phosphorylated tyrosine residues provide the interaction site for the SH2 domains of other downstream signaling
molecules like PI3K, Grb2, SHP2 etc.
References
CD Kontos, TP Stauffer, WP Yang, JD York, L Huang, MA Blanar, T Meyer, KG Peters, "Tyrosine 1101 of Tie2 is the major site of association of
p85 and is required for activation of phosphatidylinositol 3-kinase and Akt", Mol Cell Biol, 18, 1998, 4131-40.
N Jones, SH Chen, C Sturk, Z Master, J Tran, RS Kerbel, DJ Dumont, "A unique autophosphorylation site on Tie2/Tek mediates Dok-R
phosphotyrosine binding domain binding and function", Mol Cell Biol, 23, 2003, 2658-68.
Source reaction
This reaction was inferred from the corresponding reaction "Trans-phosphorylation of Tie2" in species Mus musculus.
Reaction
12.4.22.4 Interaction of Tie2 and p85 of PI3K
Authors
Reviewers
Description
The p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) associates with Tie2, most likely at phosphotyrosine 1102. This association leads
on to the activation of Akt/PKB, a process linked to cell survival and antiapoptosis, and that may in part account for Tie2's role in vascular growth
and maintenance.
References
N Jones, Z Master, J Jones, D Bouchard, Y Gunji, H Sasaki, R Daly, K Alitalo, DJ Dumont, "Identification of Tek/Tie2 binding partners. Binding to
a multifunctional docking site mediates cell survival and migration.", J Biol Chem, 274, 1999, 30896-905.
Reaction
12.4.22.5 Interaction of Tie2 and Grb2
Authors
Reviewers
Description
Tie2/Tek provide mitogenic signals to endothelial cells by promoting the association of Grb2 to one of their phosphotyrosines. Grb2 is an adaptor
protein that has been linked to activation of Ras and mitogen activated protein kinase (MAPK) cell growth signaling pathways. Grb2 also binds to
the Y1102 of the kinase domain of Tie2 with one of its SH2 doamins.
References
L Huang, CW Turck, P Rao, KG Peters, "GRB2 and SH-PTP2: potentially important endothelial signaling molecules downstream of the
TEK/TIE2 receptor tyrosine kinase", Oncogene, 11, 1995, 2097-103.
Reaction
12.4.22.6 Interaction of SOS-1 to Tie2 bound Grb2
Authors
Reviewers
Description
Grb2 binds directly to autophosphorylated Tie2 receptor. GRB2 also contains two SH3 domains, which bring various ligands to the sites of active
signaling. One of the SH3 domains on Tie2-bound Grb2 recruits SOS1, an activating nucleotide exchange factor for Ras. This interaction of
Sos1 to Grb2 brings Sos1 towards Ras molecules leading to Ras activation. Ras is implicated in the MAP kinase cascade, a pathway in cell
growth stimulation, migration and differentiation.
References
N Li, A Batzer, R Daly, V Yajnik, E Skolnik, B Margolis, J Schlessinger, "Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links
receptor tyrosine kinases to Ras signalling.", Nature, 363, 1993, 85-8.
Reaction
12.4.22.7 Sos-mediated nucleotide exchange of Ras (Tie2 receptor:Grb2:Sos)
Authors
Reviewers
Description
Sos-1 bound to Grb2:Tie2 complex promotes the exchange of inactive Ras-GDP to active Ras-GTP.
Reaction
12.4.22.8 Interaction of Tie2 and Shc1
Authors
Reviewers
Description
ShcA, an SH2-containing adapter protein, acts as a scaffold for the assembly of signaling proteins involved in the activation of the Ras-MAPK
pathway, and potentially other signaling pathways.
ShcA is one of the binding partners of endogenous Tie2 receptor on vascular endothelial cells. After Tie2 stimulation by Ang-1 interaction, ShcA
associates with Tie2 and becomes tyrosine-phosphorylated. ShcA interacts with the cytoplasmic domain of Tie2 and Y1102 of Tie2 was
identified as the primary binding site for the SH2 domain of ShcA. ShcA leads to a reduction of tyrosine phosphorylation of p85 subunit of
PI3-kinase and is involved in the inhibition of endothelial cell migration and survival.
References
E Audero, I Cascone, F Maniero, L Napione, M Arese, L Lanfrancone, F Bussolino, "Adaptor ShcA protein binds tyrosine kinase Tie2 receptor
and regulates migration and sprouting but not survival of endothelial cells", J Biol Chem, 279, 2004, 13224-33.
Reaction
12.4.22.9 Interaction of Tie2 and Dok-2
Authors
Reviewers
Description
Dok-2 is a member of a docking proteins class, termed the DOK family. The DOK family members are characterized by an N-terminal pleckstrin
homology (PH) domain followed by a central PTB domain and a proline- and tyrosine-rich C-terminal tail. Dok-2 is recruited to activated Tie2 via
its PTB domain, which results in its subsequent tyrosine phosphorylation, thereby establishing binding sites for the small GTPase-activating
protein for Ras, p120RasGAP (RasGAP) and the adapter protein Nck. The binding of DOK to the receptor leads to Nck recruitment and
subsequent phosphorylation. Binding of Pak to Nck follows. this brings about the Ang-1-dependent phosphorylation of Pak in endothelial cells.
References
Z Master, N Jones, J Tran, J Jones, RS Kerbel, DJ Dumont, "Dok-R plays a pivotal role in angiopoietin-1-dependent cell migration through
recruitment and activation of Pak", EMBO J, 20, 2001, 5919-28.
Reaction
Authors
Reviewers
Description
The major ligands for Tie2 are Ang1 and Ang2. Ang1 has been considered as the primary activating ligand of Tie2 whereas role of Ang2 remains
controversial. Ang2 acts as stimulating in some studies and inhibiting in others. The activity of Ang2 is concentration dependent. Ang2
possesses similar receptor affinity to Ang1 and they both share the same binding site on Tie2. The Ang2 fibrinogen domain is solely responsible
for receptor recognition and binding, the coiled-coil motif mediates its oligomerization.
References
E Bogdanovic, VP Nguyen, DJ Dumont, "Activation of Tie2 by angiopoietin-1 and angiopoietin-2 results in their release and receptor
internalization", J Cell Sci, 119, 2006, 3551-60.
WA Barton, D Tzvetkova, DB Nikolov, "Structure of the angiopoietin-2 receptor binding domain and identification of surfaces involved in Tie2
recognition", Structure, 13, 2005, 825-32.
Reaction
12.4.22.11 Interaction of Tie2 and Ang4
Authors
Reviewers
Description
Ang4 represents a third protein of the Ang family that binds to the Tie2 receptor. The mouse Ang3 and human Ang4 are interspecies orthologs.
Ang4 acts as an activating ligand and induces phosphorylation in Tie2.
References
2002, 19-27.
HJ Lee, CH Cho, SJ Hwang, HH Choi, KT Kim, SY Ahn, JH Kim, JL Oh, GM Lee, GY Koh, "Biological characterization of angiopoietin-3 and
angiopoietin-4", FASEB J, 18, 2004, 1200-8.
DM Valenzuela, JA Griffiths, J Rojas, TH Aldrich, PF Jones, H Zhou, J McClain, NG Copeland, DJ Gilbert, NA Jenkins, T Huang, N
Papadopoulos, PC Maisonpierre, S Davis, GD Yancopoulos, "Angiopoietins 3 and 4: diverging gene counterparts in mice and humans", Proc
Natl Acad Sci U S A, 96, 1999, 1904-9.
Reaction
12.4.23 PECAM1 interactions
Authors
de Bono, B, Garapati, Phani Vijay, 2008-02-26.
Reviewers
Description
PECAM-1/CD31 is a member of the immunoglobulin superfamily (IgSF) and has been implicated to mediate the adhesion and trans-endothelial
migration of T-lymphocytes into the vascular wall, T cell activation and angiogenesis. It has six Ig homology domains within its extracellularly and
an ITIM motif within its cytoplasmic region. PECAM-1 mediates cellular interactions by both homophilic and heterophilic interactions. The
cytoplasmic domain of PECAM-1 contains tyrosine residues which serves as docking sites for recruitment of cytosolic signaling molecules.
Under conditions of platelet activation, PECAM-1 is phosphorylated by Src kinase members. The tyrosine residues 663 and 686 are required for
recruitment of the SH2 domain containing PTPs.
References
DE Jackson, "The unfolding tale of PECAM-1", FEBS Lett, 540, 2003, 7-14.
N Gong, S Chatterjee, "Platelet endothelial cell adhesion molecule in cell signaling and thrombosis", Mol Cell Biochem, 253, 2003, 151-8.
12.4.23.1 Trans-homophilic interaction of PECAM-1
Authors
Reviewers
Description
PECAM-mediated adhesion is complex, because it is capable of binding both to itself (homophilic adhesion) and to non-PECAM ligands
(heterophilic adhesion). The trans-homophilic interaction between the two PECAM-1 molecules is mediated by their NH2-terminal membrane
distal Ig homology domains 1 and 2 plus the proper spacing formed by the six Ig-homology domains.
References
JP Newton, AP Hunter, DL Simmons, CD Buckley, DJ Harvey, "CD31 (PECAM-1) exists as a dimer and is heavily N-glycosylated", Biochem
JP Newton, CD Buckley, EY Jones, DL Simmons, "Residues on both faces of the first immunoglobulin fold contribute to homophilic binding sites
of PECAM-1/CD31", J Biol Chem, 272, 1997, 20555-63.
Reaction
12.4.23.2 Heterophilic interaction of PECAM-1 and Integrin alpha-v beta-3
Authors
Reviewers
Description
Alpha v beta 3 integrin is one of the potential heterophilic ligands of PECAM-1 that is involved in down-regulation of T-cell responses. The
heterophilic interaction of alpha v beta 3 integrin on endothelial cells with PEACAM-1 on leukocytes increases the adhesive function of beta
integrins on T cells, monocytes, neutrophils and NK cells suggesting that leukocyte PEACAM-1 act as a signaling molecule.
References
CD Buckley, R Doyonnas, JP Newton, SD Blystone, EJ Brown, SM Watt, DL Simmons, "Identification of alpha v beta 3 as a heterotypic ligand
for CD31/PECAM-1", J Cell Sci, 109, 1996, 437-45.
L Piali, P Hammel, C Uherek, F Bachmann, RH Gisler, D Dunon, BA Imhof, "CD31/PECAM-1 is a ligand for alpha v beta 3 integrin involved in
adhesion of leukocytes to endothelium", J Cell Biol, 130, 1995, 451-60.
Reaction
12.4.23.3 Phoshorylation of PECAM-1 by Fyn or Lyn or c-Src
Authors
Reviewers
Description
PECAM-1 is capable of transmitting information into the cell following its engagement and becomes tyrosine-phosphorylated during the platelet
aggregation process. The Src family of tyrosine kinases (more specifically, Src, Lyn, and c-src) has been widely implicated in the
phosphorylation of PECAM-1. Conserved tyrosine residues (Tyr663 and Tyr686) within the PECAM-1 cytoplasmic ITIM motif have been shown
to become phosphorylated. Tyrosine phosphorylation of PECAM-1 prompts its association with intracellular signal transduction molecules.
References
MY Cao, M Huber, N Beauchemin, J Famiglietti, SM Albelda, A Veillette, "Regulation of mouse PECAM-1 tyrosine phosphorylation by the Src
and Csk families of protein-tyrosine kinases", J Biol Chem, 273, 1998, 15765-72.
M Cicmil, JM Thomas, T Sage, FA Barry, M Leduc, C Bon, JM Gibbins, "Collagen, convulxin, and thrombin stimulate aggregation-independent
tyrosine phosphorylation of CD31 in platelets. Evidence for the involvement of Src family kinases.", J Biol Chem, 275, 2000, 27339-47.
Reaction
12.4.23.4 Interaction of PECAM-1 and SHIP
Authors
Reviewers
Description
PECAM/CD31 is a member of the immunoglobulin superfamily (IgSF) and has been implicated to mediate the adhesion and trans-endothelial
an ITIM motif within its cytoplasmic region.
References
NJ Pumphrey, V Taylor, S Freeman, MR Douglas, PF Bradfield, SP Young, JM Lord, MJ Wakelam, IN Bird, M Salmon, CD Buckley, "Differential
association of cytoplasmic signalling molecules SHP-1, SHP-2, SHIP and phospholipase C-gamma1 with PECAM-1/CD31", FEBS Lett, 450,
1999, 77-83.
Reaction
12.4.23.5 Interaction of PECAM-1 and SHP-2
Authors
Reviewers
Description
PECAM-1 becomes tyrosine-phosphorylated during the platelet aggregation process and that this creates docking sites for the protein-tyrosine
phosphatase SHP-2. The interaction between SHP-2 and PECAM-1 is dependent upon integrin-mediated platelet/platelet interactions and
occurs via the Src homology 2 (SH2) domains of the phosphatase and highly conserved phosphatase-binding motifs encompassing
phosphotyrosines 663 and 686 within the cytoplasmic domain of PECAM-1.
References
DE Jackson, CM Ward, R Wang, PJ Newman, "The protein-tyrosine phosphatase SHP-2 binds platelet/endothelial cell adhesion molecule-1
(PECAM-1) and forms a distinct signaling complex during platelet aggregation. Evidence for a mechanistic link between PECAM-1- and
integrin-mediated cellular signaling.", J Biol Chem, 272, 1997, 6986-93.
Reaction
12.4.23.6 Interaction of PECAM-1 and PLC gamma1
Authors

Reviewers
Description
Like SHP-1 and SHP-2, PLC-gamma 1 also interacts with PECAM-1. PLC-gamma 1 binds with both the tyrosine residues (Y663 and Y686).
Unlike the N-SH2 domain, the C-SH2 domain on PLC-gamma 1 can only bind phosphotyrosine 663. The engagement of PECAM-1 with
PLC-gamma 1 may lead to PLC-gamma 1 activation and subsequent calcium influx.
References
1999, 77-83.
Reaction
Authors
Reviewers
Description
The phosphorylation of two tandem tyrosine residues (Y663 and Y686) within the cytoplasmic domain of PECAM-1 is required for the
downstream signalling events observed following PECAM-1 ligation. Both SH2 domains of SHP-1 are required in tandem to bind PECAM-1.
References
1999, 77-83.
CT Hua, JR Gamble, MA Vadas, DE Jackson, "Recruitment and activation of SHP-1 protein-tyrosine phosphatase by human platelet endothelial
cell adhesion molecule-1 (PECAM-1). Identification of immunoreceptor tyrosine-based inhibitory motif-like binding motifs and substrates.", J Biol
Chem, 273, 1998, 28332-40.
Reaction
12.4.24 Basigin interactions
Authors
Reviewers
Description
Basigin is a widely expressed transmembrane glycoprotein that belongs to the Ig superfamily and is highly enriched on the surface of epithelial
cells. Basigin is involved in intercellular interactions involved in various immunologic phenomena, differentiation, and development, but a major
function of basigin is stimulation of synthesis of several matrix metalloproteinases. Basigin also induces angiogenesis via stimulation of VEGF
production.
Basigin has an extracellular region with two Ig-like domains of which the N-term Ig-like domain is involved in interactions. It undergoes
interactions between basigin molecules on opposing cells or on neighbouring cells. It also interacts with a variety of other proteins like
caveolin-1, cyclophilins, integrins and annexin II that play important roles in cell proliferation, energy metabolism, migration, adhesion and
motion, especially in cancer metastasis.
References
JL Jiang, J Tang, "CD147 and its interacting proteins in cellular functions", Sheng Li Xue Bao, 59, 2007, 517-23.
12.4.24.1 CyP60 chaperones Basigin
Authors
Reviewers
Description
Basigin serves as a signaling receptor for extracellular cyclophilins. Its been reported that cyclophilin 60 (Cyp60), a distinct member of the
cyclophilin family is involved in the regulation of intracellular transport of basigin. The mechanism of this activity involves interaction of Cyp60
with the proline-containing region within or adjacent to the predicted transmembrane domain basigin. Cyp60 is co-localized with basigin at the
plasma membrane suggesting that Cyp60 may function as a chaperone escorting basigin through the secretory pathway.
References
T Pushkarsky, V Yurchenko, C Vanpouille, B Brichacek, I Vaisman, S Hatakeyama, KI Nakayama, B Sherry, MI Bukrinsky, "Cell surface
expression of CD147/EMMPRIN is regulated by cyclophilin 60", J Biol Chem, 280, 2005, 27866-71.
Reaction
12.4.24.2 Basigin homodimerises

Authors
Reviewers
Description
Basigin (Bsg) is a highly glycosylated transmembrane protein belonging to the Ig superfamily with two Ig domains. Bsg forms homo-oligomers on
the plasma membrane in a cis-dependent manner. The N-terminal Ig-like domain is functionally important in oligomer formation.
References
S Yoshida, M Shibata, S Yamamoto, M Hagihara, N Asai, M Takahashi, S Mizutani, T Muramatsu, K Kadomatsu, "Homo-oligomer formation by
basigin, an immunoglobulin superfamily member, via its N-terminal immunoglobulin domain", Eur J Biochem, 267, 2000, 4372-80.
Reaction
12.4.24.3 Caveolin-1 binds Basigin
Authors
Reviewers
Description
Stromal fibroblasts secrete multiple matrix metalloproteinases (MMP)1 that can promote tumor cell growth, survival, invasion, angiogenesis, and
metastasis. Basigin on the surface of carcinoma cells, stimulates production of MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), and
MMP-3 (stromelysin). Basigin has been shown to co-immunoprecipitate with caveolin-1. The second Ig domain of Basigin is required for this
association, which leads to decreased Besigin self-association on the cell surface. Therefore, caveolin-1 is a negative regulator of CD147
self-association, and its MMP-inducing activity.
References
W Tang, SB Chang, ME Hemler, "Links between CD147 function, glycosylation, and caveolin-1", Mol Biol Cell, 15, 2004, 4043-50.
W Tang, ME Hemler, "Caveolin-1 regulates matrix metalloproteinases-1 induction and CD147/EMMPRIN cell surface clustering", J Biol Chem,
279, 2004, 11112-8.
Reaction
12.4.24.4 Basigin binds CyPA
Authors
Reviewers
Description
Cyclophilin A (CyPA)1 is an intracellular protein belonging to the immunophilin family and is recognized as the major target for the potent
immunosuppressive drug cyclosporin A. CD147 is the natural cell surface receptor for CyPA. It is demonstrated that CD147 is an essential
component in the CyPA-initiated signaling cascade that culminates in ERK activation.
References
V Yurchenko, G Zybarth, M O'Connor, WW Dai, G Franchin, T Hao, H Guo, HC Hung, B Toole, P Gallay, B Sherry, M Bukrinsky, "Active site
residues of cyclophilin A are crucial for its signaling activity via CD147", J Biol Chem, 277, 2002, 22959-65.
Reaction
12.5 Further platelet releasate
Authors
Editors
Reviewers
Description
Platelets contain a number of distinguishable storage granules; alpha granules, dense granules and lysosomes. On activation platelets release a
variety of proteins from these storage granules that act in an autocrine or paracrine fashion to modulate cell signaling and play a key role in
hemostasis.
The platelet releasate as well contains a range of proteins released from different secretory organelles other than the storage granules within the
platelets. In some cases platelet lysis may also contribute to the presence of these proteins in the platelet relesate.
References
2096-104.
12.5.1 Release of Serotransferrin

Authors
Editors
Reviewers
Description
After Platelet activation Serotransferrin from secretory granules is released in to the extracellular platelet releasate.
References
2096-104.
Reaction
12.5.2 Release of Tubulin alpha-4A chain
Authors

Editors
Reviewers
Description
After Platelet activation Tubulin alpha-4A chain from cytosol is released in to the extracellular platelet releasate.
References
2096-104.
Reaction
12.5.3 Release of Apolipoprotein A-I
Authors
Editors

Reviewers
Description
After Platelet activation Apolipoprotein from the secretory granule is released in to the extracellular platelet releasate.
References
2096-104.
Reaction
12.5.4 Release of Calmodulin
Authors
Editors
Reviewers

Description
After Platelet activation Calmodulin from the cytosol is released in to the extracellular platelet releasate.
References
2096-104.
Reaction
12.5.5 Release of Pleckstrin
Authors
Editors
Reviewers
Description
After Platelet activation Pleckstrin from the cytosol is released in to the extracellular platelet releasate.
References
2096-104.
Reaction
12.5.6 Release of Vinculin
Authors
Editors
Reviewers
Description
After Platelet activation Vinculin from the cytosol is released in to the extracellular platelet releasate.
References
2096-104.
Reaction
12.5.7 Release of WD repeat-containing protein 1
Authors
Editors
Reviewers
Description
After Platelet activation WD repeat-containing protein from the cytosol is released in to the extracellular platelet releasate.
References
2096-104.
Reaction
12.5.8 Release of Superoxide dismutase [Cu-Zn]
Authors
Editors
Reviewers
Description
After Platelet activation Superoxide dismutase from the cytosol is released in to the extracellular platelet releasate.
References
2096-104.
Reaction
12.5.9 Release of Bromodomain and PHD finger-containing protein 3

Authors
Editors
Reviewers
Description
After Platelet activation 'Bromodomain and PHD finger-containing protein-3' from the cytosol is released in to the extracellular platelet releasate.
References
2096-104.
Reaction
12.5.10 Release of Titin
Authors

Editors
Reviewers
Description
After Platelet activation 'Titin' from the cytosol is released in to the extracellular platelet releasate.
References
2096-104.
Reaction
12.5.11 Release of Kappa-actin
Authors
Editors

Reviewers
Description
After Platelet activation 'Kappa-actin' from the cytosol is released in to the extracellular platelet releasate.
References
2096-104.
Reaction
12.5.12 Release of Intracellular hyaluronan-binding protein 4
Authors
Editors
Reviewers

Description
After Platelet activation 'Intracellular hyaluronan-binding protein 4' from the cytosol is released in to the extracellular platelet releasate.
References
2096-104.
Reaction
12.5.13 Release of Filamin-A
Authors
Editors
Reviewers
Description
After Platelet activation 'Filamin-A' from the cytosol is released in to the extracellular platelet releasate.
References
2096-104.
Reaction
12.5.14 Release of Talin-1
Authors
Editors
Reviewers
Description
After Platelet activation 'Talin-1' from the cytosol is released in to the extracellular platelet releasate.
References
2096-104.
Reaction
12.5.15 Release of Peptidyl-prolyl cis-trans isomerase A
Authors
Editors
Reviewers
Description
After Platelet activation 'CypA protein' from the cytosol is released in to the extracellular platelet releasate.
References
2096-104.
Source reaction
This reaction was inferred from the corresponding reaction "Release of Peptidyl-prolyl cis-trans isomerase A" in species Mus musculus.
2096-104.
Reaction
12.5.16 Release of Calumenin
Authors
Editors
Reviewers
Description
After Platelet activation 'Calumenin' from the endoplasmic reticulum lumen is released in to the extracellular platelet releasate.
References
2096-104.
Source reaction
This reaction was inferred from the corresponding reaction "Release of Calumenin " in species Mus musculus.
2096-104.
Reaction
12.5.17 Release of Adenylyl cyclase-associated protein 1
Authors
Editors
Reviewers
Description
After Platelet activation 'Adenylyl cyclase-associated protein 1' from the cytosol is released in to the extracellular platelet releasate.
References
2096-104.
Source reaction
This reaction was inferred from the corresponding reaction "Release of Adenylyl cyclase-associated protein 1" in species Mus musculus.
2096-104.
Reaction
12.5.18 Release of Cofilin-1
Authors
Editors
Reviewers
Description
After Platelet activation 'Cofilin-1' from the cytosol is released in to the extracellular platelet releasate.
References
2096-104.
Source reaction
This reaction was inferred from the corresponding reaction "Release of Cofilin-1" in species Mus musculus.
Reaction
12.5.19 Release of Profilin-1
Authors
Editors
Reviewers

References
Source reaction
This reaction was inferred from the corresponding reaction "Release of Profilin-1" in species Mus musculus.
2096-104.
Reaction
12.5.20 Release of Secretogranin-3
Authors
Editors
Reviewers

Description
After Platelet activation 'Secretogranin-3' from the secretory granules is released in to the extracellular platelet releasate.
References
2096-104.
Source reaction
This reaction was inferred from the corresponding reaction "Release of Secretogranin-3" in species Mus musculus.
2096-104.
Reaction
12.5.21 Release of 78 kDa glucose-regulated protein
Authors

Editors
Reviewers
Description
After Platelet activation '78 kDa glucose-regulated protein' from the endoplasmic reticulum lumen is released in to the extracellular platelet
releasate.
References
2096-104.
Source reaction
This reaction was inferred from the corresponding reaction "Release of 78 kDa glucose-regulated protein" in species Mus musculus.
2096-104.
Reaction
12.5.22 Exocytosis of Proactivator polypeptide
Editors
Reviewers
Description
After Platelet activation 'Proactivator polypeptide' from the lysosomal lumen is released in to the extracellular platelet releasate.
References
2096-104.
Reaction
13 Influenza Infection
Authors
Luo, F, Squires, B, Scheuermann, RH, 2006-01-05.
Editors
D'Eustachio, P, Gillespie, ME, 2006-01-07.
Reviewers
Garcia-Sastre, A, Squires, B, 2006-10-30.
Description
For centuries influenza epidemics have plagued man, and influenza was probably the disease described by Hippocrates in 412 BC. Today it
remains a major cause of morbidity and mortality worldwide with large segments of the human population affected every year. Many animal
species can be infected by influenza viruses, often with catastrophic consequences. A continuing threat is the possibility of a pandemic similar to
that experienced in 1918, estimated to have been responsible for 50 million deaths worldwide.
Influenza viruses belong to the family of Orthomyxoviridae; viruses with segmented RNA genomes that are negative sense and single-stranded
(Baltimore 1971).
Influenza virus strains are named according to their type (A, B, or C), the species from which the virus was isolated (omitted if human), location
of isolate, the number of the isolate, the year of isolation, and in the case of influenza A viruses, the hemagglutinin (H) and neuraminidase (N)
subtype. For example, the virus of H5N1 subtype isolated from chickens in Hong Kong in 1997 is: influenza A/chicken/Hong Kong/220/97(H5N1)
virus. Currently 16 different hemagglutinin (H1 to H16) subtypes and 9 different neuraminidase (N1 to N9) subtypes are known for influenza A
viruses. Most human disease is due to Influenza viruses of the A type, so the events of Influenza infection have been annotated in Reactome
with reference to this type.
References
RM Krug, RA Lamb, "Orthomyxoviridae: The Viruses and Their Replication", Fields Virology. 4th edition, editors: Knipe DM, Howley PM,.
Philadelphia: Lippincott Williams & Wilkins. ISBN: 0-7817-1832-5, 2001.
13.1 Influenza Life Cycle
Reviewers
Garcia-Sastre, A, 2020-05-12, Garcia-Sastre, A, Squires, B, 2006-10-30, Squires, B, Rush, MG, 2007-04-30.
Description
The virus particle initially associates with a human host cell by binding to sialic acid-containing receptors on the host cell surface. The bound
virus is endocytosed by one of four distinct mechanisms. The low endosomal pH sets in motion a number of steps that lead to viral membrane
fusion mediated by the viral hemagglutinin (HA) protein, and the eventual release of the uncoated viral ribonucleoprotein complex into the
cytosol of the host cell. The ribonucleoprotein complex is transported through the nuclear pore into the nucleus. Once in the nucleus, the
incoming negative-sense viral RNA (vRNA) is transcribed into messenger RNA (mRNA) by a primer-dependent mechanism. Replication occurs
via a two step process. A full-length complementary RNA (cRNA), a positive-sense copy of the vRNA, is first made and this in turn is used as a
template to produce more vRNA. The viral proteins are expressed and processed and eventually assemble with vRNAs at budding sites within
the host cell membrane. The viral protein complexes and ribonucleoproteins are assembled into viral particles and bud from the host cell,
enveloped in the host cell's membrane.
This release contains a framework for the further annotation of the viral life-cycle.
References
13.1.1 Binding of the influenza virion to the host cell
Description
Influenza viruses bind via their surface HA (hemagglutinin) to sialic acid in alpha 2,3 or alpha 2,6 linkage with galactose on the host cell surface.
Sialic acid in 2,6 linkages is characteristic of human cells while 2,3 linkages are characteristic of avian cells. The specificity of influenza HA for
sialic acid in alpha 2,6 or alpha 2,3 linkages is a feature restricting the transfer of influenza viruses between avian species and humans. This
species barrier can be overcome, however. Notably, passaged viruses adapt to their host through mutation in the receptor binding site of the
viral HA gene.
References
L Mochalova, A Gambaryan, J Romanova, A Tuzikov, A Chinarev, D Katinger, H Katinger, A Egorov, N Bovin, "Receptor-binding properties of
modern human influenza viruses primarily isolated in Vero and MDCK cells and chicken embryonated eggs", Virology, 313, 2003, 473-80.
RJ Connor, Y Kawaoka, RG Webster, JC Paulson, "Receptor specificity in human, avian, and equine H2 and H3 influenza virus isolates",
Virology, 205, 1994, 17-23.
AS Gambaryan, JS Robertson, MN Matrosovich, "Effects of egg-adaptation on the receptor-binding properties of human influenza A and B
viruses", Virology, 258, 1999, 232-9.
Reaction
13.1.2 Entry of Influenza Virion into Host Cell via Endocytosis
Description
Virus particles bound to the cell surface can be internalized by four mechanisms. Most internalization appears to be mediated by clathrin-coated
pits, but internalization via caveolae, macropinocytosis, and by non-clathrin, non-caveolae pathways has also been described for influenza
viruses.
References
13.1.2.1 Clathrin-Mediated Pit Formation And Endocytosis Of The Influenza Virion
Description
Virus particles bound to the cell surface can be internalized by four mechanisms. Most internalization appears to be mediated by clathrin-coated
pits.
References
KS Matlin, H Reggio, A Helenius, K Simons, "Infectious entry pathway of influenza virus in a canine kidney cell line", J Cell Biol, 91, 1981,
601-13.
Reaction
13.1.3 Fusion and Uncoating of the Influenza Virion
Description
Uncoating of viral particles takes place in the host cell endosome. Acidification of the endosome promotes fusion of the viral and endosomal
membranes, causing a structural change in the viral hemagglutinin (HA) and freeing the fusion peptide of its HA2 subunit to interact with the
endosome membrane. The concerted structural change of several HA molecules opens up a pore through which the viral RNP passes into the
cytosol of the cell. The precise timing and the location of uncoating (early vs. late endosomes) depends on the pH-mediated transition of the
specific HA molecule involved. The virus-associated M2 ion channel protein allows the influx of H+ ions into the virion, which disrupts
protein-protein interactions, resulting in the release of RNP free of the viral M1 matrix protein. Thus the HA mediated fusion of the viral
membrane with the endosomal membrane and the M2-mediated release of the RNP results in the release of the RNP complex into the cytosol.
Amantadine and rimantadine have been shown to block the ion channel activity of the M2 protein and thus interfere with uncoating.
References
IV Chizhmakov, FM Geraghty, DC Ogden, A Hayhurst, M Antoniou, AJ Hay, "Selective proton permeability and pH regulation of the influenza
virus M2 channel expressed in mouse erythroleukaemia cells", J Physiol, 494, 1996, 329-36.
T Stegmann, HW Morselt, J Scholma, J Wilschut, "Fusion of influenza virus in an intracellular acidic compartment measured by fluorescence
dequenching", Biochim Biophys Acta, 904, 1987, 165-70.
M Marsh, A Helenius, "Virus entry into animal cells", Adv Virus Res, 36, 1989, 107-51.
JJ Skehel, AJ Hay, JA Armstrong, "On the mechanism of inhibition of influenza virus replication by amantadine hydrochloride", J Gen Virol, 38,
1978, 97-110.
13.1.3.1 Fusion of the Influenza Virion to the Host Cell Endosome
Description
After the virus binds to the target cell surface and is endocytosed, the low pH of the endosome causes the viral HA (hemagglutinin) to undergo a
structural change which frees the fusion peptide of its HA2 subunit allowing it to interact with the endosome membrane. The transmembrane
domain of the HA2 (inserted into the viral membrane) and the fusion peptide (inserted into the endosomal membrane) are in juxtaposition in the
acidic pH structure of HA. The concerted structural change of several hemagglutinin molecules then opens a pore through which the viral RNP
will be able to pass into the host cell cytosol.
References
M Marsh, A Helenius, "Virus entry into animal cells", Adv Virus Res, 36, 1989, 107-51.
SB Sieczkarski, GR Whittaker, "Viral entry", Curr Top Microbiol Immunol, 285, 2005, 1-23.
13.1.3.1.1 Conformation change in hemagglutinin freeing the fusion peptide of HA2
Description
The low pH of the endosome causes the viral HA (hemagglutinin) to undergo a structural change which frees the fusion peptide of its HA2
subunit.
References
T Stegmann, "Membrane fusion mechanisms: the influenza hemagglutinin paradigm and its implications for intracellular fusion", Traffic, 1, 2000,
598-604.
Reaction
13.1.3.1.2 Fusion of the influenza virion HA2 protein transmembrane domain to the host cell endosome membrane
Description
The fusion peptide of its HA2 subunit interacts with the endosome membrane. The transmembrane domain of the HA2 is inserted into the viral
membrane and the fusion peptide is inserted into the endosomal membrane. In the acidic pH structure of HA the two ends of the HA complex
are in juxtaposition.
References
598-604.
Reaction
13.1.3.1.3 Concerted hemagglutinin pore formation
Description
The concerted structural change of several hemagglutinin molecules opens a pore through which the viral RNP will be able to pass into the host
cell cytosol.
References
598-604.
Reaction
13.1.3.2 Uncoating of the Influenza Virion
Description
The precise timing and location of uncoating (early vs. late endosomes) depends on the pH-mediated transition of the specific viral
hemagglutinin (HA) molecule involved. The uncoating of influenza viruses in endosomes is blocked by changes in pH caused by weak bases
(e.g. ammonium chloride and chloroquine) or ionophores (e.g. monensin). Effective uncoating is also dependent on the presence of the viral M2
ion channel protein. Early on it was recognized that amantadine and rimantadine inhibit replication immediately following virus infection. Later it
was found that the virus-associated M2 protein allows the influx of H+ ions into the virion, which disrupts protein-protein interactions, resulting in
the release of viral RNP free of the viral matrix (M1) protein. Amantadine and rimantadine have been shown to block the ion channel activity of
the M2 protein and thus uncoating. The HA mediated fusion of the viral membrane with the endosomal membrane and the M2-mediated release
of the RNP results in the appearance of free RNP complexes in the cytosol. This completes the uncoating process. The time frame for the
uncoating process has been examined by inhibiting virus penetration with ammonium chloride. Typically, virus particles show a penetration half
time of about 25 minutes after viral adsorption. Ten minutes later (half time of 34 minutes after adsorption) RNP complexes are found in the
nucleus. Uptake of RNP molecules through nuclear pores is an active process, involving the nucleo-cytoplasmic trafficking machinery of the host
cell.
References
K Martin, A Helenius, "Transport of incoming influenza virus nucleocapsids into the nucleus", J Virol, 65, 1991, 232-44.
C Wang, K Takeuchi, LH Pinto, RA Lamb, "Ion channel activity of influenza A virus M2 protein: characterization of the amantadine block", J Virol,
67, 1993, 5585-94.
13.1.3.2.1 Virion-associated M2 protein mediated ion infusion
Description
The uncoating of influenza viruses in endosomes is blocked by changes in pH caused by weak bases (e.g. ammonium chloride and chloroquine)
or ionophores (e.g. monensin). Effective uncoating is also dependent on the presence of the viral M2 ion channel protein. Early on it was
recognized that amantadine and rimantadine inhibit replication immediately following virus infection. Later it was found that the virus-associated
M2 protein allows the influx of H+ ions from the endosome into the virion. This disrupts protein-protein interactions, resulting in the release of
viral RNP free of the viral matrix (M1) protein. Amantadine and rimantadine have been shown to block the ion channel activity of the M2 protein
and thus uncoating.
References
JJ Skehel, AJ Hay, JA Armstrong, "On the mechanism of inhibition of influenza virus replication by amantadine hydrochloride", J Gen Virol, 38,
1978, 97-110.
C Wang, K Takeuchi, LH Pinto, RA Lamb, "Ion channel activity of influenza A virus M2 protein: characterization of the amantadine block", J Virol,
67, 1993, 5585-94.
Reaction
13.1.3.2.2 Ribonucleoprotein release from M1 proteins
Description
The influx of H+ ions into the virion disrupts protein-protein interactions, resulting in the release of the viral RNP from the viral matrix (M1)
protein. The uncoating process is complete with the appearance of free RNP complexes in the cytosol.
References
K Martin, A Helenius, "Transport of incoming influenza virus nucleocapsids into the nucleus", J Virol, 65, 1991, 232-44.
Reaction
13.1.4 Transport of Ribonucleoproteins into the Host Nucleus
Authors

Reviewers
Squires, B, 2006-10-29.
Description
An unusual characteristic of the influenza virus life cycle is its dependence on the nucleus. Trafficking of the viral genome into and out of the
nucleus is a tightly regulated process with all viral RNA synthesis occurring in the nucleus. The eight influenza virus genome segments never
exist as naked RNA but are associated with four viral proteins to form viral ribonucleoprotein complexes (vRNPs). The major viral protein in the
RNP complex is the nucleocapsid protein (NP), which coats the RNA. The remaining proteins PB1, PB2 and PA bind to the partially
complementary ends of the viral RNA, creating the distinctive panhandle structure. These RNPs (10-20nm wide) are too large to passively
diffuse into the nucleus and therefore, once released from an incoming particle must rely on the active import mechanism of the host cell nuclear
pore complex. All proteins in the RNP complex can independently localize to the nucleus due to the presence of nuclear localization signals
(NLSs) which mediate their interaction with the nuclear import machinery, including the RanGTPase (Fodor, 2004; Deng et al., 2006). However
the signals on NP have been shown to be both sufficient and necessary for the import of viral RNA.
References
T Deng, OG Engelhardt, B Thomas, AV Akoulitchev, GG Brownlee, E Fodor, "Role of ran binding protein 5 in nuclear import and assembly of the
influenza virus RNA polymerase complex", J Virol, 80, 2006, 11911-9.
JF Cros, A Garcia-Sastre, P Palese, "An unconventional NLS is critical for the nuclear import of the influenza A virus nucleoprotein and
ribonucleoprotein", Traffic, 6, 2005, 205-13.
E Fodor, M Smith, "The PA subunit is required for efficient nuclear accumulation of the PB1 subunit of the influenza A virus RNA polymerase
complex", J Virol, 78, 2004, 9144-53.
ST Nath, DP Nayak, "Function of two discrete regions is required for nuclear localization of polymerase basic protein 1 of A/WSN/33 influenza
virus (H1 N1)", Mol Cell Biol, 10, 1990, 4139-45.
A Nieto, S de la Luna, J Barcena, A Portela, J Ortin, "Complex structure of the nuclear translocation signal of influenza virus polymerase PA
subunit", J Gen Virol, 75, 1994, 29-36.
13.1.4.1 Recognition of the Nuclear Localization Signal (NLS) by a Karyopherin Alpha Family Protein
Authors
Reviewers
Squires, B, 2006-10-29.
Description
The eight influenza virus genome segments never exist as naked RNA but are associated with four viral proteins to form viral ribonucleoprotein
complexes (vRNPs). The major viral protein in the RNP complex is the nucleocapsid protein (NP), which coats the RNA. The remaining proteins
PB1, PB2 and PA bind to the partially complementary ends of the viral RNA, creating the distinctive panhandle structure. The influenza viral NP
behaves like a nuclear localization sequence (NLS) containing protein. The RNP docks at the nuclear envelope only in the presence of the
heterodimeric karyopherin alpha and beta complex. Here karyopherin alpha recognizes the RNP.
References
RE O'Neill, R Jaskunas, G Blobel, P Palese, J Moroianu, "Nuclear import of influenza virus RNA can be mediated by viral nucleoprotein and
transport factors required for protein import", J Biol Chem, 270, 1995, 22701-4.
Reaction
13.1.4.2 Recruitment of Karyopherin Beta to form a Trimeric Complex
Authors
Reviewers
Squires, B, 2006-10-29.
Description
The eight influenza virus genome segments are associated with four viral proteins to form viral ribonucleoprotein complexes (vRNPs). The major
viral protein in the RNP complex is the nucleocapsid protein (NP), which coats the RNA. The remaining proteins PB1, PB2 and PA bind to the
partially complementary ends of the viral RNA. The influenza viral NP behaves like a nuclear localization sequence (NLS) containing protein.
The RNP docks at the nuclear envelope only in the presence of the heterodimeric karyopherin alpha and beta complex. Once the NLS is
recognized by karyopherin alpha the karyopherin beta subunit joins the complex.
References
Reaction
13.1.4.3 Docking and transport of the RNP:Karyopherin complex through the nuclear pore
Authors
Reviewers
Squires, B, 2006-10-29.
Description
These RNPs (10-20nm wide) are too large to passively diffuse into the nucleus and therefore, once released from an incoming particle they must
rely on the active import mechanism of the host cell nuclear pore complex (NPC). Once the RNP heterodimeric karyopherin complex docks at
the NPC, it is transported into the nucleus.
References
J Mukaigawa, DP Nayak, "Two signals mediate nuclear localization of influenza virus (A/WSN/33) polymerase basic protein 2", J Virol, 65, 1991,
245-53.
Reaction
13.1.4.4 Release of the RNP into the host cell nucleus
Authors
Reviewers
Squires, B, 2006-10-29.
Description
Once the viral RNP and heterodimeric karyopherin complex has been transported into the nucleus the RNP dissasociates from the heterodimeric
karyopherins.
References
Reaction
13.1.5 Influenza Viral RNA Transcription and Replication
Authors
Bortz, E, Garcia-Sastre, A, 2007-02-12.
Reviewers
Squires, B, 2007-02-12.
Description
In the host cell nucleus, the viral negative-strand RNA (vRNA) serves as a template for the synthesis both of capped, polyadenylated viral
messenger RNA and of full-length positive-strand RNA or complementary RNA (cRNA). The cRNA is associated with the same viral proteins as
the vRNA. It serves as a template for the synthesis of new vRNA molecules, which in turn serve as a template for mRNA particularly early in
infection, and cRNA. Viral RNA polymerase subunits (PB1, PB2, and PA) and nucleoprotein (NP) enter the host cell nucleus and catalyze all
three of these reactions. During initial infection, these proteins enter the nucleus as part of the viral RNP complex. After the first round of viral
mRNA synthesis (primary transcription) and translation, newly synthesized viral polymerase proteins and NP localize to the nucleus to catalyze
further mRNA transcription and vRNA/cRNA replication. Late in the infection process, the synthesis of vRNA and nuclear export of newly
synthesized vRNP (vRNA complexed with NP and viral polymerase) is increased relative to transcription (Krug, 1981; Braam, 1983; Kawakami,
1983; Huang, 1990; Cros, 2003; Fodor, 2004; Deng, 2005; Amorim, 2006; reviewed in Neumann, 2004; Engelhardt, 2006; Buolo, 2006).
References
TS Huang, P Palese, M Krystal, "Determination of influenza virus proteins required for genome replication", J Virol, 64, 1990, 5669-73.
OG Engelhardt, E Fodor, "Functional association between viral and cellular transcription during influenza virus infection", Rev Med Virol, 16,
2006, 329-45.
complex", J Virol, 78, 2004, 9144-53.
T Deng, J Sharps, E Fodor, GG Brownlee, "In vitro assembly of PB2 with a PB1-PA dimer supports a new model of assembly of influenza A
virus polymerase subunits into a functional trimeric complex", J Virol, 79, 2005, 8669-74.
JF Cros, P Palese, "Trafficking of viral genomic RNA into and out of the nucleus: influenza, Thogoto and Borna disease viruses", Virus Res, 95,
2003, 3-12.
S Boulo, H Akarsu, RW Ruigrok, F Baudin, "Nuclear traffic of influenza virus proteins and ribonucleoprotein complexes", Virus Res, 124, 2006,
12-21.
G Neumann, GG Brownlee, E Fodor, Y Kawaoka, "Orthomyxovirus replication, transcription, and polyadenylation", Curr Top Microbiol Immunol,
283, 2004, 121-43.
RM Krug, "Priming of influenza viral RNA transcription by capped heterologous RNAs", Curr Top Microbiol Immunol, 93, 1981, 125-49.
J Braam, I Ulmanen, RM Krug, "Molecular model of a eucaryotic transcription complex: functions and movements of influenza P proteins during
capped RNA-primed transcription", Cell, 34, 1983, 609-18.
MJ Amorim, P Digard, "Influenza A virus and the cell nucleus", Vaccine, 24, 2006, 6651-5.
K Kawakami, A Ishihama, "RNA polymerase of influenza virus. III. Isolation of RNA polymerase-RNA complexes from influenza virus PR8.", J
Biochem (Tokyo), 93, 1983, 989-96.
13.1.5.1 vRNP Assembly
Authors
Reviewers
Squires, B, 2007-02-12.
Description
For each of eight gene segments, a viral ribonucleoprotein (vRNP), containing a viral negative-sense RNA (vRNA) segment complexed with
nucleoprotein (NP) and the trimeric influenza polymerase (PB1, PB2, and PA), is assembled in the nucleus (Braam, 1983; Jones, 1986; Cros,
2003; reviewed in Buolo, 2006). The vRNP functions in three modes (reviewed in Mikulasova, 2000; Neumann, 2004): (1) transcription, which
synthesizes viral messenger RNA from the vRNA template using as primers 5' ends of cellular mRNAs containing the cap; (2) replication, which
produces positive-sense complementary RNA (cRNA) and subsequently vRNA, both complexed with NP and the trimeric polymerase; or (3), the
vRNP is exported from the nucleus into the cytoplasm and is incorporated into assembling virions at the plasma membrane.
References
IM Jones, PA Reay, KL Philpott, "Nuclear location of all three influenza polymerase proteins and a nuclear signal in polymerase PB2", EMBO J,
5, 1986, 2371-6.
JF Cros, P Palese, "Trafficking of viral genomic RNA into and out of the nucleus: influenza, Thogoto and Borna disease viruses", Virus Res, 95,
2003, 3-12.
12-21.
283, 2004, 121-43.
A Mikulasova, E Vareckova, E Fodor, "Transcription and replication of the influenza a virus genome", Acta Virol, 44, 2000, 273-82.
13.1.5.1.1 Viral Polymerase Assembly
Authors

Reviewers
Squires, B, 2007-02-12.
Description
The mature ternary influenza viral polymerase complex consists of PB1, PB2, and PA. The N-terminus of PB1 (residues 1-48) interacts with
PB2, and amino acids 506-659 in PB1 interact with the PA subunit (Gonzalez, 1996; Perez, 2001). Although monomeric PB1, PB2 and PA, as
well as PB1-PB2 and PB1-PA dimers are likely to exist in infected cells, it is believed that most of the polymerase proteins are assembled into
the trimeric PB1-PB2-PA complex (Detjen, 1987). Newly synthesized subunits of the polymerase are imported into the nucleus through nuclear
localization signals (NLS), which interact with cellular importin family proteins (Jones, 1986; Buolo, 2006). Importin beta-3 (Ran binding protein 5)
facilitates nuclear import of PB1 and a PB1-PA dimer (Deng, 2006); coexpression of PA with PB1 was shown to enhance the import of PB1
(Fodor, 2004). A PB1-PB2 dimer has been found to interact with the molecular chaperone heat shock protein 90 (HSP90) to facilitate import
(Naito, 2007). The three subunits assembled in the nucleus form a mature ternary polymerase complex that binds viral vRNA or cRNA (Jones,
1986; Buolo, 2006).
References
S Gonzalez, T Zurcher, J Ortin, "Identification of two separate domains in the influenza virus PB1 protein involved in the interaction with the PB2
and PA subunits: a model for the viral RNA polymerase structure", Nucleic Acids Res, 24, 1996, 4456-63.
T Naito, F Momose, A Kawaguchi, K Nagata, "Involvement of hsp90 in assembly and nuclear import of influenza virus RNA polymerase
subunits", J Virol, 81, 2007, 1339-49.
T Deng, OG Engelhardt, B Thomas, AV Akoulitchev, GG Brownlee, E Fodor, "Role of ran binding protein 5 in nuclear import and assembly of the
influenza virus RNA polymerase complex", J Virol, 80, 2006, 11911-9.
IM Jones, PA Reay, KL Philpott, "Nuclear location of all three influenza polymerase proteins and a nuclear signal in polymerase PB2", EMBO J,
5, 1986, 2371-6.
complex", J Virol, 78, 2004, 9144-53.
12-21.
DR Perez, RO Donis, "Functional analysis of PA binding by influenza a virus PB1: effects on polymerase activity and viral infectivity", J Virol, 75,
2001, 8127-36.
BM Detjen, Angelo C St, MG Katze, RM Krug, "The three influenza virus polymerase (P) proteins not associated with viral nucleocapsids in the
infected cell are in the form of a complex", J Virol, 61, 1987, 16-22.
Reaction
13.1.5.1.2 NP binds vRNA
Authors
Reviewers
Squires, B, 2007-02-12.
Description
Viral genomic RNA (vRNA) and complementary RNA (cRNA) are likely bound by the influenza nucleoprotein (NP) immediately upon synthesis.
Although two nuclear localization signals have been mapped in the NP, an unconventional N-terminal NLS and a bipartite NLS within amino
acids 198-216 (Wang, 1997; Neumann, 1997; Ozawa, 2007), the crystal structure of the NP suggests that only the unconventional NLS is
exposed and can be used as a functional NLS (Ye, 2006). This unconvenetional NLS interacts with importins alpha-1 and -2 (Cros et al., 2005;
Wang et al., 1997; Buolo et al., 2006). The three-dimensional structure of NP has revealed that NP molecules associate as a trimer, interacting
through beta-sheets b5, b6, and b7 in the C-terminal domain of the protein; the viral RNA likely wraps around the outside of the complex (Ye,
2006).
References
P Wang, P Palese, RE O'Neill, "The NPI-1/NPI-3 (karyopherin alpha) binding site on the influenza a virus nucleoprotein NP is a nonconventional
nuclear localization signal", J Virol, 71, 1997, 1850-6.
12-21.
G Neumann, MR Castrucci, Y Kawaoka, "Nuclear import and export of influenza virus nucleoprotein", J Virol, 71, 1997, 9690-700.
Q Ye, RM Krug, YJ Tao, "The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA", Nature, 444, 2006,
1078-82.
M Ozawa, K Fujii, Y Muramoto, S Yamada, S Yamayoshi, A Takada, H Goto, T Horimoto, Y Kawaoka, "Contributions of two nuclear localization
signals of influenza A virus nucleoprotein to viral replication", J Virol, 81, 2007, 30-41.
Reaction
13.1.5.2 Viral Messenger RNA Synthesis
Authors
Reviewers
Squires, B, 2007-02-12.
Description
Like the mRNAs of the host cell, influenza virus mRNAs are capped and polyadenylated (reviewed in Neumann, 2004). The methylated caps,
however, are scavenged from host cell mRNAs and serve as primers for viral RNA synthesis, a process termed 'cap-snatching' (Krug, 1981;
Hagen, 1994). The PB2 polymerase protein binds the cap, activating endonucleolytic cleavage of the host mRNA by PB1. The 3' poly-A tracts on
viral messages are generated by polymerase stuttering on poly-U tracts near the 5' end of the template vRNA (Robertson, 1981; Zheng, 1999).
The second process allows polyadenylation of viral mRNAs when the host cell polyadenylation process has been inhibited (Engelhardt, 2006;
Amorim, 2006). Notably, early transcripts (including NP and NS1) accumulate in the cytoplasm before late transcripts (M1, HA, and NS2), and in
varying abundances, suggesting additional control mechanisms regulating viral gene expression (Shapiro, 1987; Hatada, 1989; Amorim, 2006).
References
2006, 329-45.
GI Shapiro, T Jr Gurney, RM Krug, "Influenza virus gene expression: control mechanisms at early and late times of infection and
nuclear-cytoplasmic transport of virus-specific RNAs", J Virol, 61, 1987, 764-73.
H Zheng, HA Lee, P Palese, A Garcia-Sastre, "Influenza A virus RNA polymerase has the ability to stutter at the polyadenylation site of a viral
RNA template during RNA replication", J Virol, 73, 1999, 5240-3.
283, 2004, 121-43.
M Hagen, TD Chung, JA Butcher, M Krystal, "Recombinant influenza virus polymerase: requirement of both 5' and 3' viral ends for endonuclease
activity", J Virol, 68, 1994, 1509-15.
JS Robertson, M Schubert, RA Lazzarini, "Polyadenylation sites for influenza virus mRNA", J Virol, 38, 1981, 157-63.
E Hatada, M Hasegawa, J Mukaigawa, K Shimizu, R Fukuda, "Control of influenza virus gene expression: quantitative analysis of each viral
RNA species in infected cells", J Biochem (Tokyo), 105, 1989, 537-46.
13.1.5.2.1 Assembly of an Active Transcription Complex

Authors
Reviewers
Squires, B, 2007-02-12.
Description
The 5' end of the vRNA associates with a binding site on the PB1 subunit of the viral RNA polymerase, distinct from the 3' vRNA binding site,
which is subsequenty bound forming a loop. These binding events set off allosteric conformational changes in the trimeric polymerase complex
that induce PB2 binding of the methylated cap on a host pre-mRNA (Plotch, 1981; Cianci, 1995; Li, 1998; Brownlee, 2002; Kolpashchikov,
2004). PB2 amino acids 242-282 and 538-577 are involved in cap binding (Honda, 1999). Direct or indirect interaction with active, transcribing
host RNA polymerase II is thought to supply host mRNA for the caps (Bouloy, 1978; Engelhardt, 2005).
References
C Cianci, L Tiley, M Krystal, "Differential activation of the influenza virus polymerase via template RNA binding", J Virol, 69, 1995, 3995-9.
GG Brownlee, JL Sharps, "The RNA polymerase of influenza a virus is stabilized by interaction with its viral RNA promoter", J Virol, 76, 2002,
7103-13.
OG Engelhardt, M Smith, E Fodor, "Association of the influenza A virus RNA-dependent RNA polymerase with cellular RNA polymerase II", J
Virol, 79, 2005, 5812-8.
ML Li, BC Ramirez, RM Krug, "RNA-dependent activation of primer RNA production by influenza virus polymerase: different regions of the same
protein subunit constitute the two required RNA-binding sites", EMBO J, 17, 1998, 5844-52.
A Honda, K Mizumoto, A Ishihama, "Two separate sequences of PB2 subunit constitute the RNA cap-binding site of influenza virus RNA
polymerase", Genes Cells, 4, 1999, 475-85.
DM Kolpashchikov, A Honda, A Ishihama, "Structure-function relationship of the influenza virus RNA polymerase: primer-binding site on the PB1
subunit", Biochemistry, 43, 2004, 5882-7.
M Bouloy, SJ Plotch, RM Krug, "Globin mRNAs are primers for the transcription of influenza viral RNA in vitro", Proc Natl Acad Sci U S A, 75,
1978, 4886-90.
SJ Plotch, M Bouloy, I Ulmanen, RM Krug, "A unique cap(m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to
generate the primers that initiate viral RNA transcription", Cell, 23, 1981, 847-58.
Reaction
13.1.5.2.2 Priming and Initiation of Transcription
Authors
Reviewers
Squires, B, 2007-02-12.
Description
The host cell mRNA bound to viral RNA polymerase PB2 subunit is cleaved by the viral RNA polymerase PB1 subunit's endonuclease activity,
and the capped 5' end plus 10-13 nucleotides of the host mRNA remains bound to the polymerase complex (Plotch, 1981; Krug, 1981; Hagen,
1994; Cianci, 1995, Li, 1998; Li, 2001). Viral mRNA may be protected against cap-snatching by the polymerase complex itself, which tightly
binds capped viral mRNA (Shih, 1996). A guanine residue, complementary to a cytosine in the vRNA, is added to the host-derived cap,
catalyzed by the RNA polymerase activity of the PB1 viral RNA polymerase subunit (Beaton, 1981; Toyoda, 1986).
References
ML Li, P Rao, RM Krug, "The active sites of the influenza cap-dependent endonuclease are on different polymerase subunits", EMBO J, 20,
2001, 2078-86.
T Toyoda, M Kobayashi, S Nakada, A Ishihama, "Molecular dissection of influenza virus RNA polymerase: PB1 subunit alone is able to catalyze
RNA synthesis", Virus Genes, 12, 1996, 155-63.
SR Shih, RM Krug, "Surprising function of the three influenza viral polymerase proteins: selective protection of viral mRNAs against the
cap-snatching reaction catalyzed by the same polymerase proteins", Virology, 226, 1996, 430-5.
ML Li, BC Ramirez, RM Krug, "RNA-dependent activation of primer RNA production by influenza virus polymerase: different regions of the same
protein subunit constitute the two required RNA-binding sites", EMBO J, 17, 1998, 5844-52.
M Hagen, TD Chung, JA Butcher, M Krystal, "Recombinant influenza virus polymerase: requirement of both 5' and 3' viral ends for endonuclease
activity", J Virol, 68, 1994, 1509-15.
AR Beaton, RM Krug, "Selected host cell capped RNA fragments prime influenza viral RNA transcription in vivo", Nucleic Acids Res, 9, 1981,
4423-36.
SJ Plotch, M Bouloy, I Ulmanen, RM Krug, "A unique cap(m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to
generate the primers that initiate viral RNA transcription", Cell, 23, 1981, 847-58.
Reaction
13.1.5.2.3 Elongation of viral mRNA
Authors
Reviewers
Squires, B, 2007-02-12.
Description
Catalyzed by the RNA polymerase activity of the viral PB1 subunit, an mRNA complementary to the bound vRNA is synthesized (Plotch, 1977).
PA and PB2 move down the growing mRNA in complex with PB1, with PB2 possibly dissociating from the cap (Braam, 1983). However, the
5â€™ end of the vRNA may remain bound during elongation as the template is threaded through in a 3â€™ to 5â€™ direction until a
polyadenylation signal is encountered (Poon, 1998; Zheng, 1999).
References
LL Poon, DC Pritlove, J Sharps, GG Brownlee, "The RNA polymerase of influenza virus, bound to the 5' end of virion RNA, acts in cis to
polyadenylate mRNA", J Virol, 72, 1998, 8214-9.
SJ Plotch, RM Krug, "Influenza virion transcriptase: synthesis in vitro of large, polyadenylic acid-containing complementary RNA", J Virol, 21,
1977, 24-34.
Reaction
13.1.5.2.4 Polyadenylation and Termination
Authors
Reviewers
Squires, B, 2007-02-12.
Description
A poly-uridine sequence motif, consisting in most cases of 5-7 U residues, abuts the "panhandle" duplex structure in the vRNA; this sequence is
approximately 16 nucleotides from the 5' end of this RNA duplex structure within the vRNA promoter. Encountering this signal, the viral RNA
polymerase stutters, leading to the synthesis of a poly-A tail on the viral mRNA (Robertson, 1981; Luo, 1991; Li,1994; Poon, 1998; Zheng et al.
1999).
References
LL Poon, DC Pritlove, J Sharps, GG Brownlee, "The RNA polymerase of influenza virus, bound to the 5' end of virion RNA, acts in cis to
polyadenylate mRNA", J Virol, 72, 1998, 8214-9.
GX Luo, W Luytjes, M Enami, P Palese, "The polyadenylation signal of influenza virus RNA involves a stretch of uridines followed by the RNA
duplex of the panhandle structure", J Virol, 65, 1991, 2861-7.
JS Robertson, M Schubert, RA Lazzarini, "Polyadenylation sites for influenza virus mRNA", J Virol, 38, 1981, 157-63.
X Li, P Palese, "Characterization of the polyadenylation signal of influenza virus RNA", J Virol, 68, 1994, 1245-9.
Reaction
13.1.5.2.5 Viral mRNA Splicing (M, NS segments)
Authors
Reviewers
Squires, B, 2007-02-12.
Description
The viral polymerase complex produces positive-sense viral mRNA with host-cell derived 5' methyl caps. Alternately spliced mRNA transcribed
from M and NS vRNA segments 7 and 8, producing the spliced mRNA for M2 and NEP/NS2, respectively, are thought to be coupled to the
cellular splicing and export mechanisms (Lamb, 1980; Lamb, 1981; Chen, 2000; Li, 2001). As segments 7 and 8 each encode two proteins,
splicing must be regulated allowing for alternative mRNAs, with the spliced products in the minority (approximately 10%). M1 splicing may be
regulated by the viral polymerase and the cellular SR splicing protein SF2/ASF (Shih, 1995; Shih, 1996); while NS1 splicing appears to be
regulated by the viral mRNA intrinsically (Alonso-Caplen, 1991; Valcarel, 1991).
References
Z Chen, RM Krug, "Selective nuclear export of viral mRNAs in influenza-virus-infected cells", Trends Microbiol, 8, 2000, 376-83.
Y Li, ZY Chen, W Wang, CC Baker, RM Krug, "The 3'-end-processing factor CPSF is required for the splicing of single-intron pre-mRNAs in
vivo", RNA, 7, 2001, 920-31.
J Valcarcel, A Portela, J Ortin, "Regulated M1 mRNA splicing in influenza virus-infected cells", J Gen Virol, 1991, 1301-8.
RA Lamb, CJ Lai, PW Choppin, "Sequences of mRNAs derived from genome RNA segment 7 of influenza virus: colinear and interrupted
mRNAs code for overlapping proteins", Proc Natl Acad Sci U S A, 78, 1981, 4170-4.
RA Lamb, PW Choppin, RM Chanock, CJ Lai, "Mapping of the two overlapping genes for polypeptides NS1 and NS2 on RNA segment 8 of
influenza virus genome", Proc Natl Acad Sci U S A, 77, 1980, 1857-61.
FV Alonso-Caplen, RM Krug, "Regulation of the extent of splicing of influenza virus NS1 mRNA: role of the rates of splicing and of the
nucleocytoplasmic transport of NS1 mRNA", Mol Cell Biol, 11, 1991, 1092-8.
SR Shih, RM Krug, "Novel exploitation of a nuclear function by influenza virus: the cellular SF2/ASF splicing factor controls the amount of the
essential viral M2 ion channel protein in infected cells", EMBO J, 15, 1996, 5415-27.
SR Shih, ME Nemeroff, RM Krug, "The choice of alternative 5' splice sites in influenza virus M1 mRNA is regulated by the viral polymerase
complex", Proc Natl Acad Sci U S A, 92, 1995, 6324-8.
Reaction
13.1.5.2.6 Export of Spliced Viral mRNA
Authors
Reviewers
Squires, B, 2007-02-12.
Description
In the cases of spliced, polyadenylated mRNA transcribed from M (segment 7) and NS (segment 8) vRNA templates (producing the spliced
mRNA for M2 and NS2/NEP, respectively), export may be coupled to aspects of the cellular splicing and export mechanisms (Chen, 2000;
Alonso-Caplan et al, 1992; Amorim, 2006). Simultaneously, the export of cellular mRNA appear to be inhibited by the viral NS1 protein, which
binds to the cellular cleavage and polyadenylation specificity factor (CPSF), preventing polyadenylation and completion of pre-mRNA processing
(Nemerof et al., 1998; Fortes, 1994; Lu, 1994; Li, 2001).
References
vivo", RNA, 7, 2001, 920-31.
Y Lu, XY Qian, RM Krug, "The influenza virus NS1 protein: a novel inhibitor of pre-mRNA splicing", Genes Dev, 8, 1994, 1817-28.
P Fortes, A Beloso, J Ortin, "Influenza virus NS1 protein inhibits pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport", EMBO J,
13, 1994, 704-12.
FV Alonso-Caplen, ME Nemeroff, Y Qiu, RM Krug, "Nucleocytoplasmic transport: the influenza virus NS1 protein regulates the transport of
spliced NS2 mRNA and its precursor NS1 mRNA", Genes Dev, 6, 1992, 255-67.
ME Nemeroff, SM Barabino, Y Li, W Keller, RM Krug, "Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and inhibits
3'end formation of cellular pre-mRNAs", Mol Cell, 1, 1998, 991-1000.
Reaction
13.1.5.2.7 Viral mRNA Export
Authors
Reviewers
Squires, B, 2007-02-12.
Description
The viral polymerase complex produces positive-sense viral mRNA with host-cell derived 5' methyl caps. Capped viral mRNAs are selectively
exported from the host cell nucleus through a currently unclear mechanism that may rely on components of the host cell mRNA export
machinery (Chen, 2000; Engelhardt, 2006). Polyadenylation of viral mRNA appears be required for influenza mRNA export (Poon, 2000). A
coupling of viral mRNA export with cellular pre-mRNA processing complexes, recruited by phosphorylation of host RNA polymerase II C-terminal
domain which interacts with the viral polymerase (Engelhardt, 2005), has been proposed as controlling the export of a subset (M1, HA, and NS1,
but not NP) of viral mRNA from the nucleus (Amorim, 2007).
References
MJ Amorim, EK Read, RM Dalton, L Medcalf, P Digard, "Nuclear Export of Influenza A Virus mRNAs Requires Ongoing RNA Polymerase II
Activity", Traffic, 8, 2007, 1-11.
2006, 329-45.
OG Engelhardt, M Smith, E Fodor, "Association of the influenza A virus RNA-dependent RNA polymerase with cellular RNA polymerase II", J
Virol, 79, 2005, 5812-8.
LL Poon, E Fodor, GG Brownlee, "Polyuridylated mRNA synthesized by a recombinant influenza virus is defective in nuclear export", J Virol, 74,
2000, 418-27.
Reaction
13.1.5.3 Viral mRNA Translation
Authors
Reviewers
Squires, B, 2007-02-12.
Description
Spliced and unspliced viral mRNA in the cytoplasm are translated by host cell ribosomal translation machinery (reviewed in Kash, 2006). At least
ten viral proteins are synthesized: HA, NA, PB1, PB2, PA, NP, NS1, NEP/NS2, M1, and M2. Viral mRNA translation is believed to be enhanced
by conserved 5'UTR sequences that interact with the ribosomal machinery and at least one cellular RNA-binding protein, G-rich sequence factor
1 (GRSF-1), has been found to specifically interact with the viral 5' UTRs. (Park, 1995; Park, 1999). The viral NS1 protein and the cellular protein
P58(IPK) enhance viral translation indirectly by preventing the activation of the translational inhibitor PKR (Salvatore, 2002; Goodman, 2006).
The viral NS1 protein has also been proposed to specifically enhance translation through interaction with host poly(A)-binding protein 1 (PABP1)
(Burgui, 2003). Simultaneously, host cell protein synthesis is downregulated in influenza virus infection through still uncharacterized mechanisms
(Katze, 1986; Garfinkel, 1992; Kash, 2006). In most human influenza A strains (such as PR8), the PB1 mRNA segment is capable of producing
a second protein, PB1-F2, from a short +1 open reading frame initiating downstream of the PB1 ORF initiation codon (Chen, 2001).
References
AG Goodman, JA Smith, S Balachandran, O Perwitasari, SC Proll, MJ Thomas, MJ Korth, GN Barber, LA Schiff, MG Katze, "The cellular protein
P58IPK regulates influenza virus mRNA translation and replication through a PKR-mediated mechanism", J Virol, 81, 2007, 2221-30.
M Salvatore, CF Basler, JP Parisien, CM Horvath, S Bourmakina, H Zheng, T Muster, P Palese, A Garcia-Sastre, "Effects of influenza A virus
NS1 protein on protein expression: the NS1 protein enhances translation and is not required for shutoff of host protein synthesis", J Virol, 76,
2002, 1206-12.
JC Kash, DM Cunningham, MW Smit, Y Park, D Fritz, J Wilusz, MG Katze, "Selective translation of eukaryotic mRNAs: functional molecular
analysis of GRSF-1, a positive regulator of influenza virus protein synthesis", J Virol, 76, 2002, 10417-26.
W Chen, PA Calvo, D Malide, J Gibbs, U Schubert, I Bacik, S Basta, R O'Neill, J Schickli, P Palese, P Henklein, JR Bennink, JW Yewdell, "A
novel influenza A virus mitochondrial protein that induces cell death", Nat Med, 7, 2001, 1306-12.
MG Katze, D DeCorato, RM Krug, "Cellular mRNA translation is blocked at both initiation and elongation after infection by influenza virus or
adenovirus", J Virol, 60, 1986, 1027-39.
JC Kash, AG Goodman, MJ Korth, MG Katze, "Hijacking of the host-cell response and translational control during influenza virus infection", Virus
Res, 119, 2006, 111-20.
I Burgui, T Aragon, J Ortin, A Nieto, "PABP1 and eIF4GI associate with influenza virus NS1 protein in viral mRNA translation initiation
complexes", J Gen Virol, 84, 2003, 3263-74.
YW Park, J Wilusz, MG Katze, "Regulation of eukaryotic protein synthesis: selective influenza viral mRNA translation is mediated by the cellular
RNA-binding protein GRSF-1", Proc Natl Acad Sci U S A, 96, 1999, 6694-9.
YW Park, MG Katze, "Translational control by influenza virus. Identification of cis-acting sequences and trans-acting factors which may regulate
selective viral mRNA translation.", J Biol Chem, 270, 1995, 28433-9.
MS Garfinkel, MG Katze, "Translational control by influenza virus. Selective and cap-dependent translation of viral mRNAs in infected cells.", J
Biol Chem, 267, 1992, 9383-90.
13.1.5.3.1 Synthesis of PB1-F2
Authors
Reviewers
Squires, B, 2007-02-12.
Description
For most influenza A strains (such as PR8), the PB1 mRNA segment produces a second protein, PB1-F2, from the +1 open reading frame
(Chen, 2001). PB1-F2 is a pro-apoptotic, mitochondria-localized protein (Chen, 2001; Gibbs, 2003) that oligomerizes (Bruns, 2007) and
sensitizes cells to death in concert with the mitochondrial ANT3 and VDAC proteins (Zamarin, 2005).
References
D Zamarin, A Garcia-Sastre, X Xiao, R Wang, P Palese, "Influenza virus PB1-F2 protein induces cell death through mitochondrial ANT3 and
VDAC1", PLoS Pathog, 1, 2005, e4.
K Bruns, N Studtrucker, A Sharma, T Fossen, D Mitzner, A Eissmann, U Tessmer, R Roder, P Henklein, V Wray, U Schubert, "Structural
characterization and oligomerization of PB1-F2, a proapoptotic influenza A virus protein", J Biol Chem, 282, 2007, 353-63.
JS Gibbs, D Malide, F Hornung, JR Bennink, JW Yewdell, "The influenza A virus PB1-F2 protein targets the inner mitochondrial membrane via a
predicted basic amphipathic helix that disrupts mitochondrial function", J Virol, 77, 2003, 7214-24.
Reaction
13.1.5.3.2 Viral Protein Synthesis
Authors
Reviewers
Squires, B, 2007-02-12.
Description
Spliced and unspliced viral mRNA exported into the cytoplasm are translated by the host cell ribosomal translation machinery (reviewed in Kash,
2006). At least ten viral proteins are synthesized: HA, NA, PB1, PB2, PA, NP, NS1, NEP/NS2 (from spliced NS mRNA), M1, and M2 (from
spliced M mRNA). The abundance of each of these proteins is thought to be controlled by differential mRNA abundances and stability (Tekamp,
1980; Hatada, 1989). As the localization of the nascent polypeptides is different between viral proteins with transmembrane domains (HA, NA
and M2, which translocate to the ER and are transported through the Golgi to the plasma membrane) and soluble viral proteins (such as NP, the
polymerase subunits, and NS1), mechanisms linking the translation of particular viral mRNA with subsequent protein localization rely on signal
sequences recognized by the cell.
References
AG Goodman, JA Smith, S Balachandran, O Perwitasari, SC Proll, MJ Thomas, MJ Korth, GN Barber, LA Schiff, MG Katze, "The cellular protein
P58IPK regulates influenza virus mRNA translation and replication through a PKR-mediated mechanism", J Virol, 81, 2007, 2221-30.
JC Kash, AG Goodman, MJ Korth, MG Katze, "Hijacking of the host-cell response and translational control during influenza virus infection", Virus
Res, 119, 2006, 111-20.
E Hatada, M Hasegawa, J Mukaigawa, K Shimizu, R Fukuda, "Control of influenza virus gene expression: quantitative analysis of each viral
RNA species in infected cells", J Biochem (Tokyo), 105, 1989, 537-46.
PA Tekamp, EE Penhoet, "Quantification of influenza virus messenger RNAs", J Gen Virol, 47, 1980, 449-59.
Reaction
13.1.5.4 cRNA Synthesis
Authors
Reviewers
Squires, B, 2007-02-12.
Description
Synthesis of full length complementary viral RNA (cRNA) requires that vRNA transcription initiates without the help of a host cell methyl RNA cap
as a primer (Crow, 2004; Vreede, 2004; Deng, 2006), and that it proceeds to the 5' end of the vRNA template without stuttering on the
sub-terminal poly-U sequence. Free viral NP protein appears to play a central role in enabling both of these features of cRNA synthesis,
although the molecular details of its role remain unclear (Shapiro, 1988; Medcalf, 1999; Mullin, 2004).
References
M Crow, T Deng, M Addley, GG Brownlee, "Mutational analysis of the influenza virus cRNA promoter and identification of nucleotides critical for
GI Shapiro, RM Krug, "Influenza virus RNA replication in vitro: synthesis of viral template RNAs and virion RNAs in the absence of an added
primer", J Virol, 62, 1988, 2285-90.
AE Mullin, RM Dalton, MJ Amorim, D Elton, P Digard, "Increased amounts of the influenza virus nucleoprotein do not promote higher levels of
viral genome replication", J Gen Virol, 85, 2004, 3689-98.
FT Vreede, TE Jung, GG Brownlee, "Model suggesting that replication of influenza virus is regulated by stabilization of replicative intermediates",
J Virol, 78, 2004, 9568-72.
T Deng, FT Vreede, GG Brownlee, "Different de novo initiation strategies are used by influenza virus RNA polymerase on its cRNA and viral
RNA promoters during viral RNA replication", J Virol, 80, 2006, 2337-48.
L Medcalf, E Poole, D Elton, P Digard, "Temperature-sensitive lesions in two influenza A viruses defective for replicative transcription disrupt
RNA binding by the nucleoprotein", J Virol, 73, 1999, 7349-56.
13.1.5.4.1 Initiation of cRNA Synthesis
Authors

Reviewers
Squires, B, 2007-02-12.
Description
Viral vRNA, complexed with NP protein, is bound by the trimeric viral polymerase complex in a stable secondary structure-dependent manner,
referred to as a panhandle, fork or cork-screw (Fodor, 1994; Brownlee, 2002; Park, 2003; Crow, 2004). This RNA structure is made of both the
5Ã¢â‚¬â„¢ and 3' ends of the vRNA. The polymerase is thought to first bind the 5' end of the vRNA and then the 3' end. Synthesis of cRNA
initiates without a host cell methylated RNA cap as a primer (Beaton, 1986; Galarza, 1996; Deng, 2006; Engehardt, 2006).
References
CJ Park, SH Bae, MK Lee, G Varani, BS Choi, "Solution structure of the influenza A virus cRNA promoter: implications for differential recognition
of viral promoter structures by RNA-dependent RNA polymerase", Nucleic Acids Res, 31, 2003, 2824-32.
2006, 329-45.
GG Brownlee, JL Sharps, "The RNA polymerase of influenza a virus is stabilized by interaction with its viral RNA promoter", J Virol, 76, 2002,
7103-13.
E Fodor, DC Pritlove, GG Brownlee, "The influenza virus panhandle is involved in the initiation of transcription", J Virol, 68, 1994, 4092-6.
JM Galarza, Q Peng, L Shi, DF Summers, "Influenza A virus RNA-dependent RNA polymerase: analysis of RNA synthesis in vitro", J Virol, 70,
1996, 2360-8.
AR Beaton, RM Krug, "Transcription antitermination during influenza viral template RNA synthesis requires the nucleocapsid protein and the
absence of a 5' capped end", Proc Natl Acad Sci U S A, 83, 1986, 6282-6.
Reaction
13.1.5.4.2 cRNA Extension

Authors
Reviewers
Squires, B, 2007-02-12.
Description
Virion vRNP is capable of synthesizing cRNA immediately following entry into the cell nucleus (Vreede, 2006). The PB1 subunit principally
catalyzes extension (Nakagawa, 1996). However, cRNA does not accumulate until later in the infection process, possiby requiring NP and the
trimeric polymerase for stabilization (Vreede, 2004). The vRNA template is released.
References
Y Nakagawa, K Oda, S Nakada, "The PB1 subunit alone can catalyze cRNA synthesis, and the PA subunit in addition to the PB1 subunit is
required for viral RNA synthesis in replication of the influenza virus genome", J Virol, 70, 1996, 6390-4.
FT Vreede, GG Brownlee, "Influenza virion-derived viral ribonucleoproteins synthesize both mRNA and cRNA in vitro", J Virol, 2006.
J Virol, 78, 2004, 9568-72.
Reaction
13.1.5.5 vRNA Synthesis
Authors

Reviewers
Squires, B, 2007-02-12.
Description
The synthesis of full-length negative strand viral RNA from a cRNA template is believed to follow the same principles as the synthesis of cRNA
from a vRNA template. The cRNA, complexed with viral nucleocapsid (NP) protein, is used as template by the trimeric viral polymerase (Pritlove,
1995; Vreede, 2004; Crow, 2004), and newly synthesized vRNA molecules are immediately packaged with NP molecules to form
ribonucleoprotein complexes (Vreede, 2004). There is some evidence that the production of vRNA-containing vRNP occurs in the nuclear matrix
as well as the nucleoplasm (Takizawa, 2006).
References
DC Pritlove, E Fodor, BL Seong, GG Brownlee, "In vitro transcription and polymerase binding studies of the termini of influenza A virus cRNA:
evidence for a cRNA panhandle", J Gen Virol, 1995, 2205-13.
J Virol, 78, 2004, 9568-72.
N Takizawa, K Watanabe, K Nouno, N Kobayashi, K Nagata, "Association of functional influenza viral proteins and RNAs with nuclear chromatin
and sub-chromatin structure", Microbes Infect, 8, 2006, 823-33.
13.1.5.5.1 Initiation of vRNA Synthesis
Authors
Reviewers
Squires, B, 2007-02-12.
Description
Initiation of synthesis of the viral genomic RNA (vRNA) is thought to require hairpin (or panhandle/corkscrew) RNA loop structures formed by
both the 5' and 3' ends of the cRNA (Pritlove, 1995; Crow, 2004; Park, 2003; Deng, 2006). The cRNA promoter has a similar structure to the
vRNA promoter, but slight sequence differences are believed to result in a stronger cRNA promoter. As with the vRNA promoter, the polymerase
is thought to first bind to the 5' end of the cRNA, then to the 3' end, and subsequently initiate RNA synthesis.
References
CJ Park, SH Bae, MK Lee, G Varani, BS Choi, "Solution structure of the influenza A virus cRNA promoter: implications for differential recognition
of viral promoter structures by RNA-dependent RNA polymerase", Nucleic Acids Res, 31, 2003, 2824-32.
DC Pritlove, E Fodor, BL Seong, GG Brownlee, "In vitro transcription and polymerase binding studies of the termini of influenza A virus cRNA:
evidence for a cRNA panhandle", J Gen Virol, 1995, 2205-13.
Reaction
13.1.5.5.2 vRNA Extension
Authors
Reviewers
Squires, B, 2007-02-12.
Description
vRNA is synthesized from the complementary cRNA strand by the trimeric polymerase complex, and bound by free NP protein (Honda, 1988;
Mikulasova, 2000; Neumann, 2004). The PB1 subunit, with PA, catalyzes extension (Nakagawa, 1996). The cRNA is released.
References
283, 2004, 121-43.
Y Nakagawa, K Oda, S Nakada, "The PB1 subunit alone can catalyze cRNA synthesis, and the PA subunit in addition to the PB1 subunit is
required for viral RNA synthesis in replication of the influenza virus genome", J Virol, 70, 1996, 6390-4.
A Honda, K Ueda, K Nagata, A Ishihama, "RNA polymerase of influenza virus: role of NP in RNA chain elongation", J Biochem (Tokyo), 104,
1988, 1021-6.
A Mikulasova, E Vareckova, E Fodor, "Transcription and replication of the influenza a virus genome", Acta Virol, 44, 2000, 273-82.
Reaction
13.1.6 Export of Viral Ribonucleoproteins from Nucleus
Authors
Reviewers
Squires, B, 2007-02-12.
Description
Influenza genomic RNA (vRNA), synthesized in the nucleus of the infected host cell, is packaged into ribonucleoprotein (RNP) complexes
containing viral polymerase proteins and NP (nucleocapsid). NP trimers bind the sugar phosphate backbone of the vRNA. As influenza viral RNP
complexes are too large for passive diffusion out of the nucleus, utilization of the cellular nuclear export machinery is achieved by viral adaptor
proteins. Matrix protein (M1) is critical for export of the complex from the nucleus, mediating the interaction of the RNP complex with the viral
NEP/NS2 protein, which in turn interacts with host cell CRM1/exportin-1 nuclear export protein (Martin, 1991; O'Neill, 1998; Neumann et al.,
2000; Elton, 2001; Cros, 2003; Ye, 2006; reviewed in Boulo, 2006).
References
12-21.
G Neumann, MT Hughes, Y Kawaoka, "Influenza A virus NS2 protein mediates vRNP nuclear export through NES-independent interaction with
hCRM1", EMBO J, 19, 2000, 6751-8.
D Elton, M Simpson-Holley, K Archer, L Medcalf, R Hallam, J McCauley, P Digard, "Interaction of the influenza virus nucleoprotein with the
cellular CRM1-mediated nuclear export pathway", J Virol, 75, 2001, 408-19.
13.1.6.1 Viral RNP Complexes in the Host Cell Nucleus
Authors
Reviewers
Squires, B, 2007-02-12.
Description
Viral RNP is assembled in the host cell nucleus through the interaction of full-length negative strand viral RNA (vRNA) and the viral nucleocapsid
(NP) and matrix (M1) proteins. Studies of interactions of the purified components in vitro and of tissue culture model systems expressing various
combinations of the components have established roles for both NP and M1 proteins in the assembly of a complex that has the physical
properties of vRNP purified from virions and that can be exported from the host cell nucleus (Whittaker, 1996; Huang, 2001; Baudin, 2001). Viral
RNP complexes have been found in the nucleoplasm, and also in the nuclear periphery, associated with the nuclear matrix or chromatin,
particularly for vRNA-containing complexes and M1 protein (Elton, 2005; Garcia-Robles, 2005; Takizawa et al., 2006).
References
X Huang, T Liu, J Muller, RA Levandowski, Z Ye, "Effect of influenza virus matrix protein and viral RNA on ribonucleoprotein formation and
nuclear export", Virology, 287, 2001, 405-16.
D Elton, MJ Amorim, L Medcalf, P Digard, "'Genome gating'; polarized intranuclear trafficking of influenza virus RNPs", Biol Lett, 1, 2005, 113-7.
I Garcia-Robles, H Akarsu, CW Muller, RW Ruigrok, F Baudin, "Interaction of influenza virus proteins with nucleosomes", Virology, 332, 2005,
329-36.
13.1.6.1.1 Newly synthesized vRNP for export
Authors
Reviewers
Squires, B, 2007-02-12.
Description
The nascent vRNP complexes, one for each gene segment, contain the negative-sense viral RNA and polymerase proteins (PB1, PB2, PA, and
NP). In a model using negative-sense viral RNP reconstituted from transfected cells, there are multiple NP complexes and one polymerase
complex arranged along a closed vRNA loop (Area et al., 2004). The three-dimensional structure of NP has revealed that three NP molecules
form a stable trimer, interacting through beta-sheets b5, b6, and b7 in the C-terminal domain of the protein (Ye, 2006), with the viral RNA
wrapping around the outside of the complex. Viral RNA from purified virions is present in an RNase-sensitive complex with NP and PB1, PB2,
and PA, consistent with this structural model (Baudin et al, 1994; Ruigrok et al., 1995; Klumpp et al., 1997). It is not clear what controls the fate
of vRNP, whether it is destined to become a template for transcription, for replication, or for export into the cytoplasm for packaging into virions
at the plasma membrane, nor how distinct sub-nuclear localization and NP distribution at the nuclear matrix might mark, or polarize, a vRNP for
export (Elton, 2005; Takizawa et al., 2006).
References
E Area, J Martin-Benito, P Gastaminza, E Torreira, JM Valpuesta, JL Carrascosa, J Ortin, "3D structure of the influenza virus polymerase
complex: localization of subunit domains", Proc Natl Acad Sci U S A, 101, 2004, 308-13.
Q Ye, RM Krug, YJ Tao, "The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA", Nature, 444, 2006,
1078-82.
RW Ruigrok, F Baudin, "Structure of influenza virus ribonucleoprotein particles. II. Purified RNA-free influenza virus ribonucleoprotein forms
structures that are indistinguishable from the intact influenza virus ribonucleoprotein particles.", J Gen Virol, 1995, 1009-14.
D Elton, MJ Amorim, L Medcalf, P Digard, "'Genome gating'; polarized intranuclear trafficking of influenza virus RNPs", Biol Lett, 1, 2005, 113-7.
K Klumpp, RW Ruigrok, F Baudin, "Roles of the influenza virus polymerase and nucleoprotein in forming a functional RNP structure", EMBO J,
16, 1997, 1248-57.
F Baudin, C Bach, S Cusack, RW Ruigrok, "Structure of influenza virus RNP. I. Influenza virus nucleoprotein melts secondary structure in
panhandle RNA and exposes the bases to the solvent.", EMBO J, 13, 1994, 3158-65.
Reaction
13.1.6.1.2 Binding of M1 to vRNP
Authors
Reviewers
Squires, B, 2007-02-12.
Description
M1 protein binds to viral RNP through its C-terminal domain (Baudin, 2001). The influenza M1 protein accumulates in the infected cell nucleus
through a nuclear localization signal (NLS) RKLKR (residues 101-105) in its N-terminus (Ye, 1999). A host cell protein, HSP70, is thought to
inhibit M1 binding at nonpermissive temperatures (Hirayama et al., 2004).
References
T Liu, J Muller, Z Ye, "Association of influenza virus matrix protein with ribonucleoproteins may control viral growth and morphology", Virology,
304, 2002, 89-96.
E Hirayama, H Atagi, A Hiraki, J Kim, "Heat shock protein 70 is related to thermal inhibition of nuclear export of the influenza virus
ribonucleoprotein complex", J Virol, 78, 2004, 1263-70.
X Huang, T Liu, J Muller, RA Levandowski, Z Ye, "Effect of influenza virus matrix protein and viral RNA on ribonucleoprotein formation and
nuclear export", Virology, 287, 2001, 405-16.
Z Ye, T Liu, DP Offringa, J McInnis, RA Levandowski, "Association of influenza virus matrix protein with ribonucleoproteins", J Virol, 73, 1999,
7467-73.
F Baudin, I Petit, W Weissenhorn, RW Ruigrok, "In vitro dissection of the membrane and RNP binding activities of influenza virus M1 protein",
Virology, 281, 2001, 102-8.
M Bui, EG Wills, A Helenius, GR Whittaker, "Role of the influenza virus M1 protein in nuclear export of viral ribonucleoproteins", J Virol, 74,
2000, 1781-6.
Reaction
13.1.6.1.3 Binding of NEP/NS2 to vRNP:M1
Authors
Reviewers
Squires, B, 2007-02-12.
Description
Structural characterization of NEP/NS2 suggests that acidic residues in the C-terminus of NEP/NS2 bind to M1, with Trp78 critical for interaction
(Ward, 1995; Yasuda, 1993; Akarsu, 2003).
References
J Yasuda, S Nakada, A Kato, T Toyoda, A Ishihama, "Molecular assembly of influenza virus: association of the NS2 protein with virion matrix",
Virology, 196, 1993, 249-55.
AC Ward, LA Castelli, AC Lucantoni, JF White, AA Azad, IG Macreadie, "Expression and analysis of the NS2 protein of influenza A virus", Arch
Virol, 140, 1995, 2067-73.
Reaction
13.1.6.2 NEP/NS2 Interacts with the Cellular Export Machinery
Authors
Reviewers
Squires, B, 2007-02-12.
Description
The viral RNP complex is exported from the nucleus via the host cell CRM1 export pathway (Fukuda, 1997; Neumann, 2000; reviewed in Buolo,
2006). The vRNP complex does not interact directly with CRM1 to form an export complex. Rather, an additional viral protein, nuclear export
protein (NEP/NS2), acts as an adaptor, binding the viral matrix M1 protein and CRM1, thus linking the viral RNP with CRM1 (Martin, 1991;
O'Neill, 1998; Neumann, 2000; Akarsu, 2003). The CRM1/exportin-1 complex recruits additional host cell proteins, and traverses the nuclear
pore into the cytosol.
References
12-21.
RE O'Neill, J Talon, P Palese, "The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins", EMBO J, 17,
1998, 288-96.
H Akarsu, WP Burmeister, C Petosa, I Petit, CW Muller, RW Ruigrok, F Baudin, "Crystal structure of the M1 protein-binding domain of the
influenza A virus nuclear export protein (NEP/NS2)", EMBO J, 22, 2003, 4646-55.
hCRM1", EMBO J, 19, 2000, 6751-8.
K Martin, A Helenius, "Nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (M1) promotes export and inhibits import",
Cell, 67, 1991, 117-30.
13.1.6.2.1 Binding of vRNP:M1:NEP complex to CRM1 export receptor
Description
Virus NEP/NS2 interacts with human CRM1 (hCRM1), possibly dependent on a nuclear export signal (NES) motif in the NEP/NS2 N-terminal
region (O'Neill, 1998; Neumann, 2000). The CRM1/exportin-1 pathway is a cellular mechanism for nuclear export, with CRM1 interacting with the
Ran small GTPase and a cargo molecule's leucine-rich NES (Fukuda, 1997; Petosa, 2004). Leptomycin B, which specifically inhibits hCRM1,
blocks export of viral RNP (Elton, 2001; Ma, 2001; Watanabe, 2001). Thus, NEP/NS2 interaction with cellular nuclear export machinery is
essential for nuclear export of vRNP complexes and influenza virus release. A role for NP protein interaction with export machinery has also
been proposed (Elton, 2001).
References
K Ma, AM Roy, GR Whittaker, "Nuclear export of influenza virus ribonucleoproteins: identification of an export intermediate at the nuclear
periphery", Virology, 282, 2001, 215-20.
K Watanabe, N Takizawa, M Katoh, K Hoshida, N Kobayashi, K Nagata, "Inhibition of nuclear export of ribonucleoprotein complexes of influenza
virus by leptomycin B", Virus Res, 77, 2001, 31-42.
hCRM1", EMBO J, 19, 2000, 6751-8.
D Elton, M Simpson-Holley, K Archer, L Medcalf, R Hallam, J McCauley, P Digard, "Interaction of the influenza virus nucleoprotein with the
cellular CRM1-mediated nuclear export pathway", J Virol, 75, 2001, 408-19.
Reaction
13.1.6.2.2 vRNP Export through the nuclear pore
Description
Viral RNP, bound by M1 and NEP/NS2 interacting with CRM1, are shuttled through the nuclear pore into the cytoplasm (Martin, 1991; O'Neill,
1998; Buolo, 2006). This mechanism may resemble export of HIV-1 ribonucleoprotein, where the HIV-1 Rev export protein interacts with CRM1
(Askjaer, 1998). A number of cofactors are implicated in CRM1-mediated export, including the small GTPase Ran, Ran-binding proteins 1 and 3,
and a guanine nucleotide exchange factor (Nilsson, 2001; Nemergut, 2002; Petosa, 2004). Ternary CRM1-cofactor-cargo complexes likely
interact transiently with nuclear pore proteins (nucleoporins) as they traverse the pore (reviewed in Suntharalingam, 2003). RanGTP is
hydrolyzed to RanGDP in the cytoplasm, an activity that can be stimulated by NEP/NS2 (Akarsu, 2003). Influenza infection activates
Raf/MEK/ERK signaling, which is necessary for NEP/NS2-mediated export of viral RNP (Pleschka, 2001; Marjuki, 2006). Influenza vRNP
complexes released into the cytoplasm do not re-enter the nucleus, as they are thought to remain bound by M1, preventing re-import (Martin,
1991). It has been suggested that M1 binding of zinc cations could distinguish M1 bound to the vRNP from polymerized, matrix M1 present in
nascent virions (Elster, 1994).
References
K Ma, AM Roy, GR Whittaker, "Nuclear export of influenza virus ribonucleoproteins: identification of an export intermediate at the nuclear
periphery", Virology, 282, 2001, 215-20.
J Nilsson, P Askjaer, J Kjems, "A role for the basic patch and the C terminus of RanGTP in regulating the dynamic interactions with importin
beta, CRM1 and RanBP1", J Mol Biol, 305, 2001, 231-43.
S Pleschka, T Wolff, C Ehrhardt, G Hobom, O Planz, UR Rapp, S Ludwig, "Influenza virus propagation is impaired by inhibition of the
Raf/MEK/ERK signalling cascade", Nat Cell Biol, 3, 2001, 301-5.
12-21.
C Elster, E Fourest, F Baudin, K Larsen, S Cusack, RW Ruigrok, "A small percentage of influenza virus M1 protein contains zinc but zinc does
not influence in vitro M1-RNA interaction", J Gen Virol, 1994, 37-42.
C Petosa, G Schoehn, P Askjaer, U Bauer, M Moulin, U Steuerwald, M Soler-Lopez, F Baudin, IW Mattaj, CW Muller, "Architecture of
CRM1/Exportin1 suggests how cooperativity is achieved during formation of a nuclear export complex", Mol Cell, 16, 2004, 761-75.
ME Nemergut, ME Lindsay, AM Brownawell, IG Macara, "Ran-binding protein 3 links Crm1 to the Ran guanine nucleotide exchange factor", J
Biol Chem, 277, 2002, 17385-8.
K Watanabe, N Takizawa, M Katoh, K Hoshida, N Kobayashi, K Nagata, "Inhibition of nuclear export of ribonucleoprotein complexes of influenza
virus by leptomycin B", Virus Res, 77, 2001, 31-42.
H Marjuki, MI Alam, C Ehrhardt, R Wagner, O Planz, HD Klenk, S Ludwig, S Pleschka, "Membrane accumulation of influenza A virus
hemagglutinin triggers nuclear export of the viral genome via protein kinase Calpha-mediated activation of ERK signaling", J Biol Chem, 281,
2006, 16707-15.
Reaction
13.1.7 Virus Assembly and Release
Description
Influenza viruses assemble and bud from the apical plasma membrane of polarized cells e.g. lung epithelial cells of the infected host. This
asymmetrical process (i.e. apical [Influenza virus] or basolateral [Marburg virus]) is thought to have an important role in viral pathogenesis and
tissue tropism. In most cases the individual viral envelope proteins are seen to accumulate at the same polar surface from which virus budding
occurs, suggesting that they determine the maturation site
References
AP Schmitt, RA Lamb, "Influenza virus assembly and budding at the viral budozone", Adv Virus Res, 64, 2005, 383-416.
ER Boulan, DD Sabatini, "Asymmetric budding of viruses in epithelial monlayers: a model system for study of epithelial polarity", Proc Natl Acad
Sci U S A, 75, 1978, 5071-5.
13.1.7.1 Assembly of Viral Components at the Budding Site
Authors
Steel, J, 2007-04-30.
Reviewers
Squires, B, Rush, MG, 2007-04-30.
Description
Following synthesis on membrane-bound ribosomes, the three viral integral membrane proteins, HA (hemagglutinin), NA (neuraminidase) and
M2 (ion channel) enter the host endoplasmic reticulum (ER) where all three are folded and HA and NA are glycosylated. Subsequently HA is
assembled into a trimer. HA, NA and M2 are transported to the Golgi apparatus where cysteine residues on HA and M2 are palmitoylated. Furin
cleaves HA into HA1 and HA2 subunits and all three proteins are directed to the virus assembly site on the apical plasma membrane via apical
sorting signals. The signals for HA and NA reside on the transmembrane domains (TMD) while the sorting signal for M2 is not yet characterized.
The TMDs of HA and NA also contain the signals for lipid raft association. Lipid rafts are non-ionic detergent-resistant lipid microdomains within
the plasma membrane that are rich in sphingolipids and cholesterol. Examination of purified virus particles indicates that influenza virus buds
preferentially from these microdomains.
References
P Palese, ML Shaw, "Orthomyxoviridae: The Viruses and Their Replication", Fields Virology, 5th edition D.M. Knipe and P.M. Howley, Editors.
2006, Lippencott Williams and Wilkins: Philadelphia ISBN-10: 0-7817-6060-7, 2001.
13.1.7.1.1 Entry of M2 into the endoplasmic reticulum
Authors
Steel, J, 2007-04-30.
Reviewers
Description
The integral membrane protein M2 is synthesized on membrane-bound ribosomes and subsequently transported across the ER, where it is
folded and assembled into a tetramer.
References
RW Doms, RA Lamb, JK Rose, A Helenius, "Folding and assembly of viral membrane proteins", Virology, 193, 1993, 545-62.
Reaction
13.1.7.1.2 Assembly of M2 tetramers
Authors
Steel, J, 2007-04-30.
Reviewers
Description
The M2 from influenza A virus is a 97-residue protein with a single transmembrane helix that associates to form a tetramer in the endoplasmic
reticulum (Salom et al, 2000). A 15-20-residue segment C-terminal to the membrane-spanning region has been postulated to aid in the
stabilization of the tetrameric assembly (Kochendoerfer et al 1999).
References
D Salom, BR Hill, JD Lear, WF DeGrado, "pH-dependent tetramerization and amantadine binding of the transmembrane helix of M2 from the
influenza A virus", Biochemistry, 39, 2000, 14160-70.
GG Kochendoerfer, D Salom, JD Lear, R Wilk-Orescan, SB Kent, WF DeGrado, "Total chemical synthesis of the integral membrane protein
influenza A virus M2: role of its C-terminal domain in tetramer assembly", Biochemistry, 38, 1999, 11905-13.
Reaction
13.1.7.1.3 Entry of NA into the endoplasmic reticulum
Authors
Steel, J, 2007-04-30.
Reviewers
Description
The integral membrane protein NA is synthesized on membrane-bound ribosomes and subsequently transported across the ER where it is
folded and glycosylated. Subsequently NA is assembled into a tetramer.
References
Reaction
13.1.7.1.4 Glycosylation of NA
Authors
Steel, J, 2007-04-30.
Reviewers

Description
Glycosylation of NA occurs within the endoplasmic reticulum and is believed to be neccessary for proper tetramerization of the NA dimers. Sugar
residues become attached to four of the five potential glycosylation sites in the head of N1 neuraminidase (Hausman et al., 1997).
References
L Markoff, BC Lin, MM Sveda, CJ Lai, "Glycosylation and surface expression of the influenza virus neuraminidase requires the N-terminal
hydrophobic region", Mol Cell Biol, 4, 1984, 8-16.
DJ Hulse, RG Webster, RJ Russell, DR Perez, "Molecular determinants within the surface proteins involved in the pathogenicity of H5N1
influenza viruses in chickens", J Virol, 78, 2004, 9954-64.
T Saito, G Taylor, RG Webster, "Steps in maturation of influenza A virus neuraminidase", J Virol, 69, 1995, 5011-7.
J Hausmann, E Kretzschmar, W Garten, HD Klenk, "Biosynthesis, intracellular transport and enzymatic activity of an avian influenza A virus
neuraminidase: role of unpaired cysteines and individual oligosaccharides", J Gen Virol, 1997, 3233-45.
Reaction
13.1.7.1.5 Assembly of NA tetramers
Authors
Steel, J, 2007-04-30.
Reviewers
Description
Tetramerisation of the NA occurs in the ER following an initial dimerisation step. Tetramerisation is believed to be dependant on glycosylation of
the NA molecules
References
T Saito, G Taylor, RG Webster, "Steps in maturation of influenza A virus neuraminidase", J Virol, 69, 1995, 5011-7.
J Hausmann, E Kretzschmar, W Garten, HD Klenk, "Biosynthesis, intracellular transport and enzymatic activity of an avian influenza A virus
neuraminidase: role of unpaired cysteines and individual oligosaccharides", J Gen Virol, 1997, 3233-45.
Reaction
13.1.7.1.6 Entry of HA into the endoplasmic reticulum
Authors
Steel, J, 2007-04-30.
Reviewers
Description
The integral membrane protein HA is synthesized on membrane-bound ribosomes and subsequently transported across the endoplasmic
reticulum, where it is folded, glycosylated, and assembled into a trimer.
References
Reaction
13.1.7.1.7 Glycosylation and Folding of HA
Authors
Steel, J, 2007-04-30.
Reviewers
Description
The ectodomain of HA is translocated into the ER lumen, where it undergoes a series of folding events mediated by the formation of disulfide
bonds and glycosylation reactions. The formation of a discrete intermediate species of highly folded monomeric protein preceeds trimerisation.
The folding process is efficient and rapid, with greater than 90% of the protein trafficked to the golgi apparatus; and mature HA0 subunits
appearing in a matter of a few minutes. Calnexin and calreticulin have been identified as cellular lectins which interact transiently with newly
synthesized HA by attaching to partially trimmed N-linked oligosaccharides (Herbert et al., 1997), facilitating correct folding of the HA molecule.
References
PC Roberts, W Garten, HD Klenk, "Role of conserved glycosylation sites in maturation and transport of influenza A virus hemagglutinin", J Virol,
67, 1993, 3048-60.
R Daniels, B Kurowski, AE Johnson, DN Hebert, "N-linked glycans direct the cotranslational folding pathway of influenza hemagglutinin", Mol
Cell, 11, 2003, 79-90.
PJ Gallagher, JM Henneberry, JF Sambrook, MJ Gething, "Glycosylation requirements for intracellular transport and function of the
hemagglutinin of influenza virus", J Virol, 66, 1992, 7136-45.
W Chen, A Helenius, "Role of ribosome and translocon complex during folding of influenza hemagglutinin in the endoplasmic reticulum of living
cells", Mol Biol Cell, 11, 2000, 765-72.
W Chen, J Helenius, I Braakman, A Helenius, "Cotranslational folding and calnexin binding during glycoprotein synthesis", Proc Natl Acad Sci U
S A, 92, 1995, 6229-33.
DN Hebert, JX Zhang, W Chen, B Foellmer, A Helenius, "The number and location of glycans on influenza hemagglutinin determine folding and
association with calnexin and calreticulin", J Cell Biol, 139, 1997, 613-23.
M Molinari, A Helenius, "Chaperone selection during glycoprotein translocation into the endoplasmic reticulum", Science, 288, 2000, 331-3.
CS Copeland, KP Zimmer, KR Wagner, GA Healey, I Mellman, A Helenius, "Folding, trimerization, and transport are sequential events in the
biogenesis of influenza virus hemagglutinin", Cell, 53, 1988, 197-209.
I Singh, RW Doms, KR Wagner, A Helenius, "Intracellular transport of soluble and membrane-bound glycoproteins: folding, assembly and
secretion of anchor-free influenza hemagglutinin", EMBO J, 9, 1990, 631-9.
MS Segal, JM Bye, JF Sambrook, MJ Gething, "Disulfide bond formation during the folding of influenza virus hemagglutinin", J Cell Biol, 118,
1992, 227-44.
JR Peterson, A Ora, PN Van, A Helenius, "Transient, lectin-like association of calreticulin with folding intermediates of cellular and viral
glycoproteins", Mol Biol Cell, 6, 1995, 1173-84.
C Hammond, I Braakman, A Helenius, "Role of N-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and
quality control", Proc Natl Acad Sci U S A, 91, 1994, 913-7.
Reaction
13.1.7.1.8 Trimerization of HA
Authors
Steel, J, 2007-04-30.
Reviewers
Description
Trimerisation of the fully folded and fully oxidised HA monomer is thought to occur in the endoplasmic reticulum and ERGIC compartment,
following dissociation of HA from calnexin. Trimerisation is generally thought to be the final step in HA maturation occurring in the endoplasmic
reticulum before transport to the Golgi apparatus, although Yewdell et al (1988) provide data suggesing that trimerisation may occur within the
Golgi.
References
CS Copeland, RW Doms, EM Bolzau, RG Webster, A Helenius, "Assembly of influenza hemagglutinin trimers and its role in intracellular
transport", J Cell Biol, 103, 1986, 1179-91.
U Tatu, C Hammond, A Helenius, "Folding and oligomerization of influenza hemagglutinin in the ER and the intermediate compartment", EMBO
J, 14, 1995, 1340-8.
J Hearing, MJ Gething, J Sambrook, "Addition of truncated oligosaccharides to influenza virus hemagglutinin results in its
temperature-conditional cell-surface expression", J Cell Biol, 108, 1989, 355-65.
CS Copeland, KP Zimmer, KR Wagner, GA Healey, I Mellman, A Helenius, "Folding, trimerization, and transport are sequential events in the
biogenesis of influenza virus hemagglutinin", Cell, 53, 1988, 197-209.
A Ceriotti, A Colman, "Trimer formation determines the rate of influenza virus haemagglutinin transport in the early stages of secretion in
Xenopus oocytes", J Cell Biol, 111, 1990, 409-20.
MJ Gething, K McCammon, J Sambrook, "Expression of wild-type and mutant forms of influenza hemagglutinin: the role of folding in intracellular
transport", Cell, 46, 1986, 939-50.
JW Yewdell, A Yellen, T Bachi, "Monoclonal antibodies localize events in the folding, assembly, and intracellular transport of the influenza virus
hemagglutinin glycoprotein", Cell, 52, 1988, 843-52.
Reaction
13.1.7.1.9 Transport of HA trimer, NA tetramer and M2 tetramer from the endoplasmic reticulum to the Golgi Apparatus
Authors
Steel, J, 2007-04-30.
Reviewers
Description
Processed viral proteins are transported from the endoplasmic reticulum to the Golgi apparatus.
References
13.1.7.1.9.1 Budding of vesicle with the HA trimer, NA tetramer and M2 tetramer from the endoplasmic reticulum into the cytosol
Authors
Steel, J, 2007-04-30.
Reviewers

Description
Viral proteins are packaged into a golgi apparatus bound transport vesicle.
References
Reaction
13.1.7.1.9.2 Fusion of vesicle containing the HA trimer, NA tetramer and M2 tetramer to the Golgi apparatus
Authors
Steel, J, 2007-04-30.
Reviewers
Description
Once the tranport vesicle arrives at the golgi apparatus, it docks and dumps its contents into the golgi lumen.
References
Reaction
13.1.7.1.10 Palmitoylation of cysteine residues on HA in the cis-Golgi network
Authors
Steel, J, 2007-04-30.
Reviewers
Description
The hemagglutinin of influenza virus is palmitoylated with long-chain fatty acids.
Palmitoylation of HA is believed to occur in the cis golgi network (Veit 1993), shortly after trimerisation of the molecule, and before cleavage of
the HA into HA1 and HA2. HA is palmitoylated through thioester linkages at three cysteine residues located in the cytoplasmic domain and at the
carboxy-terminal end of the transmembrane region. Lack of acylation has no obvious influence on the biological activities of HA.
References
E Ponimaskin, MF Schmidt, "Domain-structure of cytoplasmic border region is main determinant for palmitoylation of influenza virus
hemagglutinin (H7)", Virology, 249, 1998, 325-35.
G Russ, JR Bennink, T Bachi, JW Yewdell, "Influenza virus hemagglutinin trimers and monomers maintain distinct biochemical modifications and
intracellular distribution in brefeldin A-treated cells", Cell Regul, 2, 1991, 549-63.
M Veit, H Reverey, MF Schmidt, "Cytoplasmic tail length influences fatty acid selection for acylation of viral glycoproteins", Biochem J, 1996,
163-72.
DA Steinhauer, SA Wharton, DC Wiley, JJ Skehel, "Deacylation of the hemagglutinin of influenza A/Aichi/2/68 has no effect on membrane fusion
properties", Virology, 184, 1991, 445-8.
H Jin, K Subbarao, S Bagai, GP Leser, BR Murphy, RA Lamb, "Palmitylation of the influenza virus hemagglutinin (H3) is not essential for virus
assembly or infectivity", J Virol, 70, 1996, 1406-14.
HY Naim, B Amarneh, NT Ktistakis, MG Roth, "Effects of altering palmitylation sites on biosynthesis and function of the influenza virus
hemagglutinin", J Virol, 66, 1992, 7585-8.
M Veit, E Kretzschmar, K Kuroda, W Garten, MF Schmidt, HD Klenk, R Rott, "Site-specific mutagenesis identifies three cysteine residues in the
cytoplasmic tail as acylation sites of influenza virus hemagglutinin", J Virol, 65, 1991, 2491-500.
BJ Chen, M Takeda, RA Lamb, "Influenza virus hemagglutinin (H3 subtype) requires palmitoylation of its cytoplasmic tail for assembly: M1
proteins of two subtypes differ in their ability to support assembly", J Virol, 79, 2005, 13673-84.
M Veit, MF Schmidt, "Timing of palmitoylation of influenza virus hemagglutinin", FEBS Lett, 336, 1993, 243-7.
P Portincasa, G Conti, C Chezzi, "Role of acylation of viral haemagglutinin during the influenza virus infectious cycle", Res Virol, 143, 1992,
401-6.
M Veit, G Herrler, MF Schmidt, R Rott, HD Klenk, "The hemagglutinating glycoproteins of influenza B and C viruses are acylated with different
fatty acids", Virology, 177, 1990, 807-11.
Reaction
13.1.7.1.11 Palmitoylation of cysteine residues on M2 in the cis-golgi network
Authors
Steel, J, 2007-04-30.
Reviewers
Description
Palmitoylation of influenza A M2 occurs in the ER, or cis golgi network, following tetramerisation. The palmitoylation reaction proceeds via a
labile thioester type bond at a specific residue of M2 (Sugrue et al., 1990).
References
RJ Sugrue, RB Belshe, AJ Hay, "Palmitoylation of the influenza A virus M2 protein", Virology, 179, 1990, 51-6.
M Veit, HD Klenk, A Kendal, R Rott, "The M2 protein of influenza A virus is acylated", J Gen Virol, 72, 1991, 1461-5.
Reaction
13.1.7.1.12 Association of HA into rafts
Authors
Steel, J, 2007-04-30.
Reviewers
Description
Influenza virus buds preferentially from lipid rafts (Scheiffele et al, 1999). NA protein individually accumulates at, and is selectively incorporated
into rafts (Kundu et al., 1996). The signals for raft association lie within the transmembranse domain (TMD), (Barman et al., 2001, Barman et al.,
2004), and raft association of NA has been shown to be essential for efficient virus replication. This is believed to be due to a requirement for a
concentration of NA at specific areas of the plasma membrane to support a level of NA incorporation into budding particles sufficient to allow for
efficient virus release (Barman et al., 2004).
References
S Barman, A Ali, EK Hui, L Adhikary, DP Nayak, "Transport of viral proteins to the apical membranes and interaction of matrix protein with
glycoproteins in the assembly of influenza viruses", Virus Res, 77, 2001, 61-9.
P Scheiffele, A Rietveld, T Wilk, K Simons, "Influenza viruses select ordered lipid domains during budding from the plasma membrane", J Biol
Chem, 274, 1999, 2038-44.
S Barman, L Adhikary, AK Chakrabarti, C Bernas, Y Kawaoka, DP Nayak, "Role of transmembrane domain and cytoplasmic tail amino acid
sequences of influenza a virus neuraminidase in raft association and virus budding", J Virol, 78, 2004, 5258-69.
P Scheiffele, MG Roth, K Simons, "Interaction of influenza virus haemagglutinin with sphingolipid-cholesterol membrane domains via its
transmembrane domain", EMBO J, 16, 1997, 5501-8.
DP Nayak, EK Hui, S Barman, "Assembly and budding of influenza virus", Virus Res, 106, 2004, 147-65.
A Kundu, RT Avalos, CM Sanderson, DP Nayak, "Transmembrane domain of influenza virus neuraminidase, a type II protein, possesses an
apical sorting signal in polarized MDCK cells", J Virol, 70, 1996, 6508-15.
M Takeda, GP Leser, CJ Russell, RA Lamb, "Influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion", Proc
Natl Acad Sci U S A, 100, 2003, 14610-7.
Reaction
13.1.7.1.13 Association of NP into rafts
Authors
Steel, J, 2007-04-30.
Reviewers
Description
There is evidence that NP alone is intrinsically targeted to the apical plasma membrane and associates with lipid rafts in a cholesterol-dependent
manner, which suggests that RNPs could reach the assembly site independently of the other viral components.
References
M Carrasco, MJ Amorim, P Digard, "Lipid raft-dependent targeting of the influenza A virus nucleoprotein to the apical plasma membrane",
Traffic, 5, 2004, 979-92.
M Simpson-Holley, D Ellis, D Fisher, D Elton, J McCauley, P Digard, "A functional link between the actin cytoskeleton and lipid rafts during
budding of filamentous influenza virions", Virology, 301, 2002, 212-25.
RT Avalos, Z Yu, DP Nayak, "Association of influenza virus NP and M1 proteins with cellular cytoskeletal elements in influenza virus-infected
cells", J Virol, 71, 1997, 2947-58.
Reaction
13.1.7.1.14 Association of NA into rafts
Authors
Steel, J, 2007-04-30.
Reviewers
Description
Influenza virus buds preferentially from lipid rafts (Scheiffele et al, 1999). NA protein individually accumulates at, and is selectively incorporated
into rafts (Kundu et al., 1996). The signals for raft association lie within the transmembranse domain (TMD), (Barman et al., 2001, Barman et al.,
2004), and raft association of NA has been shown to be essential for efficient virus replication. This is believed to be due to a requirement for a
concentration of NA at specific areas of the plasma membrane to support a level of NA incorporation into budding particles sufficient to allow for
efficient virus release (Barman et al., 2004).
References
S Barman, A Ali, EK Hui, L Adhikary, DP Nayak, "Transport of viral proteins to the apical membranes and interaction of matrix protein with
glycoproteins in the assembly of influenza viruses", Virus Res, 77, 2001, 61-9.
P Scheiffele, A Rietveld, T Wilk, K Simons, "Influenza viruses select ordered lipid domains during budding from the plasma membrane", J Biol
Chem, 274, 1999, 2038-44.
S Barman, L Adhikary, AK Chakrabarti, C Bernas, Y Kawaoka, DP Nayak, "Role of transmembrane domain and cytoplasmic tail amino acid
sequences of influenza a virus neuraminidase in raft association and virus budding", J Virol, 78, 2004, 5258-69.
A Kundu, RT Avalos, CM Sanderson, DP Nayak, "Transmembrane domain of influenza virus neuraminidase, a type II protein, possesses an
apical sorting signal in polarized MDCK cells", J Virol, 70, 1996, 6508-15.
Reaction
13.1.7.1.15 Transport of processed viral proteins to the cell membrane
Authors
Steel, J, 2007-04-30.
Reviewers

Description
Once processed, the viral proteins are transported from the golgi apparatus to the plasma membrane.
References
S Heino, S Lusa, P Somerharju, C Ehnholm, VM Olkkonen, E Ikonen, "Dissecting the role of the golgi complex and lipid rafts in biosynthetic
transport of cholesterol to the cell surface", Proc Natl Acad Sci U S A, 97, 2000, 8375-80.
Reaction
13.1.7.1.16 Accumulation of M1 at the inner leaflet of the lipid bilayer
Authors
Steel, J, 2007-04-30.
Reviewers
Description
There is evidence for the association of M1 with lipid rafts in influenza infected cells, whereas M1 expressed alone remains soluble (Ali et al.,
2000; Zhang and Lamb, 1996), suggesting association of M1 with other viral proteins in targetting to the cell membrane. Coexpression of HA and
NA together with M1 has been shown to promote raft association of M1. This association requires the TMD and cytoplasmic tails of HA and NA
(Ali et al, 2000; Zhang et al, 2000). This is consistent with M1 becoming associated with HA and NA during their passage through the exocytic
pathway to raft domains in the apical membrane. alternatively M1 may use the cytoskeleton to reach the virus assembly site, as M1 interacts
with cytoskeletal components (Alvalos et al., 1997). The M1 interaction depends on the presence of RNP and is most likely mediated by direct
binding of F-actin by NP (Digard et al., 1999).
References
J Zhang, A Pekosz, RA Lamb, "Influenza virus assembly and lipid raft microdomains: a role for the cytoplasmic tails of the spike glycoproteins", J
Virol, 74, 2000, 4634-44.
J Zhang, RA Lamb, "Characterization of the membrane association of the influenza virus matrix protein in living cells", Virology, 225, 1996,
255-66.
RT Avalos, Z Yu, DP Nayak, "Association of influenza virus NP and M1 proteins with cellular cytoskeletal elements in influenza virus-infected
cells", J Virol, 71, 1997, 2947-58.
P Digard, D Elton, K Bishop, E Medcalf, A Weeds, B Pope, "Modulation of nuclear localization of the influenza virus nucleoprotein through
interaction with actin filaments", J Virol, 73, 1999, 2222-31.
A Ali, RT Avalos, E Ponimaskin, DP Nayak, "Influenza virus assembly: effect of influenza virus glycoproteins on the membrane association of M1
protein", J Virol, 74, 2000, 8709-19.
Reaction
13.1.7.2 Packaging of Eight RNA Segments
Authors
Marsh, G, 2007-04-30.
Reviewers
Description
For a budding influenza virus to be fully infectious is it essential that it contains a full complement of the eight vRNA segments. Two different
models have been proposed for packaging of the vRNPs into newly assembling virus particles; the random incorporation model and the selective
incorporation model.
The random incorporation model as its name suggests proposes that there is no selection at all on which vRNPs are packaged. It is assumed
that each vRNP has equal probability of being packaged, and that if enough vRNPS are packaged a particular percentage of budding virions will
receive at least one copy of each genome segment. This model is supported by evidence that infectious virions may possess more than eight
vRNPs assuring the presence of a full complement of eight vRNPs in a significant percentage of virus particles. Mathematical analysis of
packaging suggested that twelve RNA segments would need to be packaged in order to obtain approximately 10% of virus particles that are fully
infectious (Enami, 1991), a number that is compatible with experimental data (Donald, 1954). Due to the low amount of RNA per virion
(estimated at 1-2% w/w), enumeration of the precise number of RNAs packaged in a virion is difficult.
The selective incorporation model, suggests that each vRNA segment contains a unique "packaging signal" allowing it to act independently, with
each vRNA segment being packaged selectively. There is increasing evidence to support the theory of a packaging signal within the coding
regions at both the 5' and 3' end of the genomic RNA, with signals being reported for all segments except segment 7 (Ozawa 2007, Muramoto
2006, Fujii 2005, Fujii 2003, Watanabe 2003, Liang 2005). The exact method by which individual vRNP segments are packaged is not known but
it has been hypothesized to occur via specific RNA-RNA or protein-RNA interactions. This model is also supported by thin section electron
microscopy images of influenza particles that show eight distinct "dots", presumably vRNPs within virus particles (Noda 2006).
References
Y Muramoto, A Takada, K Fujii, T Noda, K Iwatsuki-Horimoto, S Watanabe, T Horimoto, H Kida, Y Kawaoka, "Hierarchy among viral RNA
(vRNA) segments in their role in vRNA incorporation into influenza A virions", J Virol, 80, 2006, 2318-25.
Y Liang, Y Hong, TG Parslow, "cis-Acting packaging signals in the influenza virus PB1, PB2, and PA genomic RNA segments", J Virol, 79, 2005,
10348-55.
T Noda, H Sagara, A Yen, A Takada, H Kida, RH Cheng, Y Kawaoka, "Architecture of ribonucleoprotein complexes in influenza A virus
particles", Nature, 439, 2006, 490-2.
K Fujii, Y Fujii, T Noda, Y Muramoto, T Watanabe, A Takada, H Goto, T Horimoto, Y Kawaoka, "Importance of both the coding and the
segment-specific noncoding regions of the influenza A virus NS segment for its efficient incorporation into virions", J Virol, 79, 2005, 3766-74.
T Watanabe, S Watanabe, T Noda, Y Fujii, Y Kawaoka, "Exploitation of nucleic acid packaging signals to generate a novel influenza virus-based
vector stably expressing two foreign genes", J Virol, 77, 2003, 10575-83.
Y Fujii, H Goto, T Watanabe, T Yoshida, Y Kawaoka, "Selective incorporation of influenza virus RNA segments into virions", Proc Natl Acad Sci
U S A, 100, 2003, 2002-7.
HB DONALD, A ISAACS, "Counts of influenza virus particles", J Gen Microbiol, 10, 1954, 457-64.
M Ozawa, K Fujii, Y Muramoto, S Yamada, S Yamayoshi, A Takada, H Goto, T Horimoto, Y Kawaoka, "Contributions of two nuclear localization
signals of influenza A virus nucleoprotein to viral replication", J Virol, 81, 2007, 30-41.
M Enami, G Sharma, C Benham, P Palese, "An influenza virus containing nine different RNA segments", Virology, 185, 1991, 291-8.
13.1.7.2.1 RNP association
Authors
Marsh, G, 2007-04-30.
Reviewers
Description
The random incorporation model as its name suggests proposes that there is no selection at all on which vRNPs are packaged. It is assumed
that each vRNP has equal probability of being packaged, and that if enough vRNPS are packaged a particular percentage of budding virions will
receive at least one copy of each genome segment. This model is supported by evidence that infectious virions may possess more than eight
vRNPs assuring the presence of a full complement of eight vRNPs in a significant percentage of virus particles. Mathematical analysis of
packaging suggested that twelve RNA segments would need to be packaged in order to obtain approximately 10% of virus particles that are fully
infectious (Enami, 1991), a number that is compatible with experimental data (Donald, 1954). Due to the low amount of RNA per virion
(estimated at 1-2% w/w), enumeration of the precise number of RNAs packaged in a virion is difficult.
References
HB DONALD, A ISAACS, "Counts of influenza virus particles", J Gen Microbiol, 10, 1954, 457-64.
M Enami, G Sharma, C Benham, P Palese, "An influenza virus containing nine different RNA segments", Virology, 185, 1991, 291-8.
Reaction
13.1.7.2.2 Association with M1 at cell membrane
Authors
Marsh, G, 2007-04-30.
Reviewers
Description
As influenza viruses bud from the plasma membrane of infected cells, complete virions are not seen inside cells. In polarized epithelial cells,
assembly and budding of influenza occurs from the apical plasma membrane (Schmitt, 2004). For efficient assembly, all virion components must
accumulate at the budding site, and it is believed that the viral glycoprotein accumulation determines the site of virus assembly and budding
(Nayak, 2004). M1 is thought to be the bridge between the envelope glycoproteins and the RNPs for assembly (Schmitt, 2004). M2 is also
required, because if it is not present RNPs are not packaged into budding virions (McCown, 2005), however it role is not known.
References
MF McCown, A Pekosz, "The influenza A virus M2 cytoplasmic tail is required for infectious virus production and efficient genome packaging", J
Virol, 79, 2005, 3595-605.
AP Schmitt, RA Lamb, "Escaping from the cell: assembly and budding of negative-strand RNA viruses", Curr Top Microbiol Immunol, 283, 2004,
145-96.
Reaction
13.1.7.3 Budding
Authors
Marsh, G, 2007-04-30.
Reviewers

Description
The process by which influenza virus particles bud from an infected cell is not very well understood. Accumulation of M1 at the inner leaflet of the
plasma membrane is thought to be the trigger for the initiation of bud formation. This bud formation continues until the inner core of the virus is
completely enveloped. Completion of the budding process requires the membrane at the base of the bud to fuse. Although M1 is thought to be
the driving force for bud formation, other viral and cellular proteins have been demonstrated to affect size and shape of the virus particle.
Generally, influenza virus particles are either spherical or filamentous and this characteristic morphology is genetically linked to the M segment
(Bourmakina, 2003; Roberts, 1998). Host factors such as polarization and the actin cytoskeleton play a critical role in determining the shape of
filamentous particles (Roberts, 1998; Simpson-Holley, 2002).
References
SV Bourmakina, A Garcia-Sastre, "Reverse genetics studies on the filamentous morphology of influenza A virus", J Gen Virol, 84, 2003, 517-27.
PC Roberts, RW Compans, "Host cell dependence of viral morphology", Proc Natl Acad Sci U S A, 95, 1998, 5746-51.
PC Roberts, RA Lamb, RW Compans, "The M1 and M2 proteins of influenza A virus are important determinants in filamentous particle
formation", Virology, 240, 1998, 127-37.
M Simpson-Holley, D Ellis, D Fisher, D Elton, J McCauley, P Digard, "A functional link between the actin cytoskeleton and lipid rafts during
budding of filamentous influenza virions", Virology, 301, 2002, 212-25.
13.1.7.3.1 Membrane fusion
Authors
Marsh, G, 2007-04-30.
Reviewers
Description
The final step in the budding process is the fusion of the lipid membrane surrounding the virion core, producing an extracellular enveloped virus
particle.
Reaction
13.1.7.4 Release
Authors
Marsh, G, 2007-04-30.
Reviewers
Description
Once the viral envelope has separated from the cell membrane Influenza virus particles are actively released to complete the budding process.
HA (hemagglutinin) anchors the virus to the cell by binding to sialic acid-containing receptors on the cell surface. The enzymatic activity of the
neuraminidase (NA) protein removes the sialic acid and releases the virus from the host cell. NA activity is also required to remove sialic acid
from the carbohydrates present on the viral glycoproteins to prevent the viral particles from aggregating.
References
13.1.7.4.1 Neuraminidase enzymatic release from sialic acid
Authors
Marsh, G, 2007-04-30.
Reviewers
Description
The release of influenza virus particles after seperation of the virus and infected cell membrane is an active process. During the budding
process, HA on the surface of the newly budding virion binds to cell surface molecules containing sialic acid residues as seen during attachment.
The NA glycoproteins neuraminidase activity is essential to cleave the link between the HA and sialic acid on the surface of the host cell from
which the budding virus is emeging from. Thus the NA mediated cleavage of sialic acid residues terminally linked to glycoproteins and glycolipids
is the final step in releasing the virus particle from the host cell. This essential role of NA in release of virus particle has been demonstrated with
the use of NA inhibitors (Palese, 1976; Luo, 1999; Garman, 2004), ts NA mutant viruses (Palese, 1974) and with viruses lacking NA activity (Liu,
1995). In all cases, viruses remain bound to the cell surface in clumps in the absence of NA enzymatic activity, resulting in loss of infectivity.
Addition of exogenous sialidase results in virus release and recovery of infectivity. The sialidase activity of the NA is also important for removing
sialic acid from the HA on virus particles, if this is not removed, virus particles aggregate.
References
P Palese, K Tobita, M Ueda, RW Compans, "Characterization of temperature sensitive influenza virus mutants defective in neuraminidase",
Virology, 61, 1974, 397-410.
P Palese, RW Compans, "Inhibition of influenza virus replication in tissue culture by 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid
(FANA): mechanism of action", J Gen Virol, 33, 1976, 159-63.
E Garman, G Laver, "Controlling influenza by inhibiting the virus's neuraminidase", Curr Drug Targets, 5, 2004, 119-36.
C Liu, MC Eichelberger, RW Compans, GM Air, "Influenza type A virus neuraminidase does not play a role in viral entry, replication, assembly,
or budding", J Virol, 69, 1995, 1099-106.
C Luo, E Nobusawa, K Nakajima, "An analysis of the role of neuraminidase in the receptor-binding activity of influenza B virus: the inhibitory
effect of Zanamivir on haemadsorption", J Gen Virol, 1999, 2969-76.
Reaction
13.2 Host Interactions with Influenza Factors
Reviewers
Gale M, Jr, 2020-05-12.
Description
Infection of a human host cell with influenza virus triggers an array of host processes that interfere with viral replication, notably the production of
type I interferon. The viral NS1 protein plays a central role in these virus-host interactions.
References
13.2.1 NS1 Mediated Effects on Host Pathways
Description
Viral NS1 protein is a nuclear, dimeric protein that is highly expressed in infected cells and has dsRNA-binding activity. The RNA-binding domain
lies within the N-terminal portion of the protein. The NS1 RNA-binding domain forms a symmetric homodimer with a six-helical fold. Mutational
analysis has demonstrated that dimer formation is crucial for RNA-binding. The basic residues are believed to make contact with the phosphate
backbone of the RNA which is consistent with an observed lack of sequence specificity. Neither NS1 nor its bound RNA undergo any significant
structural changes upon binding. The NS1 dimer spans the minor groove of canonical A-form dsRNA. The non-RNA binding portion of NS1 has
been termed the effector domain and includes binding sites for host cell poly (A)-binding protein II (PABII) and the 30kDa subunit of cleavage
and polyadenylation specificity factor (CPSF).
References
ME Nemeroff, XY Qian, RM Krug, "The influenza virus NS1 protein forms multimers in vitro and in vivo", Virology, 212, 1995, 422-8.
XY Qian, CY Chien, Y Lu, GT Montelione, RM Krug, "An amino-terminal polypeptide fragment of the influenza virus NS1 protein possesses
specific RNA-binding activity and largely helical backbone structure", RNA, 1, 1995, 948-56.
13.2.1.1 Inhibition of Host mRNA Processing and RNA Silencing

Description
The Influenza Virus NS1 protein inhibits the cleavage and polyadenylation specificity factor CPSF and the PABII components of the host cell 3'
end processing machinery, preventing efficient 3' end processing of host pre-mRNAs. NS1 also inhibits the splicing of pre-mRNAs, resulting in
their retention within the host cell nucleus.
References
Y Lu, XY Qian, RM Krug, "The influenza virus NS1 protein: a novel inhibitor of pre-mRNA splicing", Genes Dev, 8, 1994, 1817-28.
P Fortes, A Beloso, J Ortin, "Influenza virus NS1 protein inhibits pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport", EMBO J,
13, 1994, 704-12.
Z Chen, Y Li, RM Krug, "Influenza A virus NS1 protein targets poly(A)-binding protein II of the cellular 3'-end processing machinery", EMBO J,
18, 1999, 2273-83.
13.2.1.1.1 Binding of NS1 to cleavage and host polyadenylation specificity factor (CPSF)
Description
Influenza virus's non-structural protein (NS1) binds to the host cell's cleavage and host polyadenylation specificity factor (CPSF), inhibiting the
ability of CPSF to bind to pre-mRNAs and thus preventing efficient 3' end processing and export of host cell mRNAs out of the nucleus.
References
vivo", RNA, 7, 2001, 920-31.
DL Noah, KY Twu, RM Krug, "Cellular antiviral responses against influenza A virus are countered at the posttranscriptional level by the viral
NS1A protein via its binding to a cellular protein required for the 3' end processing of cellular pre-mRNAS", Virology, 307, 2003, 386-95.
K Shimizu, A Iguchi, R Gomyou, Y Ono, "Influenza virus inhibits cleavage of the HSP70 pre-mRNAs at the polyadenylation site", Virology, 254,
1999, 213-9.
ME Nemeroff, SM Barabino, Y Li, W Keller, RM Krug, "Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and inhibits
3'end formation of cellular pre-mRNAs", Mol Cell, 1, 1998, 991-1000.
Reaction
13.2.1.1.2 Binding of NS1 to poly(A)-binding protein II (PABII)
Description
The influenza virus non-structural protein 1 (NS1) binds to the host cell's poly(A)-binding protein II (PABII) thus preventing PABII from properly
extending the poly-A tail of pre-mRNA within the host cell nucleus. These pre-mRNAs are then prevented from exiting the nucleus.
References
Z Chen, Y Li, RM Krug, "Influenza A virus NS1 protein targets poly(A)-binding protein II of the cellular 3'-end processing machinery", EMBO J,
18, 1999, 2273-83.
Reaction
13.2.1.2 Inhibition of Interferon Synthesis
Description
Interferon Synthesis is inhibited.
13.2.1.2.1 Inhibition of IFN-beta
Description
Since the presence of intracellular dsRNA serves as the signal for virus infection and triggers host interferon (IFN) synthesis the simplest model
for viral NS1 protein function is that it sequesters dsRNA and thus prevents the downstream signaling required to activate IRF-3, NF-kB and
AP-1. These findings are strongly supported by mutational analyses of NS1 that indicate that the IFN antagonist properties of NS1 depend on its
ability to bind dsRNA. However, a compensatory mutation (S42G), which was acquired during the passaging of the mutant RNA-binding virus,
results in partial restoration of wild-type phenotype but does not restore RNA binding. This indicates that the ability of NS1 to inhibit IFN
synthesis is not solely dependent on dsRNA binding and that additional mechanisms may be involved.
References
NR Donelan, CF Basler, A Garcia-Sastre, "A recombinant influenza A virus expressing an RNA-binding-defective NS1 protein induces high
levels of beta interferon and is attenuated in mice", J Virol, 77, 2003, 13257-66.
13.2.1.2.1.1 Binding of NS1 to dsRNA
Description
The ability of viral non-structural protein 1 (NS1) to sequester dsRNA is believed to be one of the primary mechanisms by which NS1 prevents
activation of downstream anti-viral signaling pathways.
References
Reaction
13.2.1.3 Inhibition of PKR
Description
The key role played by PKR in the innate response to virus infection is emphasized by the large number of viruses that encode PKR inhibitors.
13.2.1.3.1 Binding of NS1 to dsRNA
Description
The ability of viral non-structural protein 1 (NS1) to sequester dsRNA is believed to be one of the primary mechanisms by which NS1 prevents
activation of downstream anti-viral signaling pathways.
References
Reaction
13.2.1.3.2 Binding of NS1 to PKR
Description
At the beginning of this reaction, 1 molecule of 'NS1 Homodimer', and 1 molecule of 'PKR human' are present. At the end of this reaction, 1
molecule of 'NS1 Homodimer:PKR Complex' is present.
Reaction
13.2.2 Influenza Virus Induced Apoptosis
Description
Influenza A virus induces apoptosis in a variety of ways including activation of host TGF-beta by expression of viral NA, M1 and M2 proteins, and
by the binding of viral PB1-F2 to host mitochondrial adenine nucleotide translocator 3 (ANT3).
References
T Takizawa, S Matsukawa, Y Higuchi, S Nakamura, Y Nakanishi, R Fukuda, "Induction of programmed cell death (apoptosis) by influenza virus
infection in tissue culture cells", J Gen Virol, 74, 1993, 2347-55.
WJ Wurzer, C Ehrhardt, S Pleschka, F Berberich-Siebelt, T Wolff, H Walczak, O Planz, S Ludwig, "NF-kappaB-dependent induction of tumor
necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas/FasL is crucial for efficient influenza virus propagation", J Biol Chem, 279,
2004, 30931-7.
SJ Morris, H Smith, C Sweet, "Exploitation of the Herpes simplex virus translocating protein VP22 to carry influenza virus proteins into cells for
studies of apoptosis: direct confirmation that neuraminidase induces apoptosis and indications that other proteins may have a role", Arch Virol,
147, 2002, 961-79.
S Schultz-Cherry, N Dybdahl-Sissoko, G Neumann, Y Kawaoka, VS Hinshaw, "Influenza virus ns1 protein induces apoptosis in cultured cells", J
Virol, 75, 2001, 7875-81.
SJ Morris, GE Price, JM Barnett, SA Hiscox, H Smith, C Sweet, "Role of neuraminidase in influenza virus-induced apoptosis", J Gen Virol, 80,
1999, 137-46.
OP Zhirnov, TE Konakova, T Wolff, HD Klenk, "NS1 protein of influenza A virus down-regulates apoptosis", J Virol, 76, 2002, 1617-25.
13.2.2.1 NA activation of TGF-beta
Description
Influenza A virus induces apoptosis in a variety of ways including by activation of host TGF-beta by viral neuraminidase (NA).
References
S Schultz-Cherry, VS Hinshaw, "Influenza virus neuraminidase activates latent transforming growth factor beta", J Virol, 70, 1996, 8624-9.
SJ Morris, GE Price, JM Barnett, SA Hiscox, H Smith, C Sweet, "Role of neuraminidase in influenza virus-induced apoptosis", J Gen Virol, 80,
1999, 137-46.
S Oh, JM McCaffery, MC Eichelberger, "Dose-dependent changes in influenza virus-infected dendritic cells result in increased allogeneic T-cell
proliferation at low, but not high, doses of virus", J Virol, 74, 2000, 5460-9.
Reaction
13.2.2.2 PB1-F2 binds to the mitochondrial adenine nucleotide translocator 3 ANT3, inducing apoptosis
Description
Influenza A virus induces apoptosis in a variety of ways including binding of viral PB1-F2 to host mitochondrial adenine nucleotide translocator 3
(ANT3).
References
AN Chanturiya, G Basanez, U Schubert, P Henklein, JW Yewdell, J Zimmerberg, "PB1-F2, an influenza A virus-encoded proapoptotic
mitochondrial protein, creates variably sized pores in planar lipid membranes", J Virol, 78, 2004, 6304-12.
Reaction
14 Integration of energy metabolism
Authors
Reviewers
Rush, MG, 2005-09-10.
Description
Many hormones that affect individual physiological processes including the regulation of appetite, absorption, transport, and oxidation of
foodstuffs influence energy metabolism pathways. While insulin mediates the storage of excess nutrients, glucagon is involved in the
mobilization of energy resources in response to low blood glucose levels, principally by stimulating hepatic glucose output. Small doses of
glucagon are sufficient to induce significant glucose elevations. These hormone-driven regulatory pathways enable the body to sense and
respond to changed amounts of nutrients in the blood and demands for energy.
Glucagon and Insulin act through various metabolites and enzymes that target specific steps in metabolic pathways for sugar and fatty acids.
The processes responsible for the long-term control of fat synthesis and short term control of glycolysis by key metabolic products and enzymes
are annotated in this module as six specific pathways:
Pathway 1. Glucagon signalling in metabolic pathways: In response to low blood glucose, pancreatic alpha-cells release glucagon. The binding
of glucagon to its receptor results in increased cAMP synthesis, and Protein Kinase A (PKA) activation.
Pathway 2. PKA mediated phosphorylation:PKA phosphorylates key enzymes, e.g., 6-Phosphofructo-2-kinase /Fructose-2,6-bisphosphatase
(PF2K-Pase) at serine 36, and regulatory proteins, e.g., Carbohydrate Response Element Binding Protein (ChREBP) at serine 196 and
threonine 666.
Insulin mediated responses to high blood glucose will be annotated in future versions of Reactome. In brief, the binding of insulin to its receptor
leads to increased protein phosphatase activity and to hydrolysis of cAMP by cAMP phosphodiesterase. These events counteract the regulatory
effects of glucagon.
Pathway 3: Insulin stimulates increased synthesis of Xylulose-5-phosphate (Xy-5-P). Activation of the insulin receptor results indirectly in
increased Xy-5-P synthesis from Glyceraldehyde-3-phosphate and Fructose-6-phosphate. Xy-5-P, a metabolite of the pentose phosphate
pathway, stimulates protein phosphatase PP2A.
Pathway 4: AMP Kinase (AMPK) mediated response to high AMP:ATP ratio: In response to diet with high fat content or low energy levels, the
cytosolic AMP:ATP ratio is increased. AMP triggers a complicated cascade of events. In this module we have annotated only the
phosphorylation of ChREBP by AMPK at serine 568, which inactivates this transcription factor.
Pathway 5: Dephosphorylation of key metabolic factors by PP2A: Xy-5-P activated PP2A efficiently dephosphorylates phosphorylated
PF2K-Pase resulting in the higher output of F-2,6-P2 that enhances PFK activity in the glycolytic pathway. PP2A also dephosphorylates (and
thus activates) cytosolic and nuclear ChREBP.
Pathway 6: Transcriptional activation of metabolic genes by ChREBP: Dephosphorylated ChREBP activates the transcription of genes involved
in glucose metabolism such as pyruvate kinase, and lipogenic genes such as acetyl-CoA carboxylase, fatty acid synthetase, acyl CoA synthase
and glycerol phosphate acyl transferase.
The illustration below summarizes this network of events. Black lines are metabolic reactions, red lines are negative regulatory events, and
green lines are positive regulatory events (figure reused with permission from Veech (2003) - Copyright (2003) National Academy of Sciences,
U.S.A.).
References
DG Hardie, "The AMP-activated protein kinase pathway--new players upstream and downstream", J Cell Sci, 117, 2004, 5479-87.
G Jiang, BB Zhang, "Glucagon and regulation of glucose metabolism", Am J Physiol Endocrinol Metab, 284, 2003, E671-8.
RL Veech, "A humble hexose monophosphate pathway metabolite regulates short- and long-term control of lipogenesis", Proc Natl Acad Sci U S
A, 100, 2003, 5578-80.
T Kabashima, T Kawaguchi, BE Wadzinski, K Uyeda, "Xylulose 5-phosphate mediates glucose-induced lipogenesis by xylulose
5-phosphate-activated protein phosphatase in rat liver", Proc Natl Acad Sci U S A, 100, 2003, 5107-12.
14.1 Glucagon signaling in metabolic regulation
Authors
Description
Glucagon and insulin are peptide hormones released from the pancreas into the blood, that normally act in complementary fashion to stabilize
blood glucose concentration. When blood glucose levels rise, insulin release stimulates glucose uptake from the blood, glucose breakdown
(glycolysis), and glucose storage as glycogen. When blood glucose levels fall, glucagon release stimulates glycogen breakdown and de novo
glucose synthesis (gluconeogenesis), while inhibiting glycolysis and glycogen synthesis.
At a molecular level, the binding of glucagon to the extracellular face of its receptor causes conformational changes in the receptor that allow the
dissociation and activation of subunits Gs and Gq. The activation of Gq leads to the activation of phospholipase C, production of inositol
1,4,5-triphosphate, and subsequent release of intracellular calcium. The activation of Gs leads to activation of adenylate cyclase, an increase in
intracellular cAMP levels, and activation of protein kinase A (PKA). Active PKA phosphorylates key enzymes of glycogenolysis, glycogenesis,
gluconeogenesis, and glycolysis, modifying their activities. These signal transduction events, and some of their downstream consequences, are
illustrated below (adapted from Jiang and Zhang, 2003).
References
14.1.1 G(s)-alpha mediated events in glucagon signalling
Authors
Description
Guanine nucleotide binding proteins or G proteins constitute a large family of proteins that transmit signals from membrane receptors to
downstream effector molecules. Each G protein is composed of 3 subunits: alpha, beta and gamma. The alpha subunit binds to guanine
nucleotide and is important for receptor coupling and effector activation. Each of s, i and q - forms of the alpha subunit has a functional
specificity. About 4 isoforms of the beta subunit are known. Of the 12 subunits of gamma subunits known so far, subunits 1 and 9 are active in
photoreceptor coupled signalling while others are expressed in various tissues. The beta and gamma subunits occur as dimers on the cell
surface and the specific role, tissue occurrence and the binding preferences between isoforms of these subunits are still being unraveled (Ray et
al.,1995; Cali et al.,1992).
References
JJ Cali, EA Balcueva, I Rybalkin, JD Robishaw, "Selective tissue distribution of G protein gamma subunits, including a new form of the gamma
subunits identified by cDNA cloning", J Biol Chem, 267, 1992, 24023-7.
J Codina, D Stengel, SL Woo, L Birnbaumer, "Beta-subunits of the human liver Gs/Gi signal-transducing proteins and those of bovine retinal rod
cell transducin are identical", FEBS Lett, 207, 1986, 187-92.
K Ray, C Kunsch, LM Bonner, JD Robishaw, "Isolation of cDNA clones encoding eight different human G protein gamma subunits, including
three novel forms designated the gamma 4, gamma 10, and gamma 11 subunits", J Biol Chem, 270, 1995, 21765-71.
LS Weinstein, S Yu, DR Warner, J Liu, "Endocrine manifestations of stimulatory G protein alpha-subunit mutations and the role of genomic
imprinting", Endocr Rev, 22, 2001, 675-705.
PB Wedegaertner, HR Bourne, M von Zastrow, "Activation-induced subcellular redistribution of Gs alpha", Mol Biol Cell, 7, 1996, 1225-33.
14.1.1.1 Glucagon binds to Glucagon receptor
Description
At the beginning of this reaction, 1 molecule of 'Glucagon receptor (GCGR)', and 1 molecule of 'Glucagon' are present. At the end of this
reaction, 1 molecule of 'Glucagon:GCGR' is present.
This reaction takes place on the 'plasma membrane'.
References
T Rall, BA Harris, "Identification of the lesion in the stimulatory GTP-binding protein of the uncoupled S49 lymphoma", FEBS Lett, 224, 1987,
365-71.
Q Dallas-Yang, X Shen, M Strowski, E Brady, R Saperstein, RE Gibson, D Szalkowski, SA Qureshi, MR Candelore, JE Fenyk-Melody, ER
Parmee, BB Zhang, G Jiang, "Hepatic glucagon receptor binding and glucose-lowering in vivo by peptidyl and non-peptidyl glucagon receptor
antagonists", Eur J Pharmacol, 501, 2004, 225-34.
Reaction
14.1.1.2 Glucagon:GCGR mediates GTP-GDP exchange
Description
At the beginning of this reaction, 1 molecule of 'G-protein with G(s) alpha:GDP', and 1 molecule of 'GTP' are present. At the end of this reaction,
1 molecule of 'G(s)-alpha:GTP', 1 molecule of 'G-beta:G-gamma dimer', and 1 molecule of 'GDP' are present.
This reaction takes place on the 'plasma membrane' and is mediated by the 'G-protein coupled receptor activity' of 'Glucagon:GCGR'.
Reaction
14.1.1.3 G(s)-alpha:GTP binds to Adenylate cyclase
Description
At the beginning of this reaction, 1 molecule of 'G(s)-alpha:GTP', and 1 molecule of 'Adenylate cyclase' are present. At the end of this reaction, 1
molecule of 'Activated adenylate cyclase' is present.
This reaction takes place on the 'plasma membrane'.
Reaction
14.1.2 PKA activation in glucagon signalling
Authors
Description
Adenylate cyclase catalyses the synthesis of cyclic AMP (cAMP) from ATP. In the absence of cAMP, protein kinase A (PKA) exists as inactive
tetramers of two catalytic subunits and two regulatory subunits. cAMP binding to PKA tetramers causes them to dissociate and release their
catalytic subunits as active monomers. Four isoforms of the regulatory subunit are known, that differ in their tissue specificity and functional
characteristics, but the specific isoform activated in response to glucagon signaling has not yet been identified.
14.1.2.1 Activated Adenylate cyclase catalyses cAMP synthesis
Description
At the beginning of this reaction, 1 molecule of 'ATP' is present. At the end of this reaction, 1 molecule of 'pyrophosphate', and 1 molecule of
'3',5'-Cyclic AMP' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'adenylate cyclase activity' of 'Activated adenylate cyclase'.
Reaction
14.1.2.2 cAMP induces dissociation of inactive PKA tetramers
Authors
Description
The four protein kinase A (PKA) regulatory subunit isoforms differ in their tissue specificity and functional characteristics. The specific isoform
activated in response to glucagon signalling is not known.
Reaction
14.2 PKA-mediated phosphorylation of key metabolic factors
Authors
Description
Upon dissociation of protein kinase A (PKA) tetramers in the presence of cAMP, the released PKA catalytic monomers phosphorylate specific
serine and threonine residues of several metabolic enzymes. These target enzymes include glycogen phosphorylase kinase, glycogen synthase
and PF2K-Pase. PKA also phosphorylates ChREBP (Carbohydrate Response Element Binding Protein), preventing its movement into the
nucleus and thus its function as a positive transcription factor for genes involved in glycolytic and lipogenic reactions.
References
A, 100, 2003, 5578-80.
14.2.1 PKA catalytic subunit translocates to the nucleus
Description
In this reaction, 1 molecule of 'PKA catalytic subunit' is translocated from cytosol to nucleoplasm.
This reaction takes place in the 'intracellular'.
Reaction
14.2.2 Phosphorylation of ChREBP at Thr(666) by PKA
Authors
Description
In its active (unphosphorylated) form, ChREBP (Carbohydrate Response Element Binding Protein) binds so-called ChRE (Carbohydrate
Response Element) DNA sequence motifs found upstream of several genes involved in glucose utilization and lipid synthesis, activating
transcription of these genes. Phosphorylation of ChREBP at threonine residue 666 by PKA (protein kinase A) blocks this binding activity, and
thus has the effect of down-regulating expression of the target genes. ChREBP phosphorylation can be reversed by the action of protein
phosphatase 2A (PP2A).
Source reaction
This reaction was inferred from the corresponding reaction "Phosphorylation of mChREBP at Thr (666) residue by mPKA" in species Mus
musculus.
T Kawaguchi, M Takenoshita, T Kabashima, K Uyeda, "Glucose and cAMP regulate the L-type pyruvate kinase gene by
phosphorylation/dephosphorylation of the carbohydrate response element binding protein", Proc Natl Acad Sci U S A, 98, 2001, 13710-5.
Reaction
14.2.3 PhosphoChREBP (Thr 666) is exported to cytosol
Authors
Description
ChREBP (Carbohydrate Response Element Binding Protein) doubly phosphorylated at threonine 666 and serine 196 is inactive and is localized
to the cytosol. Removal of the phosphate residue at serine 196 allows ChREBP to translocate between the cytosol and the nucleoplasm.
Reaction
14.2.4 Phosphorylation of pChREBP (Thr 666) at Ser(196) by PKA
Authors
Description
Phosphorylation of ChREBP (Carbohydrate Response Element Binding Protein) at serine 196 by PKA inhibits its nuclear translocation. This
reaction has been studied in detail using mouse proteins (Kawaguchi et al. 2001); the human version of the reaction is inferred from these
studies.
Source reaction
This reaction was inferred from the corresponding reaction "Phosphorylation of mpChREBP (Thr 666) at Ser(196) by mPKA" in species Mus
musculus.
Reaction
14.2.5 Nuclear transport of pChREBP (Thr 666) protein
Description
ChREBP (Carbohydrate Response Element Binding Protein) doubly phosphorylated at threonine 666 and serine 196 is inactive and is localized
to the cytosol. Removal of the phosphate residue at serine 196 allows ChREBP to translocate between the cytosol and the nucleoplasm.
Reaction
14.2.6 Phosphorylation of PF2K-Pase by PKA catalytic subunit
Authors
Description
Activated PKA (protein kinase A) phosphorylates serine 36 of the bifunctional 6-Phosphofructo-2-kinase /Fructose-2,6-bisphosphatase
(PF2K-Pase) enzyme. This phosphorylation inhibits the enzyme's phosphofructokinase (PFK-2) activity while activating its phosphatase activity.
As a result, cytosolic levels of Fructose-2,6-bisphosphate (F-2,6-P2) are reduced. F-2,6-P2 in turn is a key positive regulator of the committed
step of glycolysis, so the net effect of this phosphorylation event is a reduced rate of glycolysis.
References
E Furuya, M Yokoyama, K Uyeda, "Regulation of fructose-6-phosphate 2-kinase by phosphorylation and dephosphorylation: possible
mechanism for coordinated control of glycolysis and glycogenolysis", Proc Natl Acad Sci U S A, 79, 1982, 325-9.
Source reaction
This reaction was inferred from the corresponding reaction "Phosphorylation of rPF2K-Pase by rPKA" in species Rattus norvegicus.
MR el-Maghrabi, TH Claus, J Pilkis, SJ Pilkis, "Regulation of 6-phosphofructo-2-kinase activity by cyclic AMP-dependent phosphorylation", Proc
Natl Acad Sci U S A, 79, 1982, 315-9.
Reaction
14.2.7 D-fructose 2,6-bisphosphate + H2O => D-fructose 6-phosphate + orthophosphate [liver + muscle]
Description
At the beginning of this reaction, 1 molecule of 'H2O', and 1 molecule of 'D-Fructose 2,6-bisphosphate' are present. At the end of this reaction, 1
molecule of 'Orthophosphate', and 1 molecule of 'D-Fructose 6-phosphate' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'fructose-2,6-bisphosphate 2-phosphatase activity' of 'pPF2K-Pase complex'.
References
J Algaier, K Uyeda, "Molecular cloning, sequence analysis, and expression of a human liver cDNA coding for
fructose-6-P,2-kinase:fructose-2,6-bisphosphatase", Biochem Biophys Res Commun, 153, 1988, 328-33.
Reaction
14.3 Insulin effects increased synthesis of Xylulose-5-Phosphate
Authors
Description
One of the downstream effects of insulin, mediated via protein phosphatase 2A (PP2A), is increased synthesis of Fructose-2,6-bisphosphate, an
allosteric activator of phosphofructokinase 1 (PFK1). PFK1 in turn catalyzes the committed step of glycolysis so the net effect of this whole
sequence of events set off by insulin is to increase cytosolic concentrations of the small molecules formed in the course of glycolysis. This in turn
drives the increased synthesis of Xylulose-5-phosphate, itself a positive regulator of PP2A.
References
AL Marks, C Smith, M Lieberman, "Pathways of Sugar Metabolism", Basic Medical Biochemistry, 29, 1996, 527.
T Wood, "Physiological functions of the pentose phosphate pathway", Cell Biochem Funct, 4, 1986, 241-7.
K Uyeda, H Yamashita, T Kawaguchi, "Carbohydrate responsive element-binding protein (ChREBP): a key regulator of glucose metabolism and
fat storage", Biochem Pharmacol, 63, 2002, 2075-80.
14.3.1 D-fructose 6-phosphate + D-erythrose 4-phosphate <=> sedoheptulose 7-phosphate +

D-glyceraldehyde 3-phosphate
Description
At the beginning of this reaction, 1 molecule of 'D-Erythrose 4-phosphate', and 1 molecule of 'D-Fructose 6-phosphate' are present. At the end of
this reaction, 1 molecule of 'Sedoheptulose 7-phosphate', and 1 molecule of 'D-Glyceraldehyde 3-phosphate' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'transaldolase activity' of 'transaldolase'.
References
NM Verhoeven, JH Huck, B Roos, EA Struys, GS Salomons, AC Douwes, MS van der Knaap, C Jakobs, "Transaldolase deficiency: liver
cirrhosis associated with a new inborn error in the pentose phosphate pathway.", Am J Hum Genet, 68, 2001, 1086-92.
Reaction
14.3.2 D-glyceraldehyde 3-phosphate + D-fructose 6-phosphate <=> xylulose 5-phosphate + D-erythrose

4-phosphate
Description
At the beginning of this reaction, 1 molecule of 'D-Glyceraldehyde 3-phosphate', and 1 molecule of 'D-Fructose 6-phosphate' are present. At the
end of this reaction, 1 molecule of 'D-Erythrose 4-phosphate', and 1 molecule of 'D-Xylulose 5-phosphate' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'transketolase activity' of 'transketolase dimer'.
References
JJ Wang, PR Martin, CK Singleton, "Aspartate 155 of human transketolase is essential for thiamine diphosphate-magnesium binding, and
cofactor binding is required for dimer formation", Biochim Biophys Acta, 1341, 1997, 165-72.
Reaction
14.3.3 D-glyceraldehyde 3-phosphate + sedoheptulose 7-phosphate<=> xylulose 5-phosphate+ribose

5-phosphate
Description
At the beginning of this reaction, 1 molecule of 'Sedoheptulose 7-phosphate', and 1 molecule of 'D-Glyceraldehyde 3-phosphate' are present. At
the end of this reaction, 1 molecule of 'D-ribose 5-phosphate', and 1 molecule of 'D-Xylulose 5-phosphate' are present.
References
Reaction
14.4 Activation of PP2A by Xylulose-5-phosphate
Authors
Description
Xylulose-5-phosphate binds to Protein phosphatase 2A (PP2A), activating it. This regulatory step may be the crucial physiological link explaining
the coordinately increased rates of glycolysis and lipogenesis generally observed in individuals consuming high-carbohydrate diets.
References
A, 100, 2003, 5578-80.
Source reaction
This reaction was inferred from the corresponding reaction "Activation of rPP2A by Xylulose-5-phosphate" in species Rattus norvegicus.
Reaction
14.5 AMPK inhibits chREBP transcriptional activation activity
Authors
Description
AMP-activated protein kinase (AMPK) is a sensor of cellular energy levels. A high cellular ratio of AMP:ATP triggers the phosphorylation and
activation of AMPK. Activated AMPK in turn phosphorylates a wide array of target proteins, as shown in the figure below (reproduced from
(Hardie et al. 2003), with the permission of D.G. Hardie). These targets include ChREBP (Carbohydrate Response Element Binding Protein),
whose inactivation by phosphorylation reduces transcription of key enzymes of the glycolytic and lipogenic pathways.
References
DG Hardie, JW Scott, DA Pan, ER Hudson, "Management of cellular energy by the AMP-activated protein kinase system", FEBS Lett, 546,
2003, 113-20.
T Kawaguchi, K Osatomi, H Yamashita, T Kabashima, K Uyeda, "Mechanism for fatty acid sparing effect on glucose-induced transcription:
regulation of carbohydrate-responsive element-binding protein by AMP-activated protein kinase", J Biol Chem, 277, 2002, 3829-35.
PC Cheung, IP Salt, SP Davies, DG Hardie, D Carling, "Characterization of AMP-activated protein kinase gamma-subunit isoforms and their role
in AMP binding", Biochem J, 346, 2000, 659-69.
14.5.1 AMP binds to gamma subunit of AMP kinase heterotrimer
Description
At the beginning of this reaction, 1 molecule of 'AMPK heterotrimer (inactive)', and 1 molecule of 'AMP' are present. At the end of this reaction, 1
molecule of 'AMPK heterotrimer:AMP' is present.
References
PC Cheung, IP Salt, SP Davies, DG Hardie, D Carling, "Characterization of AMP-activated protein kinase gamma-subunit isoforms and their role
in AMP binding", Biochem J, 346, 2000, 659-69.
Reaction
14.5.2 LKB1 phosphorylates the alpha subunit of AMPK heterotrimer
Authors
Description
LKB1 phosphorylates threonine residue 172 of the alpha subunit of the AMPK heterotrimer, activating it. LKB1, a serine/threonine kinase, was
first identified as the gene whose mutation is associated with the Peutz-Jeghers familial cancer syndrome. This disease phenotype is consistent
with the hypothesis that the interaction between LKB1 and AMPK normally plays a key role in the negative regulation of cell growth (Hardie
2004).
References
Source reaction
This reaction was inferred from the corresponding reaction "rLkb-1 (Stk-11) activates AMPK by phosphorylation" in species Rattus norvegicus.
A Woods, SR Johnstone, K Dickerson, FC Leiper, LG Fryer, D Neumann, U Schlattner, T Wallimann, M Carlson, D Carling, "LKB1 is the
upstream kinase in the AMP-activated protein kinase cascade", Curr Biol, 13, 2003, 2004-8.
Reaction
14.5.3 Phosphorylation of ChREBP at Serine 568 by AMPK
Authors
Description
In the nucleus, activated AMPK phosphorylates serine residue 568 of ChREBP (Carbohydrate Response Element Binding Protein).
Phosphorylated ChREBP does not bind to ChRE chromosomal DNA sequence elements and thus loses its ability to promote transcription of
genes involved in glycolysis and lipogenesis.
Source reaction
This reaction was inferred from the corresponding reaction "Phosphorylation of rChREBP(Ser 568) by rAMPK" in species Rattus norvegicus.
T Kawaguchi, K Osatomi, H Yamashita, T Kabashima, K Uyeda, "Mechanism for fatty acid sparing effect on glucose-induced transcription:
regulation of carbohydrate-responsive element-binding protein by AMP-activated protein kinase", J Biol Chem, 277, 2002, 3829-35.
Reaction
14.6 PP2A-mediated dephosphorylation of key metabolic factors
Authors
Description
A member of the PP2A family of phosphatases dephosphorylates both cytosolic and nuclear forms of ChREBP (Carbohydrate Response
Elemant Binding Protein). In the nucleus, dephosphorylated ChREBP complexes with MLX protein and binds to ChRE sequence elements in
chromosomal DNA, activating transcription of genes involved in glycolysis and lipogenesis. The phosphatase is activated by
Xylulose-5-phosphate, an intermediate of the pentose phosphate pathway (Kabashima et al. 2003). The rat enzyme has been purified to
homogeneity and shown by partial amino acid sequence analysis to differ from previously described PP2A phosphatases (Nishimura and Uyeda
1995) - the human enzyme has not been characterized.
References
A, 100, 2003, 5578-80.
M Nishimura, K Uyeda, "Purification and characterization of a novel xylulose 5-phosphate-activated protein phosphatase catalyzing
dephosphorylation of fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase", J Biol Chem, 270, 1995, 26341-6.
14.6.1 Dephosphorylation of pChREBP (Ser 196) by PP2A
Description
At the beginning of this reaction, 1 molecule of 'pChREBP (Ser 196, Thr 666)' is present. At the end of this reaction, 1 molecule of
'Orthophosphate', and 1 molecule of 'pChREBP (Thr 666)' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'phosphatidate phosphatase activity' of 'PP2A-ABdeltaC complex'.
Source reaction
This reaction was inferred from the corresponding reaction "Dephosphorylation of prChREBP (Ser 196) by rPP2A" in species Rattus norvegicus.
Reaction
14.6.2 Dephosphorylation of pChREBP (Ser 568) by PP2A
Description
At the beginning of this reaction, 1 molecule of 'pChREBP(Ser 568)' is present. At the end of this reaction, 1 molecule of 'Orthophosphate', and 1
molecule of 'ChREBP protein' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'phosphatidate phosphatase activity' of 'PP2A-ABdeltaC complex'.
Source reaction
This reaction was inferred from the corresponding reaction "Dephosphorylation of prChREBP (Ser 568) by rPP2A complex" in species Rattus
norvegicus.
Reaction
14.6.3 Dephosphorylation of pChREBP (Thr 666) by PP2A
Description
At the beginning of this reaction, 1 molecule of 'pChREBP (Thr 666)' is present. At the end of this reaction, 1 molecule of 'Orthophosphate', and
1 molecule of 'ChREBP protein' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'phosphatidate phosphatase activity' of 'PP2A-ABdeltaC complex'.
Source reaction
This reaction was inferred from the corresponding reaction "Dephosphorylation of prChREBP (Thr 666) by rPP2A" in species Rattus norvegicus.
Reaction
14.6.4 ATP + D-fructose 6-phosphate => ADP + D-fructose 2,6-bisphosphate [liver + muscle]
Description
At the beginning of this reaction, 1 molecule of 'D-Fructose 6-phosphate', and 1 molecule of 'ATP' are present. At the end of this reaction, 1
molecule of 'ADP', and 1 molecule of 'D-Fructose 2,6-bisphosphate' are present.
This reaction takes place in the 'cytosol' and is mediated by the '6-phosphofructo-2-kinase activity' of 'PF2K-Pase1 homodimer'.
References
Reaction
14.6.5 Dephosphorylation of PF2K-Pase by PP2A complex
Description
At the beginning of this reaction, 1 molecule of 'pPF2K-Pase complex' is present. At the end of this reaction, 1 molecule of 'Orthophosphate', and
1 molecule of 'PF2K-Pase1 homodimer' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'phosphatidate phosphatase activity' of 'PP2A-ABdeltaC complex'.
Source reaction
This reaction was inferred from the corresponding reaction "Dephosphorylation of PF2K-Pase by rPP2A complex" in species Rattus norvegicus.
M Nishimura, K Uyeda, "Purification and characterization of a novel xylulose 5-phosphate-activated protein phosphatase catalyzing
dephosphorylation of fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase", J Biol Chem, 270, 1995, 26341-6.
Reaction
14.7 ChREBP activates metabolic gene expression
Authors
Description
ChREBP (Carbohydrate Response Element Binding Protein) is a large multidomain protein containing a nuclear localization signal near its
amino terminus, polyproline domains, a basic helix-loop-helix-leucine zipper domain, and a leucine-zipper-like domain (Uyeda et al., 2002). Its
dephosphorylation in response to molecular signals associated with the well-fed state allows it to enter the nucleus, interact with MLX protein,
and bind to ChRE DNA sequence motifs near Acetyl-CoA carboxylase, Fatty acid synthase, and Pyruvate kinase (L isoform) genes (Ishi et
al.2004). This sequence of events is outlined schematically in the picture below (adapted from Kawaguchi et al. (2001) - copyright (2001)
National Academy of Sciences, U.S.A.).
References
S Ishii, K Iizuka, BC Miller, K Uyeda, "Carbohydrate response element binding protein directly promotes lipogenic enzyme gene transcription",
K Iizuka, RK Bruick, G Liang, JD Horton, K Uyeda, "Deficiency of carbohydrate response element-binding protein (ChREBP) reduces lipogenesis
as well as glycolysis", Proc Natl Acad Sci U S A, 101, 2004, 7281-6.
L Ma, NG Tsatsos, HC Towle, "Direct role of ChREBP.Mlx in regulating hepatic glucose-responsive genes.", J Biol Chem, 280, 2005, 12019-27.
14.7.1 Formation of ChREBP:MLX heterodimer
Description
At the beginning of this reaction, 1 molecule of 'ChREBP protein', and 1 molecule of 'MLX protein' are present. At the end of this reaction, 1
molecule of 'ChREBP:MLX' is present.
Source reaction
This reaction was inferred from the corresponding reaction "Formation of mChREBP:mMlx complex" in species Mus musculus.
L Ma, NG Tsatsos, HC Towle, "Direct role of ChREBP.Mlx in regulating hepatic glucose-responsive genes.", J Biol Chem, 280, 2005, 12019-27.
AK Stoeckman, L Ma, HC Towle, "Mlx is the functional heteromeric partner of the carbohydrate response element-binding protein in glucose
regulation of lipogenic enzyme genes", J Biol Chem, 279, 2004, 15662-9.
Reaction
14.7.2 Transcriptional activation of glucose metabolism genes by ChREBP:MLX

Description
ChREBP activates the transcrition of glucose metabolis genes resulting in alterations in the glucose levels.
14.7.2.1 Transcriptional activation of pyruvate kinase gene by ChREBP:MLX
Description
At the end of this reaction, 1 molecule of 'pyruvate kinase, liver and RBC' is present.
Source reaction
This reaction was inferred from the corresponding reaction "Transcriptional activation of pyruvate kinase L isoform gene by mChREBP:mMLX" in
species Mus musculus.
H Yamashita, M Takenoshita, M Sakurai, RK Bruick, WJ Henzel, W Shillinglaw, D Arnot, K Uyeda, "A glucose-responsive transcription factor
that regulates carbohydrate metabolism in the liver", Proc Natl Acad Sci U S A, 98, 2001, 9116-21.
J Hasegawa, K Osatomi, RF Wu, K Uyeda, "A novel factor binding to the glucose response elements of liver pyruvate kinase and fatty acid
synthase genes", J Biol Chem, 274, 1999, 1100-7.
Reaction
14.7.3 Transcriptional activation of lipogenesis genes by ChREBP:MLX
Description
ChREBP activates the transcription of lipogenesis genes resulting in the biosynthesis of lipid molecules.
14.7.3.1 Transcriptional activation of Citrate lyase monomer gene by ChREBP:MLX
Description
At the end of this reaction, 1 molecule of 'citrate lyase monomer' is present.
Reaction
14.7.3.2 Transcriptional activation of FAS monomer gene by ChREBP:MLX
Description
At the end of this reaction, 1 molecule of 'Fatty acid synthase ' is present.
Reaction
14.7.3.3 Transcriptional activation of Acetyl-CoA carboxylase by ChREBP:MLX
Description
At the end of this reaction, 1 molecule of 'Acetyl-CoA carboxylase 2 ' is present.

Reaction
14.7.3.4 Transcriptional activation of GP-acyl transferase gene by ChREBP:MLX
Description
At the end of this reaction, 1 molecule of '1-acyl-sn-glycerol-3-phosphate acyltransferase alpha ' is present.
Reaction
14.8 Activated AMPK stimulates fatty-acid oxidation in muscle
Authors
Reviewers
Description
AMPK plays a central role in regulating fatty acid oxidation in muscle. In order for fatty acids taken up and converted to fatty acyl CoAâ€™s by
muscle cells to undergo beta-oxidation, they must be transported into the mitochondrial matrix by carnitine palmitoyl transferase (CPT). This
transport process is negatively regulated by malonyl CoA, synthesized by ACC2 enzyme on the outer mitochondrial membrane. Phosphorylation
and activation of AMPK as a result of signaling via leptin, adiponectin, and alpha-adrenergic receptors, however, enables this enzyme to
phosphorylate and inactivate ACC2, reducing the levels of malonyl CoA and thus facilitating CPT-mediated fatty acid transport (Kahn et
al.,2005).
The mitochondrial CPT transport system consists of the malonyl-CoA sensitive carnitine palmitoyltransferase I (CPT-I) localized in the
mitochondrial outer membrane, the carnitine:acylcarnitine translocase, an integral inner membrane protein, and carnitine palmitoyltransferase II
localized on the matrix side of the inner membrane (Kerner and Hoppel, 2000).
In this module, the effect of activated AMPK on fatty acid beta oxidation as mediated by malonyl CoA in muscle cells is annotated. The
mechanisms by which leptin and adrenergic receptors modulate AMPK activity will be annotated in the future.
References
BB Kahn, T Alquier, D Carling, DG Hardie, "AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of
metabolism", Cell Metab, 1, 2005, 15-25.
Y Minokoshi, YB Kim, OD Peroni, LG Fryer, C Muller, D Carling, BB Kahn, "Leptin stimulates fatty-acid oxidation by activating AMP-activated
protein kinase", Nature, 415, 2002, 339-43.
J Kerner, C Hoppel, "Fatty acid import into mitochondria", Biochim Biophys Acta, 1486, 2000, 1-17.
14.8.1 Formation of Malonyl-CoA from Acetyl-CoA (muscle)
Authors
Reviewers
Description
Acetyl-CoA carboxylase 2 associated with the outer mitochondrial membrane catalyzes the reaction of cytosolic bicarbonate, ATP, and
acetyl-CoA to form malonyl-CoA, ADP, and orthophosphate.
References
L Abu-Elheiga, DB Almarza-Ortega, A Baldini, SJ Wakil, "Human acetyl-CoA carboxylase 2. Molecular cloning, characterization, chromosomal
mapping, and evidence for two isoforms.", J Biol Chem, 272, 1997, 10669-77.
KW Kim, H Yamane, J Zondlo, J Busby, M Wang, "Expression, purification, and characterization of human acetyl-CoA carboxylase 2", Protein
Expr Purif, 53, 2007, 16-23.
L Abu-Elheiga, WR Brinkley, L Zhong, SS Chirala, G Woldegiorgis, SJ Wakil, "The subcellular localization of acetyl-CoA carboxylase 2", Proc
Natl Acad Sci U S A, 97, 2000, 1444-9.
Reaction
14.8.2 Activation of cytosolic AMPK by phosphorylation
Authors
Reviewers
Description
The cytosolic AMPK complex is activated by phosphorylation. Signals leading to this phosphorylation event can be mediated by exercise, leptin
and adiponectin, the hypothalamic-sympathetic nervous system (SNS), and alpha adrenergic receptors, as demonstrated in studies of rat and
human skeletal muscle (Minoksohi et al., 2002, Kahn et al., 2005). The details of AMPK activation in response to these stimuli will be annotated
in the future.
References
WW Winder, DG Hardie, "Inactivation of acetyl-CoA carboxylase and activation of AMP-activated protein kinase in muscle during exercise", Am
J Physiol, 270, 1996, E299-304.
BB Kahn, T Alquier, D Carling, DG Hardie, "AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of
metabolism", Cell Metab, 1, 2005, 15-25.
DS Rubink, WW Winder, "Effect of phosphorylation by AMP-activated protein kinase on palmitoyl-CoA inhibition of skeletal muscle acetyl-CoA
carboxylase", J Appl Physiol, 98, 2005, 1221-7.
Y Minokoshi, YB Kim, OD Peroni, LG Fryer, C Muller, D Carling, BB Kahn, "Leptin stimulates fatty-acid oxidation by activating AMP-activated
protein kinase", Nature, 415, 2002, 339-43.
JF Wojtaszewski, P Nielsen, BF Hansen, EA Richter, B Kiens, "Isoform-specific and exercise intensity-dependent activation of 5'-AMP-activated
protein kinase in human skeletal muscle", J Physiol, 528, 2000, 221-6.
Reaction
14.8.3 pAMPK inactivates ACC2 inhibiting malonyl-CoA synthesis
Authors
Reviewers
Description
The phosphorylated AMPK inactivates ACC2 in muscle cells by phophorylation. This results in decreased levels of malonyl CoA.
References
N Ruderman, M Prentki, "AMP kinase and malonyl-CoA: targets for therapy of the metabolic syndrome", Nat Rev Drug Discov, 3, 2004, 340-51.
Reaction
14.8.4 Conversion of palmitic acid to palmitoyl-CoA
Authors
Reviewers
Description
Palmitoyl-CoA is converted to palmitoyl carnitine by the cytosol. The resulting palmitoyl CoA can be transported into the mitochondria for beta
oxidation.
References
KT Malhotra, K Malhotra, BH Lubin, FA Kuypers, "Identification and molecular characterization of acyl-CoA synthetase in human erythrocytes
and erythroid precursors", Biochem J, 344, 1999, 135-43.
Reaction
14.8.5 Import of palmitoyl-CoA into the mitochondrial matrix
Authors
Reviewers
Description
The mitochondrial carnitine system catalyzes the transport of long-chain fatty acids into the mitochondrial matrix where they undergo beta
oxidation. This transport system consists of the malonyl-CoA sensitive carnitine palmitoyltransferase I (CPT-I) localized in the mitochondrial
outer membrane, the carnitine:acylcarnitine translocase, an integral inner membrane protein, and carnitine palmitoyltransferase II localized on
the matrix side of the inner membrane. (Kerner and Hoppel, 2000).
References
RR Ramsay, RD Gandour, FR van der Leij, "Molecular enzymology of carnitine transfer and transport", Biochim Biophys Acta, 1546, 2001,
21-43.
14.8.5.1 CPT1 converts palmitoyl-CoA to palmitoyl carnitine
Authors
Reviewers
Description
CPT1 associated with the outer mitochondrial membrane catalyzes the reversible reaction of palmitoyl-CoA and carnitine to form
palmitoylcarnitine and CoA-SH. This is the first step in the transfer of long chain fatty acyl CoAs like palmitoyl-CoA into mitochondria for beta
oxidation.
References
M Morillas, P Gomez-Puertas, B Rubi, J Clotet, J Arino, A Valencia, FG Hegardt, D Serra, G Asins, "Structural model of a malonyl-CoA-binding
site of carnitine octanoyltransferase and carnitine palmitoyltransferase I: mutational analysis of a malonyl-CoA affinity domain", J Biol Chem,
277, 2002, 11473-80.
VA Zammit, NT Price, F Fraser, VN Jackson, "Structure-function relationships of the liver and muscle isoforms of carnitine palmitoyltransferase
I", Biochem Soc Trans, 29, 2001, 287-92.
D Wu, L Govindasamy, W Lian, Y Gu, T Kukar, M Agbandje-McKenna, R McKenna, "Structure of human carnitine acetyltransferase. Molecular
basis for fatty acyl transfer.", J Biol Chem, 278, 2003, 13159-65.
S Gobin, L Thuillier, G Jogl, A Faye, L Tong, M Chi, JP Bonnefont, J Girard, C Prip-Buus, "Functional and structural basis of carnitine
palmitoyltransferase 1A deficiency", J Biol Chem, 278, 2003, 50428-34.
Reaction
14.8.5.2 Transport of palmitoyl carnitine into mitochondria
Authors
Reviewers
Description
The carnitine-acylcarnitine transporter (CACT), embedded in the inner mitochondrial membrane, shuttles acylcarnitine esters, in exchange for
free carnitine, across the inner mitochondrial membrane. The CACT protein is embedded in the inner mitochondrial membrane. It
governsmediates the movement of a a one-to-one exchange, between long-chain acylcarnitine ester into the mitochondrial matrix in exchange
for one s and nonesterified carnitine molecule. CACT can also mediate the, across the inner mitochondrial membrane and also a less efificient
unidirectional transport of carnitine across the inner mitochondrialis membrane (Huizing et al., 1997).
References
C Indiveri, V Iacobazzi, N Giangregorio, F Palmieri, "The mitochondrial carnitine carrier protein: cDNA cloning, primary structure and comparison
with other mitochondrial transport proteins", Biochem J, 321, 1997, 713-9.
M Huizing, V Iacobazzi, L IJlst, P Savelkoul, W Ruitenbeek, L van den Heuvel, C Indiveri, J Smeitink, F Trijbels, R Wanders, F Palmieri, "Cloning
of the human carnitine-acylcarnitine carrier cDNA and identification of the molecular defect in a patient", Am J Hum Genet, 61, 1997, 1239-45.
Reaction
14.8.5.3 CPT2 converts palmitoyl carnitine to palmitoyl-CoA
Authors
Reviewers
Description
CPT2, associated with the inner mitochondrial membrane, catalyzes the reaction of palmitoylcarnitine and CoASH to form palmitoyl-CoA and
carnitine in which fatty acid is conjugated back to CoA for subsequent beta oxidation in the mitochondrial matrix.
References
E Verderio, P Cavadini, L Montermini, H Wang, E Lamantea, G Finocchiaro, S DiDonato, C Gellera, F Taroni, "Carnitine palmitoyltransferase II
deficiency: structure of the gene and characterization of two novel disease-causing mutations", Hum Mol Genet, 4, 1995, 19-29.
Reaction
14.9 Regulation of pyruvate dehydrogenase complex (PDC)
Authors
Reviewers
Description
The mitochondrial pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate, linking glycolysis to the
tricarboxylic acid cycle and fatty acid synthesis. Regulation of PDC activity is important: PDC inactivation is crucial for glucose conservation
when glucose is scarce, while adequate PDC activity is required to allow both ATP and fatty acid production from glucose. The mechanisms that
control human PDC activity include its phosphorylation (inactivation) by a family of pyruvate dehydrogenase kinases (PDKs 1-4) and its
dephosphorylation (activation, reactivation) by the pyruvate dehydrogenase phosphate phosphatases (PDPs 1 and 2). Isoform-specific
differences in kinetic parameters, regulation, and phosphorylation site specificity of the PDKs introduce variations in the regulation of PDC
activity in differing endocrine and metabolic states (Sugden and Holness 2003).
References
MC Sugden, MJ Holness, "Recent advances in mechanisms regulating glucose oxidation at the level of the pyruvate dehydrogenase complex by
PDKs", Am J Physiol Endocrinol Metab, 284, 2003, E855-62.
14.9.1 Inactivation of PDC by phosphorylation of PDC E1 alpha component
Authors
Reviewers
Description
Pyruvate Dehydrogenase Kinase (PDK) in the mitochondrial matrix catalyzes the phosphorylation of serine residues of the E1-alpha subunit of
the pyruvate dehydrogenase complex, inactivating it. Pyruvate negatively regulates this reaction, and NADH and acetyl CoA positively regulate it
(Bao et al. 2004). Four isoforms of PDK have been identified, that differ in their expression patterns (Kortochikina and Patel, 2001) and
quantitative reponses to regulatory small molecules. All four isoforms catalyze the phosphorylation of serine residues 293 ("site 1") and 300 ("site
2"); PDK1 also catalyzes the phosphorylation of serine 232 ("site 3"). Phosphorylation of a single site in a single E1-alpha subunit is sufficient for
enzyme inactivation.
PDK2 is most widely expressed in tissues of the human body and has been most extensively characterized in vivo and in vitro. Annotation here
is based on its enzymatic properties (Bowker-Kinley et al. 1998; Sugden and Holness 2003).
References
LG Korotchkina, MS Patel, "Site specificity of four pyruvate dehydrogenase kinase isoenzymes toward the three phosphorylation sites of human
pyruvate dehydrogenase", J Biol Chem, 276, 2001, 37223-9.
MC Sugden, MJ Holness, "Recent advances in mechanisms regulating glucose oxidation at the level of the pyruvate dehydrogenase complex by
PDKs", Am J Physiol Endocrinol Metab, 284, 2003, E855-62.
H Bao, SA Kasten, X Yan, TE Roche, "Pyruvate dehydrogenase kinase isoform 2 activity limited and further inhibited by slowing down the rate of
dissociation of ADP", Biochemistry, 43, 2004, 13432-41.
MM Bowker-Kinley, WI Davis, P Wu, RA Harris, KM Popov, "Evidence for existence of tissue-specific regulation of the mammalian pyruvate
dehydrogenase complex", Biochem J, 329, 1998, 191-6.
R Gudi, MM Bowker-Kinley, NY Kedishvili, Y Zhao, KM Popov, "Diversity of the pyruvate dehydrogenase kinase gene family in humans", J Biol
Chem, 270, 1995, 28989-94.
Reaction
14.9.2 Activation of PDC by dephosphorylation of phospho-E1 alpha component
Authors
Reviewers
Description
Pyruvate dehydrogenase phosphatase (PDP) in the mitochondrial matrix catalyzes the hydrolytic removal of phosphate groups from
phosphoserine residues in the E1-alpha subunit of the pyruvate decarboxylase complex. The active form of the enzyme is a heterodimer of a
catalytic subunit and a regulatory one. The properties of the human regulatory subunit have been deduced from those of its bovine homologue
(Lawson et al. 1997). The activity of PDP1 is greatly enhanced through Ca2+ -dependent binding of the catalytic subunit (PDP1c) to the L2
(inner lipoyl) domain of dihydrolipoyl acetyltransferase (E2), which is also integrated in PDC. PDP activity requires Mg2+ (Huang et al. 1998).
While two PDP isozymes, PDP1 and 2, have been identified, only PDP1 has been characterized in detail in vitro and only PDP1 has been shown
to have physiologically relevant phosphatase activity in vivo (Maj et al. 2005).
References
DG Vassylyev, J Symersky, "Crystal structure of pyruvate dehydrogenase phosphatase 1 and its functional implications", J Mol Biol, 370, 2007,
417-26.
B Huang, R Gudi, P Wu, RA Harris, J Hamilton, KM Popov, "Isoenzymes of pyruvate dehydrogenase phosphatase. DNA-derived amino acid
sequences, expression, and regulation", J Biol Chem, 273, 1998, 17680-8.
MC Maj, N MacKay, V Levandovskiy, J Addis, ER Baumgartner, MR Baumgartner, BH Robinson, JM Cameron, "Pyruvate dehydrogenase
phosphatase deficiency: identification of the first mutation in two brothers and restoration of activity by protein complementation", J Clin
Endocrinol Metab, 90, 2005, 4101-7.
JE Lawson, SH Park, AR Mattison, J Yan, LJ Reed, "Cloning, expression, and properties of the regulatory subunit of bovine pyruvate
dehydrogenase phosphatase", J Biol Chem, 272, 1997, 31625-9.
Reaction
15 Lipid and lipoprotein metabolism
Authors
Jassal, B, Gillespie, ME, Gopinathrao, G, D'Eustachio, P, 2007-02-02.
Editors
D'Eustachio, P, Joshi-Tope, G, 0000-00-00.
Description
Lipids are hydrophobic but otherwise chemically diverse molecules that play a wide variety of roles in human biology. They include ketone
bodies, fatty acids, triacylglycerols, phospholipids and sphingolipids, eicosanoids, cholesterol, bile salts, steroid hormones, and fat-soluble
vitamins, and function as a major source of energy (fatty acids, triacylglycerols, and ketone bodies), are major constituents of cell membranes
(cholesterol and phospholipids), play a major role in their own digestion and uptake (bile salts), and participate in numerous signaling and
regulatory processes (steroid hormones and eicosanoids). Because of their poor solubility in water, most lipids in extracellular spaces in the
human body are found as complexes with specific carrier proteins. Regulation of the formation and movement of these lipoprotein complexes is
a critical aspect of human lipid metabolism, and lipoprotein abnormalities are associated with major human disease processes including
atherosclerosis and diabetes.
Aspects of lipid metabolism currently annotated in Reactome include lipid digestion, trafficking of dietary sterols, triacylglycerol synthesis (fatty
acid synthesis and triacylglycerol assembly), hormone-sensitive lipase-mediated triacylglycerol breakdown, and beta-oxidation of fatty acids,
ketone body metabolism (synthesis and utilization), and the synthesis of cholesterol, bile salts, and steroid hormones. Three aspects of
lipoprotein function are currently annotated: chylomicron-mediated lipid transport, HDL (high density lipoprotein)-mediated lipid transport, and
LDL (low density lipoprotein) endocytosis and degradation.
15.1 Digestion of dietary lipid
Authors
Description
Dietary lipids such as long-chain triacylglycerols and cholesterol esters are digested in the stomach and small intestine to yield long-chain fatty
acids, monoacylglycerols, glycerol and cholesterol through the action of a variety of lipases, and are then absorbed into enterocytes.
References
ME Lowe, "The triglyceride lipases of the pancreas", J Lipid Res, 43, 2002, 2007-16.
D Lombardo, "Bile salt-dependent lipase: its pathophysiological implications", Biochim Biophys Acta, 1533, 2001, 1-28.
15.1.1 Digestion of cholesterol esters by extracellular CEL (bile salt-dependent lipase)
Authors
Description
CEL (bile salt-dependent lipase) catalyzes the hydrolysis of extracellular cholesterol esters to yield cholesterol and a long-chain fatty acid. This
reaction, in the lumen of the small intestine, is part of the process of digestion of dietary fats.
While alternative splicing gives rise to two CEL isoforms, only the longer one encodes all of the residues that form the active site of the enzyme
(Reue et al. 1991). In vitro, monomeric CEL protein is active even in the absence of bile salts. Its activity is greatly increased when it is
complexed with two molecules of cholate, chenodeoxycholate, or their glycine or taurine conjugates (Lombardo and Guy 1980), and the
predominant form of the enzyme active on lipid micelles in the gut is a dimer of two such complexes (Aubert-Jousset et al. 2004).
CEL is synthesized in pancreatic acinar cells and released into the small intestine. It is also synthesized in the mammary gland and is a
constituent of breast milk. The milk CEL is thought to play a role in digestion of milk fat in newborn infants, whose own pancreatic synthesis of
the enzyme is low (Lombardo 2001; Bernback et al. 1990).
References
E Aubert-Jousset, V Sbarra, D Lombardo, "Site-directed mutagenesis of the distal basic cluster of pancreatic bile salt-dependent lipase", J Biol
Chem, 279, 2004, 39697-704.
S Bernback, L Blackberg, O Hernell, "The complete digestion of human milk triacylglycerol in vitro requires gastric lipase, pancreatic
colipase-dependent lipase, and bile salt-stimulated lipase", J Clin Invest, 85, 1990, 1221-6.
K Reue, J Zambaux, H Wong, G Lee, TH Leete, M Ronk, JE Shively, B Sternby, B Borgstrom, D Ameis, MC Schotz, "cDNA cloning of carboxyl
ester lipase from human pancreas reveals a unique proline-rich repeat unit", J Lipid Res, 32, 1991, 267-76.
D Lombardo, O Guy, "Studies on the substrate specificity of a carboxyl ester hydrolase from human pancreatic juice. II. Action on cholesterol
esters and lipid-soluble vitamin esters.", Biochim Biophys Acta, 611, 1980, 147-55.
Reaction
15.1.2 Digestion of monoacylglycerols by extracellular CEL (bile salt-dependent lipase)
Authors
Description
CEL (bile salt-dependent lipase) catalyzes the hydrolysis of extracellular monoacylglycerols to yield glycerol and a long-chain fatty acid. This
reaction, in the lumen of the small intestine, is essential for the complete digestion of milk-derived triacylglycerols in the nursing infant (Bernback
et al. 1990). Its importance in adult fat digestion is unclear.
(Reue et al. 1991). In vitro, monomeric CEL protein is active even in the absence of bile salts. its activity is greatly increased when it is
constituent of breast milk (Lombardo 2001; Bernback et al. 1990).
References
Chem, 279, 2004, 39697-704.
Reaction
15.1.3 Digestion of diacylglycerols by extracellular PTL:colipase
Authors
Description
Pancreatic lipase catalyzes the hydrolysis of extracellular dicylglycerols to yield monoacylglycerols and long-chain fatty acids. The enzyme is
active only when complexed with colipase protein and plays a major role in the digestion of dietary diacylglycerols in the small intestine (Carriere
et al. 2000; Giller et al. 1992).
References
F Carriere, C Renou, V Lopez, J De Caro, F Ferrato, H Lengsfeld, A De Caro, R Laugier, R Verger, "The specific activities of human digestive
lipases measured from the in vivo and in vitro lipolysis of test meals", Gastroenterology, 119, 2000, 949-60.
T Giller, P Buchwald, D Blum-Kaelin, W Hunziker, "Two novel human pancreatic lipase related proteins, hPLRP1 and hPLRP2. Differences in
colipase dependence and in lipase activity.", J Biol Chem, 267, 1992, 16509-16.
Reaction
15.1.4 Digestion of triacylglycerols by extracellular CEL (bile salt-dependent lipase)
Authors
Description
CEL (bile salt-dependent lipase) catalyzes the hydrolysis of extracellular monoacylglycerols to yield glycerol and a long-chain fatty acid. This
reaction, in the lumen of the small intestine, is essential for the complete digestion of milk-derived triacylglycerols in the nursing infant (Bernback
et al. 1990). Its importance in adult fat digestion is unclear.
(Reue et al. 1991). In vitro, monomeric CEL protein is active even in the absence of bile salts. its activity is greatly increased when it is
constituent of breast milk (Lombardo 2001; Bernback et al. 1990).
References
Chem, 279, 2004, 39697-704.
D Lombardo, J Fauvel, O Guy, "Studies on the substrate specificity of a carboxyl ester hydrolase from human pancreatic juice. I. Action on
carboxyl esters, glycerides and phospholipids.", Biochim Biophys Acta, 611, 1980, 136-46.
Reaction
15.1.5 Digestion of triacylglycerols by extracellular PTL:colipase
Authors
Description
Pancreatic lipase catalyzes the hydrolysis of extracellular triacylglycerols to yield diacylglycerols and long-chain fatty acids. The enzyme is active
only when complexed with colipase protein and plays a major role in the digestion of dietary triacylglycerols in the small intestine (Carriere et al.
2000; Giller et al. 1992).
References
F Carriere, C Renou, V Lopez, J De Caro, F Ferrato, H Lengsfeld, A De Caro, R Laugier, R Verger, "The specific activities of human digestive
lipases measured from the in vivo and in vitro lipolysis of test meals", Gastroenterology, 119, 2000, 949-60.
Reaction
15.1.6 Digestion of triacylglycerols by extracellular pancreatic lipase-related protein 2
Authors
Description
Pancreatic lipase-related protein 2 catalyzes the hydrolysis of extracellular triacylglycerols to yield diacylglycerols and free long-chain fatty acids.
This enzyme, unlike the closely related pancreatic lipase protein, does not require colipase protein for activity. The protein is synthesized in the
pancreas, but its role in the digestion of dietary fat has not been established (Giller et al. 1992).
References
Reaction
15.2 Trafficking of dietary sterols
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
NPC1L1 protein mediates the uptake of dietary sterols from the gut lumen. These include cholesterol and plant sterols (phytosterols). Half to
two-thirds of the 250-500 milligrams of cholesterol consumed daily from a typical western diet is taken up by this route and exported from
enterocytes into the lymph in chylomicrons. Of the 200-400 milligrams of dietary phytosterol, only about 5% is absorbed.The drug ezetimibe
interferes with NPC1L1-mediated sterol uptake (Oram and Vaughan 2006).
Both cholesterol and phytosterols are secreted from the liver into the bile (Salen et al. 1970). Mutations affecting the ABCG5/8 transporter
complex inhibit this process (e.g., Berge et al. 2000), while the molecular details of sterol export in human cells have not been worked out, they
can be inferred from the properties of the homologous mouse proteins (Graf et al. 2003; Wang et al. 2006).
References
JF Oram, AM Vaughan, "ATP-Binding cassette cholesterol transporters and cardiovascular disease", Circ Res, 99, 2006, 1031-43.
GA Graf, L Yu, WP Li, R Gerard, PL Tuma, JC Cohen, HH Hobbs, "ABCG5 and ABCG8 are obligate heterodimers for protein trafficking and
biliary cholesterol excretion", J Biol Chem, 278, 2003, 48275-82.
KE Berge, H Tian, GA Graf, L Yu, NV Grishin, J Schultz, P Kwiterovich, B Shan, R Barnes, HH Hobbs, "Accumulation of dietary cholesterol in
sitosterolemia caused by mutations in adjacent ABC transporters", Science, 290, 2000, 1771-5.
G Salen, Jr Ahrens EH, SM Grundy, "Metabolism of beta-sitosterol in man", J Clin Invest, 49, 1970, 952-67.
J Wang, F Sun, DW Zhang, Y Ma, F Xu, JD Belani, JC Cohen, HH Hobbs, XS Xie, "Sterol transfer by ABCG5 and ABCG8: in vitro assay and
reconstitution", J Biol Chem, 281, 2006, 27894-904.
15.2.1 NPC1L1-mediated cholesterol uptake
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
Dietary cholesterol is taken up into enterocytes from the gut lumen in a reaction mediated by plasma membrane-associated NPC1L1 protein.
This role for NPC1L1 was first observed in studies of a mouse model system (Altman et al. 2004). Subsequent studies of human cultured cells
confirmed the participation of NPC1L1 in cholesterol transport across membranes but suggested that this transport process might not be a major
factor in dietary cholesterol uptake (Davies et al. 2005). Detailed studies in Maden-Darby canine kidney cells, both mediated by endogenous
(canine) NPC1L1 and by overexpressed human protein, indicate a major role for NPC1L1 in the uptake of extracellular cholesterol. These
studies have also shown that ezetimibe binds both human and canine proteins and renders them incapable of mediating cholesterol uptake. The
molecular mechanisms of cholesterol transport and ezetimibe inhibition, however, remain unclear (Weinglass et al. 2008).
References
SW Altmann, Jr Davis HR, LJ Zhu, X Yao, LM Hoos, G Tetzloff, SP Iyer, M Maguire, A Golovko, M Zeng, L Wang, N Murgolo, MP Graziano,
"Niemann-Pick C1 Like 1 protein is critical for intestinal cholesterol absorption", Science, 303, 2004, 1201-4.
JP Davies, C Scott, K Oishi, A Liapis, YA Ioannou, "Inactivation of NPC1L1 causes multiple lipid transport defects and protects against
diet-induced hypercholesterolemia", J Biol Chem, 280, 2005, 12710-20.
AB Weinglass, MG KÃ¶hler, EO Nketiah, J Liu, W Schmalhofer, A Thomas, B Williams, L Beers, L Smith, M Hafey, K Bleasby, J Leone, YS
Tang, MP Braun, F Ujjainwalla, ME McCann, GJ Kaczorowski, ML Garcia, "Madin-Darby canine kidney II cells: a pharmacologically validated
system for NPC1L1-mediated cholesterol uptake", Mol Pharmacol, 73, 2008, 1072-84.
Source reaction
This reaction was inferred from the corresponding reaction "NPC1L1-mediated cholesterol uptake" in species Canis familiaris.
Reaction
15.2.2 NPC1L1-mediated phytosterol uptake
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
Dietary phytosterols (sterols derived from plants) are taken up into enterocytes from the gut lumen. The reaction is thought to be mediated by
plasma membrane-associated NPC1L1 protein. Detailed studies in Maden-Darby canine kidney cells, both mediated by endogenous (canine)
NPC1L1 and by overexpressed human protein, indicate a major role for NPC1L1 in the uptake of extracellular sterol. These studies have also
shown that ezetimibe binds both human and canine proteins and renders them incapable of mediating sterol uptake. The molecular mechanisms
of cholesterol transport and ezetimibe inhibition, however, remain unclear (Weinglass et al. 2008).
References
Source reaction
This reaction was inferred from the corresponding reaction "NPC1L1-mediated cholesterol uptake" in species Homo sapiens.
SW Altmann, Jr Davis HR, LJ Zhu, X Yao, LM Hoos, G Tetzloff, SP Iyer, M Maguire, A Golovko, M Zeng, L Wang, N Murgolo, MP Graziano,
"Niemann-Pick C1 Like 1 protein is critical for intestinal cholesterol absorption", Science, 303, 2004, 1201-4.
JP Davies, C Scott, K Oishi, A Liapis, YA Ioannou, "Inactivation of NPC1L1 causes multiple lipid transport defects and protects against
diet-induced hypercholesterolemia", J Biol Chem, 280, 2005, 12710-20.
Reaction
15.2.3 NPC1L1 inactivation by ezetimibe
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
Human NPC1L1, associated with the plasma membrane, binds ezetimibe and becomes incapable of mediating cholesterol uptake. The
molecular mechanisms of cholesterol transport and ezetimibe inhibition, however, remain unclear (Garcia-Calvo et al. 2005; Weinglass et al.
2008).
References
M Garcia-Calvo, J Lisnock, HG Bull, BE Hawes, DA Burnett, MP Braun, JH Crona, Jr Davis HR, DC Dean, PA Detmers, MP Graziano, M
Hughes, DE Macintyre, A Ogawa, KA O'Neill, SP Iyer, DE Shevell, MM Smith, YS Tang, AM Makarewicz, F Ujjainwalla, SW Altmann, KT
Chapman, NA Thornberry, "The target of ezetimibe is Niemann-Pick C1-Like 1 (NPC1L1)", Proc Natl Acad Sci U S A, 102, 2005, 8132-7.
Reaction
15.2.4 ABCG5:ABCG8-mediated export of cholesterol and phytosterols
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
ABCG5/8 in the plasma membrane mediates the ATP-dependent export of cytosolic sterols (cholesterol and phytosterols). Mutations affecting
the ABCG5/8 proteins are associated with the accumulation of high levels of cholesterol and phytosterols in the body, demonstrating the
specificity and physiological importance of this process (Berge et al. 2000). Human ABCG5/8 has not been studied in detail, but the homologous
mouse protein complex mediate ATP-dependent sterol export (Wang et al. 2006). The mouse proteins localize to the apical plasma membranes
of enterocytes and hepatocytes, consistent with the hypothesis that in vivo ABCG5/8 mediates sterol export into the gut lumen and from
hepatocytes into the bile (Graf et al. 2003).
References
GA Graf, L Yu, WP Li, R Gerard, PL Tuma, JC Cohen, HH Hobbs, "ABCG5 and ABCG8 are obligate heterodimers for protein trafficking and
biliary cholesterol excretion", J Biol Chem, 278, 2003, 48275-82.
KE Berge, H Tian, GA Graf, L Yu, NV Grishin, J Schultz, P Kwiterovich, B Shan, R Barnes, HH Hobbs, "Accumulation of dietary cholesterol in
sitosterolemia caused by mutations in adjacent ABC transporters", Science, 290, 2000, 1771-5.
J Wang, F Sun, DW Zhang, Y Ma, F Xu, JD Belani, JC Cohen, HH Hobbs, XS Xie, "Sterol transfer by ABCG5 and ABCG8: in vitro assay and
reconstitution", J Biol Chem, 281, 2006, 27894-904.
Reaction
15.3 Triacylglyceride Biosynthesis
Description
The overall process of triglyceride (triacylglycerol) biosynthesis consists of four biochemical pathways: fatty acyl-CoA biosynthesis, conversion of
fatty acyl-CoA to phosphatidic acid, conversion of phosphatidic acid to diacylglycerol, and conversion fo diacylglycerol to triacylglycerol.
References
J.D. McGarry, "Lipid Metabolism I", Textbook of Biochemistry with Clinical Correlations, 5 ed. (Edited by Devlin, TM), 2002, 698-723.
B.J. Rawlings, "Fatty Acid Biosynthesis", Encyclopedia of Life Sciences, 2001, 1-12.
15.3.1 Fatty Acyl-CoA Biosynthesis
Authors
Description
Fatty acyl-CoA biosynthesis involves following steps:
-Palmitate synthesis catalyzed by Acetyl-CoA carboxylase and Fatty acid synthase
-Conversion of palmitic acid to long chain fatty acids and
-Conversion of long chain fatty acids to fatty acyl-CoA by acyl-CoA synthases.
15.3.1.1 Palmitate synthesis
Authors
Description
De novo synthesis of fatty acids from acetyl CoA involves the synthesis of palmitic acid, a saturated straight-chain C16 acid from acetyl-CoA and
other small molecule intermediates originating from metabolic processes. Palmitate synthesis can be outlined as:
Step I. Synthesis of malonyl-CoA from acetyl-CoA (enzyme: Acetyl-CoA carboxylase)
Step II. -Recruitment of C2 from malonyl-thioester (loading phase)
-Condensation with growing acyl group (condensation phase)
-Reduction, hydrolysis and reduction to acyl group (reduction phase)
-Recruitment of this acyl group as the primer for next cycle of reactions (enzyme: Fatty acid synthase).
Steps I and II take place as 6 cycles in a sequential manner. In the 7th and the last cycle, the palmitoyl moiety is released into the cytosol as
palmitic acid by the action of the thiolase site on FAS (Smith, 1994; Smith et al., 2003).
References
S Smith, "The animal fatty acid synthase: one gene, one polypeptide, seven enzymes.", FASEB J, 8, 1995, 1248-59.
S Smith, A Witkowski, AK Joshi, "Structural and functional organization of the animal fatty acid synthase.", Prog Lipid Res, 42, 2003, 289-317.
Reaction
15.3.1.1.1 Butyryl-ACP biosynthesis
Authors
Description
A single gene encodes FAS in eukaryotes in contrast to the multi-enzymatic system found in prokaryotes. The functional form of FAS is
comprised of two multifunctional polypeptide chains, each chain containing 7 catalytic sites. Two separate centers for fatty acid assembly are
formed by the juxtaposition of these 7 discrete domains. In addition to providing spatial proximity of active sites, a FAS dimer structure enables
covalent connections between the constituent monomers providing stability to growing macromolecules. The seven enzyme activities associated
with this large protein are:
1. Acetyl-CoA transacylase
2. Malonyl-CoA transacylase
3. Ketoacyl-ACP synthase
4. Ketoacyl-ACP reductase
5. Beta-hydroxyacyl-ACP dehydratase
6. Enoyl-ACP reductase
7. Thiolase
8. An eighth activity site, is an Acyl Carrier Protein (ACP) domain with a phosphopantetheine prosthetic group covalently linked to a serine side
chain.
This unique dimeric enzyme complex catalyzes a series of reactions leading to the formation of palmitic acid. Only in its homodimeric form do
the subunits adopt conformations that could facilitate reactions for fatty acid synthesis at the two ACP centers. ACP of one enzyme chain
translocates a growing fatty acid moiety from one catalytic site to another, located on the other chain in a FAS dimer complex resulting in the
synthesis of two fatty acid molecules simultaneously. These cyclic events follow an initial priming reaction by the transacetylase catalytic site
(Wakil et al.,1983; Wakil, 1989; Smith,1994).
References
S Smith, "The animal fatty acid synthase: one gene, one polypeptide, seven enzymes.", FASEB J, 8, 1995, 1248-59.
SJ Wakil, "Fatty acid synthase, a proficient multifunctional enzyme.", Biochemistry, 28, 1989, 4523-30.
SJ Wakil, JK Stoops, VC Joshi, "Fatty acid synthesis and its regulation.", Annu Rev Biochem, 52, 1983, 537-79.
15.3.1.1.1.1 Generation of Cytoplasmic Acetyl CoA from Citrate
Authors
Description
While fatty acid synthesis from acetyl CoA proceeds in the cytosol, most acetyl CoA in the cell is generated within the mitochondria, by oxidative
decarboxylation of the pyruvate derived from glycolysis, as well as from a number of reactions of amino acid catabolism.
Mitochondrial Acetyl-CoA is transported to cytoplasm as citrate to participate in fatty acid biosynthesis. Mitochondrial citrate synthase, a
tricarboxylic acid transporter in the inner mitochondrial membrane, and cytosolic citrate lyase are involved in this transport.
References
NA Elshourbagy, JC Near, PJ Kmetz, TN Wells, PH Groot, BA Saxty, SA Hughes, M Franklin, IS Gloger, "Cloning and expression of a human
ATP-citrate lyase cDNA", Eur J Biochem, 204, 1992, 491-9.
Reaction
15.3.1.1.1.2 Formation of Acetyl-FAS on one FAS monomer
Description
Acetyl-FAS is synthesized in two steps. An acetyl moiety is first transferred from acetyl-CoA to acyl carrier protein (ACP), and then from ACP to
a cysteine residue of the FAS enzyme.
15.3.1.1.1.2.1 Formation of Acetyl-ACP(intermediate)
Authors
Description
This is the first step in the initial priming reaction. Acetyl-ACP attached to the phosphopantetheine group is considered to be an intermediate
form which is immediately translocated to the catalytic site.
References
A Jayakumar, MH Tai, WY Huang, W al-Feel, M Hsu, L Abu-Elheiga, SS Chirala, SJ Wakil, "Human fatty acid synthase: properties and
molecular cloning", Proc Natl Acad Sci U S A, 92, 1995, 8695-9.
Reaction
15.3.1.1.1.2.2 Acetyl-ACP(intermediate) => Acetyl-synthase
Authors
Description
The intermediate Acetyl-ACP is translocated to the active cysteine site of the synthase site catalyzed by the domain itself. This acetyl group
contributes the initial pair of carbon atoms for palmitate synthesis. Translocation of acetyl group to the synthase site results in a free ACP site
capable of subsequent fatty acid synthesis.
References
Reaction
15.3.1.1.1.3 Formation of Malonyl-ACP on the other FAS monomer
Description
Malonyl-CoA is synthesized from acetyl-CoA, then conjugated with acyl carrier protein (ACP).
15.3.1.1.1.3.1 Formation of Malonyl-CoA from Acetyl-CoA (liver)
Authors
Description
Acetyl-CoA carboxylase 2 associated with the outer mitochondrial membrane catalyzes the reaction of cytosolic bicarbonate, ATP, and
acetyl-CoA to form malonyl-CoA, ADP, and orthophosphate.
References
L Abu-Elheiga, A Jayakumar, A Baldini, SS Chirala, SJ Wakil, "Human acetyl-CoA carboxylase: characterization, molecular cloning, and
evidence for two isoforms", Proc Natl Acad Sci U S A, 92, 1995, 4011-5.
D Cheng, CH Chu, L Chen, JN Feder, GA Mintier, Y Wu, JW Cook, MR Harpel, GA Locke, Y An, JK Tamura, "Expression, purification, and
characterization of human and rat acetyl coenzyme A carboxylase (ACC) isozymes", Protein Expr Purif, 51, 2007, 11-21.
Reaction
15.3.1.1.1.3.2 Conversion of Malonyl-CoA to Malonyl-ACP

Authors
Description
A free CoA-SH is released during the translocation of malonyl CoA to the ACP domain. This transfer results in a FAS complex with an acetyl and
a malonyl group ready for condensation.
References
Reaction
15.3.1.1.1.4 Condensation of Malonyl-ACP and Acetate on different monomers of FAS dimer to Acetoacetyl-ACP
Description
At the beginning of this reaction, 1 molecule of 'Malonyl-ACP', and 1 molecule of 'Acetyl-synthase' are present. At the end of this reaction, 1
molecule of 'Acetoacetyl-ACP', and 1 molecule of 'CO2' are present.
This reaction takes place in the 'cytosol' and is mediated by the '3-oxoacyl-[acyl-carrier protein] synthase activity' of 'FAS dimer'.
References
Reaction
15.3.1.1.1.5 Reduction of Acetoacetyl-ACP to beta-Hydroxybutyryl-ACP
Description
At the beginning of this reaction, 1 molecule of 'H+', 1 molecule of 'NADPH', and 1 molecule of 'Acetoacetyl-ACP' are present. At the end of this
reaction, 1 molecule of 'beta-Hydroxybutyryl-ACP', and 1 molecule of 'NADP+' are present.
This reaction takes place in the 'cytosol' and is mediated by the '3-oxoacyl-[acyl-carrier protein] reductase activity' of 'FAS dimer'.
References
Reaction
15.3.1.1.1.6 Dehydration of beta-Hydroxybutyryl-ACP to Crotonoyl-ACP

Description
At the beginning of this reaction, 1 molecule of 'beta-Hydroxybutyryl-ACP' is present. At the end of this reaction, 1 molecule of 'H2O', and 1
molecule of 'Crotonoyl-ACP' are present.
This reaction takes place in the 'cytosol' and is mediated by the '3-hydroxyacyl-[acyl-carrier protein] dehydratase activity' of 'FAS dimer'.
References
Reaction
15.3.1.1.1.7 Reduction of Crotonoyl-ACP to Butyrl-ACP
Authors
Description
Enoyl-ACP reductase domain catalyzes this reaction using an NADPH molecule to produce acyl-ACP.
References
Reaction
15.3.1.1.2 Conversion of Butyryl-ACP to Palmitoyl-ACP
Authors
Joshi-Tope, G, 2003-10-23.
Description
This pathway includes 6 spirals of reactions successively adding two carbons in each spiral.
References
S Smith, A Witkowski, AK Joshi, "Structural and functional organization of the animal fatty acid synthase.", Prog Lipid Res, 42, 2003, 289-317.
Reaction
15.3.1.1.3 Hydrolysis of Palmitoyl-ACP to Palmitic Acid
Authors
Description
Palmitic acid may also be available as palmitate-CoA in the cytoplasm. The resulting free ACP site may be utilized for further fatty acid
synthesis.
References
Reaction
15.3.1.1.4 Formation of fatty acid synthase (FAS) dimer
Description
Two molecules of 'Fatty acid synthase ' form an active FAS dimer. In each of the monomers, a phosphopantheine group is attached to a serine
residue at 2156.
Reaction
15.3.1.2 Conversion of Palmitic Acid to Long Chain Fatty Acids
Authors
Description
All of the fatty acids needed by the body can be synthesized from palmitate except the essential, polyunsaturated fatty acids such as linoleate,
linolenate etc. Palmitic acid is subjected to enzymatic reactions by reductases, mixed function oxidases etc. "Mixed function oxidases " are
capable of simultaneous oxidation of two products, one being fatty acid and the other usually, NADPH.
There are 3 major processes that modify palmitic acid: elongation, desaturation and hydroxylation. Elongation of fatty acids may occur at
endoplasmic reticulum where fatty acid molecules of length up to C24 may be produced. Mitochondrial elongation may result in fatty acids up to
C16 in length. Desaturation may take place involving a mixture of enzymes consisting of desaturase enzyme, cytochrome b5, and
NADPH-cytochrome b5 reductase. Biosynthesis of sphingolipids containing hydroxylated fatty acids in an example of fatty acid hydroxylation in
humans (Rawlings,2002). The results of these modification processes are fatty acids with different C numbers and side chains that contribute to
their functional and structural specificities.
References
B.J. Rawlings, "Fatty Acid Biosynthesis", Encyclopedia of Life Sciences, 2001, 1-12.
Reaction
15.3.1.3 Conversion of Long Chain Fatty Acids to Fatty Acyl-CoAs
Description
At the beginning of this reaction, 1 molecule of 'Long-chain fatty acid', 1 molecule of 'CoA-SH', and 1 molecule of 'ATP' are present. At the end of
this reaction, 1 molecule of 'Fatty acyl CoA', 1 molecule of 'pyrophosphate', 1 molecule of 'AMP', and 1 molecule of 'H2O' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'long-chain-fatty-acid-CoA ligase activity' of 'Acyl-CoA synthase '.
Reaction
15.3.2 Conversion of Fatty Acyl-CoA to Phosphatidic Acid
Description
In successive cytosolic reactions, two molecules of acyl CoA react with glycerol 3-phosphate to form phosphatidic acid.
15.3.2.1 Synthesis of lysophosphatidic acid from glycerol-3-phosphate
Authors
Description
Esterification of glycerol derivatives by fatty acyl-CoA results in the synthesis of triacylglycerol via various intermediates. Phosphatidic acid, an
intermediate in triacylglycerol synthesis is formed by the addition of acyl groups from fatty acyl-CoA to lysophosphatidic acid. Lysophosphatidic
acid can be derived from two different sources: dihydroxyacetone phosphate (DHAP) and glycerol-3- phosphate. Glycerol-3-phosphate can be
derived from two sources: glycerol and dihydroxyacetone phosphate.
15.3.2.1.1 Formation of Cytosolic Glycerol-3-phosphate
Description
Cytosolic glycerol 3-phosphate can be formed from dihydroxyacetone phosphate or by phosphorylation of glycerol. The first reaction occurs in
both the liver and adipose tissue and depends on glycolysis as a source of dihydroxyacetone phosphate. The second reaction does not occur in
adipose tissue, which lacks glycerol kinase. As a result, triacylglycerol synthesis in adipose tissue (but not in liver) is coupled to glycolysis.
15.3.2.1.1.1 Conversion of Dihydroxyacetone Phosphate to Glycerol -3- phosphate
Authors
Description
This reaction may be found in white adipose tissues where glycerol-3-kinase activity is not observed in sufficient levels. Glycerol-3-phosphate
dehydrogenase reduces dihydroxyacetone phosphate. NADH donates electrons to this reaction.
References
X Ou, C Ji, X Han, X Zhao, X Li, Y Mao, LL Wong, M Bartlam, Z Rao, "Crystal structures of human glycerol 3-phosphate dehydrogenase 1
(GPD1)", J Mol Biol, 357, 2006, 858-69.
Reaction
15.3.2.1.1.2 Conversion of Glycerol to Glycerol-3-phosphate

Authors
Description
Glycerol can be a source for glycerol-3-phosphate, in which case, a phosphate form ATP is transferred to glycerol by glycerol kinase forming
glycerol-3-phosphate and ADP.
References
RH Ohira, KM Dipple, YH Zhang, ER McCabe, "Human and murine glycerol kinase: influence of exon 18 alternative splicing on function",
Reaction
15.3.2.1.2 Conversion of glycerol-3-phosphate to lysophosphatidic acid
Authors
Description
A fatty acid moiety is transferred to glycerol-3-phosphate from fatty acid-CoA by glycerol-3-phosphate acyltransferase to form lysophosphatidic
acid. Generally saturated fatty acids are used in this pathway.
References
RA Igal, S Wang, M Gonzalez-Baro, RA Coleman, "Mitochondrial glycerol phosphate acyltransferase directs the incorporation of exogenous fatty
acids into triacylglycerol", J Biol Chem, 276, 2001, 42205-12.
Reaction
15.3.2.2 Synthesis of phosphatidic acid from lysophosphatidic acid
Authors
Description
1-acylglycerol-3-phosphate acyltransferase transfers a second fatty acid from either fatty acyl-CoA or fatty acyl-ACP to form phosphatidate or
phosphatidic acid.
15.3.2.2.1 lysophosphatic acid + fatty acyl CoA => phosphatidic acid + CoA (1)
Description
At the beginning of this reaction, 1 molecule of 'Lysophosphatidic acid', and 1 molecule of 'Fatty acyl CoA' are present. At the end of this
reaction, 1 molecule of 'Phosphatidic acid', and 1 molecule of 'CoA-SH' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'phospholipid:diacylglycerol acyltransferase activity' of
'1-acyl-sn-glycerol-3-phosphate acyltransferase alpha '.
References
B Aguado, RD Campbell, "Characterization of a human lysophosphatidic acid acyltransferase that is encoded by a gene located in the class III
region of the human major histocompatibility complex", J Biol Chem, 273, 1998, 4096-105.
Reaction
15.3.2.2.2 lysophosphatidic acid + fatty acyl CoA => phosphatidic acid + CoA (2)
Description
'1-acyl-sn-glycerol-3-phosphate acyltransferase beta '.
References
C Eberhardt, PW Gray, LW Tjoelker, "Human lysophosphatidic acid acyltransferase. cDNA cloning, expression, and localization to chromosome
9q34.3.", J Biol Chem, 272, 1997, 20299-305.
Reaction
Description
'1-acyl-sn-glycerol-3-phosphate acyltransferase gamma '.
References
AK Agarwal, RI Barnes, A Garg, "Functional characterization of human 1-acylglycerol-3-phosphate acyltransferase isoform 8: cloning, tissue
distribution, gene structure, and enzymatic activity", Arch Biochem Biophys, 449, 2006, 64-76.
Reaction
Description
'1-acyl-sn-glycerol-3-phosphate acyltransferase delta '.
References
DW Leung, "The structure and functions of human lysophosphatidic acid acyltransferases", Front Biosci, 6, 2001, D944-53.
Reaction
Description
'1-acyl-sn-glycerol-3-phosphate acyltransferase epsilon '.
References
DW Leung, "The structure and functions of human lysophosphatidic acid acyltransferases", Front Biosci, 6, 2001, D944-53.
Reaction
15.3.3 Conversion of Phosphatidic Acid to Diacylglycerol
Authors
Description
Diacylglycerol is formed by the action of phosphatidate phosphatase on phosphatidic acid coupled with the release of a phosphate. The
phosphatase exists as 3 isozymes.
15.3.3.1 Phosphatidic acid + H2O <=> Diacylglycerol + phosphate (1)
Description
At the beginning of this reaction, 1 molecule of 'Phosphatidic acid', and 1 molecule of 'H2O' are present. At the end of this reaction, 1 molecule of
'Diacylglycerol' is present.
This reaction takes place in the 'cytosol' and is mediated by the 'phosphatidate phosphatase activity' of 'Phosphatidate phosphatase (PPAP2A)'.
References
R Roberts, VA Sciorra, AJ Morris, "Human type 2 phosphatidic acid phosphohydrolases. Substrate specificity of the type 2a, 2b, and 2c enzymes
and cell surface activity of the 2a isoform.", J Biol Chem, 273, 1998, 22059-67.
Reaction
Description
This reaction takes place in the 'cytosol' and is mediated by the 'phosphatidate phosphatase activity' of 'Phosphatidate phosphatase (PPAP2B)'.
References
Reaction
Description
This reaction takes place in the 'cytosol' and is mediated by the 'phosphatidate phosphatase activity' of 'Phosphatidate phosphatase (PPAP2C)'.
References
Reaction
15.3.4 Conversion of Diacylglycerol to Triacylglycerol
Description
At the beginning of this reaction, 1 molecule of 'Fatty acyl CoA', and 1 molecule of 'Diacylglycerol' are present. At the end of this reaction, 1
molecule of 'Triacylglycerol', and 1 molecule of 'CoA-SH' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'diacylglycerol O-acyltransferase activity' of 'Diacylglycerol O-acyltransferase 1 '.
References
D Cheng, RL Meegalla, B He, DA Cromley, JT Billheimer, PR Young, "Human acyl-CoA:diacylglycerol acyltransferase is a tetrameric protein",
Biochem J, 359, 2001, 707-14.
Reaction
15.4 Hormone-sensitive lipase (HSL)-mediated triacylglycerol hydrolysis
Authors
Editors
Description
Triacylglycerol is a major energy store in the body and its hydrolysis to yield fatty acids and glycerol is a tightly regulated part of energy
metabolism. A central part in this regulation is played by hormone-sensitive lipase (HSL), a neutral lipase abundant in adipocytes and skeletal
and cardiac muscle, but also abundant in ovarian and adrenal tissue, where it mediates cholesterol ester hydrolysis, yielding cholesterol for
steroid biosynthesis. The hormones to which it is sensitive include catecholamines (e.g., epinephrine), ACTH, and glucagon, all of which trigger
signaling cascades that lead to its phosphorylation and activation, and insulin, which sets off events leading to its dephosphorylation and
inactivation (Holm et al. 2000; Kraemer and Shen 2002).
The processes of triacylglycerol and cholesterol ester hydrolysis are also regulated by subcellular compartmentalization: these lipids are
packaged in cytosolic particles and the enzymes responsible for their hydrolysis, and perhaps for additional steps in their metabolism, are
organized at the surfaces of these particles (e.g., Brasaemle et al. 2004). This organization is dynamic: the inactive form of HSL is not
associated with the particles, but is translocated there after being phosphorylated. Conversely, perilipin, a major constituent of the particle
surface, appears to block access of enzymes to the lipids within the particle; its phosphorylation allows greater access.
Here, HSL-mediated triacylglycerol hydrolysis is described as a pathway containing twelve reactions. The first six of these involve activation:
phosphorylation of HSL, dimerization of HSL, disruption of CGI-58:perilipin complexes at the surfaces of cytosolic lipid particles, phosphorylation
of perilipin, association of phosphorylated HSL with FABP, and translocation of HSL from the cytosol to the surfaces of lipid particles. The next
four reactions are the hydrolysis reactions themselves: the hydrolysis of cholesterol esters, and the successive removal of three fatty acids from
triacylglycerol. The last two reactions, dephosphorylation of perilipin and HSL, negatively regulate the pathway. These events are outlined in the
figure below. Inputs (substrates) and outputs (products) of individual reactions are connected by black arrows; blue lines connect output
activated enzymes to the other reactions that they catalyze.
Despite the undoubted importance of these reactions in normal human energy metabolism and in the pathology of diseases such as type II
diabetes, they have been studied only to a limited extent in human cells and tissues. Most experimental data are derived instead from two rodent
model systems: primary adipocytes from rats, and mouse 3T3-L1 cells induced to differentiate into adipocytes.
References
C Holm, T Osterlund, H Laurell, JA Contreras, "Molecular mechanisms regulating hormone-sensitive lipase and lipolysis", Annu Rev Nutr, 20,
2000, 365-93.
DL Brasaemle, G Dolios, L Shapiro, R Wang, "Proteomic analysis of proteins associated with lipid droplets of basal and lipolytically stimulated
3T3-L1 adipocytes", J Biol Chem, 279, 2004, 46835-42.
FB Kraemer, WJ Shen, "Hormone-sensitive lipase: control of intracellular tri-(di-)acylglycerol and cholesteryl ester hydrolysis", J Lipid Res, 43,
2002, 1585-94.
15.4.1 hormone-sensitive lipase (HSL) + 2 ATP -> phosphorylated HSL + 2 ADP
Authors
Editors
Description
Cytosolic rat HSL is phosphorylated on serine residues 659 and 660 by protein kinase A catalytic subunit (Anthonsen et al. 1998; Su et al. 2003).
Three isoforms of protein kinase A are known, but with no known differences in substrate specificity or tissue specific expression patterns, so a
generic PKA (with all three forms as instances) is annotated as the catalyst of this reaction. Other serine residues in HSL can be phosphorylated
both in vitro and in vivo, and while these other phosphorylations appear not to affect triacylglycerol hydrolysis by HSL directly, they may affect
the efficiency with which serines 659 and 660 themselves are phosphorylated, or affect the efficiency with which HSL is translocated to cytosolic
lipid particles (Holm et al. 2000).
Phosphorylation of human HSL has not been studied in detail, so the human reaction is inferred from the well-studied rat one. By BLAST
alignment, human HSL residues 649 and 650 correspond to rat serines 659 and 660.
References
2000, 365-93.
CL Su, C Sztalryd, JA Contreras, C Holm, AR Kimmel, C Londos, "Mutational analysis of the hormone-sensitive lipase translocation reaction in
adipocytes", J Biol Chem, 278, 2003, 43615-9.
MW Anthonsen, L Ronnstrand, C Wernstedt, E Degerman, C Holm, "Identification of novel phosphorylation sites in hormone-sensitive lipase that
are phosphorylated in response to isoproterenol and govern activation properties in vitro", J Biol Chem, 273, 1998, 215-21.
Source reaction
This reaction was inferred from the corresponding reaction "hormone-sensitive lipase (HSL) + 2 ATP -> phosphorylated HSL + 2 ADP" in species
Rattus norvegicus.
2000, 365-93.
MW Anthonsen, L Ronnstrand, C Wernstedt, E Degerman, C Holm, "Identification of novel phosphorylation sites in hormone-sensitive lipase that
are phosphorylated in response to isoproterenol and govern activation properties in vitro", J Biol Chem, 273, 1998, 215-21.
Reaction
15.4.2 2 phosphorylated HSL monomers -> phosphorylated HSL dimer
Authors
Editors
Description
While both monomeric and homodimeric forms of rat HSL protein have been detected, the predominant species, and the one with substantially
greater catalytic activity when activated by phosphorylation, is the homodimer so HSL-mediated lipolysis is annotated in Reactome with dimeric
phosphorylated enzyme as the catalyst. Phosphorylation appears to be required for dimerization to proceed (Shen et al. 2000).
Dimerization of human HSL has not been studied in detail, so the human reaction is inferred from the well-studied rat one.
References
WJ Shen, S Patel, R Hong, FB Kraemer, "Hormone-sensitive lipase functions as an oligomer", Biochemistry, 39, 2000, 2392-8.
Source reaction
This reaction was inferred from the corresponding reaction "2 phosphorylated HSL monomers -> phosphorylated HSL dimer" in species Rattus
norvegicus.
WJ Shen, S Patel, R Hong, FB Kraemer, "Hormone-sensitive lipase functions as an oligomer", Biochemistry, 39, 2000, 2392-8.
Reaction
15.4.3 perilipin:CGI-58 complex -> perilipin + CGI-58
Authors
Editors
Description
In unstimulated mouse 3T3-L1 adipocytes, perilipin is localized to the surfaces of cytosolic lipid particles as a complex with CGI-58.
Catecholamine stimulation (and by inference glucagon stimulation) is associated with rapid dissociation of the complex and relocalization of the
CGI-58 protein away from the lipid particle. The stoichiometry of the complex is unknown. Dissociation of the perilipin:CGI-58 complex appears
to precede perilipin phosphorylation, although the molecular link between these two steps is unknown (Subramanian et al. 2004).
The interaction of human CGI-58 and perilipin on the lipid particle surface has not been studied in detail, so the human reaction is inferred from
the well-studied mouse one. The observation that humans homozygous for CGI-58 mutations suffer from Chanarin-Dorfman Syndrome,
characterized by the abnormal accumulation of triacylglycerol droplets in most tissues (Lefevre et al. 2001), provides indirect evidence that
human and mouse CGI-58 proteins have similar functions.
References
V Subramanian, A Rothenberg, C Gomez, AW Cohen, A Garcia, S Bhattacharyya, L Shapiro, G Dolios, R Wang, MP Lisanti, DL Brasaemle,
"Perilipin A mediates the reversible binding of CGI-58 to lipid droplets in 3T3-L1 adipocytes", J Biol Chem, 279, 2004, 42062-71.
C Lefevre, F Jobard, F Caux, B Bouadjar, A Karaduman, R Heilig, H Lakhdar, A Wollenberg, JL Verret, J Weissenbach, M Ozguc, M Lathrop, JF
Prud'homme, J Fischer, "Mutations in CGI-58, the gene encoding a new protein of the esterase/lipase/thioesterase subfamily, in
Chanarin-Dorfman syndrome", Am J Hum Genet, 69, 2001, 1002-12.
Source reaction
This reaction was inferred from the corresponding reaction "perilipin:CGI-58 complex -> perilipin + CGI-58" in species Mus musculus.
V Subramanian, A Rothenberg, C Gomez, AW Cohen, A Garcia, S Bhattacharyya, L Shapiro, G Dolios, R Wang, MP Lisanti, DL Brasaemle,
"Perilipin A mediates the reversible binding of CGI-58 to lipid droplets in 3T3-L1 adipocytes", J Biol Chem, 279, 2004, 42062-71.
C Lefevre, F Jobard, F Caux, B Bouadjar, A Karaduman, R Heilig, H Lakhdar, A Wollenberg, JL Verret, J Weissenbach, M Ozguc, M Lathrop, JF
Prud'homme, J Fischer, "Mutations in CGI-58, the gene encoding a new protein of the esterase/lipase/thioesterase subfamily, in
Chanarin-Dorfman syndrome", Am J Hum Genet, 69, 2001, 1002-12.
Reaction
15.4.4 perilipin + 2 ATP -> phosphorylated perilipin + 2 ADP
Authors
Editors
Description
Rat perilipin, the major protein at the surfaces of cytosolic lipid particles in adipocytes and steroidogenic cells (Blanchette-Mackie et al. 1995), is
phosphorylated by protein kinase A catalytic subunit (Greenberg et al. 1991) on serine residues 81, 223, and 277 (Tansey et al. 2003). All three
serine residues and the adjoining sequences that mediate phosphorylation (Cohen 1988) are conserved in mouse perilipin, while only the first
and third are conserved in human perilipin. By inference, PKA targets these three mouse and two human serines as well. Phosphorylated
perilipin is redistributed on the droplet surfaces (Souza et al. 1998). While two isoforms of rat perilipin protein are found on lipid particles in
adipocytes, only the larger isoform appears to regulate lipolysis (Tansey et al 2003). The single human and mouse isoforms of perilipin
correspond to the large rat isoform. In mouse 3T3-L1 cells, perilipin phosphorylation requires the presence of caveolin-1 at the surface of the
lipid particle (Cohen et al. 2004). This positive regulatory effect of caveolin-1 is inferred for rat and human.
References
SC Souza, LM de Vargas, MT Yamamoto, P Lien, MD Franciosa, LG Moss, AS Greenberg, "Overexpression of perilipin A and B blocks the
ability of tumor necrosis factor alpha to increase lipolysis in 3T3-L1 adipocytes", J Biol Chem, 273, 1998, 24665-9.
AW Cohen, B Razani, W Schubert, TM Williams, XB Wang, P Iyengar, DL Brasaemle, PE Scherer, MP Lisanti, "Role of caveolin-1 in the
modulation of lipolysis and lipid droplet formation", Diabetes, 53, 2004, 1261-70.
P Cohen, "Protein phosphorylation and hormone action", Proc R Soc Lond B Biol Sci, 234, 1988, 115-44.
JT Tansey, AM Huml, R Vogt, KE Davis, JM Jones, KA Fraser, DL Brasaemle, AR Kimmel, C Londos, "Functional studies on native and mutated
forms of perilipins. A role in protein kinase A-mediated lipolysis of triacylglycerols.", J Biol Chem, 278, 2003, 8401-6.
AS Greenberg, JJ Egan, SA Wek, NB Garty, EJ Blanchette-Mackie, C Londos, "Perilipin, a major hormonally regulated adipocyte-specific
phosphoprotein associated with the periphery of lipid storage droplets", J Biol Chem, 266, 1991, 11341-6.
EJ Blanchette-Mackie, NK Dwyer, T Barber, RA Coxey, T Takeda, CM Rondinone, JL Theodorakis, AS Greenberg, C Londos, "Perilipin is
located on the surface layer of intracellular lipid droplets in adipocytes", J Lipid Res, 36, 1995, 1211-26.
Source reaction
This reaction was inferred from the corresponding reaction "perilipin + 3 ATP -> phosphorylated perilipin + 3 ADP" in species Rattus norvegicus.
This reaction was inferred from the corresponding reaction "perilipin + 3 ATP -> phosphorylated perilipin + 3 ADP" in species Mus musculus.
Reaction
15.4.5 Phosphorylated HSL dimer translocates from the cytosol to the lipid particle
Authors
Editors
Description
In primary adipocytes from young rats and in adipocytes derived from 3T3-L1 cells in vitro, phosphorylated hormone-sensitive lipase translocates
from the cytosol to the surfaces of lipid particles following the phosphorylation of perilipin (Clifford et al. 2000; Su et al. 2003; Sztalryd et al. 2003)
The human reaction is inferred from the well-studied rat one.
References
C Sztalryd, G Xu, H Dorward, JT Tansey, JA Contreras, AR Kimmel, C Londos, "Perilipin A is essential for the translocation of
hormone-sensitive lipase during lipolytic activation", J Cell Biol, 161, 2003, 1093-103.
GM Clifford, C Londos, FB Kraemer, RG Vernon, SJ Yeaman, "Translocation of hormone-sensitive lipase and perilipin upon lipolytic stimulation
of rat adipocytes", J Biol Chem, 275, 2000, 5011-5.
Source reaction
This reaction was inferred from the corresponding reaction "Phosphorylated HSL dimer translocates from the cytosol to the lipid particle" in
C Sztalryd, G Xu, H Dorward, JT Tansey, JA Contreras, AR Kimmel, C Londos, "Perilipin A is essential for the translocation of
hormone-sensitive lipase during lipolytic activation", J Cell Biol, 161, 2003, 1093-103.
GM Clifford, C Londos, FB Kraemer, RG Vernon, SJ Yeaman, "Translocation of hormone-sensitive lipase and perilipin upon lipolytic stimulation
of rat adipocytes", J Biol Chem, 275, 2000, 5011-5.
Reaction
15.4.6 phosphorylated HSL dimer + FABP4 -> phosphorylated HSL dimer:FABP4 complex
Authors
Editors
Description
Rat FABPA associates with HSL and increases the rate of triacylglycerol hydrolysis, possibly by sequestering the released fatty acids (Shen et
al. 1999; Shen et al. 2001). A similar association of HSL and FABP4 at the lipid droplet surface has been demonstrated in human adipocytes
(Smith et al. 2004). The stoichiometry of the fatty acid:FABP complex is unknown. This model implies that HSL-associated FABP loaded with
fatty acid should exchange with unloaded, unassociated FABP, allowing HSL to continue to work efficiently while moving newly generated fatty
acids away from the lipid particle. To date, there is no evidence for or against such a shuttling process.
References
WJ Shen, K Sridhar, DA Bernlohr, FB Kraemer, "Interaction of rat hormone-sensitive lipase with adipocyte lipid-binding protein", Proc Natl Acad
Sci U S A, 96, 1999, 5528-32.
WJ Shen, Y Liang, R Hong, S Patel, V Natu, K Sridhar, A Jenkins, DA Bernlohr, FB Kraemer, "Characterization of the functional interaction of
adipocyte lipid-binding protein with hormone-sensitive lipase", J Biol Chem, 276, 2001, 49443-8.
AJ Smith, MA Sanders, BR Thompson, C Londos, FB Kraemer, DA Bernlohr, "Physical association between the adipocyte fatty acid-binding
protein and hormone-sensitive lipase: a fluorescence resonance energy transfer analysis", J Biol Chem, 279, 2004, 52399-405.
Source reaction
This reaction was inferred from the corresponding reaction "phosphorylated HSL dimer + FABPA -> phosphorylated HSL dimer:FABPA complex"
in species Rattus norvegicus.
WJ Shen, K Sridhar, DA Bernlohr, FB Kraemer, "Interaction of rat hormone-sensitive lipase with adipocyte lipid-binding protein", Proc Natl Acad
Sci U S A, 96, 1999, 5528-32.
WJ Shen, Y Liang, R Hong, S Patel, V Natu, K Sridhar, A Jenkins, DA Bernlohr, FB Kraemer, "Characterization of the functional interaction of
adipocyte lipid-binding protein with hormone-sensitive lipase", J Biol Chem, 276, 2001, 49443-8.
AJ Smith, MA Sanders, BR Thompson, C Londos, FB Kraemer, DA Bernlohr, "Physical association between the adipocyte fatty acid-binding
protein and hormone-sensitive lipase: a fluorescence resonance energy transfer analysis", J Biol Chem, 279, 2004, 52399-405.
Reaction
15.4.7 cholesterol ester + H2O -> cholesterol + fatty acid
Authors
Editors
Description
Activated rat HSL hydrolyzes cholesterol ester to yield cholesterol + fatty acid (Fredrikson et al. 1981). The human reaction has not been studied
in detail, and is inferred from the well-characterized rat one.
References
G Fredrikson, P Stralfors, NO Nilsson, P Belfrage, "Hormone-sensitive lipase of rat adipose tissue. Purification and some properties.", J Biol
Chem, 256, 1981, 6311-20.
Source reaction
This reaction was inferred from the corresponding reaction "cholesterol ester + H2O -> cholesterol + fatty acid" in species Rattus norvegicus.
Chem, 256, 1981, 6311-20.
Reaction
15.4.8 triacylglycerol + H2O -> diacylglycerol + fatty acid
Authors
Editors
Description
Activated rat HSL at the lipid particle hydrolyzes triacylglycerol to yield diacylglycerol + fatty acid. In vitro, activated partially purified HSL
catalyzes this reaction at only about two times the rate measured with non-activated enzyme (Fredrikson et al. 1981). The much greater rate
increase caused by HSL phosphorylation in vivo appears to be due to its phosphorylation-dependent translocation to the surface of the lipid
particle (Birnbaum 2003).
HSL-mediated triacylglycerol hydrolysis in humans has not been studied in detail, so the human reaction is inferred from the well-studied rat one.
References
Chem, 256, 1981, 6311-20.
MJ Birnbaum, "Lipolysis: more than just a lipase", J Cell Biol, 161, 2003, 1011-2.
Source reaction
This reaction was inferred from the corresponding reaction "triacylglycerol + H2O -> diacylglycerol + fatty acid" in species Rattus norvegicus.
Chem, 256, 1981, 6311-20.
MJ Birnbaum, "Lipolysis: more than just a lipase", J Cell Biol, 161, 2003, 1011-2.
Reaction
15.4.9 diacylglycerol + H2O -> 2-acylglycerol + fatty acid
Authors
Editors
Description
Rat HSL catalyzes the hydrolysis of diacylglycerol to yield 2-acylglycerol + fatty acid (Fredrikson and Belfrage 1983). The human event has not
been studied in detail and is inferred from the rat one.
References
G Fredrikson, P Belfrage, "Positional specificity of hormone-sensitive lipase from rat adipose tissue", J Biol Chem, 258, 1983, 14253-6.
Source reaction
This reaction was inferred from the corresponding reaction "diacylglycerol + H2O -> 2-acylglycerol + fatty acid" in species Rattus norvegicus.
G Fredrikson, P Belfrage, "Positional specificity of hormone-sensitive lipase from rat adipose tissue", J Biol Chem, 258, 1983, 14253-6.
Reaction
15.4.10 2-acylglycerol + H2O -> glycerol + fatty acid
Authors
Editors
Description
Rat monoacylglycerol lipase (MGLL) catalyzes the hydrolysis of 2-acylglycerol to yield glycerol + fatty acid (Tornqvist and Belfrage 1976;
Fredrikson et al. 1986). Localization of the enzyme to lipid particles is plausible, given its low solubility and its involvement in acylglycerol
metabolism, but this localization has not been directly experimentally verified. The human reaction is inferred from the well-studied rat one.
References
G Fredrikson, H Tornqvist, P Belfrage, "Hormone-sensitive lipase and monoacylglycerol lipase are both required for complete degradation of
adipocyte triacylglycerol", Biochim Biophys Acta, 876, 1986, 288-93.
H Tornqvist, P Belfrage, "Purification and some properties of a monoacylglycerol-hydrolyzing enzyme of rat adipose tissue", J Biol Chem, 251,
1976, 813-9.
Source reaction
This reaction was inferred from the corresponding reaction "2-acylglycerol + H2O -> glycerol + fatty acid" in species Rattus norvegicus.
G Fredrikson, H Tornqvist, P Belfrage, "Hormone-sensitive lipase and monoacylglycerol lipase are both required for complete degradation of
adipocyte triacylglycerol", Biochim Biophys Acta, 876, 1986, 288-93.
H Tornqvist, P Belfrage, "Purification and some properties of a monoacylglycerol-hydrolyzing enzyme of rat adipose tissue", J Biol Chem, 251,
1976, 813-9.
Reaction
15.4.11 phosphorylated HSL + H2O -> HSL + orthophosphate
Authors
Editors
Description
Rat HSL is inactivated by dephosphorylation. The catalyst of this reaction is unknown. Protein phosphatases 1 and 2A are both abundant in rat
adipocytes and both are active on HSL (Olsson and Belfrage 1987; Wood et al. 1993). Whether these enzymes act on phosphate groups
attached to serine residues 659 and 660 of HSL is unknown, however (Holm et al. 2000). Although the reaction is annotated as though the
phosphatase acts on phosphorylated HSL monomers, this also is unknown: does the HSL:FABP complex dissociate before HSL
dephosphorylation (as implied here), or does dephosphorylation of HSL drive dissociation of the complex?
Dephosphorylation of human HSL has not been studied in detail, so the human reaction is inferred from the well-studied rat one.
References
2000, 365-93.
H Olsson, P Belfrage, "The regulatory and basal phosphorylation sites of hormone-sensitive lipase are dephosphorylated by protein
phosphatase-1, 2A and 2C but not by protein phosphatase-2B", Eur J Biochem, 168, 1987, 399-405.
SL Wood, N Emmison, AC Borthwick, SJ Yeaman, "The protein phosphatases responsible for dephosphorylation of hormone-sensitive lipase in
isolated rat adipocytes", Biochem J, 295, 1993, 531-5.
Source reaction
This reaction was inferred from the corresponding reaction "phosphorylated HSL + H2O -> HSL + orthophosphate" in species Rattus norvegicus.
2000, 365-93.
H Olsson, P Belfrage, "The regulatory and basal phosphorylation sites of hormone-sensitive lipase are dephosphorylated by protein
phosphatase-1, 2A and 2C but not by protein phosphatase-2B", Eur J Biochem, 168, 1987, 399-405.
SL Wood, N Emmison, AC Borthwick, SJ Yeaman, "The protein phosphatases responsible for dephosphorylation of hormone-sensitive lipase in
isolated rat adipocytes", Biochem J, 295, 1993, 531-5.
Reaction
15.4.12 phosphorylated perilipin + H2O -> perilipin + orthophosphate
Authors
Editors
Description
Rat perilipin is dephosphorylated by protein phosphatase 1 (Clifford et al. 1998). All three protein phosphatase 1 isoforms appear competent to
carry out this reaction and there are no data to indicate which one preferentially acts on perilipin in vivo. Dephosphorylation of human perilipin
has not been studied in detail, so the human reaction is inferred from the well-studied rat one.
References
GM Clifford, DK McCormick, C Londos, RG Vernon, SJ Yeaman, "Dephosphorylation of perilipin by protein phosphatases present in rat
adipocytes", FEBS Lett, 435, 1998, 125-9.
Source reaction
This reaction was inferred from the corresponding reaction "phosphorylated perilipin + H2O -> perilipin + orthophosphate" in species Rattus
norvegicus.
GM Clifford, DK McCormick, C Londos, RG Vernon, SJ Yeaman, "Dephosphorylation of perilipin by protein phosphatases present in rat
adipocytes", FEBS Lett, 435, 1998, 125-9.
Reaction
15.5 Import of palmitoyl-CoA into the mitochondrial matrix
Authors
Reviewers
Description
The mitochondrial carnitine system catalyzes the transport of long-chain fatty acids into the mitochondrial matrix where they undergo beta
oxidation. This transport system consists of the malonyl-CoA sensitive carnitine palmitoyltransferase I (CPT-I) localized in the mitochondrial
outer membrane, the carnitine:acylcarnitine translocase, an integral inner membrane protein, and carnitine palmitoyltransferase II localized on
the matrix side of the inner membrane. (Kerner and Hoppel, 2000).
References
RR Ramsay, RD Gandour, FR van der Leij, "Molecular enzymology of carnitine transfer and transport", Biochim Biophys Acta, 1546, 2001,
21-43.
15.5.1 CPT1 converts palmitoyl-CoA to palmitoyl carnitine
Authors
Reviewers
Description
CPT1 associated with the outer mitochondrial membrane catalyzes the reversible reaction of palmitoyl-CoA and carnitine to form
palmitoylcarnitine and CoA-SH. This is the first step in the transfer of long chain fatty acyl CoAs like palmitoyl-CoA into mitochondria for beta
oxidation.
References
M Morillas, P Gomez-Puertas, B Rubi, J Clotet, J Arino, A Valencia, FG Hegardt, D Serra, G Asins, "Structural model of a malonyl-CoA-binding
site of carnitine octanoyltransferase and carnitine palmitoyltransferase I: mutational analysis of a malonyl-CoA affinity domain", J Biol Chem,
277, 2002, 11473-80.
VA Zammit, NT Price, F Fraser, VN Jackson, "Structure-function relationships of the liver and muscle isoforms of carnitine palmitoyltransferase
I", Biochem Soc Trans, 29, 2001, 287-92.
D Wu, L Govindasamy, W Lian, Y Gu, T Kukar, M Agbandje-McKenna, R McKenna, "Structure of human carnitine acetyltransferase. Molecular
basis for fatty acyl transfer.", J Biol Chem, 278, 2003, 13159-65.
S Gobin, L Thuillier, G Jogl, A Faye, L Tong, M Chi, JP Bonnefont, J Girard, C Prip-Buus, "Functional and structural basis of carnitine
palmitoyltransferase 1A deficiency", J Biol Chem, 278, 2003, 50428-34.
Reaction
15.5.2 Transport of palmitoyl carnitine into mitochondria
Authors
Reviewers
Description
The carnitine-acylcarnitine transporter (CACT), embedded in the inner mitochondrial membrane, shuttles acylcarnitine esters, in exchange for
free carnitine, across the inner mitochondrial membrane. The CACT protein is embedded in the inner mitochondrial membrane. It
governsmediates the movement of a a one-to-one exchange, between long-chain acylcarnitine ester into the mitochondrial matrix in exchange
for one s and nonesterified carnitine molecule. CACT can also mediate the, across the inner mitochondrial membrane and also a less efificient
unidirectional transport of carnitine across the inner mitochondrialis membrane (Huizing et al., 1997).
References
C Indiveri, V Iacobazzi, N Giangregorio, F Palmieri, "The mitochondrial carnitine carrier protein: cDNA cloning, primary structure and comparison
with other mitochondrial transport proteins", Biochem J, 321, 1997, 713-9.
M Huizing, V Iacobazzi, L IJlst, P Savelkoul, W Ruitenbeek, L van den Heuvel, C Indiveri, J Smeitink, F Trijbels, R Wanders, F Palmieri, "Cloning
of the human carnitine-acylcarnitine carrier cDNA and identification of the molecular defect in a patient", Am J Hum Genet, 61, 1997, 1239-45.
Reaction
15.5.3 CPT2 converts palmitoyl carnitine to palmitoyl-CoA
Authors
Reviewers
Description
CPT2, associated with the inner mitochondrial membrane, catalyzes the reaction of palmitoylcarnitine and CoASH to form palmitoyl-CoA and
carnitine in which fatty acid is conjugated back to CoA for subsequent beta oxidation in the mitochondrial matrix.
References
E Verderio, P Cavadini, L Montermini, H Wang, E Lamantea, G Finocchiaro, S DiDonato, C Gellera, F Taroni, "Carnitine palmitoyltransferase II
deficiency: structure of the gene and characterization of two novel disease-causing mutations", Hum Mol Genet, 4, 1995, 19-29.
Reaction
15.6 Mitochondrial Fatty Acid Beta-Oxidation
Authors
Editors
Description
Beta-oxidation begins once fatty acids have been imported into the mitochondrial matrix by carnitine acyltransferases. The beta-oxidation spiral
of fatty acids metabolism involves the repetitive removal of two carbon units from the fatty acyl chain. There are four steps to this process:
oxidation, hydration, a second oxidation, and finally thiolysis. The last step releases the two-carbon acetyl-CoA and a ready primed acyl-CoA
that takes another turn down the spiral. In total each turn of the beta-oxidation spiral produces one NADH, one FADH2, and one acetyl-CoA.
Further oxidation of acetyl-CoA via the tricarboxylic acid cycle generates additional FADH2 and NADH. All reduced cofactors are used by the
mitochondrial electron transport chain to form ATP. The complete oxidation of a fatty acid molecule produces numerous ATP molecules.
Palmitate, used as the model here, produces 129 ATPs.
Beta-oxidation pathways differ for saturated and unsaturated fatty acids. The beta-oxidation of saturated fatty acids requires four different
enzymatic steps. Beta-oxidation produces and consumes intermediates with a trans configuration; unsaturated fatty acids that have bonds in the
cis configuration require three separate enzymatic steps to prepare these molecules for the beta-oxidation pathway.
References
CA Stanley, DE Hale, "Genetic disorders of mitochondrial fatty acid oxidation.", Curr Opin Pediatr, 6, 1994, 476-81.
CR Roe, DS Roe, "Recent developments in the investigation of inherited metabolic disorders using cultured human cells.", Mol Genet Metab, 68,
2000, 243-57.
CR Roe, J Ding, "Mitochondrial fatty acid oxidation disorders", The Metabolic and Molecular Bases of Inherited Disease (Scriver CR, et al,
editors), 2001, 2297 Ã¢â‚¬â€œ2326.
P Rinaldo, D Matern, MJ Bennett, "Fatty acid oxidation disorders.", Annu Rev Physiol, 64, 2002, 477-502.
PM Coates, K Tanaka, "Molecular basis of mitochondrial fatty acid oxidation defects.", J Lipid Res, 33, 1992, 1099-110.
15.6.1 mitochondrial fatty acid beta-oxidation of saturated fatty acids
Authors
Editors
Description
Once fatty acids have been imported into the mitochondrial matrix by the carnitine acyltransferases, the beta-oxidation spiral begins. Each turn
of this spiral concludes with the repetitive removal of two carbon units from the fatty acyl chain. beta-oxidation of saturated fatty acids (fatty acids
with even numbered carbon chains and no double bonds) involves four different enzymatic steps: oxidation, hydration, a second oxidation, and a
concluding thiolysis step, resulting in the two-carbon acetyl-CoA and a newly CoA primed acyl-CoA for the next turn of the spiral.
References
editors), 2001, 2297 Ã¢â‚¬â€œ2326.
15.6.1.1 Beta oxidation of palmitoyl-CoA to myristoyl-CoA
Authors
Editors
Description
This first pass through the beta-oxidation spiral starts with the saturated fatty acid palmitoyl-CoA and produces myristoyl-CoA. Four enzymatic
steps are required, starting with VLCAD CoA dehydrogenase (Very Long Chain) activity, followed by three enzymatic steps, enoyl-CoA
hydratase, 3-hydroxyacyl-CoA dehydrogenase, and ketoacyl-CoA thiolase activities, all present in the mitochondrial membrane associated
trifunctional protein.
References
editors), 2001, 2297 Ã¢â‚¬â€œ2326.
15.6.1.1.1 palmitoyl-CoA+FAD<=>trans-Hexadec-2-enoyl-CoA+FADH2
Description
At the beginning of this reaction, 1 molecule of 'palmitoyl-CoA', and 1 molecule of 'FAD' are present. At the end of this reaction, 1 molecule of
'FADH2', and 1 molecule of 'trans-Hexadec-2-enoyl-CoA' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'acyl-CoA dehydrogenase activity' of 'VLCAD acyl-CoA
dehydrogenase homodimer'.
References
RI Kelley, "Beta-oxidation of long-chain fatty acids by human fibroblasts: evidence for a novel long-chain acyl-coenzyme A dehydrogenase.",
FL Crane, H Beinert, "On the mechanism of dehydrogenation of fatty acyl derivatives of coenzyme A. II. The electron-transferring flavoprotein.",
J Biol Chem, 218, 1956, 717-31.
Reaction
15.6.1.1.2 trans-Hexadec-2-enoyl-CoA+H2O<=>(S)-3-Hydroxyhexadecanoyl-CoA
Description
At the beginning of this reaction, 1 molecule of 'H2O', and 1 molecule of 'trans-Hexadec-2-enoyl-CoA' are present. At the end of this reaction, 1
molecule of '(S)-3-Hydroxyhexadecanoyl-CoA' is present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'enoyl-CoA hydratase activity' of 'Trifunctional Protein'.
References
K Carpenter, RJ Pollitt, B Middleton, "Human liver long-chain 3-hydroxyacyl-coenzyme A dehydrogenase is a multifunctional membrane-bound
beta-oxidation enzyme of mitochondria.", Biochem Biophys Res Commun, 183, 1992, 443-8.
Reaction
15.6.1.1.3 (S)-3-Hydroxyhexadecanoyl-CoA+NAD<=>3-Oxopalmitoyl-CoA+NADH+H
Description
At the beginning of this reaction, 1 molecule of 'NAD+', and 1 molecule of '(S)-3-Hydroxyhexadecanoyl-CoA' are present. At the end of this
reaction, 1 molecule of '3-Oxopalmitoyl-CoA', 1 molecule of 'H+', and 1 molecule of 'NADH' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the '3-hydroxyacyl-CoA dehydrogenase activity' of 'Trifunctional
Protein'.
References
Reaction
15.6.1.1.4 3-Oxopalmitoyl-CoA+CoA-SH<=>myristoyl-CoA
Description
At the beginning of this reaction, 1 molecule of '3-Oxopalmitoyl-CoA', and 1 molecule of 'CoA' are present. At the end of this reaction, 1 molecule
of 'Acetyl-CoA', and 1 molecule of 'myristoyl-CoA' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'transferase activity' of 'Trifunctional Protein'.
References
Reaction
15.6.1.2 Beta oxidation of myristoyl-CoA to lauroyl-CoA
Authors
Editors
Description
The second pass through the beta-oxidation spiral starts with the saturated fatty acid myristoyl-CoA (from the first swing through the beta
oxidation spiral) and produces lauroyl-CoA. Four enzymatic steps are required starting with LCAD CoA dehydrogenase (Long Chain) activity,
followed by three enzymatic steps, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and ketoacyl-CoA thiolase activities, all present in
the mitochondrial membrane associated trifunctional protein.
References
editors), 2001, 2297 Ã¢â‚¬â€œ2326.
15.6.1.2.1 myristoyl-CoA+FAD<=>trans-Tetradec-2-enoyl-CoA+FADH2
Description
At the beginning of this reaction, 1 molecule of 'myristoyl-CoA', and 1 molecule of 'FAD' are present. At the end of this reaction, 1 molecule of
'FADH2', and 1 molecule of 'trans-Tetradec-2-enoyl-CoA' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'acyl-CoA dehydrogenase activity' of 'LCAD acyl-CoA
dehydrogenase homotetramer'.
References
J Biol Chem, 218, 1956, 717-31.
Reaction
15.6.1.2.2 trans-Tetradec-2-enoyl-CoA+H2O<=>(S)-3-Hydroxytetradecanoyl-CoA
Description
At the beginning of this reaction, 1 molecule of 'trans-Tetradec-2-enoyl-CoA', and 1 molecule of 'H2O' are present. At the end of this reaction, 1
molecule of '(S)-3-Hydroxytetradecanoyl-CoA' is present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'enoyl-CoA hydratase activity' of 'Trifunctional Protein'.
References
Reaction
15.6.1.2.3 (S)-3-Hydroxytetradecanoyl-CoA+NAD<=>3-Oxotetradecanoyl-CoA+NADH+H
Description
At the beginning of this reaction, 1 molecule of 'NAD+', and 1 molecule of '(S)-3-Hydroxytetradecanoyl-CoA' are present. At the end of this
reaction, 1 molecule of 'H+', 1 molecule of '3-Oxotetradecanoyl-CoA', and 1 molecule of 'NADH' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the '3-hydroxyacyl-CoA dehydrogenase activity' of 'Trifunctional
Protein'.
References
Reaction
15.6.1.2.4 3-Oxotetradecanoyl-CoA+CoA-SH<=>Lauroyl-CoA
Description
At the beginning of this reaction, 1 molecule of '3-Oxotetradecanoyl-CoA', and 1 molecule of 'CoA' are present. At the end of this reaction, 1
molecule of 'Lauroyl-CoA', and 1 molecule of 'Acetyl-CoA' are present.
References
Reaction
15.6.1.3 Beta oxidation of lauroyl-CoA to decanoyl-CoA-CoA
Authors
Editors
Description
The third pass through the beta-oxidation spiral picks up where the last left off with the saturated fatty acid lauroyl-CoA and produces
decanoyl-CoA. Four enzymatic steps are required starting with LCAD CoA dehydrogenase (Long Chain) activity, followed by the enoyl-CoA
hydratase activity of crotonase, the 3-hydroxyacyl-CoA dehydrogenase activity of the short chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD),
and completed by the ketoacyl-CoA thiolase activity, present in the mitochondrial membrane associated trifunctional protein. Note that the
3-hydroxyacyl-CoA dehydrogenase activity of SCHAD is not actually limited to short chain fatty acids, in fact SCHAD has a broad substrate
specificity.
References
editors), 2001, 2297 Ã¢â‚¬â€œ2326.
15.6.1.3.1 lauroyl-CoA+FAD<=>2-trans-Dodecenoyl-CoA+FADH2
Description
At the beginning of this reaction, 1 molecule of 'Lauroyl-CoA', and 1 molecule of 'FAD' are present. At the end of this reaction, 1 molecule of
'FADH2', and 1 molecule of '2-trans-Dodecenoyl-CoA' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'acyl-CoA dehydrogenase activity' of 'LCAD acyl-CoA
References
J Biol Chem, 218, 1956, 717-31.
Reaction
15.6.1.3.2 2-trans-Dodecenoyl-CoA+H2O<=>(S)-3-Hydroxydodecanoyl-CoA
Description
At the beginning of this reaction, 1 molecule of '2-trans-Dodecenoyl-CoA', and 1 molecule of 'H2O' are present. At the end of this reaction, 1
molecule of '(S)-3-Hydroxydodecanoyl-CoA' is present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'enoyl-CoA hydratase activity' of 'enoyl-CoA hydratase hexamer'.
References
JR Stern, A Del Campillo, "Enzymes of fatty acid metabolism. II. Properties of crystalline crotonase.", J Biol Chem, 218, 1956, 985-1002.
Reaction
15.6.1.3.3 (S)-3-Hydroxydodecanoyl-CoA+NAD<=>3-Oxododecanoyl-CoA+NADH+H
Description
At the beginning of this reaction, 1 molecule of '(S)-3-Hydroxydodecanoyl-CoA', and 1 molecule of 'NAD+' are present. At the end of this
reaction, 1 molecule of 'H+', 1 molecule of '3-Oxododecanoyl-CoA', and 1 molecule of 'NADH' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the '3-hydroxyacyl-CoA dehydrogenase activity' of 'short chain
3-hydroxyacyl-CoA dehydrogenase homodimer'.
References
PJ Vredendaal, IE van den Berg, HE MalingrÃ©, AK Stroobants, DE Olde Weghuis, R Berger, "Human short-chain L-3-hydroxyacyl-CoA
dehydrogenase: cloning and characterization of the coding sequence.", Biochem Biophys Res Commun, 223, 1996, 718-23.
Reaction
15.6.1.3.4 3-Oxododecanoyl-CoA+CoA-SH<=>Decanoyl-CoA
Description
At the beginning of this reaction, 1 molecule of '3-Oxododecanoyl-CoA', and 1 molecule of 'CoA' are present. At the end of this reaction, 1
molecule of 'Decanoyl-CoA', and 1 molecule of 'Acetyl-CoA' are present.
References
Reaction
15.6.1.4 Beta oxidation of decanoyl-CoA to octanoyl-CoA-CoA
Authors
Editors
Description
The fourth pass through the beta-oxidation spiral picks up where the last left off with the saturated fatty acid decanoyl-CoA and produces
octanoyl-CoA. Four enzymatic steps are required starting with MCAD CoA dehydrogenase (Medium Chain) activity, followed by the enoyl-CoA
and completed by the ketoacyl-CoA thiolase activity, present in the mitochondrial membrane associated trifunctional protein. Note that the
3-hydroxyacyl-CoA dehydrogenase activity of SCHAD is not actually limited to short chain fatty acids, in fact SCHAD has a broad substrate
specificity.
References
editors), 2001, 2297 Ã¢â‚¬â€œ2326.
15.6.1.4.1 Decanoyl-CoA+FAD<=>trans-Dec-2-enoyl-CoA+FADH2
Description
At the beginning of this reaction, 1 molecule of 'Decanoyl-CoA', and 1 molecule of 'FAD' are present. At the end of this reaction, 1 molecule of
'trans-Dec-2-enoyl-CoA', and 1 molecule of 'FADH2' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'acyl-CoA dehydrogenase activity' of 'MCAD acyl-CoA
References
J Biol Chem, 218, 1956, 717-31.
G Finocchiaro, M Ito, K Tanaka, "Purification and properties of short chain acyl-CoA, medium chain acyl-CoA, and isovaleryl-CoA
dehydrogenases from human liver.", J Biol Chem, 262, 1987, 7982-9.
Reaction
15.6.1.4.2 trans-Dec-2-enoyl-CoA+H2O<=>(S)-Hydroxydecanoyl-CoA
Description
At the beginning of this reaction, 1 molecule of 'trans-Dec-2-enoyl-CoA', and 1 molecule of 'H2O' are present. At the end of this reaction, 1
molecule of '(S)-Hydroxydecanoyl-CoA' is present.
References
Reaction
15.6.1.4.3 (S)-Hydroxydecanoyl-CoA+NAD<=>3-Oxodecanoyl-CoA+NADH+H
Description
At the beginning of this reaction, 1 molecule of '(S)-Hydroxydecanoyl-CoA', and 1 molecule of 'NAD+' are present. At the end of this reaction, 1
molecule of 'H+', 1 molecule of '3-Oxodecanoyl-CoA', and 1 molecule of 'NADH' are present.
References
Reaction
15.6.1.4.4 3-Oxodecanoyl-CoA+CoA-SH<=>Octanoyl-CoA
Description
At the beginning of this reaction, 1 molecule of '3-Oxodecanoyl-CoA', and 1 molecule of 'CoA' are present. At the end of this reaction, 1 molecule
of 'Acetyl-CoA', and 1 molecule of 'Octanoyl-CoA' are present.
References
Reaction
15.6.1.5 Beta oxidation of octanoyl-CoA to hexanoyl-CoA
Authors
Editors
Description
The fifth pass through the beta-oxidation spiral picks up where the last left off with the saturated fatty acid octanoyl-CoA and produces
hexanoyl-CoA. Four enzymatic steps are required starting with MCAD CoA dehydrogenase (Medium Chain) activity, followed by the enoyl-CoA
and completed by the ketoacyl-CoA thiolase activity, present in the mitochondrial membrane associated trifunctional protein.
References
editors), 2001, 2297 Ã¢â‚¬â€œ2326.
15.6.1.5.1 Octanoyl-CoA+FAD<=>trans-Oct-2-enoyl-CoA+FADH2
Description
At the beginning of this reaction, 1 molecule of 'Octanoyl-CoA', and 1 molecule of 'FAD' are present. At the end of this reaction, 1 molecule of
'FADH2', and 1 molecule of 'trans-Oct-2-enoyl-CoA' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'acyl-CoA dehydrogenase activity' of 'MCAD acyl-CoA
References
J Biol Chem, 218, 1956, 717-31.
Reaction
15.6.1.5.2 trans-Oct-2-enoyl-CoA+H2O<=>(S)-Hydroxyoctanoyl-CoA
Description
At the beginning of this reaction, 1 molecule of 'H2O', and 1 molecule of 'trans-Oct-2-enoyl-CoA' are present. At the end of this reaction, 1
molecule of '(S)-Hydroxyoctanoyl-CoA' is present.
References
Reaction
15.6.1.5.3 (S)-Hydroxyoctanoyl-CoA+NAD<=>3-Oxooctanoyl-CoA+NADH+H
Description
At the beginning of this reaction, 1 molecule of 'NAD+', and 1 molecule of '(S)-Hydroxyoctanoyl-CoA' are present. At the end of this reaction, 1
molecule of '3-Oxooctanoyl-CoA', 1 molecule of 'H+', and 1 molecule of 'NADH' are present.
References
Reaction
15.6.1.5.4 3-Oxooctanoyl-CoA+CoA-SH<=>Hexanoyl-CoA
Description
At the beginning of this reaction, 1 molecule of '3-Oxooctanoyl-CoA', and 1 molecule of 'CoA' are present. At the end of this reaction, 1 molecule
of 'Hexanoyl-CoA', and 1 molecule of 'Acetyl-CoA' are present.
References
Reaction
15.6.1.6 Beta oxidation of hexanoyl-CoA to butanoyl-CoA
Authors
Editors
Description
The sixth pass through the beta-oxidation spiral picks up where the last left off with the saturated fatty acid hexanoyl-CoA and produces
butanoyl-CoA. Four enzymatic steps are required starting with SCAD CoA dehydrogenase (Short Chain) activity, followed by the enoyl-CoA
and completed by the ketoacyl-CoA thiolase activity, present in the mitochondrial membrane associated trifunctional protein.
References
editors), 2001, 2297 Ã¢â‚¬â€œ2326.
15.6.1.6.1 Hexanoyl-CoA+FAD<=>trans-Hex-2-enoyl-CoA+FADH2
Description
At the beginning of this reaction, 1 molecule of 'Hexanoyl-CoA', and 1 molecule of 'FAD' are present. At the end of this reaction, 1 molecule of
'trans-Hex-2-enoyl-CoA', and 1 molecule of 'FADH2' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'acyl-CoA dehydrogenase activity' of 'SCAD acyl-CoA
References
J Biol Chem, 218, 1956, 717-31.
Reaction
15.6.1.6.2 trans-Hex-2-enoyl-CoA+H2O<=>(S)-Hydroxyhexanoyl-CoA
Description
At the beginning of this reaction, 1 molecule of 'trans-Hex-2-enoyl-CoA', and 1 molecule of 'H2O' are present. At the end of this reaction, 1
molecule of '(S)-Hydroxyhexanoyl-CoA' is present.
References
Reaction
15.6.1.6.3 (S)-Hydroxyhexanoyl-CoA+NAD<=>3-Oxohexanoyl-CoA+NADH+H
Description
At the beginning of this reaction, 1 molecule of 'NAD+', and 1 molecule of '(S)-Hydroxyhexanoyl-CoA' are present. At the end of this reaction, 1
molecule of '3-Oxohexanoyl-CoA', 1 molecule of 'H+', and 1 molecule of 'NADH' are present.
References
Reaction
15.6.1.6.4 3-Oxohexanoyl-CoA+CoA-SH<=>Butanoyl-CoA
Description
At the beginning of this reaction, 1 molecule of '3-Oxohexanoyl-CoA', and 1 molecule of 'CoA' are present. At the end of this reaction, 1 molecule
of 'Acetyl-CoA', and 1 molecule of 'Butanoyl-CoA' are present.
References
Reaction
15.6.1.7 Beta oxidation of butanoyl-CoA to acetyl-CoA
Authors
Editors
Description
The seventh and final pass through the beta-oxidation spiral picks up where the last left off with the saturated fatty acid butanoyl-CoA and at the
third step produces acetoacetyl-CoA, which can be used to generate 2 acetyl-CoA molecules or can be turned toward the synthesis of ketone
bodies pathway. Four enzymatic steps are required starting with SCAD CoA dehydrogenase (Short Chain) activity, followed by the enoyl-CoA
hydratase activity of crotonase, the 3-hydroxyacyl-CoA dehydrogenase activity of the short chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD).
The final enzymatic step, creating two acetyl-CoA molecules requires a specific ketoacyl-CoA thiolase, Acetoacetyl-CoA thiolase.
References
editors), 2001, 2297 Ã¢â‚¬â€œ2326.
15.6.1.7.1 Butanoyl-CoA+FAD<=>Crotonoyl-CoA+FADH2
Description
At the beginning of this reaction, 1 molecule of 'FAD', and 1 molecule of 'Butanoyl-CoA' are present. At the end of this reaction, 1 molecule of
'Crotonoyl-CoA', and 1 molecule of 'FADH2' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'acyl-CoA dehydrogenase activity' of 'SCAD acyl-CoA
References
J Biol Chem, 218, 1956, 717-31.
Reaction
15.6.1.7.2 Crotonoyl-CoA+H2O<=>(S)-3-Hydroxybutanoyl-CoA
Description
At the beginning of this reaction, 1 molecule of 'Crotonoyl-CoA', and 1 molecule of 'H2O' are present. At the end of this reaction, 1 molecule of
'(S)-3-Hydroxybutanoyl-CoA' is present.
References
Reaction
15.6.1.7.3 (S)-Hydroxybutanoyl-CoA+NAD<=>Acetoacetyl-CoA+NADH+H
Description
At the beginning of this reaction, 1 molecule of 'NAD+', and 1 molecule of '(S)-3-Hydroxybutanoyl-CoA' are present. At the end of this reaction, 1
molecule of 'acetoacetyl-CoA', 1 molecule of 'H+', and 1 molecule of 'NADH' are present.
References
JJ Barycki, LK O'Brien, JM Bratt, R Zhang, R Sanishvili, AW Strauss, LJ Banaszak, "Biochemical characterization and crystal structure
determination of human heart short chain L-3-hydroxyacyl-CoA dehydrogenase provide insights into catalytic mechanism.", Biochemistry, 38,
1999, 5786-98.
Reaction
15.6.2 mitochondrial fatty acid beta-oxidation of unsaturated fatty acids
Authors
Editors
Description
The complete beta-oxidation spiral produces and consumes intermediates with a trans configuration. Mitochondrial beta-oxidation of unsaturated
fatty acids leads to intermediates not compatible with the four enzymatic steps responsible for the beta-oxidation of saturated fatty acids.
Unsaturated fatty acids that have bonds in the cis configuration require three separate enzymatic steps to prepare these molecules for the
beta-oxidation pathway. The further processing of these intermediates requires additional enzymes, depending on the position of the double
bonds in the original fatty acids. Described here is the beta-oxidation of linoleoyl-CoA.
References
Y Ren, H Schulz, "Metabolic functions of the two pathways of oleate beta-oxidation double bond metabolism during the beta-oxidation of oleic
acid in rat heart mitochondria", J Biol Chem, 278, 2003, 111-6.
editors), 2001, 2297 Ã¢â‚¬â€œ2326.
15.6.2.1 Removal of six carbons from Linoleoyl-CoA to form cis,cis-3,6- Dodecadienoyl-CoA
Authors
Description
Linoleoyl-CoA transits through the saturated beta-oxidation spiral three times. With each turn of the spiral two carbon atoms are removed until
the double bond is reached.
References
editors), 2001, 2297 Ã¢â‚¬â€œ2326.
Reaction
15.6.2.2 Isomerization of cis,cis-3,6-Dodecadienoyl-CoA to form trans,cis-Lauro-2,6-dienoyl-CoA
Description
At the beginning of this reaction, 1 molecule of 'cis,cis-3,6-Dodecadienoyl-CoA' is present. At the end of this reaction, 1 molecule of
'trans,cis-Lauro-2,6-dienoyl-CoA ' is present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'dodecenoyl-CoA delta-isomerase activity' of '3,2-trans-enoyl-CoA
isomerase Homodimer'.
References
editors), 2001, 2297 Ã¢â‚¬â€œ2326.
Reaction
15.6.2.3 Removal of 2 Carbon atoms from trans,cis-Lauro-2,6-dienoyl-CoA to form 4-cis-decenoyl-CoA
Authors
Description
trans,cis-Lauro-2,6-dienoyl-CoA transits through the saturated beta-oxidation spiral a single time to yield 4-cis-decenoyl-CoA.
References
editors), 2001, 2297 Ã¢â‚¬â€œ2326.
Reaction
15.6.2.4 dehydrogenation of 4-cis-decenoyl-CoA to form 2-trans-4-cis-decadienoyl-CoA
Authors
Description
4-cis-decenoyl-CoA transits through the first step of the saturated beta-oxidation spiral to yield 2-trans-4-cis-decadienoyl-CoA.
References
editors), 2001, 2297 Ã¢â‚¬â€œ2326.
Reaction
15.6.2.5 Reduction of 2-trans-4-cis-decadienoyl-CoA to form 3-trans-decenoyl-CoA
Authors
Description
The second of the two accessory enzymes, 2,4-dienoyl-CoA reductase catalyses an oxidation-reduction (redox) reaction to yield
3-trans-decenoyl-CoA.
References
editors), 2001, 2297 Ã¢â‚¬â€œ2326.
Reaction
15.6.2.6 Isomerization of 3-trans-decenoyl-CoA to form trans-dec-2-enoyl-CoA
Authors
Description
Once the second of the two double bonds has been reached 3,2-trans-enoyl-CoA isomerase, changes the spatial conformation of the second
double bond from cis to trans. This step yields trans-dec-2-enoyl-CoA, which then enters the saturated beta-oxidation pathway.
References
editors), 2001, 2297 Ã¢â‚¬â€œ2326.
Reaction
15.7 Ketone body metabolism
Authors
Description
Acetoacetate, beta-hydroxybutyrate, and acetone collectively are called ketone bodies. The first two are synthesized from acetyl-CoA, in the
mitochondria of liver cells; acetone is formed by spontaneous decarboxylation of acetoacetate. Ketone body synthesis in liver is effectively
irreversible because the enzyme that catalyzes the conversion of acetoacetate to acetoacetyl-CoA is not present in liver cells.
Ketone bodies, unlike fatty acids and triglycerides, are water-soluble. They are exported from the liver, and are taken up by other tissues, notably
brain and skeletal and cardiac muscle. There, they are broken down to acetyl-CoA which is oxidized via the TCA cycle to yield energy. In a
normal person, this pathway of ketone body synthesis and utilization is most active during extended periods of fasting. Under these conditions,
mobilization and breakdown of stored fatty acids generates abundant acetyl-CoA acetyl-CoA in liver cells for synthesis of ketone bodies, and
their utilization in other tissues minimizes the demand of these tissues for glucose.
15.7.1 Synthesis of Ketone Bodies
Authors
Joshi-Tope, G, 2003-10-19.
Description
Under normal physiological conditions, the production of ketone bodies occurs at a relatively low rate. During periods of normal physiological
responses to carbohydrate shortages, the liver increases the production of ketone bodies from acetyl-CoA generated from fatty acid oxidation.
This allows heart and skeletal muscle to use ketone bodies as the primary source of energy, thereby preserving the limited glucose supply for
use in brain tissue.
In untreated diabetes mellitus, a huge buildup of ketone bodies occurs due to an increase in fatty acid oxidation. The production of ketone bodies
exceeds the ability of peripheral tissues to oxidize them, and results in lowering the pH of blood. Blood acidification is dangerous, chiefly as it
impairs the ability of hemoglobin to bind oxygen.
15.7.1.1 Formation of Acetoacetic Acid
Description
Acetoacetic acid is synthesized in three steps from acetyl CoA. In the body, this reaction occurs in the mitochondria of liver cells.
15.7.1.1.1 2 acetyl-CoA <=> acetoacetyl-CoA+CoA
Description
At the beginning of this reaction, 1 molecule of 'Acetyl-CoA' is present. At the end of this reaction, 1 molecule of 'acetoacetyl-CoA', and 1
molecule of 'CoA' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'acetyltransferase activity' of 'acetyl-CoA acetyltransferase
tetramer'.
References
B Middleton, K Bartlett, A Romanos, J Gomez Vazquez, C Conde, RA Cannon, M Lipson, L Sweetman, WL Nyhan, "3-Ketothiolase deficiency",
Eur J Pediatr, 144, 1986, 586-9.
Reaction
15.7.1.1.2 acetoacetyl-CoA+acetyl-CoA => HMG-CoA
Description
At the beginning of this reaction, 1 molecule of 'acetoacetyl-CoA', and 1 molecule of 'Acetyl-CoA' are present. At the end of this reaction, 1
molecule of 'beta-Hydroxy-beta-methylglutaryl coenzyme A (HMG CoA)' is present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'hydroxymethylglutaryl-CoA synthase activity' of
'hydroxymethylglutaryl-CoA synthase '.
References
R Aledo, J Zschocke, J Pie, C Mir, S Fiesel, E Mayatepek, GF Hoffmann, N Casals, FG Hegardt, "Genetic basis of mitochondrial HMG-CoA
synthase deficiency", Hum Genet, 109, 2001, 19-23.
Reaction
15.7.1.1.3 HMG CoA => acetoacetic acid+ acetyl CoA
Description
At the beginning of this reaction, 1 molecule of 'beta-Hydroxy-beta-methylglutaryl coenzyme A (HMG CoA)' is present. At the end of this reaction,
1 molecule of 'acetoacetyl-CoA', and 1 molecule of 'acetoacetate' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'hydroxymethylglutaryl-CoA lyase activity' of
'hydroxymethylglutaryl-CoA lyase homodimer'.
References
GA Mitchell, MF Robert, PW Hruz, S Wang, G Fontaine, CE Behnke, LM Mende-Mueller, K Schappert, C Lee, KM Gibson, HM Miziorko,
"3-Hydroxy-3-methylglutaryl coenzyme A lyase (HL). Cloning of human and chicken liver HL cDNAs and characterization of a mutation causing
human HL deficiency.", J Biol Chem, 268, 1993, 4376-81.
Reaction
15.7.1.2 Reduction of Acetoacetate to beta-Hydroxybutyrate
Description
At the beginning of this reaction, 1 molecule of 'H+', 1 molecule of 'acetoacetate', and 1 molecule of 'NADH' are present. At the end of this
reaction, 1 molecule of 'D-beta hydroxybutyrate', and 1 molecule of 'NAD+' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the '3-hydroxybutyrate dehydrogenase activity' of
'D-beta-hydroxybutyrate dehydrogenase tetramer'.
References
AR Marks, JO McIntyre, TM Duncan, H Erdjument-Bromage, P Tempst, S Fleischer, "Molecular cloning and characterization of
(R)-3-hydroxybutyrate dehydrogenase from human heart", J Biol Chem, 267, 1992, 15459-63.
Reaction
15.7.2 Utilization of Ketone Bodies
Authors
Joshi-Tope, G, 2003-10-19.
Description
The levels of acetone in ketone bodies are much lower than those of acetoacetic acid and beta-hydroxybutyric acid. Acetone cannot be
converted back to acetyl-CoA, and is excreted in urine, or breathed out through the lungs. Extrahepatic tissues utilize ketone bodies by
converting the beta-hydroxybutyrate successively to acetoacetate, acetoacetatyl-CoA, finally to acetyl-CoA.
15.7.2.1 D-beta hydroxybutyrate+NAD+ <=> acetoacetate+NADH+H+
Description
At the beginning of this reaction, 1 molecule of 'D-beta hydroxybutyrate', and 1 molecule of 'NAD+' are present. At the end of this reaction, 1
molecule of 'H+', 1 molecule of 'acetoacetate', and 1 molecule of 'NADH' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the '3-hydroxybutyrate dehydrogenase activity' of
'D-beta-hydroxybutyrate dehydrogenase tetramer'.
References
AR Marks, JO McIntyre, TM Duncan, H Erdjument-Bromage, P Tempst, S Fleischer, "Molecular cloning and characterization of
(R)-3-hydroxybutyrate dehydrogenase from human heart", J Biol Chem, 267, 1992, 15459-63.
Reaction
15.7.2.2 acetoacetate+succinyl-CoA <=> acetoacetyl-CoA+succinate
Description
At the beginning of this reaction, 1 molecule of 'Succinyl-CoA', and 1 molecule of 'acetoacetate' are present. At the end of this reaction, 1
molecule of 'acetoacetyl-CoA', and 1 molecule of 'Succinate' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the '3-oxoacid CoA-transferase activity' of '3-oxoacid CoA-transferase
homodimer'.
References
S Kassovska-Bratinova, T Fukao, XQ Song, AM Duncan, HS Chen, MF Robert, C Perez-Cerda, M Ugarte, C Chartrand, S Vobecky, N Kondo,
GA Mitchell, "Succinyl CoA: 3-oxoacid CoA transferase (SCOT): human cDNA cloning, human chromosomal mapping to 5p13, and mutation
detection in a SCOT-deficient patient", Am J Hum Genet, 59, 1996, 519-28.
Reaction
15.7.2.3 acetoacetyl-CoA+CoA <=> 2 acetyl-CoA
Description
At the beginning of this reaction, 1 molecule of 'acetoacetyl-CoA', and 1 molecule of 'CoA' are present. At the end of this reaction, 1 molecule of
'Acetyl-CoA' is present.
tetramer'.
References
B Middleton, K Bartlett, A Romanos, J Gomez Vazquez, C Conde, RA Cannon, M Lipson, L Sweetman, WL Nyhan, "3-Ketothiolase deficiency",
Eur J Pediatr, 144, 1986, 586-9.
Reaction
15.8 Steroid metabolism
Authors
Jassal, B, 2007-01-19.
Description
Steroids are a class of lipids characterized by the structure of four fused rings shown in the figure below. The central steroid in human biology is
cholesterol, obtained from animal fats consumed in the diet or synthesized de novo from acetyl-coenzyme A. (Vegetable fats contain various
sterols but no cholesterol.) Cholesterol is an essential constituent of lipid bilayer membranes and is the starting point for the biosyntheses of bile
acids and salts, steroid hormones, and vitamin D. Bile acids and salts, e.g., taurocholate, are mostly synthesized in the liver. They are released
into the intestine and function as detergents to solubilize dietary fats. Steroid hormones are mostly synthesized in the adrenal gland and gonads.
They regulate energy metabolism and stress responses (glucocorticoids such as cortisol), salt balance (mineralocorticoids such as aldosterone),
and sexual development and function (androgens and estrogens such as estradiol). At the same time, chronically elevated cholesterol levels in
the body are associated with the formation of atherosclerotic lesions and hence increased risk of heart attacks and strokes.
Pathways of steroid metabolism annotated in Reactome are cholesterol biosynthesis, the synthesis and recycling of bile acids and salts, and the
synthesis of steroid hormones. The human body lacks a mechanism for degrading excess cholesterol, although an appreciable amount is lost
daily in the form of bile salts and acids that escape recycling.
15.8.1 Cholesterol biosynthesis
Authors
Jassal, B, 2007-01-24.
Reviewers
Description
Cholesterol is synthesized de novo from from acetyl CoA. The overall synthetic process is outlined in the figure below. Enzymes whose
regulation plays a major role in determining the rate of cholesterol synthesis in the body are highlighted in red, and connections to other
metabolic processes are indicated. The molecular details of the reactions leading from acetyl CoA to lanosterol are well known and are
annotated here; the additional nine steps from lanosterol to cholesterol are less well characterized and have not been annotated here.
References
H Rudney, RC Sexton, "Regulation of cholesterol biosynthesis", Annu Rev Nutr, 6, 1986, 245-72.
DW Russell, "Cholesterol biosynthesis and metabolism", Cardiovasc Drugs Ther, 6, 1992, 103-10.
15.8.1.1 Condensation of acetyl CoA with acetoacetyl CoA to form HMG-CoA
Authors
Jassal, B, 2007-01-24.
Reviewers
Description
3-hydroxy-3-methylglutaryl Coenzyme A synthase (HMG-CoA synthase) catalyzes the condensation of acetyl CoA with acetoacetyl CoA to
produce HMG-CoA. There are two forms of this enzyme, cytosolic and mitochondrial. The cytosolic form is ubiquitous in the body and is involved
in cholesterol biosynthesis and synthesis of other isoprenoid products. The mitochondrial form, found solely in the liver and kidney, is involved in
the ketogenic pathway.
References
LL Rokosz, DA Boulton, EA Butkiewicz, G Sanyal, MA Cueto, PA Lachance, JD Hermes, "Human cytoplasmic 3-hydroxy-3-methylglutaryl
coenzyme A synthase: expression, purification, and characterization of recombinant wild-type and Cys129 mutant enzymes", Arch Biochem
Biophys, 312, 1994, 1-13.
Reaction
15.8.1.2 Reduction of HMG-CoA produces mevalonate
Authors
Jassal, B, 2007-01-24.
Reviewers
Description
3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the four-electron reduction of HMG-CoA to mevalonate. Mevalonate
concentrations in the cell are tightly controlled through the activity of HMGR, which is one of the most highly regulated enzymes known
(Goldstein and Brown 1990).
References
ES Istvan, M Palnitkar, SK Buchanan, J Deisenhofer, "Crystal structure of the catalytic portion of human HMG-CoA reductase: insights into
regulation of activity and catalysis", EMBO J, 19, 2000, 819-30.
JL Goldstein, MS Brown, "Regulation of the mevalonate pathway", Nature, 343, 1990, 425-30.
Reaction
15.8.1.3 Mevalonate is phosphorylated to mevalonate-5-phosphate
Authors
Jassal, B, 2007-01-24.
Reviewers
Description
Mevalonate kinase (MK) catalyzes the phosphorylation of mevalonate to mevalonate-5-phosphate.
References
BL Schafer, RW Bishop, VJ Kratunis, SS Kalinowski, ST Mosley, KM Gibson, RD Tanaka, "Molecular cloning of human mevalonate kinase and
identification of a missense mutation in the genetic disease mevalonic aciduria", J Biol Chem, 267, 1992, 13229-38.
S Hogenboom, JJ Tuyp, M Espeel, J Koster, RJ Wanders, HR Waterham, "Mevalonate kinase is a cytosolic enzyme in humans", J Cell Sci, 117,
2004, 631-9.
SM Houten, RJ Wanders, HR Waterham, "Biochemical and genetic aspects of mevalonate kinase and its deficiency", Biochim Biophys Acta,
1529, 2000, 19-32.
Reaction
15.8.1.4 Mevalonate-5-phosphate is further phosphorylated.
Authors
Jassal, B, 2007-01-24.
Reviewers
Description
Phosphomevalonate kinase (PMK) catalyzes the reversible, ATP-dependent phosphorylation of mevalonate-5-phosphate, producing
mevalonate-5-pyrophosphate.
References
TJ Herdendorf, HM Miziorko, "Phosphomevalonate kinase: functional investigation of the recombinant human enzyme", Biochemistry, 45, 2006,
3235-42.
S Hogenboom, JJ Tuyp, M Espeel, J Koster, RJ Wanders, HR Waterham, "Phosphomevalonate kinase is a cytosolic protein in humans", J Lipid
Res, 45, 2004, 697-705.
Reaction
15.8.1.5 Mevalonate-5-pyrophosphate is decarboxylated
Authors
Jassal, B, 2007-01-24.
Reviewers
Description
Mevalonate pyrophosphate decarboxylase (MPD) decarboxylates mevalonate-5-pyrophosphate into isopentenyl pyrophosphate while
hydrolysing ATP to ADP and orthophosphate.
References
MJ Toth, L Huwyler, "Molecular cloning and expression of the cDNAs encoding human and yeast mevalonate pyrophosphate decarboxylase", J
Biol Chem, 271, 1996, 7895-8.
Reaction
15.8.1.6 Isopentenyl pyrophosphate rearranges to dimethylallyl pyrophosphate
Authors
Jassal, B, 2007-01-24.
Reviewers
Description
Cytosolic isopentenyl diphosphate isomerase (IPP isomerase) catalyzes an essential activation step in the isoprenoid biosynthetic pathway. It
rearranges isopentenyl pyrophosphate into its highly electrophilic isomer, dimethylallyl pyrophosphate (DMAPP). IPP isomerase may also be
located in human peroxisomes but it's function there is not clear.
References
FM Hahn, JW Xuan, AF Chambers, CD Poulter, "Human isopentenyl diphosphate: dimethylallyl diphosphate isomerase: overproduction,
purification, and characterization", Arch Biochem Biophys, 332, 1996, 30-4.
Reaction
15.8.1.7 Addition of isopentenyl pyrophosphate to DMAPP
Authors
Jassal, B, 2007-01-24.
Reviewers
Description
The family of enzymes called prenyltransferases is involved in the biosynthesis of isoprenoids. One member of this family, Geranylgeranyl
pyrophosphate synthetase (GGPP synthase), catalyzes the sequential condensation of isopentenyl pyrophosphate to DMAPP.
References
T Kuzuguchi, Y Morita, I Sagami, H Sagami, K Ogura, "Human geranylgeranyl diphosphate synthase. cDNA cloning and expression.", J Biol
Chem, 274, 1999, 5888-94.
J Ericsson, JM Greene, KC Carter, BK Shell, DR Duan, C Florence, PA Edwards, "Human geranylgeranyl diphosphate synthase: isolation of the
cDNA, chromosomal mapping and tissue expression", J Lipid Res, 39, 1998, 1731-9.
Reaction
15.8.1.8 Another isopentenyl pyrophosphate is added to geranyl pyrophosphate
Authors
Jassal, B, 2007-01-24.
Reviewers
Description
Further condensation of an isopentenyl pyrophosphate with geranyl pyrophosphate to form farnesyl pyrophosphate is catalyzed by GGPP
synthase.
References
T Kuzuguchi, Y Morita, I Sagami, H Sagami, K Ogura, "Human geranylgeranyl diphosphate synthase. cDNA cloning and expression.", J Biol
Chem, 274, 1999, 5888-94.
J Ericsson, JM Greene, KC Carter, BK Shell, DR Duan, C Florence, PA Edwards, "Human geranylgeranyl diphosphate synthase: isolation of the
cDNA, chromosomal mapping and tissue expression", J Lipid Res, 39, 1998, 1731-9.
Reaction
15.8.1.9 Two FPP molecules dimerize to form presqualene diphosphate
Authors
Jassal, B, 2007-01-24.
Reviewers
Description
Farnesyl diphosphate farnesyltransferase (FDFT; squalene synthase) catalyzes the reductive dimerization of two farnesyl diphosphate (FPP)
molecules to form squalene. This happens in two distinct steps. The first step of dimerization forms presqualene diphosphate (Pandit et al.
2000).
References
J Pandit, DE Danley, GK Schulte, S Mazzalupo, TA Pauly, CM Hayward, ES Hamanaka, JF Thompson, Jr Harwood HJ, "Crystal structure of
human squalene synthase. A key enzyme in cholesterol biosynthesis.", J Biol Chem, 275, 2000, 30610-7.
Reaction
15.8.1.10 Reduction of presqualene diphosphate to form squalene
Authors
Jassal, B, 2007-01-24.
Reviewers
Description
In the second step, FDFT catalyzes the reduction of presqualene diphosphate to squalene (Pandit et al. 2000).
References
J Pandit, DE Danley, GK Schulte, S Mazzalupo, TA Pauly, CM Hayward, ES Hamanaka, JF Thompson, Jr Harwood HJ, "Crystal structure of
human squalene synthase. A key enzyme in cholesterol biosynthesis.", J Biol Chem, 275, 2000, 30610-7.
Reaction
15.8.1.11 Squalene is oxidized to its epoxide
Authors
Jassal, B, 2007-01-24.
Reviewers
Description
Squalene monooxygenase (squalene epoxidase, SE) is located on the endoplamic reticulum. It catalyzes the oxidation of squalene to squalene
2,3-epoxide. SE seems to be an important rate-limiting enzyme in cholesterol biosynthesis.
References
BP Laden, Y Tang, TD Porter, "Cloning, heterologous expression, and enzymological characterization of human squalene monooxygenase",
Arch Biochem Biophys, 374, 2000, 381-8.
Reaction
15.8.1.12 Squalene 2,3-epoxide cyclizes, forming lanosterol
Authors
Jassal, B, 2007-01-24.
Reviewers
Description
Lanosterol synthase (LS) catalyzes the cyclization of squalene 2,3-epoxide to lanosterol, a reaction that forms the sterol nucleus.LS is located on
the ER membrane and is active as the monomer (Ruf et al, 2004).
References
CK Sung, M Shibuya, U Sankawa, Y Ebizuka, "Molecular cloning of cDNA encoding human lanosterol synthase", Biol Pharm Bull, 18, 1995,
1459-61.
EE Van Tamelen, JD Willett, RB Clayton, KE Lord, "Enzymic conversion of squalene 2,3-oxide to lanosterol and cholesterol", J Am Chem Soc,
88, 1966, 4752-4.
A Ruf, F Muller, B D'Arcy, M Stihle, E Kusznir, C Handschin, OH Morand, R Thoma, "The monotopic membrane protein human oxidosqualene
cyclase is active as monomer", Biochem Biophys Res Commun, 315, 2004, 247-54.
Reaction
15.8.1.13 Transformation of lanosterol to cholesterol

Authors
Jassal, B, 2007-01-24.
Editors
Reviewers
Description
The transformation of lanosterol into cholesterol requires multiple steps, including the removal of three methyl groups, the reduction of one
double bond and the migration of another. These reactions may not occur in a single fixed order in the body, so the linear pathway laid out here
following the work of Gaylor and colleagues (Gaylor 2002) is an oversimplification of the process that occurs in vivo. Defects in several of the
enzymes involved in this process are associated with human disease and have provided useful insights into the regulatory roles of cholesterol
and its synthetic intermediates in human development (Herman 2003; Song et al. 2005).
References
GE Herman, "Disorders of cholesterol biosynthesis: prototypic metabolic malformation syndromes", Hum Mol Genet, 12, 2003, R75-88.
BL Song, NB Javitt, RA DeBose-Boyd, "Insig-mediated degradation of HMG CoA reductase stimulated by lanosterol, an intermediate in the
synthesis of cholesterol", Cell Metab, 1, 2005, 179-89.
15.8.1.13.1 Lanosterol is oxidatively demethylated to 4,4-dimethylcholesta-8(9),14,24-trien-3beta-ol
Authors
Jassal, B, 2008-05-19.
Editors
Reviewers
Description
Lanosterol, NADPH + H+, and O2 react to form 4,4-dimethylcholesta-8(9),14,24-trien-3beta-ol, NADP+, H2O and formate. This oxidative
demethylation reaction, in the endoplasmic reticulum, is catalyzed by lanosterol 14alpha-demethylase (CYP51A1). Although the reaction is
annotated here as a single concerted event, studies with purified rat enzyme indicate that the methyl group is converted successively to an
alcohol and an aldehyde before being released as formate (Trzaskos et al. 1986; Fischer et al. 1991; Gaylor 2002).
References
RT Fischer, JM Trzaskos, RL Magolda, SS Ko, CS Brosz, B Larsen, "Lanosterol 14 alpha-methyl demethylase. Isolation and characterization of
the third metabolically generated oxidative demethylation intermediate.", J Biol Chem, 266, 1991, 6124-32.
JM Trzaskos, S Kawata, JL Gaylor, "Microsomal enzymes of cholesterol biosynthesis. Purification of lanosterol 14 alpha-methyl demethylase
cytochrome P-450 from hepatic microsomes.", J Biol Chem, 261, 1986, 14651-7.
M Stromstedt, D Rozman, MR Waterman, "The ubiquitously expressed human CYP51 encodes lanosterol 14 alpha-demethylase, a cytochrome
P450 whose expression is regulated by oxysterols", Arch Biochem Biophys, 329, 1996, 73-81.
Reaction
15.8.1.13.2 4,4-dimethylcholesta-8(9),14,24-trien-3beta-ol is reduced to 4,4-dimethylcholesta-8(9),24-dien-3beta-ol [LBR]
Authors
Editors
Reviewers
Jassal, B, 2007-04-21.
Description
4,4-dimethylcholesta-8(9),14,24-trien-3beta-ol and NADPH + H+ react to form 4,4-dimethylcholesta-8(9),24-dien-3beta-ol and NADP+, catalyzed

by LBR in the nuclear envelope. LBR protein spans the inner nuclear envelope, has an aminoterminal region with properties of a laminin
receptor and a carboxyterminal domain with sequence similarity to sterol delta14-reductases (Holmer et al. 1998). Studies of material from an
individual with HEM/Greenberg skeletal dysplasia indicate that LBR catalyzes the sterol delta14-reductase step of cholesterol biosynthesis in
vivo. DNA sequencing revealed homozygosity for a mutant LBR allele encoding a truncated protein in the affected individual, and cells from the
individual accumulated cholesta-8,14-dien-3beta-ol in culture. Transfection of wild-type LBR into the cultured cells reversed the accumulation of
cholesta-8,14-dien-3beta-ol (Waterham et al. 2003). This observation is surprising because a second gene, TM7SF2, encodes an efficient sterol
delta14-reductase that is localized to the endoplasmic reticulum whose expression is up-regulated in response to sterol depletion (Bennati et al.
2006). The physiological roles of LBR and TM7SF2 in vivo remain to be determined.
References
AM Bennati, M Castelli, MA Della Fazia, T Beccari, D Caruso, G Servillo, R Roberti, "Sterol dependent regulation of human TM7SF2 gene
expression: role of the encoded 3beta-hydroxysterol Delta14-reductase in human cholesterol biosynthesis", Biochim Biophys Acta, 1761, 2006,
677-85.
L Holmer, A Pezhman, HJ Worman, "The human lamin B receptor/sterol reductase multigene family", Genomics, 54, 1998, 469-76.
HR Waterham, J Koster, P Mooyer, G van Noort, RI Kelley, WR Wilcox, RJ Wanders, RC Hennekam, JC Oosterwijk, "Autosomal recessive
HEM/Greenberg skeletal dysplasia is caused by 3 beta-hydroxysterol delta 14-reductase deficiency due to mutations in the lamin B receptor
gene", Am J Hum Genet, 72, 2003, 1013-7.
Reaction
15.8.1.13.3 4,4-dimethylcholesta-8(9),14,24-trien-3beta-ol is reduced to 4,4-dimethylcholesta-8(9),24-dien-3beta-ol [TM7SF2]
Authors
Editors
Reviewers
Jassal, B, 2007-04-21.
Description
4,4-dimethylcholesta-8(9),14,24-trien-3beta-ol and NADPH + H+ react to form 4,4-dimethylcholesta-8(9),24-dien-3beta-ol and NADP+, catalyzed

by TM7SF2 in the endoplasmic reticulum. TM7SF2 protein has sterol delta14-reductase activity in vitro, and expression of the gene is induced
by sterol starvation in human cells, as expected for a gene involved in sterol biosynthesis (Bennati et al. 2006). However, molecular studies of
material from an individual with HEM/Greenberg skeletal dysplasia indicate that LBR, a protein that spans the inner nuclear membrane and has
both laminin receptor and sterol delta14-reductase activities, is required for normal sterol 14delta-reductase activity in human cells. It remains to
be determined whether both LBR and TM7SF2 catalyze this reaction in vivo, and whether the role of TM7SF2 is essential (Waterham et al.
2003).
References
AM Bennati, M Castelli, MA Della Fazia, T Beccari, D Caruso, G Servillo, R Roberti, "Sterol dependent regulation of human TM7SF2 gene
expression: role of the encoded 3beta-hydroxysterol Delta14-reductase in human cholesterol biosynthesis", Biochim Biophys Acta, 1761, 2006,
677-85.
HR Waterham, J Koster, P Mooyer, G van Noort, RI Kelley, WR Wilcox, RJ Wanders, RC Hennekam, JC Oosterwijk, "Autosomal recessive
HEM/Greenberg skeletal dysplasia is caused by 3 beta-hydroxysterol delta 14-reductase deficiency due to mutations in the lamin B receptor
gene", Am J Hum Genet, 72, 2003, 1013-7.
Reaction
15.8.1.13.4 4,4-dimethylcholesta-8(9),24-dien-3beta-ol is oxidized to 4-methyl,4-carboxycholesta-8(9),24-dien-3beta-ol
Authors
Editors
Reviewers
Jassal, B, 2007-04-21.
Description
4,4-dimethylcholesta-8(9),24-dien-3beta-ol, NADPH + H+, and O2 react to form 4-methyl,4-carboxycholesta-8(9),24-dien-3beta-ol, NADP+, and

H2O. This reaction, in the endoplasmic reticulum, is catalyzed by SC4MOL (C-4 methylsterol oxidase). The human enzyme has been identified
based on its sequence similarity to yeast methyl sterol oxidase (ERG25) and the ability of the cloned human gene to rescue ERG25-deficient
yeast cells (Li and Kaplan 1996). The mechanism and stoichiometry of the reaction have been inferred from studies of partially purified rat
enzyme (Gaylor et al. 1975; Fukushima et al. 1981).
References
JL Gaylor, Y Miyake, T Yamano, "Stoichiometry of 4-methyl sterol oxidase of rat liver microsomes", J Biol Chem, 250, 1975, 7159-67.
L Li, J Kaplan, "Characterization of yeast methyl sterol oxidase (ERG25) and identification of a human homologue", J Biol Chem, 271, 1996,
16927-33.
H Fukushima, GF Grinstead, JL Gaylor, "Total enzymic synthesis of cholesterol from lanosterol. Cytochrome b5-dependence of 4-methyl sterol
oxidase.", J Biol Chem, 256, 1981, 4822-6.
Reaction
15.8.1.13.5 4-methyl,4-carboxycholesta-8(9),24-dien-3beta-ol is decarboxylated and oxidized to form

4-methylcholesta-8(9),24-dien-3-one
Authors
Editors
Reviewers
Jassal, B, 2007-04-21.
Description
4-methyl,4-carboxycholesta-8(9),24-dien-3beta-ol and NAD+ react to form 4-methylcholesta-8(9),24-dien-3-one, CO2, and NADH + H+. This
reaction occurs in the endoplasmic reticulum, catalyzed by NSDHL (Caldas and Herman 2003). Defects in this enzyme are associated with
CHILD syndrome (Congenital Hemidysplasia with Ichthyosiform nevus and Limb Defects) (Konig et al. 2000), but cholesterol biosynthesis in
cells and tissues from affected individuals has not been characterized. Instead, the mechanism and stoichiometry of the reaction are inferred
from biochemical studies of partially purified rat enzyme (Rahimtula and Gaylor 1972).
References
AD Rahimtula, JL Gaylor, "Partial purification of a microsomal sterol 4 -carboxylic acid decarboxylase", J Biol Chem, 247, 1972, 9-15.
A Konig, R Happle, D Bornholdt, H Engel, KH Grzeschik, "Mutations in the NSDHL gene, encoding a 3beta-hydroxysteroid dehydrogenase,
cause CHILD syndrome", Am J Med Genet, 90, 2000, 339-46.
H Caldas, GE Herman, "NSDHL, an enzyme involved in cholesterol biosynthesis, traffics through the Golgi and accumulates on ER membranes
and on the surface of lipid droplets", Hum Mol Genet, 12, 2003, 2981-91.
Reaction
15.8.1.13.6 4-methylcholesta-8(9),24-dien-3-one is reduced to 4-methylcholesta-8(9),24-dien-3beta-ol
Authors
Editors
Reviewers
Jassal, B, 2007-04-21.
Description
4-methylcholesta-8(9),24-dien-3-one and NADPH + H+ react to form 4-methylcholesta-8(9),24-dien-3beta-ol and NADP+. This reaction takes
place in the endoplasmic reticulum, catalyzed by HSD17B7. Two isoforms of the enzyme due to alternative splicing have been identified but only
the first has been tested for enzymatic activity (Marijanovic et al. 2003). The human enzyme has not been studied extensively; molecular details
of the reaction are inferred from those worked out in studies of material from rat liver (Gaylor 2002).
References
Z Marijanovic, D Laubner, G Moller, C Gege, B Husen, J Adamski, R Breitling, "Closing the gap: identification of human 3-ketosteroid reductase,
the last unknown enzyme of mammalian cholesterol biosynthesis", Mol Endocrinol, 17, 2003, 1715-1725.
Reaction
15.8.1.13.7 4-methylcholesta-8(9),24-dien-3beta-ol is oxidized to 4-carboxycholesta-8(9),24-dien-3beta-ol
Authors
Editors
Reviewers
Jassal, B, 2007-04-21.
Description
4-methylcholesta-8(9),24-dien-3beta-ol, NADPH + H+, and O2 react to form 4-carboxycholesta-8(9),24-dien-3beta-ol, NADP+, and H2O. This
reaction, in the endoplasmic reticulum, is catalyzed by SC4MOL (C-4 methylsterol oxidase). The human enzyme has been identified based on its
sequence similarity to yeast methyl sterol oxidase (ERG25) and the ability of the cloned human gene to rescue ERG25-deficient yeast cells (Li
and Kaplan 1996). The mechanism and stoichiometry of the reaction have been inferred from studies of partially purified rat enzyme (Gaylor et
al. 1975; Fukushima et al. 1981).
References
JL Gaylor, Y Miyake, T Yamano, "Stoichiometry of 4-methyl sterol oxidase of rat liver microsomes", J Biol Chem, 250, 1975, 7159-67.
L Li, J Kaplan, "Characterization of yeast methyl sterol oxidase (ERG25) and identification of a human homologue", J Biol Chem, 271, 1996,
16927-33.
H Fukushima, GF Grinstead, JL Gaylor, "Total enzymic synthesis of cholesterol from lanosterol. Cytochrome b5-dependence of 4-methyl sterol
oxidase.", J Biol Chem, 256, 1981, 4822-6.
Reaction
15.8.1.13.8 4-carboxycholesta-8(9),24-dien-3beta-ol is decarboxylated and oxidized to form cholesta-8(9),24-dien-3-one (zymosterone)
Authors
Editors
Reviewers
Jassal, B, 2007-04-21.
Description
4-carboxycholesta-8(9),24-dien-3beta-ol and NAD+ react to form zymosterone (cholesta-8(9),24-dien-3-one), CO2, and NADH + H+. This
reaction occurs in the endoplasmic reticulum, catalyzed by NSDHL (Caldas and Herman 2003). Defects in this enzyme are associated with
CHILD syndrome (Congenital Hemidysplasia with Ichthyosiform nevus and Limb Defects) (Konig et al. 2000), but cholesterol biosynthesis in
cells and tissues from affceted individuals has not been characterized. Instead, the mechanism and stoichiometry of the reaction are inferred
from biochemical studies of partially purified rat enzyme (Rahimtula and Gaylor 1972).
References
AD Rahimtula, JL Gaylor, "Partial purification of a microsomal sterol 4 -carboxylic acid decarboxylase", J Biol Chem, 247, 1972, 9-15.
A Konig, R Happle, D Bornholdt, H Engel, KH Grzeschik, "Mutations in the NSDHL gene, encoding a 3beta-hydroxysteroid dehydrogenase,
cause CHILD syndrome", Am J Med Genet, 90, 2000, 339-46.
H Caldas, GE Herman, "NSDHL, an enzyme involved in cholesterol biosynthesis, traffics through the Golgi and accumulates on ER membranes
and on the surface of lipid droplets", Hum Mol Genet, 12, 2003, 2981-91.
Reaction
15.8.1.13.9 Zymosterone (cholesta-8(9),24-dien-3-one) is reduced to zymosterol (cholesta-8(9),24-dien-3beta-ol)
Authors
Editors
Reviewers
Jassal, B, 2007-04-21.
Description
Zymosterone (cholesta-8(9),24-dien-3-one) and NADPH + H+ react to form zymosterol (cholesta-8(9),24-dien-3beta-ol) and NADP+. This
reaction takes place in the endoplasmic reticulum, catalyzed by HSD17B7. Two isoforms of the enzyme due to alternative splicing have been
identified but only the first has been tested for enzymatic activity (Marijanovic et al. 2003). The human enzyme has not been studied extensively;
molecular details of the reaction are inferred from those worked out in studies of material from rat liver (Gaylor 2002).
References
Z Marijanovic, D Laubner, G Moller, C Gege, B Husen, J Adamski, R Breitling, "Closing the gap: identification of human 3-ketosteroid reductase,
the last unknown enzyme of mammalian cholesterol biosynthesis", Mol Endocrinol, 17, 2003, 1715-1725.
Reaction
15.8.1.13.10 Zymosterol is isomerized to cholesta-7,24-dien-3beta-ol
Authors
Editors
Reviewers
Jassal, B, 2007-04-21.
Description
Isomerization of zymosterol to cholesta-7,24-dien-3beta-ol is catalyzed by EBP in the endoplasmic reticulum. The biochemical details of the
reaction have been established through studies of purified rat EBP; the role of the human enzyme has been established through studies of
patients deficient in it (Derry et al. 1999; Braverman et al. 1999).
References
JM Derry, E Gormally, GD Means, W Zhao, A Meindl, RI Kelley, Y Boyd, GE Herman, "Mutations in a delta 8-delta 7 sterol isomerase in the
tattered mouse and X-linked dominant chondrodysplasia punctata. jderry@immunex.com.", Nat Genet, 22, 1999, 286-90.
N Braverman, P Lin, FF Moebius, C Obie, A Moser, H Glossmann, WR Wilcox, DL Rimoin, M Smith, L Kratz, RI Kelley, D Valle, "Mutations in
the gene encoding 3 beta-hydroxysteroid-delta 8, delta 7-isomerase cause X-linked dominant Conradi-Hunermann syndrome", Nat Genet, 22,
1999, 291-4.
YK Paik, JT Billheimer, RL Magolda, JL Gaylor, "Microsomal enzymes of cholesterol biosynthesis from lanosterol. Solubilization and purification
of steroid 8-isomerase.", J Biol Chem, 261, 1986, 6470-7.
Reaction
15.8.1.13.11 Cholesta-7,24-dien-3beta-ol is desaturated to form cholesta-5,7,24-trien-3beta-ol
Authors
Editors
Reviewers
Jassal, B, 2007-04-21.
Description
Cholesta-7,24-dien-3beta-ol, NADPH + H+, and O2 react to form cholesta-5,7,24-trien-3beta-ol, NADP+, and 2 H2O, catalyzed by SC5D. This
reaction takes place in the endoplasmic reticulum. Its biochemical details are inferred from those of the reaction catalyzed by the purified rat
protein (Kawata et al. 1985). The role of human SC5D in catalyzing this reaction in vivo is established from studies of patients in whom the
enzyme is defective (Brunetti-Pierri et al. 2002; Krakowiak et al. 2003).
References
PA Krakowiak, CA Wassif, L Kratz, D Cozma, M Kovarova, G Harris, A Grinberg, Y Yang, AG Hunter, M Tsokos, RI Kelley, FD Porter,
"Lathosterolosis: an inborn error of human and murine cholesterol synthesis due to lathosterol 5-desaturase deficiency", Hum Mol Genet, 12,
2003, 1631-41.
S Kawata, JM Trzaskos, JL Gaylor, "Microsomal enzymes of cholesterol biosynthesis from lanosterol. Purification and characterization of delta
7-sterol 5-desaturase of rat liver microsomes.", J Biol Chem, 260, 1985, 6609-17.
N Brunetti-Pierri, G Corso, M Rossi, P Ferrari, F Balli, F Rivasi, I Annunziata, A Ballabio, A Dello Russo, G Andria, G Parenti, "Lathosterolosis, a
novel multiple-malformation/mental retardation syndrome due to deficiency of 3beta-hydroxysteroid-delta5-desaturase", Am J Hum Genet, 71,
2002, 952-8.
Reaction
15.8.1.13.12 Cholesta-5,7,24-trien-3beta-ol is reduced to desmosterol
Authors
Editors
Reviewers
Jassal, B, 2007-04-21.
Description
Cholesta-5,7,24-trien-3beta-ol and NADPH + H+ react to form desmosterol and NADP+. This reaction is catalyzed by DHCR7, associated with
the endoplasmic reticulum membrane. The biochemical details of the reaction are inferred from those of the reaction catalyzed by the
well-studied rat enzyme (Bae et al. 1999).
References
FF Moebius, BU Fitzky, JN Lee, YK Paik, H Glossmann, "Molecular cloning and expression of the human delta7-sterol reductase", Proc Natl
Acad Sci U S A, 95, 1998, 1899-902.
SH Bae, JN Lee, BU Fitzky, J Seong, YK Paik, "Cholesterol biosynthesis from lanosterol. Molecular cloning, tissue distribution, expression,
chromosomal localization, and regulation of rat 7-dehydrocholesterol reductase, a Smith-Lemli-Opitz syndrome-related protein.", J Biol Chem,
274, 1999, 14624-31.
Reaction
15.8.1.13.13 Reduction of desmosterol to cholesterol
Authors
Editors
Reviewers
Jassal, B, 2007-04-21.
Description
Desmosterol is reduced by NADPH + H+ to form cholesterol and NADP+, catalyzed by DHCR24 associated with the endoplasmic reticulum
membrane.
References
HR Waterham, J Koster, GJ Romeijn, RC Hennekam, P Vreken, HC Andersson, DR FitzPatrick, RI Kelley, RJ Wanders, "Mutations in the
3beta-hydroxysterol Delta24-reductase gene cause desmosterolosis, an autosomal recessive disorder of cholesterol biosynthesis", Am J Hum
Genet, 69, 2001, 685-94.
Reaction
15.8.2 Metabolism of bile acids and bile salts
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
In a healthy adult human, about 500 mg of cholesterol is converted to bile salts daily. Newly synthesized bile salts are secreted into the bile and
released into the small intestine where they emulsify dietary fats (Russell 2003). About 95% of the bile salts in the intestine are recovered and
returned to the liver (Kullak-Ublick et al. 2004; Trauner and Boyer 2002). The major pathway for bile salt synthesis in the liver begins with the
conversion of cholesterol to 7alpha-hydroxycholesterol. Bile salt synthesis can also begin with the synthesis of an oxysterol -
24-hydroxycholesterol or 27-hydroxycholesterol. In the body, the initial steps of these two pathways occur in extrahepatic tissues, generating
intermediates that are transported to the liver and converted to bile salts via the 7alpha-hydroxycholesterol pathway. These extrahepatic
pathways contribute little to the total synthesis of bile salts, but are thought to play important roles in extrahepatic cholesterol homeostasis (Javitt
2002).
References
JL Boyer, M Trauner, "Bile salt transporters: molecular characterization, function, and regulation", Physiol Rev, 83, 2003, 633-671.
GA Kullak-Ublick, B Stieger, PJ Meier, "Enterohepatic bile salt transporters in normal physiology and liver disease", Gastroenterology, 126,
2004, 322-42.
15.8.2.1 Synthesis of bile acids and bile salts
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
In a healthy adult human, about 500 mg of cholesterol is converted to bile salts daily (Russell 2003). The major pathway for bile salt synthesis in
the liver begins with the conversion of cholesterol to 7alpha-hydroxycholesterol. Bile salt synthesis can also begin with the synthesis of an
oxysterol - 24-hydroxycholesterol or 27-hydroxycholesterol. In the body, the initial steps of these two pathways occur in extrahepatic tissues,
generating intermediates that are transported to the liver and converted to bile salts via the 7alpha-hydroxycholesterol pathway. These
extrahepatic pathways contribute little to the total synthesis of bile salts, but are thought to play important roles in cholesterol homeostasis (Javitt
2002).
References
15.8.2.1.1 Synthesis of bile acids and bile salts via 7alpha-hydroxycholesterol

Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
In the liver, synthesis of bile acids and bile salts is initiated with the conversion of cholesterol to 7alpha-hydroxycholesterol and of
7alpha-hydroxycholesterol to 4-cholesten-7alpha-ol-3-one. The pathway then branches: hydroxylation of 4-cholesten-7alpha-ol-3-one to
4-cholesten-7alpha, 12alpha-diol-3-one leads ultimately to the formation of cholate, while its reduction to 5beta-cholestan-7alpha-ol-3-one leads
to chenodeoxycholate formation. The amounts of substrate following each branch appear to be determined by abundance of the hydroxylase
enzyme: in human liver, cholate synthesis predominates.
In both branches, reactions in the cytosol, the mitochondrial matrix, and the peroxisomal matrix result in modifications to the ring structure,
shortening and oxidation of the side chain, conversion to a Coenzyme A derivative, and conjugation with the amino acids glycine or taurine. In
the body, glycocholate, taurocholate, glycochenodeoxycholate, and taurochenodeoxycholate are released from hepatocytes into the bile and
ultimately into the lumen of the small intestine, where they function as detergents to solubilize dietary fats. The liver synthetic pathway also yields
small amounts of bile acids, cholate and deoxycholate, which may play a feedback role in regulating the bile acid synthetic pathway (Russell
2003). These reactions are outlined in the figure below.
References
15.8.2.1.1.1 Cholesterol is hydroxylated to 7alpha-hydroxycholesterol by CYP7A1
Authors
Jassal, B, 2008-05-19.
Editors
Reviewers
Description
Cholesterol, NADPH + H+, and O2 form 7alpha-cholesterol (5-cholesten-3beta, 7alpha-diol), NADP+,and H2O, in a reaction catalyzed by
CYP7A1 (cholesterol 7alpha-hydroylase) in the endoplasmic reticulum membrane. In the body, this enzyme is expressed only in liver, and its
expression is tightly regulated at the level of transcription to determine the overall rate of bile acid and bile salt production (Russell 2003;
Pullinger et al. 2002).
References
M Noshiro, K Okuda, "Molecular cloning and sequence analysis of cDNA encoding human cholesterol 7 alpha-hydroxylase", FEBS Lett, 268,
1990, 137-40.
CR Pullinger, C Eng, G Salen, S Shefer, AK Batta, SK Erickson, A Verhagen, CR Rivera, SJ Mulvihill, MJ Malloy, JP Kane, "Human cholesterol
7alpha-hydroxylase (CYP7A1) deficiency has a hypercholesterolemic phenotype", J Clin Invest, 110, 2002, 109-17.
Reaction
15.8.2.1.1.2 7alpha-hydroxycholesterol is oxidized and isomerized to 4-cholesten-7alpha-ol-3-one
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
7alpha-hydroxycholesterol and NAD+ react to form 4-cholesten-7alpha-ol-3-one, NADH, and H+, in a reaction catalyzed by HSD3B7 (3
beta-hydroxysteroid dehydrogenase type 7) in the endoplasmic reticulum membrane. Its function in vivo has been confirmed in studies of
patients with defects in bile acid synthesis (Schwarz et al. 2000).
References
M Schwarz, AC Wright, DL Davis, H Nazer, I Bjorkhem, DW Russell, "The bile acid synthetic gene 3beta-hydroxy-Delta(5)-C(27)-steroid
oxidoreductase is mutated in progressive intrahepatic cholestasis", J Clin Invest, 106, 2000, 1175-84.
JB Cheng, E Jacquemin, M Gerhardt, H Nazer, D Cresteil, JE Heubi, KD Setchell, DW Russell, "Molecular genetics of
3beta-hydroxy-Delta5-C27-steroid oxidoreductase deficiency in 16 patients with loss of bile acid synthesis and liver disease", J Clin Endocrinol
Metab, 88, 2003, 1833-41.
Reaction
15.8.2.1.1.3 4-Cholesten-7alpha-ol-3-one is 12alpha-hydroxylated to 7-alpha,12-alpha-dihydroxycholest-4-en-3-one
Authors
Jassal, B, 2008-05-19.
Editors
Reviewers
Description
7-Alpha-hydroxycholest-4-en-3-one, NADPH + H+, and O2 form 7-alpha,12-alpha-dihydroxycholest-4-en-3-one + NADP+ + H2O. This reaction
References
Genomics, 56, 1999, 184-96.
Chem, 267, 1992, 21319-23.
Reaction
15.8.2.1.1.4 4-cholesten-7alpha, 12alpha-diol-3-one is reduced to 5beta-cholesten-7alpha, 12alpha-diol-3-one
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
4-Cholesten-7alpha, 12alpha-diol-3-one and NADPH + H+ react to form 5beta-cholesten-7alpha,12alpha-diol-3-one + NADP+. This reaction is
catalyzed by AKR1D1 (3-oxo-5-beta-steroid 4-dehydrogenase). AKR1D1 is localized to the cytosol, and in the course of the reaction its steroid
substrate moves from the endoplasmic reticulum membrane to the cytosol. It is unclear whether this translocation results simply from its
increased hydrophilicity or is mediated by the enzyme or another transport protein (Russell 2003).
References
KH Kondo, MH Kai, Y Setoguchi, G Eggertsen, P Sjoblom, T Setoguchi, K Okuda, I Bjorkhem, "Cloning and expression of cDNA of human delta
4-3-oxosteroid 5 beta-reductase and substrate specificity of the expressed enzyme", Eur J Biochem, 219, 1994, 357-63.
Reaction
15.8.2.1.1.5 4-cholesten-7alpha-ol-3-one is reduced to 5beta-cholestan-7alpha-ol-3-one
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
4-Cholesten-7alpha-ol-3-one, NADPH, and H+ react to form 5beta-cholestan-7alpha-ol-3-one and NADP+. This reaction is catalyzed by
AKR1D1 (3-oxo-5-beta-steroid 4-dehydrogenase). AKR1D1 is localized to the cytosol, and in the course of the reaction its steroid substrate
moves from the endoplasmic reticulum membrane to the cytosol. It is unclear whether this translocation results simply from its increased
hydrophilicity or is mediated by the enzyme or another transport protein (Russell 2003).
References
Reaction
15.8.2.1.1.6 5Beta-cholesten-7alpha, 12alpha-diol-3-one is reduced to 5beta-cholestan-3alpha, 7alpha, 12alpha-triol
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
5Beta-cholesten-7alpha,12alpha-diol-3-one and NADPH + H+ form 5beta-cholestan-3alpha,7alpha,12alpha-triol and NAPDP+. The reaction is

catalyzed by 3alpha-hydroxysteroid dehydrogenase (AKR1C4), a cytosolic enzyme belonging to the aldo-keto reductase family (Dufort et al.
2001). Biochemical studies with rat proteins raise the possibility that other related enzymes may also carry out this reaction in vivo (Russell
2003).
References
I Dufort, "Human types 1 and 3 3 alpha-hydroxysteroid dehydrogenases: differential lability and tissue distribution", J Clin Endocrinol Metab, 86,
2001, 841-6.
Reaction
15.8.2.1.1.7 5beta-cholestan-7alpha-ol-3-one is reduced to 5beta-cholestan-3alpha, 7alpha-diol
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
5Beta-cholesten-7alpha-ol-3-one and NADPH + H+ form 5beta-cholestan-3alpha,7alpha-diol and NAPDP+. The reaction is catalyzed by
3alpha-hydroxysteroid dehydrogenase (AKR1C4), a cytosolic enzyme belonging to the aldo-keto reductase family (Dufort et al. 2001).
Biochemical studies with rat proteins raise the possibility that other related enzymes may also carry out this reaction in vivo (Russell 2003).
References
2001, 841-6.
Reaction
15.8.2.1.1.8 5beta-cholestan-3alpha, 7alpha, 12alpha-triol is translocated from the cytosol to the mitochondrial matrix
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
5beta-cholestan-3alpha,7alpha,12alpha-triol is translocated from the cytosol to the mitochondrial matrix. The transporter that mediates its
passage across the inner mitochondrial membrane is unknown: the StAR protein that performs this function for cholesterol at the start of steroid
hormone biosynthesis is excluded as StAR is not expressed in liver. Other members of the START family of transporters are candidates,
however (Russell 2003).
References
Reaction
15.8.2.1.1.9 5beta-cholestan-3alpha, 7alpha-diol is translocated from the cytosol to the mitochondrial matrix
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
5beta-cholestan-3alpha, 7alpha-diol is transported from the cytosol to the mitochondrial matrix. The transporter that mediates its passage across
the inner mitochondrial membrane is unknown: the StAR protein that performs this function for cholesterol at the start of steroid hormone
biosynthesis is excluded as StAR is not expressed in liver. Other members of the START family of transporters are candidates, however (Russell
2003).
References
Reaction
15.8.2.1.1.10 5beta-cholestan-3alpha, 7alpha, 12alpha-triol is hydroxylated to 5beta-cholestan-3alpha, 7alpha, 12alpha, 27-tetrol
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
5beta-cholestan-3alpha, 7alpha, 12alpha-triol, NADPH + H+, and O2 react to form 5beta-cholestan-3alpha, 7alpha, 12alpha, 27-tetrol + NADP+
+ H2O. This reaction occurs in the mitochondrial matrix, catalyzed by CYP27A1.
References
JJ Cali, CL Hsieh, U Francke, DW Russell, "Mutations in the bile acid biosynthetic enzyme sterol 27-hydroxylase underlie cerebrotendinous
xanthomatosis", J Biol Chem, 266, 1991, 7779-83.
IA Pikuleva, A Babiker, MR Waterman, I Bjorkhem, "Activities of recombinant human cytochrome P450c27 (CYP27) which produce
intermediates of alternative bile acid biosynthetic pathways", J Biol Chem, 273, 1998, 18153-60.
Reaction
15.8.2.1.1.11 5beta-cholestan-3alpha, 7alpha-diol is hydroxylated to 5beta-cholestan-3alpha, 7alpha, 26-triol
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
5beta-cholestan-3alpha, 7alpha-diol, NADPH + H+, and O2 react to form 5beta-cholestan-3alpha, 7alpha, 26-triol + NADP+ + H2O. This
reaction occurs in the mitochondrial matrix, catalyzed by CYP27A1.
References
Reaction
15.8.2.1.1.12 5beta-cholestan-3alpha,7alpha,12alpha,27-tetrol is oxidized to 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestan-27-al
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
5beta-cholestan-3alpha, 7alpha, 12alpha, 27-tetrol, NADPH + H+, and O2 react to form 3alpha, 7alpha,
12alpha-trihydroxy-5beta-cholestan-27-al, NADP+, and H2O. This oxidation reaction occurs in the mitochondrial matrix, catalyzed by CYP27A1.
References
Reaction
15.8.2.1.1.13 5beta-cholestan-3alpha, 7alpha, 26-triol is oxidized to 3alpha, 7alpha-dihydroxy-5beta-cholestan-26-al
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
5beta-cholestan-3alpha, 7alpha, 26-triol, NADPH + H+, and O2 react to form 3alpha, 7alpha-dihydroxy-5beta-cholestan-26-al, NADP+, and
H2O. This oxidation reaction occurs in the mitochondrial matrix, catalyzed by CYP27A1.
References
Reaction
15.8.2.1.1.14 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestan-27-al is oxidized to

3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoate (THCA)
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
3alpha,7alpha,12alpha-trihydroxy-5beta-cholestan-27-al, NADPH + H+, and O2 react to form

3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoate (THCA), NADP+, and H2O. This oxidation reaction occurs in the mitochondrial matrix,
catalyzed by CYP27A1.
References
Reaction
15.8.2.1.1.15 3alpha, 7alpha-dihydroxy-5beta-cholestan-26-al is oxidized to 3alpha, 7alpha-dihydroxy-5beta-cholestanoate (DHCA)
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
3alpha, 7alpha-dihydroxy-5beta-cholestan-26-al, NADPH + H+, and O2 react to form 3alpha, 7alpha-dihydroxy-5beta-cholestanoate (DHCA),
NADP+ and H2O. This oxidation reaction occurs in the mitochondrial matrix, catalyzed by CYP27A1.
References
Reaction
15.8.2.1.1.16 THCA is translocated from the mitochondrial matrix to the cytosol
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
THCA (3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoate) is translocated from the mitochondrial matrix to the cytosol. The transporter that
mediates its passage across the inner mitochondrial membrane has not been identified. In particular, despite the structural and functional
similarities between THCA and long chain fatty acids, searches for carnitine - THCA have failed (Russell 2003). VLCS (SLC27A2), one of the
enzymes that catalyzes CoA conjugation of THCA may also be present in peroxisomes, and Mihalik et al. (2002) have hypothesized that THCA
could be translocated unchanged from the mitochondrial matrix to the peroxisomal matrix and undergo conjugation there.
References
SJ Mihalik, SJ Steinberg, Z Pei, J Park, DG Kim, AK Heinzer, G Dacremont, RJ Wanders, DA Cuebas, KD Smith, PA Watkins, "Participation of
two members of the very long-chain acyl-CoA synthetase family in bile acid synthesis and recycling", J Biol Chem, 277, 2002, 24771-9.
Reaction
15.8.2.1.1.17 DHCA is translocated from the mitochondrial matrix to the cytosol
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
DHCA (3alpha, 7alpha-dihydroxy-5beta-cholestanoate) is translocated from the mitochondrial matrix to the cytosol. The transporter that
mediates its passage across the inner mitochondrial membrane has not been identified. In particular, despite the structural and functional
similarities between DHCA and long chain fatty acids, searches for carnitine - DHCA have failed (Russell 2003). VLCS (SLC27A2), one of the
enzymes that catalyzes CoA conjugation of DHCA, may also be present in peroxisomes, and Mihalik et al. (2002) have hypothesized that DHCA
could be translocated unchanged from the mitochondrial matrix to the peroxisomal matrix and undergo conjugation there.
References
Reaction
15.8.2.1.1.18 THCA is conjugated with Coenzyme A (SLC27A2 VLCS)
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
THCA (25(R) 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoate), coenzyme A, and ATP react to form the CoA conjugate of 25(R) THCA,
AMP, pyrophosphate and water. This cytosolic reaction is catalyzed by SLC27A2 (VLCS). SLC27A5 (BACS) also catalyzes this reaction; the
relative contributions of the two enzymes to de novo bile acid synthesis in vivo are not certain (Mihalik et al. 2002).
References
SJ Steinberg, SJ Mihalik, DG Kim, DA Cuebas, PA Watkins, "The human liver-specific homolog of very long-chain acyl-CoA synthetase is
cholate:CoA ligase", J Biol Chem, 275, 2000, 15605-8.
SJ Steinberg, SJ Wang, MC McGuinness, PA Watkins, "Human liver-specific very-long-chain acyl-coenzyme A synthetase: cDNA cloning and
characterization of a second enzymatically active protein", Mol Genet Metab, 68, 1999, 32-42.
Reaction
15.8.2.1.1.19 DHCA is conjugated with Coenzyme A (SLC27A2 VLCS)
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
DHCA (25(R) 3alpha,7alpha-dihydroxy-5beta-cholestanoate), coenzyme A, and ATP react to form the CoA conjugate of 25(R) DHCA, AMP,
pyrophosphate and water. This cytosolic reaction is catalyzed by SLC27A2 (VLCS). SLC27A5 (BACS) also catalyzes this reaction; the relative
contributions of the two enzymes to de novo bile acid synthesis in vivo are not certain (Mihalik et al. 2002).
References
Reaction
15.8.2.1.1.20 THCA is conjugated with Coenzyme A (SLC27A5 BACS)
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
THCA (25(R) 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoate) , coenzyme A, and ATP react to form the CoA conjugate of 25(R) THCA,
AMP, pyrophosphate and water. This cytosolic reaction is catalyzed by SLC27A5 (BACS). SLC27A2 (VLCS) also catalyzes this reaction; the
relative contributions of the two enzymes to de novo bile acid synthesis in vivo are not certain (Mihalik et al. 2002).
References
Reaction
15.8.2.1.1.21 DHCA is conjugated with Coenzyme A (SLC27A5 BACS)
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
DHCA (25(R) 3alpha,7alpha-dihydroxy-5beta-cholestanoate) , coenzyme A, and ATP react to form the CoA conjugate of 25(R) DHCA, AMP,
pyrophosphate and water. This cytosolic reaction is catalyzed by SLC27A5 (BACS). SLC27A2 (VLCS) also catalyzes this reaction; the relative
contributions of the two enzymes to de novo bile acid synthesis in vivo are not certain (Mihalik et al. 2002).
References
Reaction
15.8.2.1.1.22 25(R) THCA-CoA is translocated from the cytosol to the peroxisome
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
25(R) THCA-CoA is transported from the cytosol into the peroxisome. Indirect evidence suggests that a member of the ABC class of transporters
mediates this reaction.
References
M Une, Y Iguchi, T Sakamoto, T Tomita, Y Suzuki, M Morita, T Imanaka, "ATP-dependent transport of bile acid intermediates across rat liver
peroxisomal membranes", J Biochem (Tokyo), 134, 2003, 225-30.
Reaction
15.8.2.1.1.23 25(R) DHCA-CoA is translocated from the cytosol to the peroxisome
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
25(R) DHCA-CoA is transported from the cytosol into the peroxisome. Indirect evidence suggests that a member of the ABC class of
transporters mediates this reaction.
References
Reaction
15.8.2.1.1.24 Isomerization of 25(R) THCA-CoA to 25(S) THCA-CoA
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
The isomerization of 25(R) THCA-CoA to 25(S) THCA-CoA, catalyzed by 2-methylacyl-CoA racemase, occurs in the peroxisomal matrix.
References
S Ferdinandusse, S Denis, PT Clayton, A Graham, JE Rees, JT Allen, BN McLean, AY Brown, P Vreken, HR Waterham, RJ Wanders,
"Mutations in the gene encoding peroxisomal alpha-methylacyl-CoA racemase cause adult-onset sensory motor neuropathy", Nat Genet, 24,
2000, 188-91.
L Amery, M Fransen, K De Nys, GP Mannaerts, PP Van Veldhoven, "Mitochondrial and peroxisomal targeting of 2-methylacyl-CoA racemase in
humans", J Lipid Res, 41, 2000, 1752-9.
W Schmitz, C Albers, R Fingerhut, E Conzelmann, "Purification and characterization of an alpha-methylacyl-CoA racemase from human liver",
Eur J Biochem, 231, 1995, 815-22.
Reaction
15.8.2.1.1.25 Isomerization of 25(R) DHCA-CoA to 25(S) DHCA-CoA
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
The isomerization of 25(R) DHCA-CoA to 25(S) DHCA-CoA, catalyzed by 2-methylacyl-CoA racemase, occurs in the peroxisomal matrix.
References
2000, 188-91.
Eur J Biochem, 231, 1995, 815-22.
Reaction
15.8.2.1.1.26 25(S) THCA-CoA is dehydrogenated to 25(S) 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-24-enoyl-CoA
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
25(S) THCA-CoA and O2 react to form 25(S) 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-24-enoyl-CoA and H2O2. This dehydrogenation
reaction occurs in the peroxisomal matrix. It is catalyzed by the FAD-containing peroxisomal enzyme branched chain acyl-CoA oxidase
(ACOX2). The enzyme transfers electrons to molecular oxygen and hydrogen peroxide is produced as a byproduct.
References
E Baumgart, JC Vanhooren, M Fransen, P Marynen, M Puype, J Vandekerckhove, JA Leunissen, HD Fahimi, GP Mannaerts, PP Van
Veldhoven, "Molecular characterization of the human peroxisomal branched-chain acyl-CoA oxidase: cDNA cloning, chromosomal assignment,
tissue distribution, and evidence for the absence of the protein in Zellweger syndrome", Proc Natl Acad Sci U S A, 93, 1996, 13748-53.
GF Vanhove, PP Van Veldhoven, M Fransen, S Denis, HJ Eyssen, RJ Wanders, GP Mannaerts, "The CoA esters of 2-methyl-branched chain
fatty acids and of the bile acid intermediates di- and trihydroxycoprostanic acids are oxidized by one single peroxisomal branched chain
acyl-CoA oxidase in human liver and kidney", J Biol Chem, 268, 1993, 10335-44.
Reaction
15.8.2.1.1.27 25(S) DHCA-CoA is dehydrogenated to 25(S) 3alpha,7alpha-dihydroxy-5beta-cholest-24-enoyl-CoA
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
25(S) DHCA-CoA and O2 react to form 25(S) 3alpha,7alpha-dihydroxy-5beta-cholest-24-enoyl-CoA and H2O2. This dehydrogenation reaction
occurs in the peroxisomal matrix. It is catalyzed by the FAD-containing peroxisomal enzyme branched chain acyl-CoA oxidase (ACOX2). The
enzyme transfers electrons to molecular oxygen and hydrogen peroxide is produced as a byproduct.
References
E Baumgart, JC Vanhooren, M Fransen, P Marynen, M Puype, J Vandekerckhove, JA Leunissen, HD Fahimi, GP Mannaerts, PP Van
Veldhoven, "Molecular characterization of the human peroxisomal branched-chain acyl-CoA oxidase: cDNA cloning, chromosomal assignment,
tissue distribution, and evidence for the absence of the protein in Zellweger syndrome", Proc Natl Acad Sci U S A, 93, 1996, 13748-53.
GF Vanhove, PP Van Veldhoven, M Fransen, S Denis, HJ Eyssen, RJ Wanders, GP Mannaerts, "The CoA esters of 2-methyl-branched chain
fatty acids and of the bile acid intermediates di- and trihydroxycoprostanic acids are oxidized by one single peroxisomal branched chain
acyl-CoA oxidase in human liver and kidney", J Biol Chem, 268, 1993, 10335-44.
Reaction
15.8.2.1.1.28 25(S) 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-24-enoyl-CoA is hydrated to (24R, 25R)

3alpha,7alpha,12alpha,24-tetrahydroxy-5beta-cholestanoyl-CoA
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
25(S) 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-24-enoyl-CoA is hydrated to form (24R, 25R)

3alpha,7alpha,12alpha,24-tetrahydroxy-5beta-cholestanoyl-CoA. This reaction, catalyzed by the peroxisomal D-bifunctional enzyme (Huyghe et
al. 2006), occurs in the peroxisomal matrix.
References
S Huyghe, GP Mannaerts, M Baes, PP Van Veldhoven, "Peroxisomal multifunctional protein-2: the enzyme, the patients and the knockout
mouse model", Biochim Biophys Acta, 1761, 2006, 973-94.
LL Jiang, A Kobayashi, H Matsuura, H Fukushima, T Hashimoto, "Purification and properties of human D-3-hydroxyacyl-CoA dehydratase:
medium-chain enoyl-CoA hydratase is D-3-hydroxyacyl-CoA dehydratase", J Biochem (Tokyo), 120, 1996, 624-32.
Reaction
15.8.2.1.1.29 25(S) 3alpha,7alpha-dihydroxy-5beta-cholest-24-enoyl-CoA is hydrated to (24R, 25R)

3alpha,7alpha,24-trihydroxy-5beta-cholestanoyl-CoA
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
25(S) 3alpha,7alpha-dihydroxy-5beta-cholest-24-enoyl-CoA is hydrated to form (24R, 25R)

3alpha,7alpha,24-trihydroxy-5beta-cholestanoyl-CoA. This reaction, catalyzed by the peroxisomal D-bifunctional enzyme (Huyghe et al. 2006),
occurs in the peroxisomal matrix.
References
Reaction
15.8.2.1.1.30 (24R, 25R) 3alpha,7alpha,12alpha,24-tetrahydroxy-5beta-cholestanoyl-CoA is oxidized to

3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-24-one-CoA
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
(24R, 25R) 3alpha,7alpha,12alpha,24-tetrahydroxy-5beta-cholestanoyl-CoA and NAD+ react to form

3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-24-one-CoA, NADH, and H+. This oxidation reaction, catalyzed by the peroxisomal
D-bifunctional enzyme (Huyghe et al. 2006), occurs in the peroxisomal matrix.
References
Reaction
15.8.2.1.1.31 (24R, 25R) 3alpha,7alpha,24-trihydroxy-5beta-cholestanoyl-CoA is oxidized to

3alpha,7alpha-dihydroxy-5beta-cholest-24-one-CoA
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
(24R, 25R) 3alpha,7alpha,24-trihydroxy-5beta-cholestanoyl-CoA and NAD+ react to form 3alpha,7alpha-dihydroxy-5beta-cholest-24-one-CoA,

NADH, and H+. This oxidation reaction, catalyzed by the peroxisomal D-bifunctional enzyme (Huyghe et al. 2006), occurs in the peroxisomal
matrix.
References
Reaction
15.8.2.1.1.32 Thiolysis of 3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-one-CoA yields choloyl-CoA

(3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-one-CoA) and propionyl CoA
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-one-CoA and CoASH react to form choloyl-CoA

(3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-one-CoA) and propionyl CoA. This reaction, in the peroxisomal matrix, is catalyzed by
peroxisomal thiolase 2 (sterol carrier protein 2).
References
S Ferdinandusse, S Denis, E van Berkel, G Dacremont, RJ Wanders, "Peroxisomal fatty acid oxidation disorders and 58 kDa sterol carrier
protein X (SCPx). Activity measurements in liver and fibroblasts using a newly developed method.", J Lipid Res, 41, 2000, 336-42.
R Yamamoto, CB Kallen, GO Babalola, H Rennert, JT Billheimer, 3rd Strauss JF, "Cloning and expression of a cDNA encoding human sterol
carrier protein 2", Proc Natl Acad Sci U S A, 88, 1991, 463-7.
Reaction
15.8.2.1.1.33 Thiolysis of 3alpha,7alpha-dihydroxy-5beta-cholan-24-one-CoA yields chenodeoxycholoyl-CoA

(3alpha,7alpha-dihydroxy-5beta-cholan-24-one-CoA) and propionyl CoA
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
3alpha,7alpha-dihydroxy-5beta-cholan-24-one-CoA and CoASH react to form chenodeoxycholoyl-CoA

(3alpha,7alpha-dihydroxy-5beta-cholan-24-one-CoA) and propionyl CoA. This reaction, in the peroxisomal matrix, is catalyzed by peroxisomal
thiolase 2 (sterol carrier protein 2).
References
S Ferdinandusse, S Denis, E van Berkel, G Dacremont, RJ Wanders, "Peroxisomal fatty acid oxidation disorders and 58 kDa sterol carrier
protein X (SCPx). Activity measurements in liver and fibroblasts using a newly developed method.", J Lipid Res, 41, 2000, 336-42.
R Yamamoto, CB Kallen, GO Babalola, H Rennert, JT Billheimer, 3rd Strauss JF, "Cloning and expression of a cDNA encoding human sterol
carrier protein 2", Proc Natl Acad Sci U S A, 88, 1991, 463-7.
Reaction
15.8.2.1.1.34 Hydrolysis of choloyl-CoA to cholate and CoASH
Authors
Editors
Reviewers
Description
Choloyl-CoA and water react to form cholate and CoASH. This reaction, catalyzed by acyl-coenzyme A thioesterase 8, occurs in the peroxisomal
matrix (Jones et al. 1999; Hunt et al. 2002). While bile salts are the major product of the de novo biosynthetic pathway in the normal human liver,
bile acids are major feedback regulators of this pathway and this hydrolysis reaction is thought to play a role in generating them (Russell 2003).
References
JM Jones, K Nau, MT Geraghty, R Erdmann, SJ Gould, "Identification of peroxisomal acyl-CoA thioesterases in yeast and humans", J Biol
Chem, 274, 1999, 9216-23.
MC Hunt, K Solaas, BF Kase, SE Alexson, "Characterization of an acyl-coA thioesterase that functions as a major regulator of peroxisomal lipid
metabolism", J Biol Chem, 277, 2002, 1128-38.
Reaction
15.8.2.1.1.35 Cholate is translocated from the peroxisomal matrix to the cytosol
Authors
Editors
Reviewers
Description
Cholate is translocated from the peroxisomal matrix to the cytosol. The transporter that mediates this event is unknown.
References
MC Hunt, K Solaas, BF Kase, SE Alexson, "Characterization of an acyl-coA thioesterase that functions as a major regulator of peroxisomal lipid
metabolism", J Biol Chem, 277, 2002, 1128-38.
Reaction
15.8.2.1.1.36 Choloyl CoA reacts with glycine or taurine to form glycocholate or taurocholate
Authors
Jassal, B, 2007-01-31.
Editors
Reviewers
Description
Choloyl CoA reacts with glycine or taurine to form glycocholate or taurocholate, releasing CoASH. This reaction, which completes the de novo
synthesis of bile salts from cholesterol in vivo, is catalyzed by BAAT (Bile acid CoA:amino acid N-acyltransferase - Falany et al. 1994) and
occurs in the peroxisomal matrix (Solaas et al. 2000; Mihalik et al. 2002). In vivo, the relative amounts of glycocholate and taurocholate
synthesized appear to be determined solely by the intracellular abundances of glycine and taurine (Russell 2003).
References
K Solaas, A Ulvestad, O Soreide, BF Kase, "Subcellular organization of bile acid amidation in human liver: a key issue in regulating the
biosynthesis of bile salts", J Lipid Res, 41, 2000, 1154-62.
CN Falany, MR Johnson, S Barnes, RB Diasio, "Glycine and taurine conjugation of bile acids by a single enzyme. Molecular cloning and
expression of human liver bile acid CoA:amino acid N-acyltransferase.", J Biol Chem, 269, 1994, 19375-9.
Reaction
15.8.2.1.1.37 Chenodeoxycholoyl CoA reacts with glycine or taurine to form glycochenodeoxycholate or taurochenodeoxycholate
Authors
Jassal, B, 2007-01-31.
Editors
Reviewers
Description
Chenodeoxycholoyl CoA reacts with glycine or taurine to form glycochenodeoxycholate or taurochenodeoxycholate, releasing CoASH. This
reaction, which completes the de novo synthesis of bile salts from cholesterol in vivo, is catalyzed by BAAT (Bile acid CoA:amino acid
N-acyltransferase - Falany et al. 1994) and occurs in the peroxisomal matrix (Solaas et al. 2000; Mihalik et al. 2002). In vivo, the relative
amounts of glycochenodeoxycholate and taurochenodeoxycholate synthesized appear to be determined solely by the intracellular abundances
of glycine and taurine (Russell 2003).
References
Reaction
15.8.2.1.1.38 Bile salts are translocated from the peroxisomal matrix to the cytosol
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
The bile salts glycocholate, glycochenodeoxycholate, taurocholate, and taurochenodeoxycholate are translocated from the peroxisomal matrix to
the cytosol. The transporter that mediates this process is unknown (Russell 2003).
References
Reaction
15.8.2.1.1.39 Transport (efflux) of bile salts by ABCB11 (bile salt export pump)
Authors
Editors
Reviewers
Description
A molecule of glycocholate, glycochenodeoxycholate, taurocholate, or taurochenodeoxycholate is transported from the cytosol to the
extracellular space, coupled to the hydrolysis of a molecule of cytosolic ATP to ADP and orthophosphate. This reaction is mediated by ABCB11
(bile salt export pump). In the body, this reaction mediates the release of bile salts from the liver cells into the bile (Kullak-Ublick et al. 2004); the
role of ABCB11 in the reaction is confirmed by the observed failure of bile salt export in patients in whom the transporter is defective (Noe et al.
2005).
References
JA Byrne, SS Strautnieks, G Mieli-Vergani, CF Higgins, KJ Linton, RJ Thompson, "The human bile salt export pump: characterization of
substrate specificity and identification of inhibitors", Gastroenterology, 123, 2002, 1649-58.
2004, 322-42.
J Noe, GA Kullak-Ublick, W Jochum, B Stieger, R Kerb, M Haberl, B Mullhaupt, PJ Meier, C Pauli-Magnus, "Impaired expression and function of
the bile salt export pump due to three novel ABCB11 mutations in intrahepatic cholestasis", J Hepatol, 43, 2005, 536-43.
J Noe, B Stieger, PJ Meier, "Functional expression of the canalicular bile salt export pump of human liver", Gastroenterology, 123, 2002,
1659-66.
Reaction
15.8.2.1.2 Synthesis of bile acids and bile salts via 24-hydroxycholesterol
Authors
Editors
Reviewers
Description
In the body, 24-hydroxycholesterol is synthesized in the brain, exported to the liver, and converted there to bile acids and bile salts. This pathway
is only a minor source of bile acids and bile salts, but appears to be critical for the disposal of excess cholesterol from the brain (Bjorkhem et al.
1998; Javitt 2002).
In the liver, conversion of 24-hydroxycholesterol to bile acids and bile salts is initiated with hydroxylation and oxidoreductase reactions to form
4-cholesten-7alpha,24(S)-diol-3-one. The pathway then branches: hydroxylation of 4-cholesten-7alpha,24(S)-diol-3-one to
4-cholesten-7alpha,12alpha,24(S)-triol-3-one leads ultimately to the formation of cholate, while its reduction to
5beta-cholestan-7alpha,24(S)-diol-3-one leads to chenodeoxycholate formation. In both branches, reactions in the cytosol, the mitochondrial
matrix, and the peroxisomal matrix result in modifications to the ring structure, shortening and oxidation of the side chain, conversion to a
Coenzyme A derivative, and conjugation with the amino acids glycine or taurine (Russell 2003). These reactions are outlined in the figure below.
The final three reactions are identical to ones of bile salt synthesis initiated by 7alpha-hydroxylation and are shown as arrows with no substrates.
References
I Bjorkhem, D Lutjohann, U Diczfalusy, L Stahle, G Ahlborg, J Wahren, "Cholesterol homeostasis in human brain: turnover of
24S-hydroxycholesterol and evidence for a cerebral origin of most of this oxysterol in the circulation", J Lipid Res, 39, 1998, 1594-600.
15.8.2.1.2.1 Cholesterol is hydroxylated to 24-hydroxycholesterol by CYP46A1
Authors
Jassal, B, 2008-05-19.
Editors
Reviewers
Description
Cholesterol, NADPH + H+, and O2 react to form 24-hydroxycholesterol , NADP+, and H2O. This reaction is catalyzed by CYP46A1 in the
endoplasmic reticulum membrane. In the body, this enzyme is expressed predominantly in the brain and is thought to play a major role in
cholesterol turnover there (Javitt 2002).
References
EG Lund, JM Guileyardo, DW Russell, "cDNA cloning of cholesterol 24-hydroxylase, a mediator of cholesterol homeostasis in the brain", Proc
Natl Acad Sci U S A, 96, 1999, 7238-43.
N Mast, R Norcross, U Andersson, M Shou, K Nakayama, I Bjorkhem, IA Pikuleva, "Broad substrate specificity of human cytochrome P450 46A1
which initiates cholesterol degradation in the brain", Biochemistry, 42, 2003, 14284-92.
Reaction
15.8.2.1.2.2 Efflux of 24-hydroxycholesterol
Authors
Editors
Reviewers
Description
24-hydroxycholesterol is transported from the endoplasmic reticulum to the extracellular space. In humans, this event is the major source of
24-hydroxycholesterol in the blood and is the means by which the molecule, generated from cholesterol in the brain, is transported to the liver for
conversion to bile acids and bile salts. While transport proteins are likely to play a role in this process, and the 24-hydroxycholesterol is likely to
occur as part of a lipoprotein complex in the blood, the relevant proteins have not been identified (Lutjohann et al. 1996; Bjorkhem et al. 1998).
References
D Lutjohann, O Breuer, G Ahlborg, I Nennesmo, A Siden, U Diczfalusy, I Bjorkhem, "Cholesterol homeostasis in human brain: evidence for an
age-dependent flux of 24S-hydroxycholesterol from the brain into the circulation", Proc Natl Acad Sci U S A, 93, 1996, 9799-804.
Reaction
15.8.2.1.2.3 Influx of 24-hydroxycholesterol
Authors
Editors
Reviewers
Description
24-hydroxycholesterol is transported from the extracellular space to the endoplasmic reticulum. In humans, this event is the means by which the
molecule, generated from cholesterol in the brain, is taken up by liver cells for conversion to bile acids and bile salts. While transport proteins are
likely to play a role in this process, these proteins have not been identified (Lutjohann et al. 1996; Bjorkhem et al. 1998).
References
Reaction
15.8.2.1.2.4 24-hydroxycholesterol is 7alpha-hydroxylated to yield cholest-5-ene-3beta,7alpha,24-triol
Authors
Jassal, B, 2008-05-19.
Editors
Reviewers
Description
24-hydroxycholesterol, NADPH + H+, and O2 react to form cholest-5-ene-3beta,7alpha,24-triol, NADP+, and H2O. This hydroxylation reaction is
catalyzed by CYP39A1 in the membrane of the endoplasmic reticulum. In the body, expression of CYP39A1 is restricted to the liver.
References
J Li-Hawkins, EG Lund, AD Bronson, DW Russell, "Expression cloning of an oxysterol 7alpha-hydroxylase selective for 24-hydroxycholesterol",
J Biol Chem, 275, 2000, 16543-9.
Reaction
15.8.2.1.2.5 Cholest-5-ene-3beta,7alpha,24(S)-triol is oxidized and isomerized to 4-cholesten-7alpha,24(S)-diol-3-one
Authors
Editors
Reviewers
Description
Cholest-5-ene-3beta,7alpha,24(S)-triol and NAD+ react to form 4-cholesten-7alpha,24(S)-diol-3-one and NADH + H+, catalyzed by HSD3B7 (3
beta-hydroxysteroid dehydrogenase type 7) in the endoplasmic reticulum membrane. Its function in vivo has been confirmed in studies of
patients with defects in bile acid synthesis (Schwarz et al. 2000).
References
Metab, 88, 2003, 1833-41.
Reaction
15.8.2.1.2.6 4-Cholesten-7alpha,24(S)-diol-3-one is 12alpha-hydroxylated to 4-cholesten-7-alpha,12-alpha,24(S)-triol-3-one
Authors
Jassal, B, 2008-05-19.
Editors
Reviewers
Description
4-Cholesten-7alpha,24(S)-diol-3-one, NADPH + H+, and O2 form 4-cholesten-7-alpha,12-alpha,24(S)-triol-3-one + NADP+ + H2O. This reaction
References
Genomics, 56, 1999, 184-96.
Chem, 267, 1992, 21319-23.
Reaction
15.8.2.1.2.7 4-cholesten-7alpha,12alpha,24(S)-triol-3-one is reduced to 5beta-cholestan-7alpha,12alpha,24(S)-triol-3-one
Authors
Editors
Reviewers
Description
4-cholesten-7alpha,12alpha,24(S)-triol-3-one and NADPH + H+ react to form 5beta-cholesten-7alpha,12alpha,24(S)-triol-3-one and NADP+.

This reaction is catalyzed by AKR1D1 (3-oxo-5-beta-steroid 4-dehydrogenase). AKR1D1 is localized to the cytosol, and in the course of the
reaction its steroid substrate moves from the endoplasmic reticulum membrane to the cytosol. It is unclear whether this translocation results
simply from its increased hydrophilicity or is mediated by the enzyme or another transport protein (Russell 2003).
References
Reaction
15.8.2.1.2.8 4-cholesten-7alpha,24(S)-diol-3-one is reduced to 5beta-cholestan-7alpha,24(S)-diol-3-one
Authors
Editors
Reviewers
Description
4-Cholesten-7alpha,24(S)-diol-3-one, NADPH, and H+ react to form 5beta-cholestan-7alpha,24(S)-diol-3-one and NADP+. This reaction is
catalyzed by AKR1D1 (3-oxo-5-beta-steroid 4-dehydrogenase). AKR1D1 is localized to the cytosol, and in the course of the reaction its steroid
substrate moves from the endoplasmic reticulum membrane to the cytosol. It is unclear whether this translocation results simply from its
increased hydrophilicity or is mediated by the enzyme or another transport protein (Russell 2003).
References
Reaction
15.8.2.1.2.9 5Beta-cholestan-7alpha,12alpha,24(S)-triol-3-one is reduced to 5beta-cholestan-3alpha,7alpha,12alpha,24(S)-tetrol
Authors
Editors
Reviewers
Description
5Beta-cholesten-7alpha,12alpha,24(S)-triol-3-one and NADPH + H+ form 5beta-cholestan-3alpha,7alpha,12alpha,24(S)-tetrol and NAPDP+.

The reaction is catalyzed by 3alpha-hydroxysteroid dehydrogenase (AKR1C4), a cytosolic enzyme belonging to the aldo-keto reductase family
(Dufort et al. 2001). Biochemical studies with rat proteins raise the possibility that other related enzymes may also carry out this reaction in vivo
(Russell 2003).
References
2001, 841-6.
Reaction
15.8.2.1.2.10 5beta-cholestan-7alpha,24(S)-diol-3-one is reduced to 5beta-cholestan-3alpha,7alpha,24(S)-triol
Authors
Editors
Reviewers
Description
5Beta-cholesten-7alpha,24(S)-diol-3-one and NADPH + H+ form 5beta-cholestan-3alpha,7alpha,24(S)-triol and NAPDP+. The reaction is

2003).
References
2001, 841-6.
Reaction
15.8.2.1.2.11 5beta-cholestan-3alpha,7alpha,12alpha,24(S)-tetrol is translocated from the cytosol to the mitochondrial matrix
Authors
Editors
Reviewers
Description
5beta-cholestan-3alpha,7alpha,12alpha,24(S)-tetrol is translocated from the cytosol to the mitochondrial matrix. The transporter that mediates its
References
Reaction
15.8.2.1.2.12 5beta-cholestan-3alpha,7alpha,24(S)-triol is translocated from the cytosol to the mitochondrial matrix
Authors
Editors
Reviewers
Description
5beta-cholestan-3alpha,7alpha,24(S)-triol is transported from the cytosol to the mitochondrial matrix. The transporter that mediates its passage
across the inner mitochondrial membrane is unknown: the StAR protein that performs this function for cholesterol at the start of steroid hormone
2003).
References
Reaction
15.8.2.1.2.13 5beta-cholestan-3alpha,7alpha,12alpha,24(S)-tetrol is hydroxylated to 5beta-cholestan-3alpha,7alpha,12alpha,24(S),

27-pentol
Authors
Editors
Reviewers
Description
5beta-cholestan-3alpha,7alpha,12alpha,24(S)-tetrol, NADPH + H+, and O2 react to form

5beta-cholestan-3alpha,7alpha,12alpha,24(S),27-pentol, NADP+, and H2O. This reaction occurs in the mitochondrial matrix, catalyzed by
CYP27A1.
References
Reaction
15.8.2.1.2.14 5beta-cholestan-3alpha,7alpha,24(S)-triol is hydroxylated to 5beta-cholestan-3alpha,7alpha,24(S), 27-tetrol
Authors
Editors
Reviewers
Description
5beta-cholestan-3alpha,7alpha,24(S)-triol, NADPH + H+, and O2 react to form 5beta-cholestan-3alpha,7alpha,24(S),27-tetrol, NADP+, and

H2O. This reaction occurs in the mitochondrial matrix, catalyzed by CYP27A1.
References
Reaction
15.8.2.1.2.15 5beta-cholestan-3alpha,7alpha,12alpha,24(S),27-pentol is oxidized to

3alpha,7alpha,12alpha,24(S)-tetrahydroxy-5beta-cholestan-27-al
Authors
Editors
Reviewers
Description
5beta-cholestan-3alpha,7alpha,12alpha,24(S),27-pentol, NADPH + H+, and O2 react to form

3alpha,7alpha,12alpha,24(S)-tetrahydroxy-5beta-cholestan-27-al, NADP+, and H2O. This oxidation reaction occurs in the mitochondrial matrix,
catalyzed by CYP27A1.
References
Reaction
15.8.2.1.2.16 5beta-cholestan-3alpha,7alpha,24(S),27-tetrol is oxidized to 3alpha,7alpha,24(S)-trihydroxy-5beta-cholestan-27-al
Authors
Editors
Reviewers
Description
5beta-cholestan-3alpha,7alpha,24(S),27-tetrol, NADPH + H+, and O2 react to form 3alpha,7alpha,24(S)-trihydroxy-5beta-cholestan-27-al,

NADP+, and H2O. This oxidation reaction occurs in the mitochondrial matrix, catalyzed by CYP27A1.
References
Reaction
15.8.2.1.2.17 3alpha,7alpha,12alpha,24(S)-tetrahydroxy-5beta-cholestan-27-al is oxidized to

3alpha,7alpha,12alpha,24(S)-tetrahydroxy-5beta-cholestanoate (TetraHCA)
Authors
Editors
Reviewers
Description
3alpha,7alpha,12alpha,24(S)-tetrahydroxy-5beta-cholestan-27-al, NADPH + H+, and O2 react to form

3alpha,7alpha,12alpha,24(S)-tetrahydroxy-5beta-cholestanoate (TetraHCA), NADP+, and H2O. This oxidation reaction occurs in the
mitochondrial matrix, catalyzed by CYP27A1.
References
Reaction
15.8.2.1.2.18 3alpha,7alpha,24(S)-trihydroxy-5beta-cholestan-27-al is oxidized to 3alpha,7alpha,24(S)-trihydroxy-5beta-cholestanoate

(3,7,24THCA)
Authors
Editors
Reviewers
Description
3alpha,7alpha,24(S)-trihydroxy-5beta-cholestan-27-al, NADPH + H+, and O2 react to form 3alpha,7alpha,24(S)-trihydroxy-5beta-cholestanoate

(3,7,24THCA), NADP+, and H2O. This oxidation reaction occurs in the mitochondrial matrix, catalyzed by CYP27A1.
References
Reaction
15.8.2.1.2.19 TetraHCA is translocated from the mitochondrial matrix to the cytosol
Authors
Editors
Reviewers
Description
TetraHCA (3alpha,7alpha,12alpha,24(S)-tetrahydroxy-5beta-cholestanoate) is translocated from the mitochondrial matrix to the cytosol. The
transporter that mediates its passage across the inner mitochondrial membrane has not been identified (Russell 2003). VLCS (SLC27A2), one of
the enzymes that catalyzes CoA conjugation of TetraHCA, may also be present in peroxisomes, and Mihalik et al. (2002) have hypothesized that
TetraHCA could be translocated unchanged from the mitochondrial matrix to the peroxisomal matrix and undergo conjugation there.
References
Reaction
15.8.2.1.2.20 3,7,24THCA is translocated from the mitochondrial matrix to the cytosol
Authors
Editors
Reviewers
Description
3,7,24THCA (3alpha,7alpha,24(S)-trihydroxy-5beta-cholestanoate) is translocated from the mitochondrial matrix to the cytosol. The transporter
that mediates its passage across the inner mitochondrial membrane has not been identified (Russell 2003). VLCS (SLC27A2), one of the
enzymes that catalyzes CoA conjugation of 3,7,24THCA, may also be present in peroxisomes, and Mihalik et al. (2002) have hypothesized that
3,7,24THCA could be translocated unchanged from the mitochondrial matrix to the peroxisomal matrix and undergo conjugation there.
References
Reaction
15.8.2.1.2.21 TetraHCA is conjugated with Coenzyme A (SLC27A5 BACS)
Authors
Editors
Reviewers
Description
TetraHCA (25(R) 3alpha,7alpha,12alpha,24(S)-tetrahydroxy-5beta-cholestanoate), coenzyme A, and ATP react to form the CoA conjugate of
25(R) TetraHCA, AMP, pyrophosphate and water. This cytosolic reaction is catalyzed by SLC27A5 (BACS). SLC27A2 (VLCS) also catalyzes
this reaction; the relative contributions of the two enzymes to de novo bile acid synthesis in vivo are not certain (Mihalik et al. 2002).
References
Reaction
15.8.2.1.2.22 3,7,24THCA is conjugated with Coenzyme A (SLC27A5 BACS)
Authors
Editors
Reviewers
Description
3,7,24THCA (25(R) 3alpha,7alpha,24(S)-trihydroxy-5beta-cholestanoate), coenzyme A, and ATP react to form the CoA conjugate of
3,7,24THCA, AMP, pyrophosphate and water. This cytosolic reaction is catalyzed by SLC27A5 (BACS). SLC27A2 (VLCS) also catalyzes this
reaction; the relative contributions of the two enzymes to de novo bile acid synthesis in vivo are not certain (Mihalik et al. 2002).
References
Reaction
15.8.2.1.2.23 TetraHCA is conjugated with Coenzyme A (SLC27A2 VLCS)
Authors
Editors
Reviewers
Description
TetraHCA (25(R) 3alpha,7alpha,12alpha,24(S)-tetrahydroxy-5beta-cholestanoate), coenzyme A, and ATP react to form the CoA conjugate of
25(R) TetraHCA, AMP, pyrophosphate and water. This cytosolic reaction is catalyzed by SLC27A2 (VLCS). SLC27A5 (BACS) also catalyzes
this reaction; the relative contributions of the two enzymes to de novo bile acid synthesis in vivo are not certain (Mihalik et al. 2002).
References
Reaction
15.8.2.1.2.24 3,7,24THCA is conjugated with Coenzyme A (SLC27A2 VLCS)
Authors
Editors
Reviewers
Description
3,7,24THCA (25(R) 3alpha,7alpha,24(S)-trihydroxy-5beta-cholestanoate), coenzyme A, and ATP react to form the CoA conjugate of
3,7,24THCA, AMP, pyrophosphate and water. This cytosolic reaction is catalyzed by SLC27A2 (VLCS). SLC27A5 (BACS) also catalyzes this
reaction; the relative contributions of the two enzymes to de novo bile acid synthesis in vivo are not certain (Mihalik et al. 2002).
References
Reaction
15.8.2.1.2.25 25(R) TetraHCA-CoA is translocated from the cytosol to the peroxisome
Authors
Editors
Reviewers
Description
25(R) TetraHCA-CoA is transported from the cytosol into the peroxisome.
References
Reaction
15.8.2.1.2.26 3,7,24THCA-CoA is translocated from the cytosol to the peroxisome
Authors
Editors
Reviewers
Description
3,7,24THCA-CoA is transported from the cytosol into the peroxisome.

References
Reaction
15.8.2.1.2.27 Isomerization of 25(R) TetraHCA-CoA to (24R, 25R) 3alpha,7alpha,12alpha,24-tetrahydroxy-5beta-cholestanoyl-CoA
Authors
Editors
Reviewers
Description
The isomerization of 25(R) TetraHCA-CoA to (24R, 25R) 3alpha,7alpha,12alpha,24-tetrahydroxy-5beta-cholestanoyl-CoA, catalyzed by

2-methylacyl-CoA racemase, occurs in the peroxisomal matrix.
References
2000, 188-91.
Eur J Biochem, 231, 1995, 815-22.
Reaction
15.8.2.1.2.28 Isomerization of 3,7,24THCA-CoA to (24R, 25R) 3alpha,7alpha,24-trihydroxy-5beta-cholestanoyl-CoA
Authors
Editors
Reviewers
Description
The isomerization of 3,7,24THCA-CoA to (24R, 25R) 3alpha,7alpha,24-trihydroxy-5beta-cholestanoyl-CoA, catalyzed by 2-methylacyl-CoA

racemase, occurs in the peroxisomal matrix.
References
2000, 188-91.
Eur J Biochem, 231, 1995, 815-22.
Reaction
15.8.2.1.3 Synthesis of bile acids and bile salts via 27-hydroxycholesterol
Authors
Editors
Reviewers
Description
In the body, 27-hydroxycholesterol is synthesized in multiple tissues, exported to the liver, and converted there to bile acids and bile salts. This
pathway is only a minor source of bile acids and bile salts, but may play a significant role particularly in the mobilization of cholesterol from lung
phagocytes (Bjorkhem et al. 1994; Babiker et al. 1999; Javitt 2002).
In the liver, conversion of 27-hydroxycholesterol to bile acids and bile salts is initiated with hydroxylation and oxidoreductase reactions to form
4-cholesten-7alpha,27-diol-3-one. The pathway then branches: hydroxylation of 4-cholesten-7alpha,27-diol-3-one to
4-cholesten-7alpha,12alpha,27-triol-3-one leads ultimately to the formation of cholate, while its reduction to
5beta-cholestan-7alpha,27-diol-3-one leads to chenodeoxycholate formation. In both branches, reactions in the cytosol, the mitochondrial matrix,
and the peroxisomal matrix result in modifications to the ring structure, shortening and oxidation of the side chain, conversion to a Coenzyme A
derivative, and conjugation with the amino acids glycine or taurine (Russell 2003). These reactions are outlined in the figure below. The final nine
reactions are identical to ones of bile salt synthesis initiated by 7alpha-hydroxylation and are shown as arrows with no substrates.
References
40, 1999, 1417-25.
I Bjorkhem, O Andersson, U Diczfalusy, B Sevastik, RJ Xiu, C Duan, E Lund, "Atherosclerosis and sterol 27-hydroxylase: evidence for a role of
this enzyme in elimination of cholesterol from human macrophages", Proc Natl Acad Sci U S A, 91, 1994, 8592-6.
15.8.2.1.3.1 Cholesterol is hydroxylated to 27-hydroxycholesterol by CYP27
Authors
Jassal, B, 2008-05-19.
Editors
Reviewers
Description
Cholesterol, NADPH + H+, and O2 react to form 27-hydroxycholesterol, H2O, and NADP+, in the mitochondrial matrix, catalyzed by CYP27A1.
27-Hydroxycholesterol is the most abundant oxysterol in the plasma in humans; its formation is thought to play a central role in the mobilization
of cholesterol from non-hepatic tissues.
References
40, 1999, 1417-25.
Reaction
15.8.2.1.3.2 Efflux of 27-hydroxycholesterol
Authors
Editors
Reviewers
Description
27-hydroxycholesterol is transported from the mitochondrial matrix to the extracellular space. In humans, this event is the major source of
27-hydroxycholesterol in the blood and is the means by which the molecule, generated from cholesterol in a variety of cell types, notably
macrophages, is transported to the liver for conversion to bile acids and bile salts (Babiker et al. 1999; Bjorkhem et al. 1994). While transport
proteins are likely to play a role in this process, the relevant proteins have not been identified.
References
40, 1999, 1417-25.
Reaction
15.8.2.1.3.3 Influx of 27-hydroxycholesterol
Authors
Editors
Reviewers
Description
27-hydroxycholesterol is transported from the extracellular space to the endoplasmic reticulum. In humans, this event is the means by which the
molecule, generated from cholesterol in the brain, is taken up by liver cells for conversion to bile acids and bile salts (Babiker et al. 1999;
Bjorkhem et al. 1994). While transport proteins are likely to play a role in this process, these proteins have not been identified.
References
40, 1999, 1417-25.
Reaction
15.8.2.1.3.4 27-hydroxycholesterol is 7alpha-hydroxylated
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
27-hydroxycholesterol, NADPH + H+, and O2 react to form cholest-5-ene-3beta,7alpha,27-triol, H2O, and NADP+. This reaction is catalyzed by
CYP7B1 in the endoplasmic reticulum membrane. Defects in CYB7B1 are associated with failure of 7alpha-hydroxylation in vivo, and with liver
damage, confirming both the function of the enzyme and the central role of the liver in this metabolic process (Setchell et al. 1998).
References
KD Setchell, M Schwarz, NC O'Connell, EG Lund, DL Davis, R Lathe, HR Thompson, R Weslie Tyson, RJ Sokol, DW Russell, "Identification of a
new inborn error in bile acid synthesis: mutation of the oxysterol 7alpha-hydroxylase gene causes severe neonatal liver disease", J Clin Invest,
102, 1998, 1690-703.
Res, 40, 1999, 2195-203.
Reaction
15.8.2.1.3.5 Cholest-5-ene-3beta,7alpha,27-triol is oxidized and isomerized to 4-cholesten-7alpha,27-diol-3-one
Authors
Editors
Reviewers
Description
Cholest-5-ene-3beta,7alpha,27-triol and NAD+ react to form 4-cholesten-7alpha,27-diol-3-one and NADH + H+, in a reaction catalyzed by
HSD3B7 (3 beta-hydroxysteroid dehydrogenase type 7) in the endoplasmic reticulum membrane. Its function in vivo has been confirmed in
studies of patients with defects in bile acid synthesis (Schwarz et al. 2000).
References
Metab, 88, 2003, 1833-41.
Reaction
15.8.2.1.3.6 4-Cholesten-7alpha,27-diol-3-one is 12alpha-hydroxylated to 4-Cholesten-7alpha,12alpha,27-triol-3-one
Authors
Jassal, B, 2008-05-19.
Editors
Reviewers
Description
4-Cholesten-7alpha,27-diol-3-one, NADPH + H+, and O2 form 4-Cholesten-7alpha,12alpha,27-triol-3-one + NADP+ + H2O. This reaction is
catalyzed by sterol 12alpha hydroxylase (CYP8B1), an enzyme associated with the endoplasmic reticulum membrane. While the human gene
References
Genomics, 56, 1999, 184-96.
Chem, 267, 1992, 21319-23.
Reaction
15.8.2.1.3.7 4-cholesten-7alpha,12alpha,27-triol-3-one is reduced to 5beta-cholestan-7alpha,12alpha,27-triol-3-one
Authors
Editors
Reviewers
Description
4-Cholesten-7alpha,12alpha,27-triol-3-one and NADPH + H+ react to form 5beta-cholesten-7alpha,12alpha,27-triol-3-one + NADP+. This

reaction is catalyzed by AKR1D1 (3-oxo-5-beta-steroid 4-dehydrogenase). AKR1D1 is localized to the cytosol, and in the course of the reaction
its steroid substrate moves from the endoplasmic reticulum membrane to the cytosol. It is unclear whether this translocation results simply from
its increased hydrophilicity or is mediated by the enzyme or another transport protein (Russell 2003).
References
Reaction
15.8.2.1.3.8 4-cholesten-7alpha,27-diol-3-one is reduced to 5beta-cholestan-7alpha,27-diol-3-one
Authors
Editors
Reviewers
Description
4-Cholesten-7alpha,27-diol-3-one, NADPH, and H+ react to form 5beta-cholestan-7alpha,27-diol-3-one and NADP+. This reaction is catalyzed
by AKR1D1 (3-oxo-5-beta-steroid 4-dehydrogenase). AKR1D1 is localized to the cytosol, and in the course of the reaction its steroid substrate
moves from the endoplasmic reticulum membrane to the cytosol. It is unclear whether this translocation results simply from its increased
hydrophilicity or is mediated by the enzyme or another transport protein (Russell 2003).
References
Reaction
15.8.2.1.3.9 5Beta-cholestan-7alpha,12alpha,27-triol-3-one is reduced to 5beta-cholestan-3alpha,7alpha,12alpha,27-tetrol
Authors
Editors
Reviewers
Description
5Beta-cholesten-7alpha,12a,27-triol-3-one and NADPH + H+ form 5beta-cholestan-3alpha,7alpha,12a,27-tetrol and NAPDP+. The reaction is

2003).
References
2001, 841-6.
Reaction
15.8.2.1.3.10 5beta-cholestan-7alpha,27-diol-3-one is reduced to 5beta-cholestan-3alpha,7alpha,27-triol
Authors
Editors
Reviewers
Description
5Beta-cholesten-7alpha,27-diol-3-one and NADPH + H+ form 5beta-cholestan-3alpha,7alpha,27-triol and NAPDP+. The reaction is catalyzed by
3alpha-hydroxysteroid dehydrogenase (AKR1C4), a cytosolic enzyme belonging to the aldo-keto reductase family (Dufort et al. 2001).
Biochemical studies with rat proteins raise the possibility that other related enzymes may also carry out this reaction in vivo (Russell 2003).
References
2001, 841-6.
Reaction
15.8.2.1.3.11 5beta-cholestan-3alpha,7alpha,12alpha,27-tetrol is translocated from the cytosol to the mitochondrial matrix
Authors
Editors
Reviewers
Description
5beta-cholestan-3alpha,7alpha,12alpha,27-tetrol is translocated from the cytosol to the mitochondrial matrix. The transporter that mediates its
References
Reaction
15.8.2.1.3.12 5beta-cholestan-3alpha,7alpha,27-triol is translocated from the cytosol to the mitochondrial matrix
Authors
Editors
Reviewers
Description
5beta-cholestan-3alpha,7alpha,27-triol is transported from the cytosol to the mitochondrial matrix. The transporter that mediates its passage
across the inner mitochondrial membrane is unknown: the StAR protein that performs this function for cholesterol at the start of steroid hormone
2003).
References
Reaction
15.8.2.1.4 Cholesterol is hydroxylated to 25-hydroxycholesterol
Authors
Jassal, B, 2007-01-19.
Editors
Reviewers
Description
The microsomal enzyme cholesterol 25-hydroxylase is a member of a lipid metabolizing enzyme family that utilizes oxygen and diiron-oxygen
cofactor to hydroxylate, desaturate, epoxidate and acetylate substrates.
References
EG Lund, TA Kerr, J Sakai, WP Li, DW Russell, "cDNA cloning of mouse and human cholesterol 25-hydroxylases, polytopic membrane proteins
that synthesize a potent oxysterol regulator of lipid metabolism", J Biol Chem, 273, 1998, 34316-27.
Reaction
15.8.2.1.5 25-hydroxycholesterol is 7alpha-hydroxylated by CYP7B1
Authors
Jassal, B, 2008-05-19.
Editors
Reviewers
Description
25-hydroxycholesterol is 7alpha-hydroxylated to cholest-5-ene-3beta,7alpha,25-triol by CYP7B1.
References
Res, 40, 1999, 2195-203.
Reaction
15.8.2.2 Recycling of bile acids and salts
Authors
Jassal, B, 2005-03-09.
Editors
Reviewers
Description
Of the 20-40 grams of bile salts released daily by the liver, all but approximately 0.5 grams are reabsorbed from the intestine, returned to the
liver, and re-used. This recycling involves a series of transport processes: uptake by enterocytes mediated by ASBT (SLC10A2), traversal of the
enterocyte cytosol mediated by ileal bile acid binding protein (I-BABP - FABP6), efflux from enterocytes mediated by MRP3 (ABCC3), travel
through the portal blood as a complex with albumin, and uptake by hepatocytes mediated by Na+-taurocholate transporting protein (NTPC -
SLC10A1) and, to a lesser extent by organic anion transporting proteins A, C, and 8 (OATPA - SLCO1A2, OATPC - SLCO1B1, and OATP-8 -
SLCO1B3). Once returned to the hepatocyte cytosol, bile acids (generated in the intestine by the action of bacteria on secreted bile salts) are
activated by conjugation with coenzyme A, then coupled to glycine or taurine, regenerating bile salts for re-export into the bile, mediated by the
bile salt export pump, ABCB11 (Kullak-Ublick et al. 2004; Mihalik et al. 2002; Trauner and Boyer 2003). Unmodified bile salts returned to the
hepatocyte cytosol can be re-exported by ABCB11 without further modification.
References
2004, 322-42.
PG Killenberg, "Measurement and subcellular distribution of choloyl-CoA synthetase and bile acid-CoA:amino acid N-acyltransferase activities in
rat liver", J Lipid Res, 19, 1978, 24-31.
15.8.2.2.1 Co-transport (influx) of bile salts and acids and sodium ions by ASBT
Authors
Editors
Reviewers
Description
A molecule of extracellular bile salt or bile acid (cholate, chenodeoxycholate, or their glycine or taurine conjugates) and a sodium ion are
transported into the cytosol, mediated by ASBT (apical sodium-dependent bile acid transporter; SLC10A2) in the plasma membrane. Within the
cytosol, bile salts and acids are bound to a carrier protein, FABP6 (I-BABP) (Fujita et al. 1995). Studies in a rabbit model system suggest that
translocation and FABP6 binding occur as a single concerted event (Kramer et al. 1995). In the body, ASBT is expressed on the apical surfaces
of enterocytes, and this reaction is the first step in the process by which bile salts and acids are reaborbed from the intestinal lumen and returned
to the liver (Kullak-Ublick et al. 2004; Trauner and Boyer 2002).
References
M Fujita, H Fujii, T Kanda, E Sato, K Hatakeyama, T Ono, "Molecular cloning, expression, and characterization of a human intestinal 15-kDa
protein", Eur J Biochem, 233, 1995, 406-13.
P Oelkers, LC Kirby, JE Heubi, PA Dawson, "Primary bile acid malabsorption caused by mutations in the ileal sodium-dependent bile acid
transporter gene (SLC10A2)", J Clin Invest, 99, 1997, 1880-7.
2004, 322-42.
W Kramer, F Girbig, U Gutjahr, S Kowalewski, "Radiation-inactivation analysis of the Na+/bile acid co-transport system from rabbit ileum",
Biochem J, 306, 1995, 241-6.
AL Craddock, MW Love, RW Daniel, LC Kirby, HC Walters, MH Wong, PA Dawson, "Expression and transport properties of the human ileal and
renal sodium-dependent bile acid transporter", Am J Physiol, 274, 1998, G157-69.
Reaction
15.8.2.2.2 Transport (efflux) of bile salts by ABCC3 (MRP3)
Authors
Editors
Reviewers
Description
A molecule of glycocholate, taurocholate, or taurochenodeoxycholate is transported from the cytosol to the extracellular space, coupled to the
hydrolysis of a molecule of cytosolic ATP to ADP and orthophosphate. This reaction is mediated by ABCB3 (MRP3). In the body, this reaction
mediates the release of bile salts from enterocytes into the blood (Kullak-Ublick et al. 2004; Trauner and Boyer 2002). In the cytosol, bile salts
and acids are bound to a carrier protein, FABP6 (I-BABP) (Fujita et al. 1995), and in the blood these molecules are complexed with albumin. The
mechanisms by which transport across the plasma membrane is coupled to release FABP6 and binding to albumin are unknown, so the entire
process is annotated as a single concerted event.
References
M Fujita, H Fujii, T Kanda, E Sato, K Hatakeyama, T Ono, "Molecular cloning, expression, and characterization of a human intestinal 15-kDa
protein", Eur J Biochem, 233, 1995, 406-13.
N Zelcer, T Saeki, I Bot, A Kuil, P Borst, "Transport of bile acids in multidrug-resistance-protein 3-overexpressing cells co-transfected with the
ileal Na+-dependent bile-acid transporter", Biochem J, 369, 2003, 23-30.
2004, 322-42.
H Zeng, G Liu, PA Rea, GD Kruh, "Transport of amphipathic anions by human multidrug resistance protein 3", Cancer Res, 60, 2000, 4779-84.
A Bodo, E Bakos, F Szeri, A Varadi, B Sarkadi, "Differential modulation of the human liver conjugate transporters MRP2 and MRP3 by bile acids
and organic anions", J Biol Chem, 278, 2003, 23529-37.
Reaction
15.8.2.2.3 Co-transport (influx) of bile salts and sodium ions by NTCP
Authors
Editors
Reviewers
Description
A molecule of extracellular bile salt (glyco- or taurocholate, or glyco- or taurochenodeoxycholate) and two sodium ions are transported into the
cytosol, mediated by NTCP (Na+ / taurocholate cotransporter) in the plasma membrane. Bile salts exist in the blood as complexes with serum
albumin, and their uptake by NTCP must involve disruption of this complex, but the molecular mechanism of the coupling of the release of a bile
salt from albumin to its uptake by NTCP is unknown. In the body, NTCP is expressed on the basolateral surfaces of hepatocytes, and this
reaction is the major route by which bile salts reaborbed from the intestinal lumen into the portal circulation are recovered by the liver
(Kullak-Ublick et al. 2004; Trauner and Boyer 2002).
References
B Hagenbuch, PJ Meier, "Molecular cloning, chromosomal localization, and functional characterization of a human liver Na+/bile acid
cotransporter", J Clin Invest, 93, 1994, 1326-31.
2004, 322-42.
Reaction
15.8.2.2.4 Transport (influx) of bile salts and acids by OATP-A
Authors
Editors
Reviewers
Description
A molecule of extracellular bile salt (glyco- or taurocholate or taurochenodeoxycholate) or bile acid (cholate or chenodeoxycholate) is transported
into the cytosol, mediated by OATP-A (SLCO1A2) in the plasma membrane. Bile salts and acids exist in the blood as complexes with serum
albumin, and their uptake by OATP-A must involve disruption of this complex, but the molecular mechanism coupling release of a bile salt or
acid from albumin to its uptake by OATP-A is unknown. In the body, OATP-A is expressed only at low levels on the basolateral surfaces of
hepatocytes and may play only a minor role in the uptake of bile salts and acids by the liver (Kullak-Ublick et al. 2004; Trauner and Boyer 2002).
References
2004, 322-42.
GA Kullak-Ublick, B Hagenbuch, B Stieger, CD Schteingart, AF Hofmann, AW Wolkoff, PJ Meier, "Molecular and functional characterization of
an organic anion transporting polypeptide cloned from human liver", Gastroenterology, 109, 1995, 1274-82.
Reaction
15.8.2.2.5 Transport (influx) of glycocholate and taurocholate by OATP-C
Authors
Editors
Reviewers
Description
A molecule of extracellular glycocholate or taurocholate is transported into the cytosol, mediated by OATP-C (SLCO1B1) in the plasma
membrane. Glyco- and taurocholate exist in the blood as complexes with serum albumin, and its uptake by OATP-C must involve disruption of
this complex, but the molecular mechanism coupling disruption and uptake is unknown. In the body, OATP-C is expressed on the basolateral
surfaces of hepatocytes and may play a role in the uptake of glyco- and taurocholate by the liver under physiological conditions (Kullak-Ublick et
al. 2004; Trauner and Boyer 2002).
References
J Konig, Y Cui, AT Nies, D Keppler, "A novel human organic anion transporting polypeptide localized to the basolateral hepatocyte membrane",
Am J Physiol Gastrointest Liver Physiol, 278, 2000, G156Ã¢â‚¬â€œG164.
T Abe, M Kakyo, T Tokui, R Nakagomi, T Nishio, D Nakai, H Nomura, M Unno, M Suzuki, T Naitoh, S Matsuno, H Yawo, "Identification of a
novel gene family encoding human liver-specific organic anion transporter LST-1", J Biol Chem, 274, 1999, 17159-63.
2004, 322-42.
GA Kullak-Ublick, MG Ismair, B Stieger, L Landmann, R Huber, F Pizzagalli, K Fattinger, PJ Meier, B Hagenbuch, "Organic anion-transporting
polypeptide B (OATP-B) and its functional comparison with three other OATPs of human liver", Gastroenterology, 120, 2001, 525-33.
Reaction
15.8.2.2.6 Transport (influx) of glycocholate and taurocholate by OATP-8
Authors
Editors
Reviewers
Description
A molecule of extracellular glycocholate or taurocholate is transported into the cytosol, mediated by OATP-8 (SLCO1B3) in the plasma
membrane. Glycocholate and taurocholate exist in the blood as complexes with serum albumin, and their uptake by OATP-8 must involve
disruption of these complexes, but the molecular mechanism coupling disruption and uptake is unknown. In the body, OATP-8 is expressed on
the basolateral surfaces of hepatocytes and may play a role in the uptake of glycocholate and taurocholate by the liver under physiological
conditions (Kullak-Ublick et al. 2004; Trauner and Boyer 2002).
References
2004, 322-42.
GA Kullak-Ublick, MG Ismair, B Stieger, L Landmann, R Huber, F Pizzagalli, K Fattinger, PJ Meier, B Hagenbuch, "Organic anion-transporting
polypeptide B (OATP-B) and its functional comparison with three other OATPs of human liver", Gastroenterology, 120, 2001, 525-33.
Reaction
15.8.2.2.7 Cytosolic cholate and chenodeoxycholate are conjugated with Coenzyme A (SLC27A5 BACS)
Authors
Jassal, B, 2005-03-09.
Editors
Reviewers
Description
Cholate or chenodeoxycholate, coenzyme A, and ATP react to form their CoA conjugates, AMP, pyrophosphate and water. This reaction is
catalyzed by SLC27A5 (BACS) associated with the endoplasmic reticulum membrane, but the substrates and products are cytosolic (Mihalik et
al. 2002).
References
Reaction
15.8.2.2.8 Cytosolic chenodeoxycholoyl-CoA or choloyl-CoA are conjugated with glycine or taurine
Authors
Jassal, B, 2005-03-09.
Editors
Reviewers
Description
Cytosolic bile acid-CoA conjugates (choloyl-CoA; chenodeoxycholoyl-CoA) react with the amino acids glycine and taurine, generating the
corresponding bile salts and coenzyme A, catalyzed by BAAT (bile acid-CoA:amino acid N-acetyltransferase). In the body, this reaction occurs in
hepatocytes and is the means by which bile acids recovered from the intestine are converted to bile salts before being released again into the
bile (Kullak-Ublick et al. 2004; Trauner and Boyer 2002).
References
2004, 322-42.
MR Johnson, S Barnes, JB Kwakye, RB Diasio, "Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from human
liver", J Biol Chem, 266, 1991, 10227-33.
Reaction
15.8.2.2.9 Transport (efflux) of bile salts by ABCB11 (bile salt export pump)
Authors
Editors
Reviewers
Description
A molecule of glycocholate, glycochenodeoxycholate, taurocholate, or taurochenodeoxycholate is transported from the cytosol to the
extracellular space, coupled to the hydrolysis of a molecule of cytosolic ATP to ADP and orthophosphate. This reaction is mediated by ABCB11
(bile salt export pump). In the body, this reaction mediates the release of bile salts from the liver cells into the bile (Kullak-Ublick et al. 2004); the
role of ABCB11 in the reaction is confirmed by the observed failure of bile salt export in patients in whom the transporter is defective (Noe et al.
2005).
References
JA Byrne, SS Strautnieks, G Mieli-Vergani, CF Higgins, KJ Linton, RJ Thompson, "The human bile salt export pump: characterization of
substrate specificity and identification of inhibitors", Gastroenterology, 123, 2002, 1649-58.
2004, 322-42.
J Noe, GA Kullak-Ublick, W Jochum, B Stieger, R Kerb, M Haberl, B Mullhaupt, PJ Meier, C Pauli-Magnus, "Impaired expression and function of
the bile salt export pump due to three novel ABCB11 mutations in intrahepatic cholestasis", J Hepatol, 43, 2005, 536-43.
J Noe, B Stieger, PJ Meier, "Functional expression of the canalicular bile salt export pump of human liver", Gastroenterology, 123, 2002,
1659-66.
Reaction
15.8.3 Steroid hormone biosynthesis
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
Steroid hormones are synthesized primarily in the adrenal gland and gonads. They regulate energy metabolism and stress responses
(glucocorticoids), salt balance (mineralocorticoids), and sexual development and function (androgens and estrogens). All steroids are
synthesized from cholesterol. Steroid hormone synthesis is largely regulated at the initial steps of cholesterol mobilization and transport into the
mitochondrial matrix for conversion to pregnenolone. In the body, the fate of pregnenolone is tissue-specific: in the zona fasciculata of the
adrenal cortex it is converted to cortisol, in the zona glomerulosa to aldosterone, and in the gonads to testosterone and then to estrone and
estradiol. These pathways are outlined in the figure below, which also diagrams the sites on the cholesterol molecule that undergo modification
in the course of these reactions.
References
AH Payne, DB Hales, "Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones", Endocr Rev, 25, 2004,
947-70.
15.8.3.1 Pregnenolone biosynthesis
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
The first process in the synthesis of all steroid hormones is the synthesis of pregnenolone from cholesterol. In this process, cholesterol mobilized
from cytosolic lipid droplets or from lysosomes is transported to mitochondria and becomes localized to the inner mitochondrial membrane.
Cholesterol transport appears to be rate-limiting for steroid hormone synthesis and its regulation, at the step of StAR-mediated traversal of the
mitochondrial membrane, plays a central role in determining the amounts and identities of steroid hormones made in the body. In the inner
mitochondrial membrane, cholesterol is converted to pregnenolone in a sequence of three reactions, all catalyzed by CYP11A (side chain
cleavage enzyme). Finally, pregnenolone re-enters the cytosol.
References
DM Stocco, "StAR protein and the regulation of steroid hormone biosynthesis", Annu Rev Physiol, 63, 2001, 193-213.
947-70.
15.8.3.1.1 Cholesterol translocates to the inner mitochondrial membrane
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
Cholesterol traverses the cytosol and the mitochondrial intermembrane space complexed with carrier proteins. This process is essential for the
synthesis of steroid hormones in humans. Nevertheless, molecular details of the transport process remain incompletely understood. A plausible
model, supported by studies in vitro and in cells overexpressing cloned human proteins, is that cytosolic STAR-related proteins STARD4, 5, and
6 bind cholesterol liberated from lysosomes or cytosolic lipid droplets and carry it to the outer mitochondrial membrane (Rodriguez-Aguado et al.
2005; Soccio et al. 2002), where it is transferred to STAR protein and carried across the mitochondrial intermembrane space (Miller 2007).
Mutations in the gene encoding STAR block synthesis of all steroid hormones in humans, indicating the critical importance of this transport step
in the biosynthetic process (Bose et al. 1996). The transport step is also a key site for normal regulation of steroid hormone synthesis, as STAR
protein is unstable and its synthesis is up-regulated in response to signals such as the binding of ACTH to its receptors on adrenal cells (Stocco
2001).
References
HS Bose, T Sugawara, JF Strauss III, WL Miller, "The pathophysiology and genetics of congenital lipoid adrenal hyperplasia. International
Congenital Lipoid Adrenal Hyperplasia Consortium.", N Engl J Med, 335, 1996, 1870-8.
DM Stocco, "StAR protein and the regulation of steroid hormone biosynthesis", Annu Rev Physiol, 63, 2001, 193-213.
WL Miller, "StAR search--what we know about how the steroidogenic acute regulatory protein mediates mitochondrial cholesterol import", Mol
Endocrinol, 21, 2007, 589-601.
D Rodriguez-Agudo, S Ren, PB Hylemon, K Redford, R Natarajan, A Del Castillo, G Gil, WM Pandak, "Human StarD5, a cytosolic StAR-related
lipid binding protein", J Lipid Res, 46, 2005, 1615-23.
RE Soccio, RM Adams, MJ Romanowski, E Sehayek, SK Burley, JL Breslow, "The cholesterol-regulated StarD4 gene encodes a StAR-related
lipid transfer protein with two closely related homologues, StarD5 and StarD6", Proc Natl Acad Sci U S A, 99, 2002, 6943-8.
Reaction
15.8.3.1.2 Cholesterol is released into the inner mitochondrial membrane
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
Cholesterol is released from its complex with STAR in the mitochondrial intermembrane space. The mechanism of this process in vivo remains
incompletely understood.
References
WL Miller, "StAR search--what we know about how the steroidogenic acute regulatory protein mediates mitochondrial cholesterol import", Mol
Endocrinol, 21, 2007, 589-601.
Reaction
15.8.3.1.3 Oxidation of cholesterol to 22beta-hydroxycholesterol
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
Cholesterol and NADPH + H+ react to form 22beta-hydroxycholesterol, NADP+, and H2O, catalyzed by CYP11A (P450scc) associated with the
References
Reaction
15.8.3.1.4 Oxidation of 22beta-hydroxycholesterol to 20alpha,22beta-hydroxycholesterol
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
22beta-hydroxycholesterol, NADPH + H+, and O2 react to form 20alpha,22beta-hydroxycholesterol, NADP+ and H2O, catalyzed by CYP11A
(P450scc) associated with the inner mitochondrial membrane.
References
Reaction
15.8.3.1.5 20alpha,22beta-hydroxycholesterol is cleaved by CYP11A1 to yield pregnenolone and isocaproaldehyde
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2007-04-20.
Reviewers

Description
20alpha,22beta-hydroxycholesterol, NADPH + H+, and O2 react to form pregnenolone, isocaproaldehyde, NADP+ and H2O. This cleavage
reaction is catalyzed by CYP11A (P450scc) associated with the inner mitochondrial membrane. Pregnenolone is substantially more hydrophilic
than cholesterol and hydroxycholesterol and is released into the mitochondrial matrix.
References
Reaction
15.8.3.1.6 Pregnenolone translocates from the mitochondrial matrix to the cytosol
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
Pregenolone is translocated from the mitochondrial matrix to the cytosol. Neither transport proteins to mediate its movement across the inner
mitochondrial membrane nor carrier proteins to facilitate its movement in the cytosol have been identified, and the mechanism of this
translocation is unknown.
Reaction
15.8.3.2 Glucocorticoid biosynthesis
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
Cortisol, the major human glucocorticoid, is synthesized in the zona fasciculata of the adrenal cortex from pregnenolone. Pregnenolone is
converted to 17alpha-hydoxyprogesterone in two reactions, both catalyzed by 3-beta-hydroxysteroid dehydrogenase/isomerase.
17Alpha-hydroxyprogesterone is hydroxylated by CYP21A2 to form 11-deoxycortisol, which in turn is converted to cortisol by CYP11B1. The
conversion of the active steroid hormone, cortisol, to inactive cortisone occurs in many tissues, notably the liver.
References
947-70.
Authors
Jassal, B, 2007-02-13.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
References
Reaction
15.8.3.2.2 17-Hydroxypregnenolone is dehydrogenated to form pregn-5-ene-3,20-dione-17-ol
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
17-Hydroxypregnenolone and NAD+ react to form pregn-5-ene-3,20-dione-17-ol and NADH + H+. This reaction is catalyzed by the 3
beta-hydroxysteroid activity of 3-beta-hydroxysteroid dehydrogenase/isomerase (HSD3B) enzyme associated with the endoplasmic reticulum
membrane. The active form of the enzyme is a homodimer. The enzyme occurs in two isoforms that are similar in their biochemical properties
but differ in their tissue expression: type I (HSD3B1) is found in placenta and skin, while type II (HSD3B2) is found in the adrenal glands and
gonads.
References
E Rheaume, Y Lachance, HF Zhao, N Breton, M Dumont, Y de Launoit, C Trudel, V Luu-The, J Simard, F Labrie, "Structure and expression of a
new complementary DNA encoding the almost exclusive 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase in human adrenals
and gonads", Mol Endocrinol, 5, 1991, 1147-57.
JL Thomas, RP Myers, RC Strickler, "Human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase:
purification from mitochondria and kinetic profiles, biophysical characterization of the purified mitochondrial and microsomal enzymes", J Steroid
Biochem, 33, 1989, 209-17.
Y Lachance, V Luu-The, C Labrie, J Simard, M Dumont, Y de Launoit, S Guerin, G Leblanc, F Labrie, "Characterization of human 3
beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase gene and its expression in mammalian cells", J Biol Chem, 265, 1990, 20469-75.
JL Thomas, WL Duax, A Addlagatta, S Brandt, RR Fuller, W Norris, "Structure/function relationships responsible for coenzyme specificity and
the isomerase activity of human type 1 3 beta-hydroxysteroid dehydrogenase/isomerase", J Biol Chem, 278, 2003, 35483-90.
Reaction
15.8.3.2.3 Pregn-5-ene-3,20-dione-17-ol isomerizes to 17-hydroxyprogesterone
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
Pregn-5-ene-3,20-dione-17-ol isomerizes to 17-hydroxyprogesterone. This reaction is catalyzed by the isomerase activity of 3 beta-HSD,
associated with the endoplasmic reticulum membrane. The active form of the enzyme is a homodimer. The enzyme occurs in two isoforms that
are similar in their biochemical properties but differ in their tissue expression: type I (HSD3B1) is found in placenta and skin, while type II
(HSD3B2) is found in the adrenal glands and gonads.
References
Reaction
15.8.3.2.4 Hydroxylation of 17-hydroxyprogesterone to form 11-deoxycortisol
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
17-Hydroxyprogesterone, NADPH + H+, and O2 react to form 11-deoxycortisol, NADP+, and H2O. This reaction is catalyzed by CYP21A2
(steroid 21-hydroxylase) associated with the endoplasmic reticulum membrane.
References
Reaction
15.8.3.2.5 11-deoxycortisol translocates to the mitochondrion
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
11-deoxycortisol is synthesized in a reaction at the endoplasmic reticulum membrane and is further metabolized to cortisol in a reaction
catalyzed by mitochondrial enzymes. The means by which 11-deoxycortisol translocates into the mitochondrial matrix, however, is unknown.
References
947-70.
Reaction
15.8.3.2.6 11-deoxycortisol is oxidised to cortisol by CYP11B1
Authors
Jassal, B, 2008-05-19.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
11-Deoxycortisol, NADPH + H+, and O2 react to form cortisol, NADP+, and H2O. This reaction is catalyzed by CYP11B1 associated with the
References
Sci U S A, 89, 1992, 1458-62.
Chem, 264, 1989, 20961-7.
Reaction
15.8.3.2.7 Cortisol translocates from the mitochondrial matrix to the cytosol
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
Cortisol is translocated from the mitochondrial matrix into the cytosol by an unknown mechanism.
Reaction
15.8.3.2.8 Oxidation of cortisol to yield cortisone
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
Cortisol and NADP+ react to form cortisone, NADPH, and H+. This reaction is catalyzed by 11beta-hydroxysteroid dehydrogenase
(11beta-HSD), associated with the endoplasmic reticulum membrane. The conversion of cortisol, an active hormone, into inactive cortisone
occurs in many tissues in the body, notably in the liver, and appears to play a role in regulating cortison activity.
References
GM Tannin, AK Agarwal, C Monder, MI New, PC White, "The human gene for 11 beta-hydroxysteroid dehydrogenase. Structure, tissue
distribution, and chromosomal localization.", J Biol Chem, 266, 1991, 16653-8.
Reaction
15.8.3.3 Mineralocorticoid biosynthesis
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
Aldosterone, the major human mineralocorticoid, is synthesized in the zona glomerulosa of the adrenal cortex from pregnenolone. Pregnenolone
is converted to progesterone in two reactions, both catalyzed by 3-beta-hydroxysteroid dehydrogenase/isomerase. Progesterone is hydroxylated
by CYP21A2 to form deoxycorticosterone, which in turn is converted to aldosterone in a three-reaction sequence catalyzed by CYP11B2.
References
947-70.
15.8.3.3.1 Pregnenolone is dehydrogenated to form pregn-5-ene-3,20-dione
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
Pregnenolone and NAD+ react to form pregn-5-ene-3,20-dione and NADH + H+. This reaction is catalyzed by the 3 beta-hydroxysteroid activity
of 3-beta-hydroxysteroid dehydrogenase/isomerase (HSD3B) enzyme associated with the endoplasmic reticulum membrane. The active form of
the enzyme is a homodimer. The enzyme occurs in two isoforms that are similar in their biochemical properties but differ in their tissue
expression: type I (HSD3B1) is found in placenta and skin, while type II (HSD3B2) is found in the adrenal glands and gonads.
References
Biochem, 33, 1989, 209-17.
Reaction
15.8.3.3.2 Pregn-5-ene-3,20-dione isomerizes to progesterone
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
Pregn-5-ene-3,20-dione isomerizes to progesterone. This reaction is catalyzed by the isomerase activity of 3 beta-HSD, associated with the
endoplasmic reticulum membrane. The active form of the enzyme is a homodimer. The enzyme occurs in two isoforms that are similar in their
biochemical properties but differ in their tissue expression: type I (HSD3B1) is found in placenta and skin, while type II (HSD3B2) is found in the
adrenal glands and gonads.
References
Biochem, 33, 1989, 209-17.
Reaction
15.8.3.3.3 21-hydroxylation of progesterone to form 11-deoxycorticosterone
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
Progesterone, NAPDH + H+, and O2 react to form 11-deoxycorticosterone, NADP+ and H2O. This reaction is catalyzed by CYP21A2 associated
with the endoplasmic reticulum membrane.
References
Reaction
15.8.3.3.4 11-deoxycorticosterone translocates from the cytosol to the mitochondrial matrix
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
11-deoxycorticosterone is synthesized in a reaction at the endoplasmic reticulum membrane and is further metabolized, ultimately to yield
aldosterone, in reactions catalyzed by mitochondrial enzymes. The means by which 11-deoxycorticosterone translocates into the mitochondrial
matrix, however, is unknown.
References
947-70.
Reaction
15.8.3.3.5 Hydroxylation of 11-deoxycorticosterone to form corticosterone
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
11-Deoxycorticosterone, NADPH + H+, and O2 react to form corticosterone, H2O, and NADP+. This reaction is catalyzed by CYP11B2
associated with the inner mitochondrial membrane.
References
Sci U S A, 89, 1992, 1458-62.
Chem, 264, 1989, 20961-7.
Reaction
15.8.3.3.6 Hydroxylation of corticosterone to form 18-hydroxycorticosterone
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
Corticosterone, NADPH + H+, and O2 react to form 18-hydroxycorticosterone, NADP+, and H2O. This reaction is catalyzed by CYP11B2
associated with the inner mitochondrial membrane.
References
T Kawainoto, Y Mitsuuchi, T Ohnishi, Y Ichikawa, Y Yokoyama, H Sumimoto, K Toda, K Miyahara, I Kuribayashi, K Nakao, K Hosoda, Y
Yamamoto, H Imura, Y Shizuta, "Cloning and expression of a cDNA for human cytochrome P-450aldo as related to primary aldosteronism",
Chem, 264, 1989, 20961-7.
Reaction
15.8.3.3.7 Conversion of 18-hydroxycorticosterone to aldosterone
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
18-Hydroxycorticosterone and NADPH + H+ react to form aldosterone, NADP+, and H2O. This reaction is catalyzed by CYP11B2 associated
with the inner mitochondrial membrane.
References
T Kawainoto, Y Mitsuuchi, T Ohnishi, Y Ichikawa, Y Yokoyama, H Sumimoto, K Toda, K Miyahara, I Kuribayashi, K Nakao, K Hosoda, Y
Yamamoto, H Imura, Y Shizuta, "Cloning and expression of a cDNA for human cytochrome P-450aldo as related to primary aldosteronism",
Chem, 264, 1989, 20961-7.
Reaction
15.8.3.4 Androgen biosynthesis
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
Androgens are the determining factors for male development and behaviour in vertebrates.
Authors
Jassal, B, 2007-02-13.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
References
Reaction
15.8.3.4.2 Side chain cleavage of 17alpha-hydroxyprenenolone to yield DHA
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
17-alpha-hydroxypregnenolone, NADPH + H+, and O2 react to form DHA (dehydroepiandrostenedione), NADP+, H2O, and acetaldehyde.
CYP17 (which also catalyzes 17-alpha-hydroxylation) catalyzes this lyase reaction. There are marked species differences in which substrate is
used for this lyase activity. The human enzyme prefers 17alpha-pregnenolone (delta5 steroid) as substrate (Brock, BJ, Waterman, MR, 1999).
References
BJ Brock, MR Waterman, "Biochemical differences between rat and human cytochrome P450c17 support the different steroidogenic needs of
these two species", Biochemistry, 38, 1999, 1598-606.
Reaction
15.8.3.4.3 DHA isomerizes to 4-Androstene3,17-dione
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
In this two-step reaction catalyzed by 3beta-HSD associated with the endoplasmic reticulum membrane, the 3-hydroxyl group of DHA is oxidized
to a keto group and the double bond in the steroid nucleus is then isomerized from the five to the 4 position.
References
Reaction
15.8.3.4.4 Hydroxylation of progesterone to form 17alpha-hydroxyprogesterone
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
Progesterone, NADPH + H+, and O2 react to form 17alpha-hydroxyprogesterone, NADP+, and H2O, catalyzed by CYP17 (steroid
17alpha-monooxygenase), associated with the endoplasmic reticulum membrane.
References
Reaction
15.8.3.4.5 Side chain cleavage of 17alpha-hydroxyprogesterone to form 4-Androstene-3, 17-dione
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
17Alpha-hydroxyprogesterone, NADPH + H+, and O2 react to form 4-Androstene-3, 17-dione, NADP+, H2O, and acetaldehyde. CYP17 (which
also catalyzes 17-alpha-hydroxylation) catalyzes this lyase reaction. There are marked species differences in which substrate is used for this
lyase activity.
References
Reaction
15.8.3.4.6 Reduction of androstenedione to testosterone
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
The 17HSD family of enzymes catalyze the final step in the synthesis of estradiol and testosterone. They convert inactive 17-ketosteroids to their
active 17beta-hydroxy forms. Androstenedione, a ketosteroid, is reduced to testosterone, a highly potent androgen, by the enzyme
17beta-hydroxysteroid dehydrogenase isoform III (17HSD3). The other human isoforms of 17HSDs to take part in the final steps of active steroid
biosynthesis are types 1 and VII, which reduce estrone to estradiol.
References
V Luu-The, Y Zhang, D Poirier, F Labrie, "Characteristics of human types 1, 2 and 3 17 beta-hydroxysteroid dehydrogenase activities:
oxidation/reduction and inhibition", J Steroid Biochem Mol Biol, 55, 1995, 581-7.
V Luu-The, C Labrie, HF Zhao, J Couet, Y Lachance, J Simard, J Cote, G Leblanc, L Lagace, D Berube, R Gagne, F Labrie, "Purification,
cloning, complementary DNA structure, and predicted amino acid sequence of human estradiol 17 beta-dehydrogenase", Ann N Y Acad Sci,
595, 1990, 40-52.
WM Geissler, DL Davis, L Wu, KD Bradshaw, S Patel, BB Mendonca, KO Elliston, JD Wilson, DW Russell, S Andersson, "Male
pseudohermaphroditism caused by mutations of testicular 17 beta-hydroxysteroid dehydrogenase 3", Nat Genet, 7, 1994, 34-9.
Reaction
15.8.3.5 Estrogen biosynthesis
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
In female vertebrates, estrogens control reproductive system development and reproductive functions.
15.8.3.5.1 Testosterone is converted to estradiol
Authors
Jassal, B, 2007-04-20.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
The conversion of testosterone to estradiol is catalyzed by aromatase (CYP19A1) associated with the endoplasmic reticulum membrane.
References
Reaction
15.8.3.5.2 Adrostenedione is converted to estrone by Aromatase (CYP19A1)

Authors
Jassal, B, 2007-02-13.
Editors
Jassal, B, 2007-04-20.
Reviewers
Description
The conversion of androstenedione to estrone is catalyzed by aromatase (CYP19A1) associated with the endoplasmic reticulum membrane.
References
Reaction
15.9 Lipoprotein metabolism
Editors
Description
Because of their hydrophobicity, lipids are found in the extracellular spaces of the human body primarily in the form of lipoprotein complexes.
Chylomicrons form in the small intestine and transport dietary lipids to other tissues in the body. Very low density lipoproteins (VLDL) form in the
liver and transport triacylglycerol synthesized there to other tissues of the body. As they circulate, VLDL are acted on by lipoprotein lipases on
the endothelial surfaces of blood vessels, liberating fatty acids and glycerol to be taken up by tissues and converting the VLDL first to
intermediate density lipoproteins (IDL) and then to low density lipoproteins (LDL). IDL and LDL are cleared from the circulation via a specific cell
surface receptor, found in the body primarily on the surfaces of liver cells. High density lipoprotein (HDL) particles, initially formed primarily by
the liver, shuttle several kinds of lipids between tissues and other lipoproteins. Notably, they are responsible for the so-called reverse transport of
cholesterol from peripheral tissues to LDL for return to the liver.
Three aspects of lipoprotein function are currently annotated in Reactome: chylomicron-mediated lipid transport, LDL endocytosis and
degradation, and HDL-mediated lipid transport.
15.9.1 Chylomicron-mediated lipid transport
Authors
Editors
Description
Chylomicrons transport triacylglycerol, phospholipid, and cholesterol derived from dietary lipid from the small intestine to other tissues of the
body. Each chylomicron assembles around a single molecule of apolipoprotein B-48 (Phillips et al. 1997) which at the time the particle leaves the
intestine and enters the lymphatic circulation is complexed with >200,000 molecules of triacylglycerol (TG), ~35,000 of phospholipid, ~11,000 of
cholesterol ester, ~8,000 of free cholesterol, ~60 copies of apolipoprotein A-I, ~15 copies of apolipoprotein A-IV, and copies of apolipoprotein
A-II (Bhattacharya and Redgrave 1981; Havel and Kane 2001). As chylomicrons circulate in the body, they acquire molecules of apolipoproteins
C and E, and through interaction with endothelial lipases can lose a large fraction of their triacylglycerol. These changes convert them to
chylomicron remnants which bind to LDL receptors, primarily on the surfaces of liver cells, clearing them from the circulation. This whole
sequence of events is rapid: the normal lifespan of a chylomicron is 30 - 60 minutes (Havel and Kane 2001).
References
RJ Havel, JP Kane, "Introduction: Structure and metabolism of plasma lipoproteins", The Metabolic and Molecular Bases of Inherited Disease,
8th ed (Scriver CR, et al., editors), 2, 2001, 2705-2716.
S Bhattacharya, TG Redgrave, "The content of apolipoprotein B in chylomicron particles", J Lipid Res, 22, 1981, 820-8.
ML Phillips, C Pullinger, I Kroes, J Kroes, DA Hardman, G Chen, LK Curtiss, MM Gutierrez, JP Kane, VN Schumaker, "A single copy of
apolipoprotein B-48 is present on the human chylomicron remnant", J Lipid Res, 38, 1997, 1170-7.
15.9.1.1 ApoB-48 + 40 triacylglycerol + 60 phospholipid => ApoB-48:TG:PL complex
Authors
Editors
Description
Phospholipid (PL) and triacylglycerol (TG) associate with the apo B-48 polypeptide as it is translated. This process is mediated by MTP
(microsomal triacylglycerol transfer protein) in the form of a MTP:PDI (protein disulfide isomerase) heterodimer. MTP in vitro binds small
amounts of PL and TG (annotated here as as one molecule of each) and efficiently transfers the bound lipid between membranes (Atzel and
Wetterau 1994). In vivo, MTP:PDI directly interacts with apoB-48 polypeptide (Wu et al. 1996), and is thought to transfer lipid from the
endoplasmic reticulum membrane to nascent apoB-48. While some of the molecular details of MTP function remain unclear, this function is
clearly essential in vivo, as patients who lack MTP cannot produce chylomicrons (e.g., Wetterau et al. 1992; Narcisi et al. 1995).
References
DA Gordon, JR Wetterau, RE Gregg, "Microsomal triglyceride transfer protein: a protein complex required for the assembly of lipoprotein
particles", Trends Cell Biol, 5, 1995, 317-21.
JR Wetterau, LP Aggerbeck, ME Bouma, C Eisenberg, A Munck, M Hermier, J Schmitz, G Gay, DJ Rader, RE Gregg, "Absence of microsomal
triglyceride transfer protein in individuals with abetalipoproteinemia", Science, 258, 1992, 999-1001.
X Wu, M Zhou, LS Huang, JR Wetterau, HN Ginsberg, "Demonstration of a physical interaction between microsomal triglyceride transfer protein
and apolipoprotein B during the assembly of ApoB-containing lipoproteins", J Biol Chem, 271, 1996, 10277-81.
A Atzel, JR Wetterau, "Identification of two classes of lipid molecule binding sites on the microsomal triglyceride transfer protein", Biochemistry,
33, 1994, 15382-8.
TM Narcisi, CC Shoulders, SA Chester, J Read, DJ Brett, GB Harrison, TT Grantham, MF Fox, S Povey, TW de Bruin, DW Erkelens, DR Muller,
JK Lloyd, J Scott, "Mutations of the microsomal triglyceride-transfer-protein gene in abetalipoproteinemia", Am J Hum Genet, 57, 1995,
1298-310.
Reaction
15.9.1.2 Degradation of newly synthesized ApoB-48
Authors
Editors
Description
Newly synthesized apoB-48 that is not complexed with lipid is rapidly degraded (Dixon_ea_1991). The mechanism and site of this degradation
(within the endoplasmic reticulum or in cytosolic proteasomes) is unclear.
References
JL Dixon, S Furukawa, HN Ginsberg, "Oleate stimulates secretion of apolipoprotein B-containing lipoproteins from Hep G2 cells by inhibiting
early intracellular degradation of apolipoprotein B", J Biol Chem, 266, 1991, 5080-6.
Reaction
15.9.1.3 ApoB-48:TG:PL complex + 100 triacylglycerols + ApoA-I + ApoA-IV => nascent chylomicron
Authors
Editors
Description
The second phase of chylomicron assembly takes place in the lumen of the endoplasmic reticulum. ApoB-48 continues to bind triacylglycerol, as
well as cholesterol, cholesterol esters, and molecules of apolipoproteins A-I, A-II, and A-IV. The reaction is annotated here to involve small
numbers of these molecules, but the true numbers in vivo are much greater - a nascent chylomicron entering the lymphatic circulation contains
>200,000 molecules of triacylglycerol (TG), ~35,000 of phospholipid, ~11,000 of cholesterol ester, ~8,000 of free cholesterol, ~60 copies of
apolipoprotein A-I, ~15 copies of apolipoprotein A-IV, and copies of apolipoprotein A-II (Bhattacharya and Redgrave 1981; Havel and Kane
2001).
The presence of MTP:PDI (microsomal triacylglycerol transfer protein:protein disulfide isomerase) is required for lipid addition both in vitro and in
vivo, but its molecular role at this stage of chylomicron formation is unclear and may be indirect (Gordon et al. 1995; Hussain et al. 2003).
References
MM Hussain, J Shi, P Dreizen, "Microsomal triglyceride transfer protein and its role in apoB-lipoprotein assembly", J Lipid Res, 44, 2003, 22-32.
DA Gordon, JR Wetterau, RE Gregg, "Microsomal triglyceride transfer protein: a protein complex required for the assembly of lipoprotein
particles", Trends Cell Biol, 5, 1995, 317-21.
Reaction
15.9.1.4 nascent chylomicron [endoplasmic reticulum lumen] => nascent chylomicron [extracellular]
Authors
Editors
Description
While the export pathway for nascent chylomicrons has not been directly characterized in human cells, the requirement for SAR1B protein for
normal chylomicron export in vivo (Jones et al. 2003) indicates that nascent chylomicrons are exported from the endoplasmic reticulum via the
Golgin apparatus in COPII vesicles.
References
B Jones, EL Jones, SA Bonney, HN Patel, AR Mensenkamp, S Eichenbaum-Voline, M Rudling, U Myrdal, G Annesi, S Naik, N Meadows, A
Quattrone, SA Islam, RP Naoumova, B Angelin, R Infante, E Levy, CC Roy, PS Freemont, J Scott, CC Shoulders, "Mutations in a Sar1 GTPase
of COPII vesicles are associated with lipid absorption disorders", Nat Genet, 34, 2003, 29-31.
Reaction
15.9.1.5 nascent chylomicron + spherical HDL:apoC-II:apoC-III:apoE =>spherical HDL + chylomicron
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
Circulating nascent chylomicrons acquire copies of apolipoproteins C-II, C-III, and E from circulating spherical (mature) high-density lipoprotein
particles, becoming mature chylomicrons (Havel et al. 1973; Bisgaier and Glickman 1983). Here, this interaction is annotated to involve the
transfer of a single copy of each lipoprotein, but a mature chylomicron in fact contains approximately 25 copies of apolipoprotein E and 180
copies of C apolipoproteins (Bhattacharya and Redgrave 1981).
References
RJ Havel, JP Kane, ML Kashyap, "Interchange of apolipoproteins between chylomicrons and high density lipoproteins during alimentary lipemia
in man", J Clin Invest, 52, 1973, 32-8.
CL Bisgaier, RM Glickman, "Intestinal synthesis, secretion, and transport of lipoproteins", Annu Rev Physiol, 45, 1983, 625-36.
Reaction
15.9.1.6 chylomicron => TG-depleted chylomicron + 50 long-chain fatty acids + 50 diacylglycerols
Authors
Editors
Description
Lipoprotein lipase (LPL) dimers are tethered to heparan sulfate proteoglycans (HSPG) at endothelial cell surfaces (Fernandez-Borja et al. 1996;
Peterson et al. 1992). Both syndecan 1 (Rosenberg et al. 1997) and perlecan (Goldberg 1996) HSPG molecules are capable of tethering LPL.
The LPL enzyme catalyzes the hydrolysis and release of triacylglycerols (TG) associated with circulating chylomicrons. This reaction is
annotated here as causing the hydrolysis and release of 50 molecules of TG. In vivo, the number is much larger, and TG depletion probably
occurs in the course of multiple encounters between a chylomicron and endothelial LPL. This reaction is strongly activated by
chylomicron-associated apo C-II protein both in vivo and in vitro (Jackson et al. 1986). Chylomicron-associated apoC-II protein inhibits LPL
activity in vitro (Brown and Baginsky 1972), and recent studies have indicated a positive regulatory role for apoA-5 protein, though its molecular
mechanism of action remains unclear (Marcais et al. 2005; Merkel and Heeren 2005).
References
M Merkel, J Heeren, "Give me A5 for lipoprotein hydrolysis!", J Clin Invest, 115, 2005, 2694-6.
C Marcais, B Verges, S Charriere, V Pruneta, M Merlin, S Billon, L Perrot, J Drai, A Sassolas, LA Pennacchio, J Fruchart-Najib, JC Fruchart, V
Durlach, P Moulin, "Apoa5 Q139X truncation predisposes to late-onset hyperchylomicronemia due to lipoprotein lipase impairment", J Clin
Invest, 115, 2005, 2862-9.
M Fernandez-Borja, D Bellido, E Vilella, G Olivecrona, S Vilaro, "Lipoprotein lipase-mediated uptake of lipoprotein in human fibroblasts: evidence
for an LDL receptor-independent internalization pathway", J Lipid Res, 37, 1996, 464-81.
J Peterson, WY Fujimoto, JD Brunzell, "Human lipoprotein lipase: relationship of activity, heparin affinity, and conformation as studied with
monoclonal antibodies", J Lipid Res, 33, 1992, 1165-70.
WV Brown, ML Baginsky, "Inhibition of lipoprotein lipase by an apoprotein of human very low density lipoprotein", Biochem Biophys Res
Commun, 46, 1972, 375-82.
RD Rosenberg, NW Shworak, J Liu, JJ Schwartz, L Zhang, "Heparan sulfate proteoglycans of the cardiovascular system. Specific structures
emerge but how is synthesis regulated?", J Clin Invest, 99, 1997, 2062-70.
RL Jackson, S Tajima, T Yamamura, S Yokoyama, A Yamamoto, "Comparison of apolipoprotein C-II-deficient triacylglycerol-rich lipoproteins
and trioleoylglycerol/phosphatidylcholine-stabilized particles as substrates for lipoprotein lipase", Biochim Biophys Acta, 875, 1986, 211-9.
IJ Goldberg, "Lipoprotein lipase and lipolysis: central roles in lipoprotein metabolism and atherogenesis", J Lipid Res, 37, 1996, 693-707.
Reaction
15.9.1.7 TG-depleted chylomicron + spherical HDL => chylomicron remnant + spherical HDL:apoA-I:apoA-II:apoA-IV:apoC-II:apoC-III
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
Triacylglycerol (TG)-depleted chylomicrons transfer A and C apoproteins to spherical (mature) HDL, generating chylomicron remnants (Havel
and Kane 2001). This transfer is arbitrarily annotated here as involving one molecule of each apolipoprotein. The molecular difference that
enables nascent chylomicrons to accept apolipoproteins from sperical HDL and TG-depleted chylomicrons to donate them is unclear.
References
Reaction
15.9.1.8 chylomicron remnant + apoE => chylomicron remnant:apoE complex
Authors
Editors
Description
In the body, this binding involved apoE synthesized by hepatocytes and concentrated in the space of Disse, an extracellular compartment
adjacent to the hepatocytes to which blood-borne lipoprotein particles have free access (Ji_ea_1994).
References
ZS Ji, S Fazio, YL Lee, RW Mahley, "Secretion-capture role for apolipoprotein E in remnant lipoprotein metabolism involving cell surface
heparan sulfate proteoglycans", J Biol Chem, 269, 1994, 2764-72.
Reaction
15.9.1.9 chylomicron remnant:apoE complex + LDLR => chylomicron remnant:apoE:LDLR complex
Authors
Editors
Description
Chylomicron micron remnants containing apolipoprotein E associate with the surfaces of cells in a process that probably involves several steps
and that requires the presence (but not the catalytic activity) of heparan sulfate proteoglycan (HSPG)-associated hepatic lipase (HL). This
process ultimately results in binding of the remnant to cell-surface LDL receptors (LDLR) (Ji et al. 1994). The molecular details of LDLR binding,
and of the following steps of remnant endocytosis, are inferred from those of the coorresponding step of LDLR-mediated low-density lipoprotein
(LDL) endocytosis. In the body, this process occurs in the liver.
References
ZS Ji, S Fazio, YL Lee, RW Mahley, "Secretion-capture role for apolipoprotein E in remnant lipoprotein metabolism involving cell surface
heparan sulfate proteoglycans", J Biol Chem, 269, 1994, 2764-72.
Reaction
15.9.1.10 chylomicron remnant:apoE:LDLR complex [plasma membrane] => chylomicron remnant:apoE:LDLR complex
[clathrin-coated vesicle] (LDLRAP1-dependent)
Authors
Editors
Description
The molecular details of this event are inferred from those of LDLR-mediated low-density lipoprotein (LDL) endocytosis into coated vesicles.
Source reaction
This reaction was inferred from the corresponding reaction "LDL:LDLR complex [plasma membrane] => LDL:LDLR complex [clathrin-coated
vesicle] (LDLRAP1-dependent)" in species Homo sapiens.
ER Eden, DD Patel, XM Sun, JJ Burden, M Themis, M Edwards, P Lee, C Neuwirth, RP Naoumova, AK Soutar, "Restoration of LDL receptor
function in cells from patients with autosomal recessive hypercholesterolemia by retroviral expression of ARH1", J Clin Invest, 110, 2002,
1695-702.
R Garuti, C Jones, WP Li, P Michaely, J Herz, RD Gerard, JC Cohen, HH Hobbs, "The modular adaptor protein autosomal recessive
hypercholesterolemia (ARH) promotes low density lipoprotein receptor clustering into clathrin-coated pits", J Biol Chem, 280, 2005, 40996-1004.
JL Goldstein, RG Anderson, MS Brown, "Coated pits, coated vesicles, and receptor-mediated endocytosis", Nature, 279, 1979, 679-85.
G He, S Gupta, M Yi, P Michaely, HH Hobbs, JC Cohen, "ARH is a modular adaptor protein that interacts with the LDL receptor, clathrin, and
AP-2", J Biol Chem, 277, 2002, 44044-9.
P Michaely, WP Li, RG Anderson, JC Cohen, HH Hobbs, "The modular adaptor protein ARH is required for low density lipoprotein (LDL) binding
and internalization but not for LDL receptor clustering in coated pits", J Biol Chem, 279, 2004, 34023-31.
Reaction
15.9.1.11 chylomicron remnant:apoE:LDLR complex [coated vesicle membrane] => chylomicron remnant:apoE:LDLR complex
[endosome membrane]
Authors
Editors
Description
The molecular details of this event are inferred from those of LDLR-mediated low-density lipoprotein (LDL) transport from coated vesicles to
endosomes.
Source reaction
This reaction was inferred from the corresponding reaction "LDLR:LDL complex [coated vesicle membrane] => LDLR:LDL complex [endosome
membrane]" in species Homo sapiens.
Reaction
15.9.1.12 chylomicron remnant:apoE:LDLR complex => chylomicron remnant:apoE + LDLR
Authors
Editors
Description
The molecular details of this event are inferred from the dissociation of the LDL:LDLR complex in the endosome.
Source reaction
This reaction was inferred from the corresponding reaction "LDLR:LDL complex => LDLR + LDL" in species Homo sapiens.
Reaction
15.9.2 HDL-mediated lipid transport
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
HDL particles play a central role in the reverse transport of cholesterol, the process by which cholesterol in tissues other than the liver is returned
to the liver for conversion to bile salts and excretion from the body and provided to tissues such as the adrenals and gonads for steroid hormone
synthesis (Tall et al. 2008).
HDL particles are heterogeneous and can be fractionated into sub-populations based on their electrophoretic mobility, their density, or their
content of various apolipoproteins. Sub-populations yielded by any one fractionation strategy, however, remain heterogeneous with respect to
the other two parameters (Kontush and Chapman 2006). All HDL particles share two key features: they are assembled on a protein scaffold
provided by apolipoprotein A-I (apoA-I), and they are recycled to allow a net flow of lipids from peripheral tissues to the liver and steroidogenic
tissues while allowing apoA-I molecules to be re-used.
The HDL life cycle annotated here does not capture the full complexity of HDL structure and function but rather is an attempt to outline the key
steps by which HDL particles assemble and transfer their lipid content. These steps include the assembly of nascent (discoidal) HDL particles on
newly synthesized apoA-I, a process that in the body occurs primarily in the liver, the loading of discoidal HDL with additional lipid through
interaction with cells carrying excess cholesterol, the conversion of HDL-associated cholesterol to cholesterol esters, the transfer of HDL lipids to
target cells with the regeneration of pre-beta HDL (lipid-poor apoA-I), and the conversion of pre-beta HDL to discoidal HDL to complete the
cycle. Additional reactions provide houekeeping functions needed for the efficient operation of the cycle: binding and release of CETP, dispersal
of byproducts of the CETP reaction, and export and degradation of excess apoA-I protein. Finally, the function of torcetrapib as an inhibitor of
CETP function is annotated.
References
AR Tall, L Yvan-Charvet, N Terasaka, T Pagler, N Wang, "HDL, ABC Transporters, and Cholesterol Efflux: Implications for the Treatment of
Atherosclerosis", Cell Metab, 7, 2008, 365-75.
A Kontush, MJ Chapman, "Functionally defective high-density lipoprotein: a new therapeutic target at the crossroads of dyslipidemia,
inflammation, and atherosclerosis", Pharmacol Rev, 58, 2006, 342-74.
KA Rye, PJ Barter, "Formation and metabolism of prebeta-migrating, lipid-poor apolipoprotein A-I", Arterioscler Thromb Vasc Biol, 24, 2004,
421-8.
KA Rye, MA Clay, PJ Barter, "Remodelling of high density lipoproteins by plasma factors", Atherosclerosis, 145, 1999, 227-38.
P Linsel-Nitschke, AR Tall, "HDL as a target in the treatment of atherosclerotic cardiovascular disease", Nat Rev Drug Discov, 4, 2005, 193-205.
15.9.2.1 Conversion of pro-apoA-I to apoA-I
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
The six aminoterminal residues of pro-apolipoprotein A-1 are removed to generate the mature, lipid-binding form of the protein. Studies of tissue
culture systems suggest that BMP1 catalyzes this reaction (Chau et al. 2006, 2007). While BMP1 is annotated here as a monomer, its subunit
structure is not known, and its 1:1 association with Zn++ is inferred from its sequence similarity to known metallopeptidases.
Alpha2-macroglobulin at concentrations found in plasma in inflammatory responses inhibits this reaction in vitro, suggesting a possible link
between inflammation and perturbation of HDL function (Zhang et al. 2006; Chau et al. 2007).
References
P Chau, Y Nakamura, CJ Fielding, PE Fielding, "Mechanism of prebeta-HDL formation and activation", Biochemistry, 45, 2006, 3981-7.
Y Zhang, G Ge, DS Greenspan, "Inhibition of bone morphogenetic protein 1 by native and altered forms of alpha2-macroglobulin", J Biol Chem,
281, 2006, 39096-104.
P Chau, PE Fielding, CJ Fielding, "Bone morphogenetic protein-1 (BMP-1) cleaves human proapolipoprotein A1 and regulates its activation for
lipid binding", Biochemistry, 46, 2007, 8445-50.
Reaction
15.9.2.2 ABCA1 tetramer binds apoA-I
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
ABCA1 associated with the plasma membrane binds extracellular apoA-I protein, forming a membrane-associated complex. The predominant
form of ABCA1 is a heterotetramer (Denis et al. 2004), although studies in model systems in vitro are consistent with the hypothesis that the
protein may also occur as a dimer (Trompier et al. 2006).
References
D Trompier, M Alibert, S Davanture, Y Hamon, M Pierres, G Chimini, "Transition from dimers to higher oligomeric forms occurs during the
ATPase cycle of the ABCA1 transporter", J Biol Chem, 281, 2006, 20283-90.
M Denis, B Haidar, M Marcil, M Bouvier, L Krimbou, J Genest, "Characterization of oligomeric human ATP binding cassette transporter A1.
Potential implications for determining the structure of nascent high density lipoprotein particles", J Biol Chem, 279, 2004, 41529-36.
Reaction
15.9.2.3 ABCA1-mediated transport of intracellular cholesterol to the cell surface
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
In an ATP-dependent reaction, ATPA1 mediates the movement of intracellular cholesterol to the extracellular face of the plasma membrane.
Cholesterol associated with cytosolic vesicles is a substrate for this reaction. Under physiologocal conditions, the active form of ABCA1 is
predominantly a tetramer associated with apoA-I lipoprotein (Denis et al. 2004; Vedhachalam et al., 2007). The number of lipid molecules
transported per ATP consumed is not known.
References
C Vedhachalam, PT Duong, M Nickel, D Nguyen, P Dhanasekaran, H Saito, GH Rothblat, S Lund-Katz, MC Phillips, "Mechanism of ATP-binding
cassette transporter A1-mediated cellular lipid efflux to apolipoprotein A-I and formation of high density lipoprotein particles", J Biol Chem, 282,
2007, 25123-30.
Reaction
15.9.2.4 ABCA1-mediated transport of intracellular phospholipid to the cell surface
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
In an ATP-dependent reaction, ATPA1 mediates the movement of intracellular phospholipid to the extracellular face of the plasma membrane.
Cholesterol associated with cytosolic vesicles is a substrate for this reaction. Under physiologocal conditions, the active form of ABCA1 is
predominantly a tetramer associated with apoA-I lipoprotein (Denis et al. 2004; Vedhachalam et al., 2007). The number of lipid molecules
References
C Vedhachalam, PT Duong, M Nickel, D Nguyen, P Dhanasekaran, H Saito, GH Rothblat, S Lund-Katz, MC Phillips, "Mechanism of ATP-binding
cassette transporter A1-mediated cellular lipid efflux to apolipoprotein A-I and formation of high density lipoprotein particles", J Biol Chem, 282,
2007, 25123-30.
Reaction
15.9.2.5 Apolipoprotein A-I binds membrane-associated cholesterol and phospholipid to form a discoidal HDL particle
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
Extracellular apolipoprotein A-I interacts with phospholipid- and cholesterol-rich membrane patches formed through the action of ABCA1, binding
these two lipids to form a discoidal (small nascent) HDL particle (HDL 3c - Kontush and Chapman 2006). The apoA-I molecules that accept lipids
in this reaction appear to be different from the ones that activate ABCA1 at the plasma membrane (Hassan et al. 2007; Vedhachalam et al.
2007).
References
HH Hassan, M Denis, DY Lee, I Iatan, D Nyholt, I Ruel, L Krimbou, J Genest, "Identification of an ABCA1-dependent phospholipid-rich plasma
membrane apolipoprotein A-I binding site for nascent HDL formation: implications for current models of HDL biogenesis", J Lipid Res, 48, 2007,
2428-42.
C Vedhachalam, AB Ghering, WS Davidson, S Lund-Katz, GH Rothblat, MC Phillips, "ABCA1-induced cell surface binding sites for ApoA-I",
Reaction
15.9.2.6 pre-beta HDL binds membrane-associated cholesterol and phospholipid to form a discoidal HDL particle
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
Pre-beta HDL (lipid-poor apoA-I) interacts with phospholipid- and cholesterol-rich membrane patches formed through the action of ABCA1,
binding these two lipids to form a discoidal (small nascent) HDL particle (HDL 3c - Kontush and Chapman 2006).
References
C Vedhachalam, AB Ghering, WS Davidson, S Lund-Katz, GH Rothblat, MC Phillips, "ABCA1-induced cell surface binding sites for ApoA-I",
Reaction
15.9.2.7 ABCG1-mediated transport of intracellular cholesterol to the cell surface
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
In an ATP-dependent reaction, ABCG1 mediates the movement of intracellular cholesterol to the extracellular face of the plasma membrane. In
a tissue culture model system, the active form of ABCG1 is predominantly a tetramer (Vuaghan and Oram 2005). The number of lipid molecules
References
AM Vaughan, JF Oram, "ABCG1 redistributes cellular cholesterol to domains removable by high density lipoproteins but not by lipid-depleted
lipoproteins", J Biol Chem, 280, 2005, 30150-30157.
Reaction
15.9.2.8 Transformation of discoidal HDL to spherical HDL
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
The conversion of a discoidal HDL (small nascent HDL) to a spherical HDL requires acylation of free cholesterol already in the small HDL as well
as the binding of additional cholesterol, phospholipids, and apolipoproteins (Kontush and Chapman 2006).
References
Reaction
15.9.2.8.1 Discoidal HDL binds membrane-associated free cholesterol
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
Extracellular discoidal HDL particles interact with cholesterol-rich membrane patches formed through the action of ABCG1 (Vaughan and Oram
2005). In the body this reaction is a key step in the process of reverse cholesterol transport, by which excess cholesterol is recovered from cells
such a macrophages and transported ultimately to the liver. At a molecular level, it is one of the steps in the transformation of discoidal (small
nascent) HDL particles into spherical ones, distinct from the similar reaction in which cholesterol is transferred to lipid-free apoA-I protein (Oram
and Vaughan 2006; Kontush and Chapman 2006).
References
JF Oram, AM Vaughan, "ATP-Binding cassette cholesterol transporters and cardiovascular disease", Circ Res, 99, 2006, 1031-43.
AM Vaughan, JF Oram, "ABCG1 redistributes cellular cholesterol to domains removable by high density lipoproteins but not by lipid-depleted
lipoproteins", J Biol Chem, 280, 2005, 30150-30157.
Reaction
15.9.2.8.2 LCAT + discoidal HDL <=> LCAT:discoidal HDL complex
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
LCAT (lecithin-cholesterol acyltransferase) associates strongly but reversibly with discoidal HDL particles (Jonas 2000).
References
A Jonas, "Lecithin cholesterol acyltransferase", Biochim Biophys Acta, 1529, 2000, 245-56.
S Adimoolam, L Jin, E Grabbe, JJ Shieh, A Jonas, "Structural and functional properties of two mutants of lecithin-cholesterol acyltransferase
(T123I and N228K)", J Biol Chem, 273, 1998, 32561-7.
Reaction
15.9.2.8.3 cholesterol + phosphatidylcholine (lecithin) => cholesterol ester + 2-lysophosphatidylcholine (lysolecithin)
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
LCAT activated by apoA-I catalyzes the reaction of cholesterol and phosphatidylcholine to yield cholesterol esterified with a long-chain fatty acid
and 2-lysophosphatidylcholine. While this reaction was first studied in vitro using purified proteins in solution, it occurs in vivo on the surfaces of
HDL particles where transiently-bound LCAT is activated by HDL-associated apoA-I protein and consumes HDL-associated cholesterol and
phosphatidylcholine. The cholesterol ester reaction product is strongly associated with the HDL particle because of its increased hydrophobicity,
while the 2-lysophosphatidylcholine product is released from the particle (Fielding et al. 1972 [2 references]; Adimoolam et al. 1998).
References
CJ Fielding, VG Shore, PE Fielding, "A protein cofactor of lecithin:cholesterol acyltransferase", Biochem Biophys Res Commun, 46, 1972,
1493-8.
CJ Fielding, VG Shore, PE Fielding, "Lecithin: cholesterol acyltransferase: effects of substrate composition upon enzyme activity", Biochim
Biophys Acta, 270, 1972, 513-8.
Reaction
15.9.2.9 LCAT:discoidal HDL complex <=> LCAT + discoidal HDL
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
The LCAT:discoidal HDL complex dissociates reversibly to yield LCAT and a discoidal HDL particle.
References
Reaction
15.9.2.10 Remodeling of spherical HDL
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
Spherical (mature) HDL particles gain and lose apolipoproteins and lipids as they circulate. In the body, the net effect of these events is to
mediate the net uptake of cholesterol from a variety of cell types including foam cells in atherosclerotic lesions, its conversion to cholesterol
esters, and the transfer of these to LDL particles or directly to liver cells.
References
Reaction
15.9.2.10.1 TG-depleted chylomicron + spherical HDL => chylomicron remnant + spherical HDL:apoA-I:apoA-II:apoA-IV:apoC-II:apoC-III
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
Triacylglycerol (TG)-depleted chylomicrons transfer A and C apoproteins to spherical (mature) HDL, generating chylomicron remnants (Havel
and Kane 2001). This transfer is arbitrarily annotated here as involving one molecule of each apolipoprotein. The molecular difference that
enables nascent chylomicrons to accept apolipoproteins from sperical HDL and TG-depleted chylomicrons to donate them is unclear.
References
Reaction
15.9.2.10.2 nascent chylomicron + spherical HDL:apoC-II:apoC-III:apoE =>spherical HDL + chylomicron
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
Circulating nascent chylomicrons acquire copies of apolipoproteins C-II, C-III, and E from circulating spherical (mature) high-density lipoprotein
particles, becoming mature chylomicrons (Havel et al. 1973; Bisgaier and Glickman 1983). Here, this interaction is annotated to involve the
transfer of a single copy of each lipoprotein, but a mature chylomicron in fact contains approximately 25 copies of apolipoprotein E and 180
copies of C apolipoproteins (Bhattacharya and Redgrave 1981).
References
RJ Havel, JP Kane, ML Kashyap, "Interchange of apolipoproteins between chylomicrons and high density lipoproteins during alimentary lipemia
in man", J Clin Invest, 52, 1973, 32-8.
CL Bisgaier, RM Glickman, "Intestinal synthesis, secretion, and transport of lipoproteins", Annu Rev Physiol, 45, 1983, 625-36.
Reaction
15.9.2.10.3 Spherical HDL binds C and E apolipoproteins
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
Spherical HDL particles can bind apoC-II, apoC-III and and apoE proteins. The sources of these proteins and their role or roles in HDL function
under physiological conditions are not well understood, however (Kontush and Chapman 2006).
References
Reaction
15.9.2.10.4 Spherical HDL binds membrane-associated free cholesterol and phospholipids
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
Spherical (mature) HDL particles can acquire additional molecules of free cholesterol and phospholipid from cell membranes. In the body, this is
an important step in the so-called reverse cholesterol transport process in which excess cholesterol, notably in foam cells in atherosclerotic
plaques, is transferred to HDL particles and transported ultimately to the liver. While studies in vitro and in mutant mice indicate that PLTP
(phospholipid transfer protein) plays a major role in this process, its molecular details remain unclear (Oram et al. 2003).
References
JF Oram, G Wolfbauer, AM Vaughan, C Tang, JJ Albers, "Phospholipid transfer protein interacts with and stabilizes ATP-binding cassette
transporter A1 and enhances cholesterol efflux from cells", J Biol Chem, 278, 2003, 52379-85.
Reaction
15.9.2.10.5 cholesterol + phosphatidylcholine (lecithin) => cholesterol ester + 2-lysophosphatidylcholine (lysolecithin)
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
LCAT activated by apoA-I catalyzes the reaction of cholesterol and phosphatidylcholine to yield cholesterol esterified with a long-chain fatty acid
and 2-lysophosphatidylcholine. While this reaction was first studied in vitro using purified proteins in solution, it occurs in vivo on the surfaces of
HDL particles where transiently-bound LCAT is activated by HDL-associated apoA-I protein and consumes HDL-associated cholesterol and
phosphatidylcholine. The cholesterol ester reaction product is strongly associated with the HDL particle because of its increased hydrophobicity,
while the 2-lysophosphatidylcholine product is released from the particle (Fielding et al. 1972 [2 references]; Adimoolam et al. 1998).
References
CJ Fielding, VG Shore, PE Fielding, "A protein cofactor of lecithin:cholesterol acyltransferase", Biochem Biophys Res Commun, 46, 1972,
1493-8.
CJ Fielding, VG Shore, PE Fielding, "Lecithin: cholesterol acyltransferase: effects of substrate composition upon enzyme activity", Biochim
Biophys Acta, 270, 1972, 513-8.
Reaction
15.9.2.10.6 LCAT + spherical HDL <=> LCAT:spherical HDL complex
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
LCAT (lecithin-cholesterol acyltransferase) associates strongly but reversibly with spherical HDL particles (Jonas 2000).
References
A Jonas, "Lecithin cholesterol acyltransferase", Biochim Biophys Acta, 1529, 2000, 245-56.
Reaction
15.9.2.10.7 CETP-mediated lipid exchange: spherical HDL gains triacylglycerol
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
CETP (cholesterol ester transfer protein) complexed with triacylglycerol interacts with a spherical HDL (high density lipoprotein) particle,
acquiring cholesterol ester molecules and donating triacylglycerol to the HDL (Swenson et al. 1988; Morton and Zilversmit 1983). This process is
reversible but in the body proceeds in the direction annotated here. A model for the lipid exchange process has been proposed based on recent
studies of the structure of CETP:lipid complexes (Qiu et al. 2007).
References
TL Swenson, RW Brocia, AR Tall, "Plasma cholesteryl ester transfer protein has binding sites for neutral lipids and phospholipids", J Biol Chem,
263, 1988, 5150-7.
X Qiu, A Mistry, MJ Ammirati, BA Chrunyk, RW Clark, Y Cong, JS Culp, DE Danley, TB Freeman, KF Geoghegan, MC Griffor, SJ Hawrylik, CM
Hayward, P Hensley, LR Hoth, GA Karam, ME Lira, DB Lloyd, KM McGrath, KJ Stutzman-Engwall, AK Subashi, TA Subashi, JF Thompson, IK
Wang, H Zhao, AP Seddon, "Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules", Nat
Struct Mol Biol, 14, 2007, 106-13.
RE Morton, DB Zilversmit, "Inter-relationship of lipids transferred by the lipid-transfer protein isolated from human lipoprotein-deficient plasma", J
Biol Chem, 258, 1983, 11751-7.
Reaction
15.9.2.11 CETP + spherical HDL + torcetrapib => CETP:spherical HDL:torcetrapib complex
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
Torcetrapib associates with a molecule of CETP and a spherical HDL particle to form a stable complex, thus trapping CETP and inhibiting
CETP-mediated lipid transfer between HDL and LDL (Clark et al. 2006).
References
RW Clark, RB Ruggeri, D Cunningham, MJ Bamberger, "Description of the torcetrapib series of cholesteryl ester transfer protein inhibitors,
including mechanism of action", J Lipid Res, 47, 2006, 537-52.
Reaction
15.9.2.12 LCAT:spherical HDL complex <=> LCAT + spherical HDL
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
The LCAT:spherical HDL complex dissociates reversibly to yield LCAT and a spherical HDL particle.
References
Reaction
15.9.2.13 spherical HDL and SR-BI receptor form a complex at the cell surface
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
An extracellular spherical HDL particle binds to the plasma membrance-associated SR-BI receptor with high affinity (Murao et al. 1997). In the
body SR-BI receptors are abundant on the surfaces of steroidogenic cells in the adrenal glands and gonads, and on hepatocytes. SR-BI thus
appears to play a central role in cholesterol uptake for steroid hormone synthesis and for bile acid synthesis and cholesterol excretion (Rigotti et
al. 2003; Silver and Tall 2001).
References
K Murao, V Terpstra, SR Green, N Kondratenko, D Steinberg, O Quehenberger, "Characterization of CLA-1, a human homologue of rodent
scavenger receptor BI, as a receptor for high density lipoprotein and apoptotic thymocytes", J Biol Chem, 272, 1997, 17551-7.
A Rigotti, HE Miettinen, M Krieger, "The role of the high-density lipoprotein receptor SR-BI in the lipid metabolism of endocrine and other
tissues", Endocr Rev, 24, 2003, 357-87.
DL Silver, AR Tall, "The cellular biology of scavenger receptor class B type I", Curr Opin Lipidol, 12, 2001, 497-504.
Reaction
15.9.2.14 Disassembly of SR-BI-bound spherical HDL
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
Spherical HDL particles bound to the cell-surface SR-BI receptor are disassembled, with the release of pre-beta HDL (essentially apoA-I
lipoprotein with a few molecules of bound lipid) and the cellular uptake of the bulk of the HDL-associated cholesterol, cholesterol esters,
phospholipids, and triacylglycerols. The specificity and efficiency of this process has been demonstrated through a variety of studies in tissue
culture model systems (Rigotti et al. 2003; Silver and Tall 2001). The process is annotated here as a concerted event occuring at the cell surface
but its molecular details remain incompletely defined and it is possible that the HDL particle is internalized while undergoing disassembly.
References
A Rigotti, HE Miettinen, M Krieger, "The role of the high-density lipoprotein receptor SR-BI in the lipid metabolism of endocrine and other
tissues", Endocr Rev, 24, 2003, 357-87.
DL Silver, AR Tall, "The cellular biology of scavenger receptor class B type I", Curr Opin Lipidol, 12, 2001, 497-504.
Reaction
15.9.2.15 Endocytosis and degradation of apoA-I
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
ApoA-I bound to CUBN:AMN on the cell surface is endocytosed, moved to lysosomes, and degraded (Kozyraki et al. 1999). It is not known
whether the CUBN:AMN complex is also degraded or recycled to the cell surface.
References
R Kozyraki, JC Fyfe, M Kristiansen, C Gerdes, C Jacobsen, S Cui, EI Christensen, M Aminoff, A de la Chapelle, R Krahe, PJ Verroust, SK
Moestrup, "The intrinsic factor-vitamin B12 receptor, cubilin, is a high-affinity apolipoprotein A-I receptor facilitating endocytosis of high-density
lipoprotein", Nat Med, 5, 1999, 656-61.
Reaction
15.9.2.16 Serum albumin binds 2-lysophosphatidylcholine
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
Serum albumin binds 2-lysophosphatidylcholine (lysolecithin) to form a complex. Two molecules of lipid bind strongly to a molecule of albumin;
an additional five molecules bind more weakly (Nakagawa and Nishida 1973). The fate of the complex in vivo is unclear. In vitro
2-lysophosphatidylcholine can be esterified with fatty acid to generate phosphatidylcholine. Such a process could replenish the
phosphatidylcholine consumed by cholesterol esterification in HDL particles, but the extent to which it occurs in vivo is unclear (Nakagawa and
Nishida 1973; Switzer and Eder 1965).
References
S Switzer, HA Eder, "Transport of lysolecithin by albumin in human and rat plasma", J Lipid Res, 6, 1965, 506-11.
M Nakagawa, T Nishida, "Effect of lysolecithin and albumin on lecithin-cholesterol acyltransferase activity in human plasma", J Biochem, 74,
1973, 1263-6.
Reaction
15.9.2.17 apoA-I binds to CUBN:AMN
Authors
Editors
Reviewers
Jassal, B, 2008-06-13.
Description
Extracellular apoA-I protein binds to the CUBN (cubilin) subunit of the CUBN:AMN complex associated with the plasma membrane. In the body,
this complex is found on the apical surfaces of kidney glomerular cells, where it mediates binding and endocytosis of proteins in the glomerular
filtrate, and on the apical surfaces of enterocytes, where it mediates uptake of several vitamins complexed with carrier proteins (notably vitamin
B12 (cobalamin):intrinsic factor) (Kozyraki et al. 1999; Fyfe et al. 2004).
References
R Kozyraki, JC Fyfe, M Kristiansen, C Gerdes, C Jacobsen, S Cui, EI Christensen, M Aminoff, A de la Chapelle, R Krahe, PJ Verroust, SK
Moestrup, "The intrinsic factor-vitamin B12 receptor, cubilin, is a high-affinity apolipoprotein A-I receptor facilitating endocytosis of high-density
lipoprotein", Nat Med, 5, 1999, 656-61.
S DuguÃ©-Pujol, X Rousset, D ChÃ¢teau, D Pastier, C Klein, J Demeurie, C Cywiner-Golenzer, M Chabert, PJ Verroust, J Chambaz, FP
ChÃ¢telet, AD Kalopissis, "Apolipoprotein A-II is catabolized in the kidney as a function of its plasma concentration", J Lipid Res, 48, 2007,
2151-61.
Reaction
15.9.3 LDL endocytosis
Authors
Editors
Description
LDL (low density lipoproteins) are complexes of a single molecule of apoprotein B-100 (apoB-100) non-covalently associated with triacylglycerol,
free cholesterol, cholesterol esters, and phospholipids. LDL complexes contain single molecules of apoB-100, but their content of lipids is
variable (Chapman et al. 1988; Mateu et al. 1972; Tardieu et al. 1976). High levels of LDL in the blood are strongly correlated with increased risk
of atherosclerosis, and recent studies have raised the possibility that this risk is further increased in individuals whose blood LDL population is
enriched in high-density (low lipid content) LDL complexes (Rizzo and Berneis 2006). The LDL complex annotated here is given a lipid
composition that is the average of those measured in the studies by Chapman, Mateu, Tardieu and their colleagues.
As outlined in the figure below, extracellular low density lipoprotein (LDL) complexes bind to LDL receptors associated with coated pits at the cell
surface (a), forming complexes that are internalized and passed via clathrin-coated vesicles (b) to endosomes (c), where they dissociate. The
LDL particles move into lysosomes (d) and are degraded while the LDL receptors are returned to the cell surface (e). This process occurs in
most cell types but is especially prominent in hepatocytes. It plays a major role in returning cholesterol from peripheral tissues to the liver.
Familial hypercholesterolemia due to mutations affecting the LDL receptor (or, rarely, the apolipoprotein B-100 (apoB-100) component of LDL or
the LDLRAP1 accessory protein involved in receptor uptake into clathrin-coated vesicles) is one of the commonest human genetic diseases,
affecting approximately one person in 500 in many populations worldwide (Hobbs et al. 1990; Jeon and Blacklow 2005).
References
L Mateu, A Tardieu, V Luzzati, L Aggerbeck, AM Scanu, "On the structure of human serum low density lipoprotein", J Mol Biol, 70, 1972, 105-16.
M Rizzo, K Berneis, "Low-density lipoprotein size and cardiovascular risk assessment", QJM, 99, 2006, 1-14.
A Tardieu, L Mateu, C Sardet, B Weiss, V Luzzati, L Aggerbeck, AM Scanu, "Structure of human serum lipoproteins in solution. II. Small-angle
x-ray scattering study of HDL3 and LDL.", J Mol Biol, 105, 1976, 459-60.
HH Hobbs, DW Russell, MS Brown, JL Goldstein, "The LDL receptor locus in familial hypercholesterolemia: mutational analysis of a membrane
protein", Annu Rev Genet, 24, 1990, 133-70.
H Jeon, SC Blacklow, "Structure and physiologic function of the low-density lipoprotein receptor", Annu Rev Biochem, 74, 2005, 535-62.
MJ Chapman, PM Laplaud, G Luc, P Forgez, E Bruckert, S Goulinet, D Lagrange, "Further resolution of the low density lipoprotein spectrum in
normal human plasma: physicochemical characteristics of discrete subspecies separated by density gradient ultracentrifugation", J Lipid Res,
29, 1988, 442-58.
15.9.3.1 LDL + LDLR => LDL:LDLR complex
Authors
Editors
Description
Low density lipoprotein (LDL) particles associate with LDL receptors (LDLR) at the cell surface (Goldstein et al. 1979). This binding is mediated
by the apoprotein B-100 component of the LDL particle, which binds LDLR with 1:1 stoichiometry (van Driel et al. 1989).
References
IR Van Driel, MS Brown, JL Goldstein, "Stoichiometric binding of low density lipoprotein (LDL) and monoclonal antibodies to LDL receptors in a
solid phase assay", J Biol Chem, 264, 1989, 9533-8.
Reaction
15.9.3.2 LDL:LDLR complex [plasma membrane] => LDL:LDLR complex [clathrin-coated vesicle] (LDLRAP1-dependent)
Authors
Editors
Description
Low density lipoprotein (LDL) particles bound to their receptors (LDLR) in coated pits on the cell surface are taken up into clathrin-coated
vesicles (Goldstein et al. 1979). In hepatocytes and lymphocytes, but not in fibroblasts, this process requires the presence of an additional
protein, LDLRAP1 (ARH1). In human patients, LDLRAP1 deficiency is associated with hypercholesterolemia, emphasizing the central role of the
liver in clearance of circulating LDL in vivo (Eden et al. 2002; Garuti et al. 2005; He et al. 2002; Michaely et al. 2004). In vitro, LDLRAP1 protein
binds both to LDLR and to components of the clathrin coat, suggesting that it might play an essential bridging function during the movement of
LDL:LDLR complexes into clathrin-coated vesicles. This role has not yet been demonstrated in vivo, however, nor is it clear what might
substitute for such a bridging function in fibroblasts.
References
1695-702.
AP-2", J Biol Chem, 277, 2002, 44044-9.
Reaction
15.9.3.3 LDL:LDLR complex [plasma membrane] => LDL:LDLR complex [clathrin-coated vesicle] (LDLRAP1-independent)
Authors
Editors
Description
Low density lipoprotein (LDL) particles bound to their receptors (LDLR) in coated pits on the cell surface are taken up into clathrin-coated
vesicles (Goldstein et al. 1979). In hepatocytes and lymphocytes, but not in fibroblasts, this process requires the presence of an additional
protein, LDLRAP1 (ARH1). In human patients, LDLRAP1 deficiency is associated with hypercholesterolemia, emphasizing the central role of the
liver in clearance of circulating LDL in vivo (Eden et al. 2002; Garuti et al. 2005; He et al. 2002; Michaely et al. 2004). In vitro, LDLRAP1 protein
binds both to LDLR and to components of the clathrin coat, suggesting that it might play an essential bridging function during the movement of
LDL:LDLR complexes into clathrin-coated vesicles. This role has not yet been demonstrated in vivo, however, nor is it clear what might
substitute for such a bridging function in fibroblasts.
References
1695-702.
AP-2", J Biol Chem, 277, 2002, 44044-9.
Reaction
15.9.3.4 LDLR:LDL complex [coated vesicle membrane] => LDLR:LDL complex [endosome membrane]
Authors
Editors
Description
LDL:LDLR complexes move rapidly from clathrin-coated vesicles to endosomes (Goldstein et al. 1979).
References
Reaction
15.9.3.5 LDLR:LDL complex => LDLR + LDL
Authors
Editors
Description
Dissociation of the LDL:LDLR complex in the early endosome frees the LDL particle to be transferred to lysosomes for degradation while the
LDL receptor is returned to the plasma membrane (Goldstein et al. 1979).
References
Reaction
15.9.3.6 LDLR [endosome membrane] => LDLR [plasma membrane]
Authors
Editors
Description
LDL receptors in the endosome membrane are quickly returned to the cell surface (Goldstein et al. 1979).
References
Reaction
16 Membrane Trafficking
Description
The secretory membrane system allows a cell to regulate delivery of newly synthesized proteins, carbohydrates, and lipids to the cell surface, a
necessity for growth and homeostasis. The system is made up of distinct organelles, including the endoplasmic reticulum (ER), Golgi complex,
plasma membrane, and tubulovesicular transport intermediates. These organelles mediate intracellular membrane transport between
themselves and the cell surface. Membrane traffic within this system flows along highly organized directional routes. Secretory cargo is
synthesized and assembled in the ER and then transported to the Golgi complex for further processing and maturation. Upon arrival at the trans
Golgi network (TGN), the cargo is sorted and packaged into post-Golgi carriers that move through the cytoplasm to fuse with the cell surface.
This directional membrane flow is balanced by retrieval pathways that bring membrane and selected proteins back to the compartment of origin.
References
J Lippincott-Schwartz, TH Roberts, K Hirschberg, "Secretory protein trafficking and organelle dynamics in living cells", Annu Rev Cell Dev Biol,
16, 2000, 557-89.
16.1 ER to Golgi Transport
Authors
Reviewers
Rush, MG, 2008-01-11.
Description
Secretory cargo destined to be secreted or to arrive at the plasma membrane (PM) leaves the ER via distinct exit sites. This cargo is destined for
the Golgi apparatus.
References
T Kirchhausen, "Three ways to make a vesicle", Nat Rev Mol Cell Biol, 1, 2000, 187-98.
16.1.1 COPII (Coat Protein 2) Mediated Vesicle Transport
Reviewers
Rush, MG, 2008-01-11.
Description
COPII components (known as Sec13p, Sec23p, Sec24p, Sec31p, and Sar1p in yeast) traffic cargo from the endoplasmic reticulum to the Golgi
apparatus. COPII-coated vesicles were originally discovered in the yeast Saccharomyces cerevisiae using genetic approaches coupled with a
cell-free assay. The mammalian counterpart of this pathway is represented here. Newly synthesized proteins destined for secretion are sorted
into COPII-coated vesicles at specialized regions of the ER. These vesicles leave the ER, become uncoated and subsequently fuse with the
Golgi apparatus membrane.
References
K Sato, A Nakano, "Mechanisms of COPII vesicle formation and protein sorting", FEBS Lett, 581, 2007, 2076-82.
T Kirchhausen, "Three ways to make a vesicle", Nat Rev Mol Cell Biol, 1, 2000, 187-98.
16.1.1.1 Sar1p Activation And Membrane Binding
Reviewers
Rush, MG, 2008-01-11.

Description
Sar1p-GDP is recruited to the ER membrane by the transmembrane GEF (Guanine nucleotide exchange factor) Sec12, where it is converted to
Sar1p-GTP.
References
DJ Stephens, N Lin-Marq, A Pagano, R Pepperkok, JP Paccaud, "COPI-coated ER-to-Golgi transport complexes segregate from COPII in close
proximity to ER exit sites", J Cell Sci, 113, 2000, 2177-85.
AT Hammond, BS Glick, "Dynamics of transitional endoplasmic reticulum sites in vertebrate cells", Mol Biol Cell, 11, 2000, 3013-30.
Reaction
16.1.1.2 Coat Assembly
Reviewers
Rush, MG, 2008-01-11.
Description
Sar1p-GTP recruits the cytoplasmic Sec23p-Sec24p complex. Though not represented in the subsequent steps, Sec23p-Sec24p would bind to
members of the p24 protein family of possible cargo receptors, and together with Sar1p bind the appropiate v-SNARE.
References
Reaction
16.1.1.3 Loss of Sar1p GTPase
Reviewers
Rush, MG, 2008-01-11.
Description
Sar1p-GTP hydrolysis is increased 15-30-fold by Sec23p. Sar1p-GDP is released as a result of this hydrolysis and used in further vesicle
sculpting cycles. Sar1p-GTP hydrolysis occurs at two critical points during the cycle, first (as represented here) as a proofreading step, insuring
that the cargo is loaded. Later in the cycle Sar1p-GTP hydrolysis triggers the uncoating of the budded vesicle.
References
Reaction
16.1.1.4 Cargo, Sec31p:Sec13p, and v-SNARE recruitment

Reviewers
Rush, MG, 2008-01-11.
Description
Cytosolic Sec13p-Sec31p complexes bind to pre-bound Sec23p-Sec24p complexes.
References
Reaction
16.1.1.5 Vesicle Budding
Reviewers
Rush, MG, 2008-01-11.
Description
Once loaded the vesicles become fully sculpted, pinch off from the ER and bud into the cytosol.
References
Reaction
16.1.1.6 Vesicle Uncoating
Reviewers
Rush, MG, 2008-01-11.
Description
Vesicle uncoating is triggered by Sar1p-GTP hydrolysis leaving only the vesicle cargo and the v-SNARE to target the vesicle to the Golgi
membrane.
References
Reaction
16.2 Golgi to ER Retrograde Transport
Description
The anterograde membrane flow from the ER to the Golgi is compensated for by a retrograde pathway. This pathway's functions include the
recycling of membrane and proteins to the ER.
References
J Lippincott-Schwartz, TH Roberts, K Hirschberg, "Secretory protein trafficking and organelle dynamics in living cells", Annu Rev Cell Dev Biol,
16, 2000, 557-89.
16.2.1 COPI Mediated Transport
Authors
Reviewers
Lippincott-Schwartz, J, 2007-08-07.
Description
Secretory transport depends on membrane-bounded carriers to move protein and lipid between intracellular compartments. The COPI coat has
a central role in this process, creating a sorting domain on the membrane into which cargo proteins, destined to return to the endoplasmic
reticulum (ER), concentrate. The membrane domain deforms into a coated bud, pinches off the membrane as a coated carrier, and then
uncoats. Successful operation of the COPI coat system is necessary for selective retrieval of protein and lipid components back to the ER.
References
J Lippincott-Schwartz, W Liu, "Insights into COPI coat assembly and function in living cells", Trends Cell Biol, 16, 2006, e1-4.
16.2.1.1 Arf1 Activation by GBF1
Authors
Reviewers
Description
The formation of the COPI coat requires membrane recruitment and activation of Arf1. Arf1 activation is normally mediated by guanine
nucleotide exchange factors (GEFs) that convert Arf1 to its active GTP-bound state on the membrane. In this reaction that GEF is represented
by GBF1.
References
A Claude, BP Zhao, CE Kuziemsky, S Dahan, SJ Berger, JP Yan, AD Armold, EM Sullivan, P Melancon, "GBF1: A novel Golgi-associated
BFA-resistant guanine nucleotide exchange factor that displays specificity for ADP-ribosylation factor 5", J Cell Biol, 146, 1999, 71-84.
Reaction
16.2.1.2 Coat Complex Formation
Authors
Reviewers
Description
In models of COPI coat assembly and disassembly, after GBF1 activates Arf1 on membranes, the active form of Arf1 recruits coatomer
complexes from the cytoplasm.
References
R Schekman, L Orci, "Coat proteins and vesicle budding", Science, 271, 1996, 1526-33.
Reaction
16.2.1.3 GAP Recruitment to the Coatomer:Arf1-GTP Complex
Authors
Reviewers
Description
Once both coatomer and the active form of Arf1 have been recruited to the Golgi membrane surface, ArfGAP joins the complex.
References
E Cukierman, I Huber, M Rotman, D Cassel, "The ARF1 GTPase-activating protein: zinc finger motif and Golgi complex localization", Science,
270, 1995, 1999-2002.
Reaction
16.2.1.4 Coatomer:Arf1-GTP:GAP lattice formation on golgi membrane
Authors
Reviewers
Description
Together, the complex of coatomer, Arf1, and ArfGAP assembles into a lattice that concentrates cargo proteins and deforms the membrane.
Eventually this membrane pocket forms into a bud that pinches off the membrane, and is released into the cytoplasm (Lippincott-Schwartz and
Liu 2006).
References
Reaction
16.2.1.5 Sculpting and pinching-off of Golgi vessicle
Authors
Reviewers
Description
All of the key components and regulators of the COPI coat (GBF1, Arf1, ArfGAP1 and coatomer) cycle on and off the Golgi membrane. This
cycle is required for vesicle formation, but is uncoupled from the actual vesicle release. Continuous membrane binding and release of these
molecules enables the COPI lattice to be dynamically modulated. Once the vesicle is released from the Golgi apparatus, this lattice is completely
disassociated from the vesicle.
References
J Bigay, P Gounon, S Robineau, B Antonny, "Lipid packing sensed by ArfGAP1 couples COPI coat disassembly to membrane bilayer curvature",
Nature, 426, 2003, 563-6.
JS Yang, SY Lee, M Gao, S Bourgoin, PA Randazzo, RT Premont, VW Hsu, "ARFGAP1 promotes the formation of COPI vesicles, suggesting
function as a component of the coat", J Cell Biol, 159, 2002, 69-78.
Reaction
16.2.1.6 Hydrolysis of Arf1-GTP to Arf1-GDP
Authors
Reviewers
Description
The hydrolysis of Arf1-bound GTP is induced by the recruited ArfGAP.
References
W Liu, R Duden, RD Phair, J Lippincott-Schwartz, "ArfGAP1 dynamics and its role in COPI coat assembly on Golgi membranes of living cells", J
Cell Biol, 168, 2005, 1053-63.
Reaction
16.2.1.7 Diffusion of inactive Arf1-GDP from membrane
Authors
Reviewers
Description
Inactive Arf1-GDP then diffuses away from the membrane, initiating the disassembly of the lattice.
References
W Liu, R Duden, RD Phair, J Lippincott-Schwartz, "ArfGAP1 dynamics and its role in COPI coat assembly on Golgi membranes of living cells", J
Cell Biol, 168, 2005, 1053-63.
Reaction
16.2.1.8 Golgi vesicle lattice disassociation
Authors
Reviewers
Description
Ultimately the hydrolysis of Arf1-bound GTP, induced by the recruited ArfGAP, allows the inactive Arf1-GDP to diffuses away from the
membrane, initiating disassembly of the lattice from the vesicle (Lippincott-Schwartz and Liu 2006).
References
Reaction
17 Metabolism of amino acids
Authors
Editors
Description
This group of reactions is responsible for: 1) the breakdown of amino acids; 2) the synthesis of urea from ammonia and amino groups generated
by amino acid breakdown; 3) the synthesis of the ten amino acids that are not essential components of the human diet; and 4) the synthesis of
related nitrogen-containing molecules including carnitine and creatine.
17.1 Amino acid uptake across the plasma membrane
Authors
Editors
Reviewers
Jassal, B, 2008-06-03.
Description
Amino acid transport across plasma membranes is critical to the uptake of these molecules from the gut, to their reabsortion in the kidney
proximal tubulues, and to their distribution to cells in which they are required for the synthesis of proteins and of amino acid derived small
molecules such as neurotransmitters. Physiological studies have defined 18 "systems" that mediate amino acid transport, each characterized by
its amino acid substrates, as well as its pH sensitivity and its association (or not) with ion transport. More recently, molecular cloning studies
have allowed the identification of the plasma membrane transport proteins that mediate these reactions. Amino acid uptake mediated by 15 of
these transporters is annotated here (Broer 2008).
References
S BrÃ¶er, "Amino acid transport across mammalian intestinal and renal epithelia", Physiol Rev, 88, 2008, 249-86.
17.1.1 SLC16A10-mediated uptake of aromatic amino acids
Authors
Editors
Reviewers
Jassal, B, 2008-06-03.
Description
SLC16A10 mediates the reversible facilitated diffusion of phenylalanine, tyrosine, and tryptophan across the plasma membrane. The process is
Na+-independent and not coupled to H+ transport. As measured by Northern blotting SLC16A10 is widely expressed in the body but especially
abundant in kidney. In situ hybridization studies indicate that the gene product is abundant in kidney proximal tubules (Kim et al. 2001; Kim et al.
2002; Park et al. 2005).
References
SY Park, JK Kim, IJ Kim, BK Choi, KY Jung, S Lee, KJ Park, A Chairoungdua, Y Kanai, H Endou, DK Kim, "Reabsorption of neutral amino acids
mediated by amino acid transporter LAT2 and TAT1 in the basolateral membrane of proximal tubule", Arch Pharm Res, 28, 2005, 421-32.
DK Kim, Y Kanai, A Chairoungdua, H Matsuo, SH Cha, H Endou, "Expression cloning of a Na+-independent aromatic amino acid transporter
with structural similarity to H+/monocarboxylate transporters", J Biol Chem, 276, 2001, 17221-8.
DK Kim, Y Kanai, H Matsuo, JY Kim, A Chairoungdua, Y Kobayashi, A Enomoto, SH Cha, T Goya, H Endou, "The human T-type amino acid
transporter-1: characterization, gene organization, and chromosomal location", Genomics, 79, 2002, 95-103.
Reaction
17.1.2 SLC38A1 (ATA1)-mediated uptake of neutral amino acids
Authors
Editors
Reviewers
Jassal, B, 2008-06-03.
Description
SLC38A1 (ATA1), associated with the plasma membrane, mediates the uptake of neutral amino acids, especially alanine, asparagine,
glutamine, methionine, and serine in a sodium ion-dependent transport process. Northern blotting experiments indicate gene expression in
placenta and heart, and at lower levels in other tissues including brain, lung, skeletal muscle, spleen, stomach and testis, but not kidney or
intestine (Wang et al. 2000).
References
H Wang, W Huang, M Sugawara, LD Devoe, FH Leibach, PD Prasad, V Ganapathy, "Cloning and functional expression of ATA1, a subtype of
amino acid transporter A, from human placenta", Biochem Biophys Res Commun, 273, 2000, 1175-9.
Reaction
17.1.3 SLC38A2 (ATA2)-mediated uptake of neutral amino acids
Authors
Editors
Reviewers
Jassal, B, 2008-06-03.
Description
SLC38A2 (ATA2), associated with the plasma membrane, mediates the uptake of neutral amino acids, especially alanine, asparagine,
glutamine, glycine, leucine, methionine, proline, and threonine in a sodium ion-dependent transport process. Northern blotting experiments
indicate gene expression in placenta and heart, and at lower levels in other tissues including brain, lung, skeletal muscle, spleen, stomach,
testis, kidney, and intestine (Hatanaka et al. 2000).
References
T Hatanaka, W Huang, H Wang, M Sugawara, PD Prasad, FH Leibach, V Ganapathy, "Primary structure, functional characteristics and tissue
expression pattern of human ATA2, a subtype of amino acid transport system A", Biochim Biophys Acta, 1467, 2000, 1-6.
Reaction
17.1.4 SLC38A3-mediated uptake of glutamine, histidine, asparagine, and alanine
Authors
Editors
Reviewers
Jassal, B, 2008-06-03.
Description
SLC38A3 (SN1), associated with the plasma membrane, mediates the uptake of glutamine, histidine, and, with lower efficiency, alanine and
asparagine. Uptake of one molecule of amino acid is coupled to the uptake of two sodium ions and the export of one H+. Northern blotting
experiments indicate gene expression in liver and kidney, and at much lower levels in brain, lung, skeletal muscle, spleen, stomach, testis,
kidney, and intestine (Fei et al. 2000; Nakanishi et al. 2001).
References
YJ Fei, M Sugawara, T Nakanishi, W Huang, H Wang, PD Prasad, FH Leibach, V Ganapathy, "Primary structure, genomic organization, and
functional and electrogenic characteristics of human system N 1, a Na+- and H+-coupled glutamine transporter", J Biol Chem, 275, 2000,
23707-17.
T Nakanishi, M Sugawara, W Huang, RG Martindale, FH Leibach, ME Ganapathy, PD Prasad, V Ganapathy, "Structure, function, and tissue
expression pattern of human SN2, a subtype of the amino acid transport system N", Biochem Biophys Res Commun, 281, 2001, 1343-8.
Reaction
17.1.5 SLC38A4 (ATA3)-mediated uptake of arginine and lysine
Authors
Editors
Reviewers
Jassal, B, 2008-06-03.
Description
SLC38A4 (ATA3), associated with the plasma membrane, mediates the sodium-independent uptake of arginine and lysine. SLC38A4 was first
identified on the basis of its similarity to SLC38A1 and SLC38A2. Like those two transporters, it can mediate the sodium-dependent uptake of
neutral amino acids in cultured cells transfected with an expression vector, but it does so very inefficiently and its role, if any, in neutral amino
acid uptake in vivo is unclear. By Northern blotting, SLC38A4 is abundant in liver and undetectable in all other tissues tested, including heart,
placenta, kidney, and intestine (Hatanaka et al. 2001).
References
T Hatanaka, W Huang, R Ling, PD Prasad, M Sugawara, FH Leibach, V Ganapathy, "Evidence for the transport of neutral as well as cationic
amino acids by ATA3, a novel and liver-specific subtype of amino acid transport system A", Biochim Biophys Acta, 1510, 2001, 10-7.
Reaction
17.1.6 SLC38A5-mediated uptake of glutamine, histidine, asparagine, and serine
Authors
Editors
Reviewers
Jassal, B, 2008-06-03.
Description
SLC38A5 (SN2), associated with the plasma membrane, mediates the uptake of asparagine, glutamine, histidine, serine and, with lower
efficiency, alanine and glycine. Indirect assays suggest that amino acid uptake is coupled to the uptake of sodium ion(s) and the export of H+.
Northern blotting experiments indicate gene expression in brain and stomach, and at lower levels in liver, lung, and intestine (Nakanishi et al.
2001).
References
T Nakanishi, M Sugawara, W Huang, RG Martindale, FH Leibach, ME Ganapathy, PD Prasad, V Ganapathy, "Structure, function, and tissue
expression pattern of human SN2, a subtype of the amino acid transport system N", Biochem Biophys Res Commun, 281, 2001, 1343-8.
Reaction
17.1.7 SLC43A1 (LAT3)-mediated uptake of large neutral amino acids
Authors
Editors
Reviewers
Jassal, B, 2008-06-03.
Description
SLC43A1 (LAT3), associated with the plasma membrane, mediates the uptake of isoleucine, leucine, methionine, phenylalanine, and valine in a
biphasic and sodium ion-independent transport process. Northern blotting experiments indicate gene expression in liver, pancreas, and skeletal
muscle, and at lower levels in many tissues including kidney and intestine (Babu et al. 2003).
References
E Babu, Y Kanai, A Chairoungdua, DK Kim, Y Iribe, S Tangtrongsup, P Jutabha, Y Li, N Ahmed, S Sakamoto, N Anzai, S Nagamori, H Endou,
"Identification of a novel system L amino acid transporter structurally distinct from heterodimeric amino acid transporters", J Biol Chem, 278,
2003, 43838-45.
Reaction
17.1.8 SLC43A2 (LAT4)-mediated uptake of large neutral amino acids
Authors
Editors
Reviewers
Jassal, B, 2008-06-03.
Description
SLC43A2 (LAT4), associated with the plasma membrane, mediates the uptake of isoleucine, leucine, methionine, phenylalanine, and valine in a
biphasic and sodium ion-independent transport process. Northern blotting and in situ hybridization experiments indicate gene expression in
kidney and intestine (Bodoy et al. 2005).
References
S Bodoy, L MartÃ-n, A Zorzano, M PalacÃ-n, R EstÃ©vez, J Bertran, "Identification of LAT4, a novel amino acid transporter with system L
activity", J Biol Chem, 280, 2005, 12002-11.
Reaction
17.1.9 SLC6A12 (BGT-1)-mediated uptake of GABA and betaine
Authors
Editors
Reviewers
Jassal, B, 2008-06-03.
Description
The plasma membrane transport protein SLC6A12 (BGT-1) mediates the uptake of GABA (gamma-aminobutyrate) and betaine and, less
efficiently, of diminobutyrate (DABA) and beta-alanine. Together with each amino acid molecule, 3 sodium ions and 2 chloride ions are taken up.
In the body, SLC6A12 is expressed in the proximal tubules of the kidney and cells of the central nervous system (Rasola et al. 1995; Matskevitch
et al. 1999).
References
A Rasola, LJ Galietta, V Barone, G Romeo, S Bagnasco, "Molecular cloning and functional characterization of a GABA/betaine transporter from
human kidney", FEBS Lett, 373, 1995, 229-33.
I Matskevitch, CA Wagner, C Stegen, S BrÃ¶er, B Noll, T Risler, HM Kwon, JS Handler, S Waldegger, AE Busch, F Lang, "Functional
characterization of the Betaine/gamma-aminobutyric acid transporter BGT-1 expressed in Xenopus oocytes", J Biol Chem, 274, 1999, 16709-16.
Reaction
17.1.10 SLC6A15-mediated amino acid uptake
Authors
Editors
Reviewers
Jassal, B, 2008-06-03.
Description
SLC6A15, associated with the plasma membrane, mediates the uptake of a broad range of amino acids plus a sodium ion, transporting
branched-chain amiono acids and methionine most efficiently. The human protein is expressed in the brain (Takanaga et al. 2005).
References
H Takanaga, B Mackenzie, JB Peng, MA Hediger, "Characterization of a branched-chain amino-acid transporter SBAT1 (SLC6A15) that is
expressed in human brain", Biochem Biophys Res Commun, 337, 2005, 892-900.
Reaction
17.1.11 SLC6A18-mediated glycine uptake
Authors
Editors
Reviewers
Jassal, B, 2008-06-03.
Description
The protein SLC6A18 was first identified as an amino acid transporter based on sequence similarity to other members of the SLC6 protein family
(Hoglund et al. 2005). It is annotated here as mediating glycine uptake based on the phenotype of mice homozygous for a null mutation in the
homologous gene (Quan et al. 2004).
References
H Quan, K Athirakul, WC Wetsel, GE Torres, R Stevens, YT Chen, TM Coffman, MG Caron, "Hypertension and impaired glycine handling in
mice lacking the orphan transporter XT2", Mol Cell Biol, 24, 2004, 4166-73.
PJ HÃ¶glund, D Adzic, SJ Scicluna, J Lindblom, R Fredriksson, "The repertoire of solute carriers of family 6: identification of new human and
rodent genes", Biochem Biophys Res Commun, 336, 2005, 175-89.
Reaction
17.1.12 SLC6A20-mediated uptake of proline
Authors
Editors
Reviewers
Jassal, B, 2008-06-03.
Description
SLC6A20, associated with the plasma membrane, mediates the uptake of proline plus a sodium ion. The human protein is expressed in the
intestine and kidney (Takanaga et al. 2005).
References
H Takanaga, B Mackenzie, Y Suzuki, MA Hediger, "Identification of mammalian proline transporter SIT1 (SLC6A20) with characteristics of
classical system imino", J Biol Chem, 280, 2005, 8974-84.
Reaction
17.1.13 SLC6A6-mediated uptake of taurine and beta-alanine
Authors
Editors
Reviewers
Jassal, B, 2008-06-03.
Description
The plasma membrane transport protein SLC6A6 mediates the uptake of taurine and beta-alanine. Together with each amino acid molecule, 2
sodium ions and 1 chloride ion are taken up. SLC6A6 is widely expressed in the body (Ramamoorthy et al. 1994).
References
S Ramamoorthy, FH Leibach, VB Mahesh, H Han, T Yang-Feng, RD Blakely, V Ganapathy, "Functional characterization and chromosomal
localization of a cloned taurine transporter from human placenta", Biochem J, 300, 1994, 893-900.
Reaction
17.1.14 SLC7A5-mediated uptake of neutral amino acids
Authors
Editors
Reviewers
Jassal, B, 2008-06-03.
Description
SLC7A5, complexed with SLC3A2 in the plasma membrane, mediates the uptake of neutral amino acids. The process is Na+-independent and
not coupled to H+ transport. As measured by Northern blotting SLC7A5 is widely expressed in the body. In situ hybridization studies indicate that
the gene product is widely expressed in the body but not in the kidney (Pineda et al. 1999; Prasad et al. 1999).
References
PD Prasad, H Wang, W Huang, R Kekuda, DP Rajan, FH Leibach, V Ganapathy, "Human LAT1, a subunit of system L amino acid transporter:
molecular cloning and transport function", Biochem Biophys Res Commun, 255, 1999, 283-8.
M Pineda, E FernÃ¡ndez, D Torrents, R EstÃ©vez, C LÃ³pez, M Camps, J Lloberas, A Zorzano, M PalacÃ-n, "Identification of a membrane
protein, LAT-2, that Co-expresses with 4F2 heavy chain, an L-type amino acid transport activity with broad specificity for small and large
zwitterionic amino acids", J Biol Chem, 274, 1999, 19738-44.
Reaction
17.1.15 SLC7A8-mediated uptake of neutral amino acids
Authors
Editors
Reviewers
Jassal, B, 2008-06-03.
Description
SLC7A8, complexed with SLC3A2 in the plasma membrane, mediates the uptake of neutral amino acids. The process is Na+-independent and
not coupled to H+ transport. As measured by Northern blotting SLC7A8 is widely expressed in the body. In situ hybridization studies indicate that
the gene product is abundant in kidney proximal tubules (Pineda et al. 1999; Park et al. 2005)
References
SY Park, JK Kim, IJ Kim, BK Choi, KY Jung, S Lee, KJ Park, A Chairoungdua, Y Kanai, H Endou, DK Kim, "Reabsorption of neutral amino acids
mediated by amino acid transporter LAT2 and TAT1 in the basolateral membrane of proximal tubule", Arch Pharm Res, 28, 2005, 421-32.
M Pineda, E FernÃ¡ndez, D Torrents, R EstÃ©vez, C LÃ³pez, M Camps, J Lloberas, A Zorzano, M PalacÃ-n, "Identification of a membrane
protein, LAT-2, that Co-expresses with 4F2 heavy chain, an L-type amino acid transport activity with broad specificity for small and large
zwitterionic amino acids", J Biol Chem, 274, 1999, 19738-44.
Reaction
17.1.16 SLC1A4-mediated exchange of alanine, serine, threonine, and cysteine across the plasma
membrane
Authors
Editors
Reviewers
Jassal, B, 2008-06-03.
Description
SLC1A4, associated with the plasma membrane, mediates the exchange of alanine and an extracellular sodium ion for a cytosolic sodium ion
and any one of the four amino acids alanine, serine, threonine, or cysteine (Zerangue and Kavanaugh 1996).
References
JL Arriza, MP Kavanaugh, WA Fairman, YN Wu, GH Murdoch, RA North, SG Amara, "Cloning and expression of a human neutral amino acid
transporter with structural similarity to the glutamate transporter gene family", J Biol Chem, 268, 1993, 15329-32.
S Shafqat, BK Tamarappoo, MS Kilberg, RS Puranam, JO McNamara, A GuadaÃ±o-Ferraz, Jr Fremeau RT, "Cloning and expression of a novel
Na(+)-dependent neutral amino acid transporter structurally related to mammalian Na+/glutamate cotransporters", J Biol Chem, 268, 1993,
15351-5.
N Zerangue, MP Kavanaugh, "ASCT-1 is a neutral amino acid exchanger with chloride channel activity", J Biol Chem, 271, 1996, 27991-4.
Reaction
17.1.17 SLC1A5-mediated exchange of alanine and glutamine across the plasma membrane
Authors
Editors
Reviewers
Jassal, B, 2008-06-03.
Description
SLC1A5, associated with the plasma membrane, mediates the exchange of alanine, glutamine, and sodium ions across the plasma membrane
(Broer et al. 2000; Kekuda et al 1996).
References
A BrÃ¶er, C Wagner, F Lang, S BrÃ¶er, "Neutral amino acid transporter ASCT2 displays substrate-induced Na+ exchange and a
substrate-gated anion conductance", Biochem J, 346, 2000, 705-10.
R Kekuda, PD Prasad, YJ Fei, V Torres-Zamorano, S Sinha, TL Yang-Feng, FH Leibach, V Ganapathy, "Cloning of the sodium-dependent,
broad-scope, neutral amino acid transporter Bo from a human placental choriocarcinoma cell line", J Biol Chem, 271, 1996, 18657-61.
Reaction
17.2 Urea synthesis
Authors
2003-03-31.
Editors
Description
This groups of reactions yields urea, the major form in which excess nitrogen is excreted from the human body, and the amino acid arginine. The
urea cycle consists of four reactions, in which ornithine and carbamoyl phosphate react to form citrulline, citrulline and aspartate react to form
argininosuccinate, argininosuccinate is cleaved to yield fumarate and arginine, and arginine is cleaved to yield urea and ornithine. The
carbamoyl phosphate consumed in this cycle is synthesized in the mitochondria from bicarbonate and ammonia, and this synthesis in turn is
dependent on the presence of N-acetylglutamate (which allosterically activates carbamoyl synthetase I enzyme). The synthesis of
N-acetylglutamate is stimulated by high levels of arginine. Increased levels of free amino acids, indicated by elevated arginine levels, thus
stimulate urea synthesis.
References
2, 2001, 1909-1963.
17.2.1 glutamate + acetyl CoA => N-acetyl glutamate + CoA
Description
At the beginning of this reaction, 1 molecule of 'L-Glutamate', and 1 molecule of 'Acetyl-CoA' are present. At the end of this reaction, 1 molecule
of 'N-Acetyl-L-glutamate', and 1 molecule of 'CoA' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'amino-acid N-acetyltransferase activity' of 'N-acetylglutamate
synthetase'.
References
L Caldovic, H Morizono, M Gracia Panglao, R Gallegos, X Yu, D Shi, MH Malamy, NM Allewell, M Tuchman, "Cloning and expression of the
human N-acetylglutamate synthase gene.", Biochem Biophys Res Commun, 299, 2002, 581-6.
Reaction
17.2.2 2 ATP + NH4+ + HCO3- => 2 ADP + orthophosphate + carbamoyl phosphate [mitochondrial]
Description
At the beginning of this reaction, 1 molecule of 'NH4+', 1 molecule of 'HCO3-', and 1 molecule of 'ATP' are present. At the end of this reaction, 1
molecule of 'Carbamoyl phosphate', 1 molecule of 'ADP', and 1 molecule of 'Orthophosphate' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'carbamoyl-phosphate synthase (ammonia) activity' of
'carbamoyl-phosphate synthetase I dimer'.
References
2, 2001, 1909-1963.
DL Pierson, JM Brien, "Human carbamylphosphate synthetase I. Stabilization, purification, and partial characterization of the enzyme from
human liver.", J Biol Chem, 255, 1980, 7891-5.
Reaction
17.2.3 carbamoyl phosphate + ornithine => citrulline + orthophosphate
Description
At the beginning of this reaction, 1 molecule of 'L-Ornithine', and 1 molecule of 'Carbamoyl phosphate' are present. At the end of this reaction, 1
molecule of 'Orthophosphate', and 1 molecule of 'L-Citrulline' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'ornithine carbamoyltransferase activity' of 'ornithine
transcarbamylase homotrimer'.
References
AL Horwich, WA Fenton, KR Williams, F Kalousek, JP Kraus, RF Doolittle, W Konigsberg, LE Rosenberg, "Structure and expression of a
complementary DNA for the nuclear coded precursor of human mitochondrial ornithine transcarbamylase.", Science, 224, 1984, 1068-74.
Reaction
17.2.4 ATP + aspartate + citrulline <=> argininosuccinate + AMP + pyrophosphate
Description
At the beginning of this reaction, 1 molecule of 'L-Aspartate', 1 molecule of 'L-Citrulline', and 1 molecule of 'ATP' are present. At the end of this
reaction, 1 molecule of 'pyrophosphate', 1 molecule of 'N-(L-Arginino)succinate', and 1 molecule of 'AMP' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'argininosuccinate synthase activity' of 'argininosuccinate synthase
homotetramer'.
References
2, 2001, 1909-1963.
Reaction
17.2.5 argininosuccinate <=> fumarate + arginine
Description
At the beginning of this reaction, 1 molecule of 'N-(L-Arginino)succinate' is present. At the end of this reaction, 1 molecule of 'Fumarate', and 1
molecule of 'L-Arginine' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'argininosuccinate lyase activity' of 'argininosuccinate lyase tetramer'.
References
2, 2001, 1909-1963.
Reaction
17.2.6 arginine => ornithine + urea
Description
At the beginning of this reaction, 1 molecule of 'L-Arginine' is present. At the end of this reaction, 1 molecule of 'L-Ornithine', and 1 molecule of
'Urea' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'arginase activity' of 'arginase 1 homotrimer'.
References
ZF Kanyo, LR Scolnick, DE Ash, DW Christianson, "Structure of a unique binuclear manganese cluster in arginase.", Nature, 383, 1996, 554-7.
2, 2001, 1909-1963.
Reaction
17.2.7 ornithine (cytosolic) + citrulline (mitochondrial) => ornithine (mitochondrial) + citrulline (cytosolic)
Description
In this reaction, 1 molecule of 'L-Ornithine', and 1 molecule of 'L-Citrulline' are translocated from cytosol to mitochondrial matrix.
This reaction takes place in the 'cell' and is mediated by the 'L-ornithine transporter activity' of 'Mitochondrial ornithine transporter 1'.
References
JA Camacho, C Obie, B Biery, BK Goodman, CA Hu, S Almashanu, G Steel, R Casey, M Lambert, GA Mitchell, D Valle,
"Hyperornithinaemia-hyperammonaemia-homocitrullinuria syndrome is caused by mutations in a gene encoding a mitochondrial ornithine
transporter.", Nat Genet, 22, 1999, 151-8.
D Valle, O Simell, "The hyperornithinemias", The Metabolic and Molecular Bases of Inherited Disease, 8th ed (Scriver CR, et al., editors), 2,
2001, 1857-1895.
Reaction
17.3 Alanine metabolism
Authors
2003-04-29.
Editors
Description
The freely reversible interconversion of alanine and pyruvate, coupled to the interconversion of alpha-ketoglutarate and glutamate, allows the
synthesis of alanine from intermediates of glucose metabolism in a well-fed person. Under fasting conditions, alanine, derived from protein
breakdown, can be converted to pyruvate and used to synthesize glucose via the gluconeogenic pathway in liver, or fully oxidized via the TCA
cycle in other tissues.
17.3.1 alanine + alpha-ketoglutarate <=> pyruvate + glutamate
Description
At the beginning of this reaction, 1 molecule of 'L-Alanine', and 1 molecule of '2-Oxoglutarate' are present. At the end of this reaction, 1 molecule
of 'L-Glutamate', and 1 molecule of 'Pyruvate' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'alanine transaminase activity' of 'alanine aminotransferase, holoenzyme'.
References
MM Sohocki, LS Sullivan, WR Harrison, EJ Sodergren, FF Elder, G Weinstock, S Tanase, SP Daiger, "Human glutamate pyruvate transaminase
(GPT): localization to 8q24.3, cDNA and genomic sequences, and polymorphic sites.", Genomics, 40, 1997, 247-52.
Reaction
17.3.2 pyruvate + glutamate <=> alanine + alpha-ketoglutarate
Description
At the beginning of this reaction, 1 molecule of 'L-Glutamate', and 1 molecule of 'Pyruvate' are present. At the end of this reaction, 1 molecule of
'L-Alanine', and 1 molecule of '2-Oxoglutarate' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'alanine transaminase activity' of 'alanine aminotransferase, holoenzyme'.
References
MM Sohocki, LS Sullivan, WR Harrison, EJ Sodergren, FF Elder, G Weinstock, S Tanase, SP Daiger, "Human glutamate pyruvate transaminase
(GPT): localization to 8q24.3, cDNA and genomic sequences, and polymorphic sites.", Genomics, 40, 1997, 247-52.
Reaction
17.4 Aspartate, asparagine, glutamate, and glutamine metabolism
Authors
2003-04-29.
Editors
Description
This group of reactions allows the synthesis of aspartate, asparagine, glutamate, and glutamine from ammonia and intermediates of glycolysis in
well-fed humans, and allows the utilization of the carbon atoms from these four amino acids for glucose synthesis under fasting conditions.
These reactions also provide a means to collect nitrogen, both as ammonia and as amino groups, and direct it towards urea synthesis.
Transamination, the conversion of an amino acid to the corresponding alpha-keto acid, is the first step in the catabolism of most amino acids.
This reaction is always coupled to the conversion of a molecule of alpha-ketoglutarate to glutamate. A second transamination reaction, involving
this glutamate and a molecule of oxaloacetate, yields aspartate, which functions as a nitrogen donor in urea synthesis, and also regenerates
alpha-ketoglutarate.
17.4.1 alpha-ketoglutarate + NH4+ + NADPH + H+ => glutamate + NADP+
Description
At the beginning of this reaction, 1 molecule of 'H+', 1 molecule of 'NH4+', 1 molecule of '2-Oxoglutarate', and 1 molecule of 'NADPH' are
present. At the end of this reaction, 1 molecule of 'NADP+', and 1 molecule of 'L-Glutamate' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'glutamate dehydrogenase [NAD(P)+] activity' of 'glutamate
dehydrogenase 1, homohexamer'.
References
JH Julliard, EL Smith, "Partial amino acid sequence of the glutamate dehydrogenase of human liver and a revision of the sequence of the bovine
enzyme", J Biol Chem, 254, 1979, 3427-38.
Reaction
17.4.2 aspartate + alpha-ketoglutarate <=> oxaloacetate + glutamate [cytosolic]
Description
At the beginning of this reaction, 1 molecule of 'L-Aspartate', and 1 molecule of '2-Oxoglutarate' are present. At the end of this reaction, 1
molecule of 'L-Glutamate', and 1 molecule of 'Oxaloacetate' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'aspartate transaminase activity' of 'aspartate aminotransferase, cytoplasmic,
holoenzyme'.
References
JM Doyle, ME Schinina, F Bossa, S Doonan, "The amino acid sequence of cytosolic aspartate aminotransferase from human liver", Biochem J,
270, 1990, 651-7.
Reaction
17.4.3 aspartate + alpha-ketoglutarate <=> oxaloacetate + glutamate [mitochondrial]
Description
At the beginning of this reaction, 1 molecule of 'L-Aspartate', and 1 molecule of '2-Oxoglutarate' are present. At the end of this reaction, 1
molecule of 'L-Glutamate', and 1 molecule of 'Oxaloacetate' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'aspartate transaminase activity' of 'aspartate aminotransferase,
mitochondrial, holoenzyme'.
References
F Martini, S Angelaccio, D Barra, S Pascarella, B Maras, S Doonan, F Bossa, "The primary structure of mitochondrial aspartate
aminotransferase from human heart", Biochim Biophys Acta, 832, 1985, 46-51.
Reaction
17.4.4 aspartate + glutamine + ATP <=> asparagine + glutamate + AMP + pyrophosphate
Description
At the beginning of this reaction, 1 molecule of 'L-Glutamine', 1 molecule of 'L-Aspartate', and 1 molecule of 'ATP' are present. At the end of this
reaction, 1 molecule of 'pyrophosphate', 1 molecule of 'L-Glutamate', 1 molecule of 'AMP', and 1 molecule of 'L-Asparagine' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'asparagine synthase (glutamine-hydrolyzing) activity' of 'Asparagine synthetase
[glutamine-hydrolyzing] '.
References
G Van Heeke, SM Schuster, "Expression of human asparagine synthetase in Escherichia coli", J Biol Chem, 264, 1989, 5503-9.
Reaction
17.4.5 glutamate + NAD+ => alpha-ketoglutarate + NH4+ + NADH + H+
Description
At the beginning of this reaction, 1 molecule of 'L-Glutamate', and 1 molecule of 'NAD+' are present. At the end of this reaction, 1 molecule of
'H+', 1 molecule of 'NH4+', 1 molecule of '2-Oxoglutarate', and 1 molecule of 'NADH' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'glutamate dehydrogenase [NAD(P)+] activity' of 'glutamate
dehydrogenase 1, homohexamer'.
References
JH Julliard, EL Smith, "Partial amino acid sequence of the glutamate dehydrogenase of human liver and a revision of the sequence of the bovine
enzyme", J Biol Chem, 254, 1979, 3427-38.
Reaction
17.4.6 glutamate + NH4+ + ATP => glutamine + ADP + orthophosphate
Description
At the beginning of this reaction, 1 molecule of 'ATP', 1 molecule of 'NH4+', and 1 molecule of 'L-Glutamate' are present. At the end of this
reaction, 1 molecule of 'L-Glutamine', 1 molecule of 'ADP', and 1 molecule of 'Orthophosphate' are present.
This reaction takes place in the 'cytoplasm' and is mediated by the 'glutamate-ammonia ligase activity' of 'glutamine synthetase homooctamer'.
References
J Haberle, B Gorg, F Rutsch, E Schmidt, A Toutain, JF Benoist, A Gelot, AL Suc, W Hohne, F Schliess, D Haussinger, HG Koch, "Congenital
glutamine deficiency with glutamine synthetase mutations", N Engl J Med, 353, 2005, 1926-33.
IS Boksha, HJ Schonfeld, H Langen, F Muller, EB Tereshkina, GSh Burbaeva, "Glutamine synthetase isolated from human brain: octameric
structure and homology of partial primary structure with human liver glutamine synthetase", Biochemistry (Mosc), 67, 2002, 1012-20.
Reaction
17.4.7 glutamine + H2O => glutamate + NH4+ [kidney]
Description
At the beginning of this reaction, 1 molecule of 'L-Glutamine', and 1 molecule of 'H2O' are present. At the end of this reaction, 1 molecule of
'NH4+', and 1 molecule of 'L-Glutamate' are present.
This reaction takes place in the 'mitochondrion' and is mediated by the 'glutaminase activity' of 'glutaminase, kidney isoform'.
References
KM Elgadi, RA Meguid, M Qian, WW Souba, SF Abcouwer, "Cloning and analysis of unique human glutaminase isoforms generated by
tissue-specific alternative splicing", Physiol Genomics, 1, 1999, 51-62.
Reaction
17.4.8 glutamine + H2O => glutamate + NH4+ [liver]
Description
At the beginning of this reaction, 1 molecule of 'L-Glutamine', and 1 molecule of 'H2O' are present. At the end of this reaction, 1 molecule of
'NH4+', and 1 molecule of 'L-Glutamate' are present.
This reaction takes place in the 'mitochondrion' and is mediated by the 'glutaminase activity' of 'glutaminase, liver isoform'.
References
PM Gomez-Fabre, JC Aledo, A Del Castillo-Olivares, FJ Alonso, I Nunez De Castro, JA Campos, J Marquez, "Molecular cloning, sequencing
and expression studies of the human breast cancer cell glutaminase", Biochem J, 345, 2000, 365-75.
Reaction
17.4.9 oxaloacetate + glutamate <=> aspartate + alpha-ketoglutarate [cytosolic]
Description
At the beginning of this reaction, 1 molecule of 'L-Glutamate', and 1 molecule of 'Oxaloacetate' are present. At the end of this reaction, 1
molecule of 'L-Aspartate', and 1 molecule of '2-Oxoglutarate' are present.
This reaction takes place in the 'cytoplasm' and is mediated by the 'aspartate transaminase activity' of 'aspartate aminotransferase, cytoplasmic,
holoenzyme'.
References
JM Doyle, ME Schinina, F Bossa, S Doonan, "The amino acid sequence of cytosolic aspartate aminotransferase from human liver", Biochem J,
270, 1990, 651-7.
Reaction
17.4.10 oxaloacetate + glutamate <=> aspartate + alpha-ketoglutarate [mitochondrial]
Description
At the beginning of this reaction, 1 molecule of 'L-Glutamate', and 1 molecule of 'Oxaloacetate' are present. At the end of this reaction, 1
molecule of 'L-Aspartate', and 1 molecule of '2-Oxoglutarate' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'aspartate transaminase activity' of 'aspartate aminotransferase,
mitochondrial, holoenzyme'.
References
F Martini, S Angelaccio, D Barra, S Pascarella, B Maras, S Doonan, F Bossa, "The primary structure of mitochondrial aspartate
aminotransferase from human heart", Biochim Biophys Acta, 832, 1985, 46-51.
Reaction
17.5 Ornithine and proline metabolism
Authors
2003-03-31.
Editors
Description
Reactions involving ornithine are parts of the urea cycle, polyamine synthesis, creatine synthesis, and proline and glutamate metabolism.
Expression of the enzymes catalyzing these reactions is tissue-specific, giving rise to four metabolic patterns: 1) In epithelial cells of the small
intestine, ornithine is used primarily to synthesize citrulline and arginine; 2) In liver cells (hepatocytes) surrounding the portal vein, ornithine
functions primarily as an intermediate of the urea cycle; 3) In liver cells surrounding the central vein, ornithine is used to synthesize glutamate
and glutamine; and 4) In many peripheral tissues, ornithine is used for the synthesis of glutamate and proline.
The reactions prominent in the liver, patterns 2 and 3, provide an efficient sequential mechanism for clearing ammonia from blood: the urea cycle
enzymes (2) have relatively low affinity for ammonia but high capacity, while those of pattern 3 can efficiently scavenge trace ammonia. The
reactions prominent in the small intestine (1) and peripheral tissues (4) provide amino acids for protein synthesis.
References
2001, 1857-1895.
JM Phang, CA Hu, D Valle, "Disorders of proline and hydroxyproline metabolism", The Metabolic and Molecular Bases of Inherited Disease, 8th
ed (Scriver CR, et al., editors), 2, 2001.
17.5.1 Ornithine metabolism
Description
Reactions involving ornithine are parts of the urea cycle, polyamine synthesis, creatine synthesis, and proline and glutamate metabolism.
Expression of the enzymes catalyzing these reactions is tissue-specific, giving rise to four metabolic patterns: 1) In epithelial cells of the small
intestine, ornithine is used primarily to synthesize citrulline and arginine; 2) In liver cells (hepatocytes) surrounding the portal vein, ornithine
functions primarily as an intermediate of the urea cycle; 3) In liver cells surrounding the central vein, ornithine is used to synthesize glutamate
and glutamine; and 4) In many peripheral tissues, ornithine is used for the synthesis of glutamate and proline.
17.5.1.1 argininosuccinate <=> fumarate + arginine
Description
At the beginning of this reaction, 1 molecule of 'N-(L-Arginino)succinate' is present. At the end of this reaction, 1 molecule of 'Fumarate', and 1
molecule of 'L-Arginine' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'argininosuccinate lyase activity' of 'argininosuccinate lyase tetramer'.
References
2, 2001, 1909-1963.
Reaction
17.5.1.2 arginine => ornithine + urea
Description
At the beginning of this reaction, 1 molecule of 'L-Arginine' is present. At the end of this reaction, 1 molecule of 'L-Ornithine', and 1 molecule of
'Urea' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'arginase activity' of 'arginase 1 homotrimer'.
References
ZF Kanyo, LR Scolnick, DE Ash, DW Christianson, "Structure of a unique binuclear manganese cluster in arginase.", Nature, 383, 1996, 554-7.
2, 2001, 1909-1963.
Reaction
17.5.1.3 ornithine => putrescine + CO2
Reviewers
Description
L-ornithine is converted into putrescine by ODC holoenzyme complex.Putrescine is subsequent used for polyamine synthesis.
References
2001, 1857-1895.
Reaction
17.5.1.4 glutamate + L-glutamate gamma-semialdehyde <=> ornithine + alpha-ketoglutarate
Description
At the beginning of this reaction, 1 molecule of 'L-Glutamate', and 1 molecule of 'L-Glutamate 5-semialdehyde' are present. At the end of this
reaction, 1 molecule of '2-Oxoglutarate', and 1 molecule of 'L-Ornithine' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'ornithine-oxo-acid transaminase activity' of 'ornithine
aminotransferase homohexamer'.
References
2001, 1857-1895.
LC Brody, GA Mitchell, C Obie, J Michaud, G Steel, G Fontaine, MF Robert, I Sipila, M Kaiser-Kupfer, D Valle, "Ornithine delta-aminotransferase
mutations in gyrate atrophy. Allelic heterogeneity and functional consequences.", J Biol Chem, 267, 1992, 3302-7.
Reaction
17.5.1.5 carbamoyl phosphate + ornithine => citrulline + orthophosphate
Description
At the beginning of this reaction, 1 molecule of 'L-Ornithine', and 1 molecule of 'Carbamoyl phosphate' are present. At the end of this reaction, 1
molecule of 'Orthophosphate', and 1 molecule of 'L-Citrulline' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'ornithine carbamoyltransferase activity' of 'ornithine
transcarbamylase homotrimer'.
References
AL Horwich, WA Fenton, KR Williams, F Kalousek, JP Kraus, RF Doolittle, W Konigsberg, LE Rosenberg, "Structure and expression of a
complementary DNA for the nuclear coded precursor of human mitochondrial ornithine transcarbamylase.", Science, 224, 1984, 1068-74.
Reaction
17.5.1.6 ATP + aspartate + citrulline <=> argininosuccinate + AMP + pyrophosphate

Description
At the beginning of this reaction, 1 molecule of 'L-Aspartate', 1 molecule of 'L-Citrulline', and 1 molecule of 'ATP' are present. At the end of this
reaction, 1 molecule of 'pyrophosphate', 1 molecule of 'N-(L-Arginino)succinate', and 1 molecule of 'AMP' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'argininosuccinate synthase activity' of 'argininosuccinate synthase
homotetramer'.
References
2, 2001, 1909-1963.
Reaction
17.5.1.7 ornithine (cytosolic) + citrulline (mitochondrial) => ornithine (mitochondrial) + citrulline (cytosolic)
Description
In this reaction, 1 molecule of 'L-Ornithine', and 1 molecule of 'L-Citrulline' are translocated from cytosol to mitochondrial matrix.
This reaction takes place in the 'cell' and is mediated by the 'L-ornithine transporter activity' of 'Mitochondrial ornithine transporter 1'.
References
JA Camacho, C Obie, B Biery, BK Goodman, CA Hu, S Almashanu, G Steel, R Casey, M Lambert, GA Mitchell, D Valle,
"Hyperornithinaemia-hyperammonaemia-homocitrullinuria syndrome is caused by mutations in a gene encoding a mitochondrial ornithine
transporter.", Nat Genet, 22, 1999, 151-8.
2001, 1857-1895.
Reaction
17.5.1.8 ornithine + alpha-ketoglutarate <=> glutamate + L-glutamate gamma-semialdehyde

Description
At the beginning of this reaction, 1 molecule of '2-Oxoglutarate', and 1 molecule of 'L-Ornithine' are present. At the end of this reaction, 1
molecule of 'L-Glutamate', and 1 molecule of 'L-Glutamate 5-semialdehyde' are present.
References
2001, 1857-1895.
Reaction
17.5.1.9 L-glutamate gamma-semialdehyde <=> L-1-pyrroline-5-carboxylate
Description
At the beginning of this reaction, 1 molecule of 'L-Glutamate 5-semialdehyde' is present. At the end of this reaction, 1 molecule of
'(S)-1-Pyrroline-5-carboxylate' is present.
This reaction takes place in the 'mitochondrial matrix'.
References
2001, 1857-1895.
Reaction
17.5.1.10 Regulation of ornithine decarboxylase (ODC)
Authors
Reviewers
Description
Polyamines increase the production of antizyme (AZ). The carboxy-terminal half of antizyme interacts with ODC, generating an inactive AZ:ODC
heterodimer complex. A carboxy-terminal domain of ODC is exposed only within the heterodimer, and is the target for subsequent degradation.
A domain within the amino-terminal portion of antizyme provides a function needed for efficient degradation of ODC by the proteasome.
The proteasome cycle starts with the processing of AZ:ODC, sequestering ODC and then degrading it to peptides but releasing AZ. AZ
participates in additional rounds of binding and degradation. Antizyme-mediated inhibition and destruction of ODC reduces synthesis of
polyamines. Additionally, antizyme also inhibits polyamine transport into the cell. Antizyme production is reduced, completing the regulatory
circuit (Coffino, 2001).
The following illustration is adapted from a minireview by Pegg, 2006; J. Biol. Chem., Vol. 281, Issue 21, 14529-14532.
References
C Kahana, G Asher, Y Shaul, "Mechanisms of protein degradation: an odyssey with ODC", Cell Cycle, 4, 2005, 1461-4.
Y Murakami, S Matsufuji, S Hayashi, N Tanahashi, K Tanaka, "Degradation of ornithine decarboxylase by the 26S proteasome", Biochem
P Coffino, "Regulation of cellular polyamines by antizyme", Nat Rev Mol Cell Biol, 2, 2001, 188-94.
AE Pegg, "Regulation of ornithine decarboxylase", J Biol Chem, 281, 2006, 14529-32.
17.5.1.10.1 ornithine => putrescine + CO2
Reviewers
Description
L-ornithine is converted into putrescine by ODC holoenzyme complex.Putrescine is subsequent used for polyamine synthesis.
References
2001, 1857-1895.
Reaction
17.5.1.10.2 Antizyme OAZ binds to Ornithine decarboxylase
Authors
Reviewers
Description
Antizyme is a non-competitive inhibitor of ODC that is synthesized in response to an increase in polyamine concentration. Tight binding of the
antizyme to the ODC monomer forming a heterodimer prevents enzymatic activity. The region of antizyme interacting with ODC is contained in a
section involving residues 106â€"212 in the COOH-terminal half of the antizyme molecule. The induction of antizyme thus leads to a loss of
active ODC protein (Pegg, 2006 and references cited in that review).
References
DS Tewari, Y Qian, RD Thornton, J Pieringer, R Taub, E Mochan, M Tewari, "Molecular cloning and sequencing of a human cDNA encoding
ornithine decarboxylase antizyme", Biochim Biophys Acta, 1209, 1994, 293-5.
H Chen, A MacDonald, P Coffino, "Structural elements of antizymes 1 and 2 are required for proteasomal degradation of ornithine
decarboxylase", J Biol Chem, 277, 2002, 45957-61.
D Yang, T Takii, H Hayashi, S Itoh, M Hayashi, K Onozaki, "Molcecular cloning of human antizyme cDNA", Biochem Mol Biol Int, 38, 1996,
957-64.
M Zhang, CM Pickart, P Coffino, "Determinants of proteasome recognition of ornithine decarboxylase, a ubiquitin-independent substrate", EMBO
J, 22, 2003, 1488-96.
Reaction
17.5.1.10.3 26S proteosome degrades ODC holoenzyme complex
Authors
Reviewers
Description
The rapid turnover of ODC is brought about by the 26S proteasome. Proteolytic processing of ODC is highly unusual in that ubiquitination is not
required for this degradation. Instead, a non-covalent association with antizyme directs ODC to the proteasome. Antizyme increases the
degradation of ODC by enhancing its interaction with the proteasome (Pegg, 2006).
References
P Coffino, "Regulation of cellular polyamines by antizyme", Nat Rev Mol Cell Biol, 2, 2001, 188-94.
M Zhang, CM Pickart, P Coffino, "Determinants of proteasome recognition of ornithine decarboxylase, a ubiquitin-independent substrate", EMBO
J, 22, 2003, 1488-96.
S Hayashi, Y Murakami, "Rapid and regulated degradation of ornithine decarboxylase", Biochem J, 306, 1995, 1-10.
U Mangold, "The antizyme family: polyamines and beyond", IUBMB Life, 57, 2005, 671-6.
Reaction
17.5.1.10.4 Antizyme inhibitor binds to OAZ and stablizes ODC complex
Authors
Reviewers
Description
Antizyme inhibitor blocks the effects of antizyme on ODC. It has substantial similarity to ODC itself but has no ODC activity. It binds to antizyme
more tightly than ODC displacing ODC from the antizyme-ODC complex. Recent studies have shown that antizyme inhibitor is able to disrupt the
interaction between all forms of mammalian antizyme and ODC (Murakami et al., 1996, Nilsson et al., 2000, Mangold and Leberer, 2005).
References
Y Murakami, T Ichiba, S Matsufuji, S Hayashi, "Cloning of antizyme inhibitor, a highly homologous protein to ornithine decarboxylase", J Biol
Chem, 271, 1996, 3340-2.
K Koguchi, S Kobayashi, T Hayashi, S Matsufuji, Y Murakami, S Hayashi, "Cloning and sequencing of a human cDNA encoding ornithine
decarboxylase antizyme inhibitor", Biochim Biophys Acta, 1353, 1997, 209-16.
U Mangold, E Leberer, "Regulation of all members of the antizyme family by antizyme inhibitor", Biochem J, 385, 2005, 21-8.
J Nilsson, B Grahn, O Heby, "Antizyme inhibitor is rapidly induced in growth-stimulated mouse fibroblasts and releases ornithine decarboxylase
from antizyme suppression", Biochem J, 346, 2000, 699-704.
T Hayashi, S Matsufuji, S Hayashi, "Characterization of the human antizyme gene", Gene, 203, 1997, 131-9.
Z Bercovich, C Kahana, "Degradation of antizyme inhibitor, an ornithine decarboxylase homologous protein, is ubiquitin-dependent and is
inhibited by antizyme", J Biol Chem, 279, 2004, 54097-102.
K Kanerva, LT MÃ¤kitie, A Pelander, M Heiskala, LC Andersson, "Human ornithine decarboxylase paralogue (ODCp) is an antizyme inhibitor but
not an arginine decarboxylase", Biochem J, 409, 2008, 187-92.
Reaction
17.5.1.10.5 NQO1 interaction with ODC
Authors
Reviewers
Description
A novel pathway has been described for ODC degradation during oxidative stress, which is regulated by NAD(P)H quinone oxidoreductase
(NQO1). In this pathway, the 20S proteasome has been shown to degrade unfolded ODC monomers. This event does not require the
COOH-terminal domain. NQO1 binds to ODC and stabilizes it. If this interaction is disrupted with dicoumarol, it sensitizes ODC monomers to
degradation by the 20S proteasome independent of both antizyme and ubiquitin. The details of the role of this pathway remains to be
determined, but it could be involved in the nascent ODC chain turnover.
References
L Persson, A Jeppsson, S Nasizadeh, "Turnover of trypanosomal ornithine decarboxylases", Biochem Soc Trans, 31, 2003, 411-4.
G Asher, Z Bercovich, P Tsvetkov, Y Shaul, C Kahana, "20S proteasomal degradation of ornithine decarboxylase is regulated by NQO1", Mol
Cell, 17, 2005, 645-55.
Reaction
17.5.2 Proline synthesis
Description
Proline is synthesized in three steps from ornithine and alpha-ketoglutarate.
17.5.2.1 ornithine + alpha-ketoglutarate <=> glutamate + L-glutamate gamma-semialdehyde
Description
At the beginning of this reaction, 1 molecule of '2-Oxoglutarate', and 1 molecule of 'L-Ornithine' are present. At the end of this reaction, 1
molecule of 'L-Glutamate', and 1 molecule of 'L-Glutamate 5-semialdehyde' are present.
References
2001, 1857-1895.
Reaction
17.5.2.2 L-glutamate gamma-semialdehyde <=> L-1-pyrroline-5-carboxylate
Description
At the beginning of this reaction, 1 molecule of 'L-Glutamate 5-semialdehyde' is present. At the end of this reaction, 1 molecule of
'(S)-1-Pyrroline-5-carboxylate' is present.
References
2001, 1857-1895.
Reaction
17.5.2.3 L-1-pyrroline-5-carboxylate + NADPH + H+ => proline + NADP+
Description
At the beginning of this reaction, 1 molecule of 'H+', 1 molecule of 'NADPH', and 1 molecule of '(S)-1-Pyrroline-5-carboxylate' are present. At the
end of this reaction, 1 molecule of 'L-Proline', and 1 molecule of 'NADP+' are present.
This reaction takes place in the 'cytoplasm' and is mediated by the 'pyrroline-5-carboxylate reductase activity' of 'pyrroline-5-carboxylate
reductase homomultimer'.
References
MJ Merrill, GC Yeh, JM Phang, "Purified human erythrocyte pyrroline-5-carboxylate reductase. Preferential oxidation of NADPH.", J Biol Chem,
264, 1989, 9352-8.
Reaction
17.5.3 Proline catabolism
Description
Proline is catabolized in two steps to yield L-glutamate gamma-semialdehyde, which can react further with glutamate to yield ornithine and
alpha-ketoglutarate or with NAD+ to yield glutamate and NADH + H+.
17.5.3.1 proline => L-1-pyrroline-5-carboxylate
Authors
2003-03-13.
Description
The dehydrogenation of proline to L-1-pyrroline-5-carboxylate is the first step in proline breakdown. The enzyme that catalyzes this reaction is
found in liver, kidney, and brain cells, where it is tightly bound to the inner mitochondrial membrane. The enzyme has not been purified, and the
reaction itself is incompletely characterized: while the electrons generated by the oxidation of proline appear ultimately to enter the mitochondrial
electron transport chain, the immediate acceptor of these electrons is unknown.
References
Reaction
17.5.3.2 L-1-pyrroline-5-carboxylate <=> L-glutamate gamma-semialdehyde
Description
At the beginning of this reaction, 1 molecule of '(S)-1-Pyrroline-5-carboxylate' is present. At the end of this reaction, 1 molecule of 'L-Glutamate
5-semialdehyde' is present.
References
2001, 1857-1895.
Reaction
17.5.3.3 glutamate + L-glutamate gamma-semialdehyde <=> ornithine + alpha-ketoglutarate

Description
At the beginning of this reaction, 1 molecule of 'L-Glutamate', and 1 molecule of 'L-Glutamate 5-semialdehyde' are present. At the end of this
reaction, 1 molecule of '2-Oxoglutarate', and 1 molecule of 'L-Ornithine' are present.
References
2001, 1857-1895.
Reaction
17.5.3.4 L-glutamate gamma-semialdehyde + NAD+ => glutamate + NADH + H+
Authors
2003-03-13.
Description
In this reaction, L-glutamate gamma-semialdehyde is converted to glutamate. The immediately preceding step in proline catabolism is the
spontaneous conversion of L-1-pyrroline-5-carboxylate to L-glutamate gamma-semialdehyde. The enzyme that catalyzes glutamate formation,
however, P5C [pyrroline-5-carboxylate] dehydrogenase is named as if it catalyzed both steps, perhaps because pyrroline-5-carboxylate
accumulates in cells that lack the enzyme.
References
MT Geraghty, D Vaughn, AJ Nicholson, WW Lin, G Jimenez-Sanchez, C Obie, MP Flynn, D Valle, CA Hu, "Mutations in the Delta1-pyrroline
5-carboxylate dehydrogenase gene cause type II hyperprolinemia.", Hum Mol Genet, 7, 1998, 1411-5.
CM Forte-McRobbie, R Pietruszko, "Purification and characterization of human liver "high Km" aldehyde dehydrogenase and its identification as
glutamic gamma-semialdehyde dehydrogenase.", J Biol Chem, 261, 1986, 2154-63.
Reaction
17.6 Branched-chain amino acid catabolism
Authors
2003-04-29.
Editors
Description
The branched-chain amino acids, leucine, isoleucine, and valine, are all essential amino acids (i.e., ones required in the diet) whose side chains
contain a branched methyl group. They are major constituents of muscle protein. The breakdown of these amino acids starts with two common
steps, catalyzed by enzyme complex capable of acting on all three amino acids: transamination by branched-chain amino acid aminotransferase,
and oxidative decarboxylation by branched-chain ketoacid dehydrogenase. Isovaleryl-CoA is produced from leucine by these two reactions,
alpha-methylbutyryl-CoA from isoleucine, and isobutyryl-CoA from valine. These acyl-CoA's undergo dehydrogenation, catalyzed by three
different but related enzymes, and the breakdown pathways then diverge. Leucine is ultimately converted to acetyl-CoA and acetoacetate;
isoleucine to acetyl-CoA and succinyl-CoA; and valine to succinyl-CoA. Under fasting conditions, substantial amounts of all three amino acids
are generated by protein breakdown. In muscle, the final products of leucine, isoleucine, and valine catabolism can be fully oxidized via the citric
acid cycle; in liver they can be directed toward the synthesis of ketone bodies (acetoacetate and acetyl-CoA) and glucose (succinyl-CoA).
References
DT Chuang, VE Shih, "Maple syrup urine disease (branched-chain ketoaciduria)", The Metabolic and Molecular Bases of Inherited Disease, 8th
ed (Scriver CR, et al., editors), 2, 2001, 1971-2005.
L Sweetman, JC Williams, "Branched chain organic acidurias", The Metabolic and Molecular Bases of Inherited Disease, 8th ed (Scriver CR, et
al., editors), 2, 2001, 2125-2163.
17.6.1 Leucine catabolism
Description
Leucine is catabolized in six steps to beta-hydroxy-beta-methylglutaryl-CoA, which is metabolized further to acetyl-CoA and acetoacetate.
References
al., editors), 2, 2001, 2125-2163.
17.6.1.1 L-Glutamate [cytosolic] from leucine catabolism
Authors
Mahajan, SS, 2008-01-14.
Description
At the beginning of this reaction, 1 molecule of 'L-Leucine', and 1 molecule of '2-Oxoglutarate' are present. At the end of this reaction, 1 molecule
of 'L-Glutamate', and 1 molecule of 'alpha-ketoisocaproate' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'branched-chain-amino-acid transaminase activity' of 'branched-chain amino acid
aminotransferase, cytosolic, holoenzyme'.
References
Reaction
17.6.1.2 L-Glutamate [mitochondrial] from leucine catabolism
Authors
Mahajan, SS, 2008-01-14.
Description
This reaction takes place in the 'mitochondrion' and is mediated by the 'branched-chain-amino-acid transaminase activity' of 'branched-chain
amino acid aminotransferase, mitochondrial, holoenzyme'.
References
Reaction
17.6.1.3 Oxidative decarboxylation of alpha-ketoisocaproate to isovaleryl-CoA by branched-chain alpha-ketoacid dehydrogenase
Description
This process, carried out by the mitochondrial branched-chain alpha-ketoacid dehydrogenase complex, consists of five distinct reactions. First,
alpha-ketoisocaproate is oxidatively decarboxylated, catalyzed by the E1 component of the complex. Lipoamide associated with E1 is reduced at
the same time. Next, the isovaleryl group derived from alpha-ketoisocaproate is transferred to coenzyme A in two steps catalyzed by the E2
component of the complex (dihydrolipoyl transacetylase). Finally, the oxidized form of lipoamide is regenerated and electrons are transferred to
NAD+ in two steps catalyzed by the E3 component of the complex (dihydrolipoyl dehydrogenase).
References
17.6.1.3.1 alpha-ketoisocaproate + TPP => 3-methyl-1-hydroxybutyl-TPP + CO2
Description
At the beginning of this reaction, 1 molecule of 'Thiamin diphosphate', and 1 molecule of 'alpha-ketoisocaproate' are present. At the end of this
reaction, 1 molecule of 'CO2', and 1 molecule of '3-methyl-1-hydroxybutyl-TPP' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the '3-methyl-2-oxobutanoate dehydrogenase
(2-methylpropanoyl-transferring) activity' of 'branched-chain alpha-ketoacid dehydrogenase complex'.
References
Reaction
17.6.1.3.2 3-methyl-1-hydroxybutyl-TPP + lipoamide => S-isovaleryldihydrolipoamide + TPP
Description
At the beginning of this reaction, 1 molecule of '3-methyl-1-hydroxybutyl-TPP', and 1 molecule of 'Lipoamide' are present. At the end of this
reaction, 1 molecule of 'S-isovaleryldihydrolipoamide', and 1 molecule of 'Thiamin diphosphate' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'acyltransferase activity' of 'branched-chain alpha-ketoacid
dehydrogenase complex, 3-methyl-1-hydroxybutyl linked'.
References
Reaction
17.6.1.3.3 S-isovaleryldihydrolipoamide + CoA => isovaleryl-CoA + dihydrolipoamide

Description
At the beginning of this reaction, 1 molecule of 'S-isovaleryldihydrolipoamide', and 1 molecule of 'CoA' are present. At the end of this reaction, 1
molecule of 'isovaleryl-CoA', and 1 molecule of 'Dihydrolipoamide' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'dihydrolipoamide branched chain acyltransferase activity' of
'branched-chain alpha-ketoacid dehydrogenase complex, S-isovaleryldihydrolipoamide linked'.
References
Reaction
17.6.1.3.4 dihydrolipoamide + FAD => lipoamide + FADH2 [branched-chain ketoacid dehydrogenase]
Description
At the beginning of this reaction, 1 molecule of 'FAD', and 1 molecule of 'Dihydrolipoamide' are present. At the end of this reaction, 1 molecule of
'FADH2', and 1 molecule of 'Lipoamide' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'dihydrolipoyl dehydrogenase activity' of 'branched-chain
alpha-ketoacid dehydrogenase complex, dihydrolipoamide linked'.
References
Reaction
17.6.1.3.5 FADH2 + NAD+ => FAD + NADH + H+ [branched-chain ketoacid dehydrogenase]
Description
At the beginning of this reaction, 1 molecule of 'FADH2', and 1 molecule of 'NAD+' are present. At the end of this reaction, 1 molecule of 'H+', 1
molecule of 'FAD', and 1 molecule of 'NADH' are present.
alpha-ketoacid dehydrogenase complex, FADH2 linked'.
References
Reaction
17.6.1.4 isovaleryl-CoA + FAD => beta-methylcrotonyl-CoA + FADH2
Description
At the beginning of this reaction, 1 molecule of 'FAD', and 1 molecule of 'isovaleryl-CoA' are present. At the end of this reaction, 1 molecule of
'beta-methylcrotonyl-CoA', and 1 molecule of 'FADH2' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'isovaleryl-CoA dehydrogenase activity' of 'isovaleryl-CoA
dehydrogenase, holoenzyme'.
References
WJ Rhead, K Tanaka, "Demonstration of a specific mitochondrial isovaleryl-CoA dehydrogenase deficiency in fibroblasts from patients with
isovaleric acidemia.", Proc Natl Acad Sci U S A, 77, 1980, 580-3.
Reaction
17.6.1.5 beta-methylcrotonyl-CoA + ATP + CO2 <=> beta-methylglutaconyl-CoA + ADP + orthophosphate + H2O
Description
At the beginning of this reaction, 1 molecule of 'beta-methylcrotonyl-CoA', 1 molecule of 'CO2', and 1 molecule of 'ATP' are present. At the end
of this reaction, 1 molecule of 'ADP', 1 molecule of 'beta-methylglutaconyl-CoA', 1 molecule of 'Orthophosphate', and 1 molecule of 'H2O' are
present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'methylcrotonoyl-CoA carboxylase activity' of 'methylcrotonyl-CoA
carboxylase complex'.
References
A Holzinger, W RÃ¶schinger, F Lagler, PU Mayerhofer, P Lichtner, T Kattenfeld, LP Thuy, WL Nyhan, HG Koch, AC Muntau, AA Roscher,
"Cloning of the human MCCA and MCCB genes and mutations therein reveal the molecular cause of 3-methylcrotonyl-CoA: carboxylase
deficiency.", Hum Mol Genet, 10, 2001, 1299-306.
MR Baumgartner, S Almashanu, T Suormala, C Obie, RN Cole, S Packman, ER Baumgartner, D Valle, "The molecular basis of human
3-methylcrotonyl-CoA carboxylase deficiency.", J Clin Invest, 107, 2001, 495-504.
Reaction
17.6.1.6 beta-methylglutaconyl-CoA + H2O <=> beta-hydroxy-beta-methylglutaryl-CoA
Description
At the beginning of this reaction, 1 molecule of 'beta-methylglutaconyl-CoA', and 1 molecule of 'H2O' are present. At the end of this reaction, 1
molecule of 'beta-hydroxy-beta-methylglutaryl-CoA' is present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'enoyl-CoA hydratase activity' of 'methylglutaconyl-CoA hydratase'.
References
L IJlst, FJ Loupatty, JP Ruiter, M Duran, W Lehnert, RJ Wanders, "3-Methylglutaconic aciduria type I is caused by mutations in AUH.", Am J
Hum Genet, 71, 2002, 1463-6.
K Narisawa, KM Gibson, L Sweetman, WL Nyhan, M Duran, SK Wadman, "Deficiency of 3-methylglutaconyl-coenzyme A hydratase in two
siblings with 3-methylglutaconic aciduria.", J Clin Invest, 77, 1986, 1148-52.
al., editors), 2, 2001, 2125-2163.
Reaction
17.6.2 Isoleucine catabolism
Description
Isoleucine is catabolized in seven steps to propionyl CoA and acetyl CoA.
References
al., editors), 2, 2001, 2125-2163.
17.6.2.1 isoleucine + alpha-ketoglutarate <=> alpha-keto-beta-methylvalerate + glutamate [cytosolic]
Description
At the beginning of this reaction, 1 molecule of 'L-Isoleucine', and 1 molecule of '2-Oxoglutarate' are present. At the end of this reaction, 1
molecule of 'L-Glutamate', and 1 molecule of 'alpha-keto-beta-methylvalerate' are present.
References
Reaction
17.6.2.2 isoleucine + alpha-ketoglutarate <=> alpha-keto-beta-methylvalerate + glutamate [mitochondrial]
Description
At the beginning of this reaction, 1 molecule of 'L-Isoleucine', and 1 molecule of '2-Oxoglutarate' are present. At the end of this reaction, 1
molecule of 'L-Glutamate', and 1 molecule of 'alpha-keto-beta-methylvalerate' are present.
References
Reaction
17.6.2.3 Oxidative decarboxylation of alpha-keto-beta-methylvalerate to alpha-methylbutyryl-CoA by branched-chain alpha-ketoacid

dehydrogenase
Description
alpha-keto-beta-methylvalerate is oxidatively decarboxylated, catalyzed by the E1 component of the complex. Lipoamide associated with E1 is
reduced at the same time. Next, the alpha-keto-beta-methylvalerate group derived from alpha-keto-beta-methylvalerate is transferred to
coenzyme A in two steps catalyzed by the E2 component of the complex (dihydrolipoyl transacetylase). Finally, the oxidized form of lipoamide is
regenerated and electrons are transferred to NAD+ in two steps catalyzed by the E3 component of the complex (dihydrolipoyl dehydrogenase).
References
17.6.2.3.1 alpha-keto-beta-methylvalerate + TPP => 2-methyl-1-hydroxybutyl-TPP + CO2
Description
At the beginning of this reaction, 1 molecule of 'alpha-keto-beta-methylvalerate', and 1 molecule of 'Thiamin diphosphate' are present. At the end
of this reaction, 1 molecule of '2-methyl-1-hydroxybutyl-TPP', and 1 molecule of 'CO2' are present.
References
Reaction
17.6.2.3.2 2-methyl-1-hydroxybutyl-TPP + lipoamide => S-(2-methylbutanoyl)-dihydrolipoamide + TPP
Description
At the beginning of this reaction, 1 molecule of '2-methyl-1-hydroxybutyl-TPP', and 1 molecule of 'Lipoamide' are present. At the end of this
reaction, 1 molecule of 'Thiamin diphosphate', and 1 molecule of 'S-(2-methylbutanoyl)-dihydrolipoamide' are present.
dehydrogenase complex, 2-methyl-1-hydroxybutyl linked'.
References
Reaction
17.6.2.3.3 S-(2-methylbutanoyl)-dihydrolipoamide + CoA => alpha-methylbutyryl-CoA + dihydrolipoamide
Description
At the beginning of this reaction, 1 molecule of 'S-(2-methylbutanoyl)-dihydrolipoamide', and 1 molecule of 'CoA' are present. At the end of this
reaction, 1 molecule of 'alpha-methylbutyryl-CoA', and 1 molecule of 'Dihydrolipoamide' are present.
'branched-chain alpha-ketoacid dehydrogenase complex, S-2-methylbutanoyldihydrolipoamide linked'.
References
Reaction
Description
References
Reaction
Description
References
Reaction
17.6.2.4 alpha-methylbutyryl-CoA + FAD => tiglyl-CoA + FADH2

Description
At the beginning of this reaction, 1 molecule of 'alpha-methylbutyryl-CoA', and 1 molecule of 'FAD' are present. At the end of this reaction, 1
molecule of 'tiglyl-CoA', and 1 molecule of 'FADH2' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'acyl-CoA dehydrogenase activity' of '2-methyl branched chain
acyl-CoA dehydrogenase, holoenzyme'.
References
KM Gibson, TG Burlingame, B Hogema, C Jakobs, RB Schutgens, D Millington, CR Roe, DS Roe, L Sweetman, RD Steiner, L Linck, P
Pohowalla, M Sacks, D Kiss, P Rinaldo, J Vockley, "2-Methylbutyryl-coenzyme A dehydrogenase deficiency: a new inborn error of L-isoleucine
metabolism.", Pediatr Res, 47, 2000, 830-3.
BS Andresen, E Christensen, TJ Corydon, P Bross, B Pilgaard, RJ Wanders, JP Ruiter, H Simonsen, V Winter, I Knudsen, LD Schroeder, N
Gregersen, F Skovby, "Isolated 2-methylbutyrylglycinuria caused by short/branched-chain acyl-CoA dehydrogenase deficiency: identification of a
new enzyme defect, resolution of its molecular basis, and evidence for distinct acyl-CoA dehydrogenases in isoleucine and valine metabolism.",
Am J Hum Genet, 67, 2000, 1095-103.
Reaction
17.6.2.5 tiglyl-CoA + H2O <=> alpha-methyl-beta-hydroxybutyryl-CoA
Description
The reversible reaction of tiglyl-CoA and water to form alpha-methyl-beta-hydroxybutyryl-CoA takes place in the mitochondrial matrix. While
crude extracts of human liver cells have been shown to catalyze the reaction, the specific human enzyme responsible for it has not been
identified.
References
al., editors), 2, 2001, 2125-2163.
Reaction
17.6.2.6 alpha-methyl-beta-hydroxybutyryl-CoA + NAD+ <=> alpha-methylacetoacetyl-CoA + NADH + H+
Description
At the beginning of this reaction, 1 molecule of 'alpha-methyl-beta-hydroxybutyryl-CoA', and 1 molecule of 'NAD+' are present. At the end of this
reaction, 1 molecule of 'alpha-methylacetoacetyl-CoA', 1 molecule of 'H+', and 1 molecule of 'NADH' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the '3-hydroxyacyl-CoA dehydrogenase activity' of
'2-methyl-3-hydroxybutyryl-CoA dehydrogenase'.
References
R Ofman, JP Ruiter, M Feenstra, M Duran, BT Poll-The, J Zschocke, R Ensenauer, W Lehnert, JO Sass, W Sperl, RJ Wanders,
"2-Methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency is caused by mutations in the HADH2 gene.", Am J Hum Genet, 72, 2003, 1300-7.
al., editors), 2, 2001, 2125-2163.
Reaction
17.6.2.7 alpha-methyl-acetoacetyl-CoA + CoA <=> propionyl-CoA + acetyl-CoA
Description
At the beginning of this reaction, 1 molecule of 'alpha-methylacetoacetyl-CoA', and 1 molecule of 'CoA' are present. At the end of this reaction, 1
molecule of 'propionyl-CoA', and 1 molecule of 'Acetyl-CoA' are present.
tetramer'.
References
DV Forrest, S Fahn, "Tardive dysphrenia and subjective akathisia.", J Clin Psychiatry, 40, 1979, 206.
Reaction
17.6.3 Valine catabolism
Description
Valine is catabolized in eight steps to propionyl CoA, which can be metabolized further to yield succinyl CoA.
References
al., editors), 2, 2001, 2125-2163.
17.6.3.1 valine + alpha-ketoglutarate <=> alpha-ketoisovalerate + glutamate [cytosolic]
Description
At the beginning of this reaction, 1 molecule of '2-Oxoglutarate', and 1 molecule of 'L-Valine' are present. At the end of this reaction, 1 molecule
of 'L-Glutamate', and 1 molecule of 'alpha-ketoisovalerate' are present.
References
Reaction
17.6.3.2 valine + alpha-ketoglutarate <=> alpha-ketoisovalerate + glutamate [mitochondrial]
Description
At the beginning of this reaction, 1 molecule of '2-Oxoglutarate', and 1 molecule of 'L-Valine' are present. At the end of this reaction, 1 molecule
of 'L-Glutamate', and 1 molecule of 'alpha-ketoisovalerate' are present.
References
Reaction
17.6.3.3 Oxidative decarboxylation of alpha-ketoisovalerate to isobutyryl-CoA by branched-chain alpha-ketoacid dehydrogenase
Description
alpha-ketoisovalerate is oxidatively decarboxylated, catalyzed by the E1 component of the complex. Lipoamide associated with E1 is reduced at
the same time. Next, the isobutyryl group derived from alpha-ketoisovalerate is transferred to coenzyme A in two steps catalyzed by the E2
component of the complex (dihydrolipoyl transacetylase). Finally, the oxidized form of lipoamide is regenerated and electrons are transferred to
References
17.6.3.3.1 alpha-ketoisovalerate + TPP => 2-methyl-1-hydroxypropyl-TPP + CO2
Description
At the beginning of this reaction, 1 molecule of 'alpha-ketoisovalerate', and 1 molecule of 'Thiamin diphosphate' are present. At the end of this
reaction, 1 molecule of 'CO2', and 1 molecule of '2-methyl-1-hydroxypropyl-TPP' are present.
References
Reaction
17.6.3.3.2 2-methyl-1-hydroxypropyl-TPP + lipoamide => S-(isobutyryl)-dihydrolipoamide + TPP
Description
At the beginning of this reaction, 1 molecule of 'Lipoamide', and 1 molecule of '3-carboxy-1-hydroxypropyl-TPP' are present. At the end of this
reaction, 1 molecule of 'S-(isobutyryl)-dihydrolipoamide', and 1 molecule of 'Thiamin diphosphate' are present.
dehydrogenase complex, 2-methyl-1-hydroxypropyl linked'.
References
Reaction
17.6.3.3.3 S-(isobutyryl)-dihydrolipoamide + CoA => isobutyryl-CoA + dihydrolipoamide
Description
At the beginning of this reaction, 1 molecule of 'CoA', and 1 molecule of 'S-(isobutyryl)-dihydrolipoamide' are present. At the end of this reaction,
1 molecule of 'isobutyryl-CoA', and 1 molecule of 'Dihydrolipoamide' are present.
'branched-chain alpha-ketoacid dehydrogenase complex, S-isobutyryldihydrolipoamide linked'.
References
Reaction
Description
References
Reaction
Description
References
Reaction
17.6.3.4 isobutyryl-CoA + FAD => methacrylyl-CoA + FADH2

Description
At the beginning of this reaction, 1 molecule of 'isobutyryl-CoA', and 1 molecule of 'FAD' are present. At the end of this reaction, 1 molecule of
'FADH2', and 1 molecule of 'methacrylyl-CoA' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'acyl-CoA dehydrogenase activity' of 'isobutyryl-CoA
dehydrogenase, holoenzyme'.
References
TV Nguyen, BS Andresen, TJ Corydon, S Ghisla, N Abd-El Razik, AW Mohsen, SD Cederbaum, DS Roe, CR Roe, NJ Lench, J Vockley,
"Identification of isobutyryl-CoA dehydrogenase and its deficiency in humans.", Mol Genet Metab, 77, 2002, 68-79.
CR Roe, SD Cederbaum, DS Roe, R Mardach, A Galindo, L Sweetman, "Isolated isobutyryl-CoA dehydrogenase deficiency: an unrecognized
defect in human valine metabolism.", Mol Genet Metab, 65, 1999, 264-71.
Reaction
17.6.3.5 methacrylyl-CoA + H2O <=> beta-hydroxyisobutyryl-CoA
Description
The reversible reaction of methacrylyl-CoA and water to form beta-hydroxybutyryl-CoA takes place in the mitochondrial matrix. While crude
extracts of human fibroblasts and liver cells have been shown to catalyze the reaction, the specific human enzyme responsible for it has not
been identified.
References
al., editors), 2, 2001, 2125-2163.
Reaction
17.6.3.6 beta-hydroxyisobutyryl-CoA + H2O => beta-hydroxyisobutyrate + CoA
Description
At the beginning of this reaction, 1 molecule of 'beta-hydroxyisobutyryl-CoA', and 1 molecule of 'H2O' are present. At the end of this reaction, 1
molecule of '3-Hydroxypropanoate', and 1 molecule of 'CoA' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the '3-hydroxyisobutyryl-CoA hydrolase activity' of
'3-hydroxyisobutyryl-coenzyme A hydrolase'.
References
JW Hawes, J Jaskiewicz, Y Shimomura, B Huang, J Bunting, ET Harper, RA Harris, "Primary structure and tissue-specific expression of human
beta-hydroxyisobutyryl-coenzyme A hydrolase.", J Biol Chem, 271, 1996, 26430-4.
al., editors), 2, 2001, 2125-2163.
Reaction
17.6.3.7 beta-hydroxyisobutyrate + NAD+ <=> methylmalonyl semialdehyde + NADH + H+
Description
At the beginning of this reaction, 1 molecule of 'NAD+', and 1 molecule of '3-Hydroxypropanoate' are present. At the end of this reaction, 1
molecule of 'H+', 1 molecule of 'NADH', and 1 molecule of '2-Methyl-3-oxopropanoate' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the '3-hydroxyisobutyrate dehydrogenase activity' of
'3-hydroxyisobutyrate dehydrogenase, homodimer'.
References
WG Robinson, MJ Coon, "Purification and properties of beta-hydroxyisobutyric dehydrogenase", J Biol Chem, 225, 1957, 511-521.
al., editors), 2, 2001, 2125-2163.
Reaction
17.6.3.8 methylmalonate semialdehyde + NAD+ + CoA => propionyl-CoA + CO2 + NADH + H+
Description
At the beginning of this reaction, 1 molecule of '2-Methyl-3-oxopropanoate', 1 molecule of 'CoA', and 1 molecule of 'NAD+' are present. At the
end of this reaction, 1 molecule of 'H+', 1 molecule of 'CO2', 1 molecule of 'propionyl-CoA', and 1 molecule of 'NADH' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'methylmalonate-semialdehyde dehydrogenase (acylating) activity'
of 'methylmalonate semialdehyde dehydrogenase, homotetramer'.
References
al., editors), 2, 2001, 2125-2163.
Reaction
17.7 Histidine catabolism
Description
Histidine is catabolized in four steps to yield glutamate and, in the process, convert one molecule of tetrahydrofolate to
5-formiminotetrahydrofolate.
17.7.1 histidine => urocanate + NH4+
Description
At the beginning of this reaction, 1 molecule of 'L-Histidine' is present. At the end of this reaction, 1 molecule of 'Urocanate', and 1 molecule of
'NH4+' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'histidine ammonia-lyase activity' of 'Histidine ammonia-lyase '.
References
Y Kawai, A Moriyama, K Asai, CM Coleman-Campbell, S Sumi, H Morishita, M Suchi, "Molecular characterization of histidinemia: identification of
four missense mutations in the histidase gene", Hum Genet, 116, 2005, 340-6.
Reaction
17.7.2 urocanate + H2O => 4-imidazolone-5-propionate
Description
Cytosolic urocanase catalyzes the reaction of urocanate and water to form 4-Imidazolone-5-propanoate. While evidence from many
experimental systems indicates that this reaction occurs, existence of the human enzyme is inferred only from high-throughput screening studies
(e.g., Yamada et al. 2004).
References
S Yamada, M Ohira, H Horie, K Ando, H Takayasu, Y Suzuki, S Sugano, T Hirata, T Goto, T Matsunaga, E Hiyama, Y Hayashi, H Ando, S Suita,
M Kaneko, F Sasaki, K Hashizume, N Ohnuma, A Nakagawara, "Expression profiling and differential screening between hepatoblastomas and
the corresponding normal livers: identification of high expression of the PLK1 oncogene as a poor-prognostic indicator of hepatoblastomas",
Oncogene, 23, 2004, 5901-11.
Reaction
17.7.3 4-imidazolone-5-propionate + H2O => N-formiminoglutamate
Description
Cytosolic imidazolonepropionase catalyzes the reaction 4-imidazolone-5-propanoate and H2O to form N-Formimino-L-glutamate. While
evidence from many experimental systems indicates that this reaction occurs, existence of the human enzyme is inferred only from
high-throughput screening studies (e.g., Yamada et al. 2004).
References
S Yamada, M Ohira, H Horie, K Ando, H Takayasu, Y Suzuki, S Sugano, T Hirata, T Goto, T Matsunaga, E Hiyama, Y Hayashi, H Ando, S Suita,
M Kaneko, F Sasaki, K Hashizume, N Ohnuma, A Nakagawara, "Expression profiling and differential screening between hepatoblastomas and
the corresponding normal livers: identification of high expression of the PLK1 oncogene as a poor-prognostic indicator of hepatoblastomas",
Oncogene, 23, 2004, 5901-11.
Reaction
17.7.4 N-formiminoglutamate + tetrahydrofolate => glutamate + 5-formiminotetrahydrofolate
Description
At the beginning of this reaction, 1 molecule of 'N-Formimino-L-glutamate', and 1 molecule of 'tetrahydrofolate' are present. At the end of this
reaction, 1 molecule of 'L-Glutamate', and 1 molecule of '5-formiminotetrahydrofolate' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'formimidoyltetrahydrofolate cyclodeaminase activity' of
'formiminotransferase-cyclodeaminase holoenzyme octamer'.
References
A Solans, X Estivill, S de la Luna, "Cloning and characterization of human FTCD on 21q22.3, a candidate gene for glutamate
formiminotransferase deficiency.", Cytogenet Cell Genet, 88, 2000, 43-9.
LL Murley, NR Mejia, RE MacKenzie, "The nucleotide sequence of porcine formiminotransferase cyclodeaminase. Expression and purification
from Escherichia coli.", J Biol Chem, 268, 1993, 22820-4.
Reaction
17.8 Lysine catabolism
Authors
2003-03-31.
Editors
Description
In humans, most catabolism of L-lysine normally proceeds via a sequence of seven reactions, the last of which feeds into the pathway for fatty
acid catabolism. In the first two reactions, catalyzed by a single enzyme complex, lysine is combined with alpha-ketoglutarate to form
saccharopine, which in turn is cleaved and oxidized to yield glutamate and alpha-ketoadipic semialdehyde. The latter molecule is further oxidized
to alpha-ketoadipate. Alpha-ketoadipate is oxidatively decarboxylated by the alpha-ketoglutarate dehydrogenase complex (the same enzyme
complex responsible for the conversion of alpha-ketoglutarate to succinyl-CoA in the citric acid cycle), yielding glutaryl-CoA. Glutaryl-CoA is
converted to crotonyl-CoA, crotonyl-CoA is converted to beta-hydroxybutyryl-CoA, and beta-hydroxybutyryl-CoA is converted to
acetoacetyl-CoA. The products of lysine catabolism are thus exclusively ketogenic; i.e., under starvation conditions, they can be used for the
synthesis of ketone bodies, beta-hydroxybutyrate and acetoacetate, but not for the net synthesis of glucose.
References
PJ Markovitz, DT Chuang, RP Cox, "Familial hyperlysinemias. Purification and characterization of the bifunctional aminoadipic semialdehyde
synthase with lysine-ketoglutarate reductase and saccharopine dehydrogenase activities.", J Biol Chem, 259, 1984, 11643-6.
SI Goodman, FE Frerman, "Organic acidemias due to defects in lysine oxidation: 2-ketoadipic acidemia and glutaric acidemia", The Metabolic
and Molecular Bases of Inherited Disease, 8th ed (Scriver CR, et al., editors), 2, 2001, 2195-2204.
DL Goh, A Patel, GH Thomas, GS Salomons, DS Schor, C Jakobs, MT Geraghty, "Characterization of the human gene encoding
alpha-aminoadipate aminotransferase (AADAT).", Mol Genet Metab, 76, 2002, 172-80.
RP Cox, "Errors of lysine metabolism", The Metabolic and Molecular Bases of Inherited Disease, 8th ed (Scriver CR, et al., editors), 2, 2001,
1965-1970.
17.8.1 lysine + alpha-ketoglutarate +NADPH + H+ => saccharopine + NADP+ + H2O
Description
At the beginning of this reaction, 1 molecule of 'H+', 1 molecule of 'L-Lysine', 1 molecule of 'NADPH', and 1 molecule of '2-Oxoglutarate' are
present. At the end of this reaction, 1 molecule of 'H2O', 1 molecule of 'NADP+', and 1 molecule of 'N6-(L-1,3-Dicarboxypropyl)-L-lysine' are
present.
This reaction takes place in the 'mitochondrion' and is mediated by the 'saccharopine dehydrogenase (NAD+, L-lysine-forming) activity' of
'lysine-ketoglutarate reductase /saccharopine dehydrogenase homotetramer'.
References
KA Sacksteder, BJ Biery, JC Morrell, BK Goodman, BV Geisbrecht, RP Cox, SJ Gould, MT Geraghty, "Identification of the alpha-aminoadipic
semialdehyde synthase gene, which is defective in familial hyperlysinemia.", Am J Hum Genet, 66, 2001, 1736-43.
1965-1970.
Reaction
17.8.2 saccharopine + NAD+ + H2O => alpha-aminoadipic semialdehyde + glutamate + NADH + H+
Description
At the beginning of this reaction, 1 molecule of 'H2O', 1 molecule of 'NAD+', and 1 molecule of 'N6-(L-1,3-Dicarboxypropyl)-L-lysine' are present.
At the end of this reaction, 1 molecule of 'H+', 1 molecule of 'L-2-Aminoadipate 6-semialdehyde', 1 molecule of 'L-Glutamate', and 1 molecule of
'NADH' are present.
This reaction takes place in the 'mitochondrion' and is mediated by the 'saccharopine dehydrogenase (NADP+, L-glutamate-forming) activity' of
'lysine-ketoglutarate reductase /saccharopine dehydrogenase homotetramer'.
References
KA Sacksteder, BJ Biery, JC Morrell, BK Goodman, BV Geisbrecht, RP Cox, SJ Gould, MT Geraghty, "Identification of the alpha-aminoadipic
semialdehyde synthase gene, which is defective in familial hyperlysinemia.", Am J Hum Genet, 66, 2001, 1736-43.
1965-1970.
Reaction
17.8.3 alpha-aminoadipic semialdehyde + NAD+ => alpha-aminoadipate + NADH + H+
Description
At the beginning of this reaction, 1 molecule of 'NAD+', and 1 molecule of 'L-2-Aminoadipate 6-semialdehyde' are present. At the end of this
reaction, 1 molecule of 'L-2-Aminoadipate', 1 molecule of 'H+', and 1 molecule of 'NADH' are present.
References
1965-1970.
Reaction
17.8.4 alpha-aminoadipate + alpha-ketoglutarate => alpha-ketoadipate + glutamate
Description
At the beginning of this reaction, 1 molecule of 'L-2-Aminoadipate', and 1 molecule of '2-Oxoglutarate' are present. At the end of this reaction, 1
molecule of 'alpha-ketoadipate', and 1 molecule of 'L-Glutamate' are present.
This reaction takes place in the 'cytoplasm' and is mediated by the 'kynurenine-oxoglutarate transaminase activity' of
'L-kynurenine/alpha-aminoadipate aminotransferase'.
References
DL Goh, A Patel, GH Thomas, GS Salomons, DS Schor, C Jakobs, MT Geraghty, "Characterization of the human gene encoding
alpha-aminoadipate aminotransferase (AADAT).", Mol Genet Metab, 76, 2002, 172-80.
Reaction
17.8.5 Oxidative decarboxylation of alpha-ketoadipate to glutaryl CoA by alpha-ketoglutarate

dehydrogenase
Description
This process, carried out by the mitochondrial alpha-ketoglutarate dehydrogenase complex, consists of five distinct reactions. First,
alpha-ketoadipate is oxidatively decarboxylated, catalyzed by the E1 component of the complex. Lipoamide associated with E1 is reduced at the
same time. Next, the glutaryl group derived from alpha-ketoadipate is transferred to coenzyme A in two steps catalyzed by the E2 component of
the complex (dihydrolipoyl transacetylase). Finally, the oxidized form of lipoamide is regenerated and electrons are transferred to NAD+ in two
steps catalyzed by the E3 component of the complex (dihydrolipoyl dehydrogenase).
References
1965-1970.
17.8.5.1 alpha-ketoadipate + TPP => 4-carboxy-1-hydroxybutyl-TPP + CO2
Description
At the beginning of this reaction, 1 molecule of 'alpha-ketoadipate', and 1 molecule of 'Thiamin diphosphate' are present. At the end of this
reaction, 1 molecule of 'CO2', and 1 molecule of '4-carboxy-1-hydroxybutyl-TPP' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'unknown activity' of 'alpha-ketoglutarate dehydrogenase complex'.
References
LJ Reed, ML Hackert, "Structure-function relationships in dihydrolipoamide acyltransferases.", J Biol Chem, 265, 1990, 8971-4.
Reaction
17.8.5.2 4-carboxy-1-hydroxybutyl-TPP + lipoamide => S-glutaryldihydrolipoamide + TPP
Description
At the beginning of this reaction, 1 molecule of 'Lipoamide', and 1 molecule of '4-carboxy-1-hydroxybutyl-TPP' are present. At the end of this
reaction, 1 molecule of 'S-glutaryldihydrolipoamide', and 1 molecule of 'Thiamin diphosphate' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'succinyltransferase activity' of 'alpha-ketoglutarate dehydrogenase
complex, 4-carboxy-1-hydroxybutyl linked'.
References
Reaction
17.8.5.3 S-glutaryldihydrolipoamide + CoA => glutaryl-CoA + dihydrolipoamide
Description
At the beginning of this reaction, 1 molecule of 'S-glutaryldihydrolipoamide', and 1 molecule of 'CoA' are present. At the end of this reaction, 1
molecule of 'glutaryl-CoA', and 1 molecule of 'Dihydrolipoamide' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'dihydrolipoyllysine-residue succinyltransferase activity' of
'alpha-ketoglutarate dehydrogenase complex, S-glutaryldihydrolipoamide linked'.
References
Reaction
17.8.5.4 dihydrolipoamide + FAD => lipoamide + FADH2 [alpha-ketoglutarate dehydrogenase]
Description
'Lipoamide', and 1 molecule of 'FADH2' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'dihydrolipoyl dehydrogenase activity' of 'alpha-ketoglutarate
dehydrogenase complex, dihydrolipoamide linked'.
References
RG McCartney, JE Rice, SJ Sanderson, V Bunik, H Lindsay, JG Lindsay, "Subunit interactions in the mammalian alpha-ketoglutarate
dehydrogenase complex. Evidence for direct association of the alpha-ketoglutarate dehydrogenase and dihydrolipoamide dehydrogenase
components.", J Biol Chem, 273, 1998, 24158-64.
Reaction
17.8.5.5 FADH2 + NAD+ => FAD + NADH + H+ [alpha-ketoglutarate dehydrogenase]
Description
dehydrogenase complex, FADH2 linked'.
References
Reaction
17.8.6 glutaryl-CoA + FAD => crotonyl-CoA + FADH2 + CO2
Description
At the beginning of this reaction, 1 molecule of 'FAD', and 1 molecule of 'glutaryl-CoA' are present. At the end of this reaction, 1 molecule of
'CO2', 1 molecule of 'FADH2', and 1 molecule of 'crotonyl-CoA' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'glutaryl-CoA dehydrogenase activity' of 'glutaryl-CoA
dehydrogenase holoenzyme tetramer'.
References
SI Goodman, FE Frerman, "Organic acidemias due to defects in lysine oxidation: 2-ketoadipic acidemia and glutaric acidemia", The Metabolic
and Molecular Bases of Inherited Disease, 8th ed (Scriver CR, et al., editors), 2, 2001, 2195-2204.
Reaction
17.8.7 Crotonoyl-CoA+H2O<=>(S)-3-Hydroxybutanoyl-CoA
Description
At the beginning of this reaction, 1 molecule of 'Crotonoyl-CoA', and 1 molecule of 'H2O' are present. At the end of this reaction, 1 molecule of
'(S)-3-Hydroxybutanoyl-CoA' is present.
References
Reaction
17.8.8 (S)-Hydroxybutanoyl-CoA+NAD<=>Acetoacetyl-CoA+NADH+H
Description
At the beginning of this reaction, 1 molecule of 'NAD+', and 1 molecule of '(S)-3-Hydroxybutanoyl-CoA' are present. At the end of this reaction, 1
molecule of 'acetoacetyl-CoA', 1 molecule of 'H+', and 1 molecule of 'NADH' are present.
References
JJ Barycki, LK O'Brien, JM Bratt, R Zhang, R Sanishvili, AW Strauss, LJ Banaszak, "Biochemical characterization and crystal structure
determination of human heart short chain L-3-hydroxyacyl-CoA dehydrogenase provide insights into catalytic mechanism.", Biochemistry, 38,
1999, 5786-98.
Reaction
17.9 Phenylalanine and tyrosine catabolism
Authors
2003-04-29.
Editors
Description
The first reaction in this pathway converts phenylalanine to tyrosine, coupled to the conversion of tetrahydrobiopterin to
4a-hydroxytetrahydrobiopterin, catalyzed by phenylalanine hydroxylase. (Deficiencies in this enzyme are responsible for the commonest form of
phenylketonuria (PKU) in humans.) This reaction functions both as the first step in the pathway by which the body disposes of excess
phenylalanine, and as a source of the amino acid tyrosine. The next two reactions are responsible for the regeneration of tetrahydrobiopterin
from 4a-hydroxytetrahydrobiopterin. The following five reactions convert tyrosine to fumarate, an intermediate in the citric acid cycle, and
acetoacetate, a ketone body.
References
N Blau, B Thony, RG Cotton, K Hyland, "Disorders of tetrahydrobiopterin and related biogenic amines", The Metabolic and Molecular Bases of
Inherited Disease, 8th ed (Scriver CR, et al., editors), 2, 2001, 1725-1776.
GA Mitchell, M Grompe, M Lambert, RM Tanguay, "Hypertyrosinemia", The Metabolic and Molecular Bases of Inherited Disease, 8th ed (Scriver
CR, et al., editors), 2, 2001, 1777-1805.
17.9.1 phenylalanine + tetrahydrobiopterin + O2 => tyrosine + 4a-hydroxytetrahydrobiopterin + H2O
Description
At the beginning of this reaction, 1 molecule of 'Oxygen', 1 molecule of 'Tetrahydrobiopterin', and 1 molecule of 'L-Phenylalanine' are present. At
the end of this reaction, 1 molecule of 'L-Tyrosine', 1 molecule of 'H2O', and 1 molecule of '4a-hydroxytetrahydrobiopterin' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'phenylalanine 4-monooxygenase activity' of 'phenylalanine hydroxylase
holoenzyme tetramer'.
References
F Fusetti, H Erlandsen, T Flatmark, RC Stevens, "Structure of tetrameric human phenylalanine hydroxylase and its implications for
phenylketonuria.", J Biol Chem, 273, 1998, 16962-7.
Reaction
17.9.2 4a-hydroxytetrahydrobiopterin => q-dihydrobiopterin + H2O
Description
At the beginning of this reaction, 1 molecule of '4a-hydroxytetrahydrobiopterin' is present. At the end of this reaction, 1 molecule of
'q-dihydrobiopterin', and 1 molecule of 'H2O' are present.
This reaction takes place in the 'cytosol' and is mediated by the '4-alpha-hydroxytetrahydrobiopterin dehydratase activity' of
'pterin-4-alpha-carbinolamine dehydratase homotetramer'.
References
BA Citron, S Kaufman, S Milstien, EW Naylor, CL Greene, MD Davis, "Mutation in the 4a-carbinolamine dehydratase gene leads to mild
hyperphenylalaninemia with defective cofactor metabolism.", Am J Hum Genet, 53, 1993, 768-74.
Reaction
17.9.3 q-dihydrobiopterin + NADH + H+ => tetrahydrobiopterin + NAD+
Description
At the beginning of this reaction, 1 molecule of 'q-dihydrobiopterin', 1 molecule of 'H+', and 1 molecule of 'NADH' are present. At the end of this
reaction, 1 molecule of 'NAD+', and 1 molecule of 'Tetrahydrobiopterin' are present.
This reaction takes place in the 'cytosol' and is mediated by the '6,7-dihydropteridine reductase activity' of 'dihydropteridine reductase
homodimer'.
References
J Lockyer, RG Cook, S Milstien, S Kaufman, SL Woo, FD Ledley, "Structure and expression of human dihydropteridine reductase.", Proc Natl
Acad Sci U S A, 84, 1987, 3329-33.
Reaction
17.9.4 tyrosine + alpha-ketoglutarate => p-hydroxyphenylpyruvate + glutamate
Description
At the beginning of this reaction, 1 molecule of 'L-Tyrosine', and 1 molecule of '2-Oxoglutarate' are present. At the end of this reaction, 1
molecule of '3-(4-Hydroxyphenyl)pyruvate', and 1 molecule of 'L-Glutamate' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'tyrosine transaminase activity' of 'tyrosine aminotransferase holoenzyme'.
References
CR, et al., editors), 2, 2001, 1777-1805.
Reaction
17.9.5 p-hydroxyphenylpyruvate + O2 => homogentisate + CO2

Description
At the beginning of this reaction, 1 molecule of '3-(4-Hydroxyphenyl)pyruvate', and 1 molecule of 'Oxygen' are present. At the end of this
reaction, 1 molecule of 'Homogentisate', and 1 molecule of 'CO2' are present.
This reaction takes place in the 'cytosol' and is mediated by the '4-hydroxyphenylpyruvate dioxygenase activity' of '4-hydroxyphenylpyruvate
dioxygenase holoenzyme dimer'.
References
CR, et al., editors), 2, 2001, 1777-1805.
Reaction
17.9.6 homogentisate + O2 => maleylacetoacetate
Description
At the beginning of this reaction, 1 molecule of 'Homogentisate', and 1 molecule of 'Oxygen' are present. At the end of this reaction, 1 molecule
of '4-Maleylacetoacetate' is present.
This reaction takes place in the 'cytosol' and is mediated by the 'homogentisate 1,2-dioxygenase activity' of 'homogentisic acid oxidase
holoenzyme hexamer'.
References
GP Titus, HA Mueller, J Burgner, S RodrÃ-guez De CÃ³rdoba, MA PeÃ±alva, DE Timm, "Crystal structure of human homogentisate
dioxygenase.", Nat Struct Biol, 7, 2000, 542-6.
CR, et al., editors), 2, 2001, 1777-1805.
Reaction
17.9.7 maleylacetoacetate => fumarylacetoacetate
Description
At the beginning of this reaction, 1 molecule of '4-Maleylacetoacetate' is present. At the end of this reaction, 1 molecule of
'4-Fumarylacetoacetate' is present.
This reaction takes place in the 'cytosol' and is mediated by the 'maleylacetoacetate isomerase activity' of 'maleylacetoacetic acid isomerase
holoenzyme dimer'.
References
G Polekhina, PG Board, AC Blackburn, MW Parker, "Crystal structure of maleylacetoacetate isomerase/glutathione transferase zeta reveals the
molecular basis for its remarkable catalytic promiscuity.", Biochemistry, 40, 2001, 1567-76.
Reaction
17.9.8 fumarylacetoacetate => fumarate + acetoacetate
Description
At the beginning of this reaction, 1 molecule of '4-Fumarylacetoacetate' is present. At the end of this reaction, 1 molecule of 'Fumarate', and 1
molecule of 'Acetoacetate' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'fumarylacetoacetase activity' of 'fumarylacetoacetate hydrolase dimer'.
References
Y Labelle, D Phaneuf, B Leclerc, RM Tanguay, "Characterization of the human fumarylacetoacetate hydrolase gene and identification of a
missense mutation abolishing enzymatic activity.", Hum Mol Genet, 2, 1993, 941-6.
Reaction
17.10 Tryptophan catabolism
Description
Tryptophan is catabolized in seven steps to yield aminomuconate.
17.10.1 tryptophan + O2 => N-formylkynurenine [TDO]
Description
Cytosolic tryptophan 2,3-dioxygenase (TDO) tetramer catalyzes the conversion of L-tryptophan and oxygen to formylkynurenine. The structure
and catalytic properties of the human enzyme are inferred from the close similarity of its predicted amino acid sequence (Comings et al. 1995) to
that of the well-studied rat enzyme (Dick et al. 2001). In the body, TDO is found predominantly in the liver and is induced by metabolites such as
tryptophan and histidine, and by glucocorticoids. These properties, together with TDO's narrow substrate specificity, are consistent with the
hypothesis that the enzyme functions functions primarily in tryptophan catabolism and NAD biosynthesis (Taylor and Feng 1991).
References
MW Taylor, G Feng, "Relationship between interferon-gamma, indoleamine 2,3-dioxygenase, and tryptophan catabolism", FASEB J, 5, 1991,
2516-2522.
DE Comings, G Dietz, GL Forest, D Muhleman, M Sherman, "Sequence of human tryptophan 2,3-dioxygenase (TDO2): presence of a
glucocorticoid response-like element composed of a GTT repeat and an intronic CCCCT repeat", Genomics, 29, 1995, 390-396.
R Dick, BP Murray, MJ Reid, MA Correia, "Structure-function relationships of rat hepatic tryptophan 2,3-dioxygenase: identification of the
putative heme-ligating histidine residues", Arch Biochem Biophys, 392, 2001, 71-78.
Reaction
17.10.2 N-formylkynurenine + H2O => kynurenine + formate
Description
In this cytosolic reaction, arylformamidase catalyzes the hydrolysis of formylkynurenine to yield formate and L-kynurenine.
Source reaction
This reaction was inferred from the corresponding reaction "N-formylkynurenine + H2O => kynurenine + formate [mouse]" in species Mus
musculus.
MK Pabarcus, JE Casida, "Kynurenine formamidase: determination of primary structure and modeling-based prediction of tertiary structure and
catalytic triad.", Biochim Biophys Acta, 1596, 2002, 201-11.
Reaction
17.10.3 kynurenine + O2 + NADPH + H+ => 3-hydroxykynurenine + NADP+ + H2O
Description
Cytosolic kynurenine 3-monooxygenase catalyzes the reaction of L-kynurenine, NADPH + H+, and O2 to form 3-hydroxy-L-kynurenine, NADP+,
and H2O.
References
J Breton, N Avanzi, S Magagnin, N Covini, G Magistrelli, L Cozzi, A Isacchi, "Functional characterization and mechanism of action of
recombinant human kynurenine 3-hydroxylase", Eur J Biochem, 267, 2000, 1092-1099.
Reaction
17.10.4 3-hydroxykynurenine + H2O => 3-hydroxyanthranilate + alanine
Description
Cytosolic kynureninase catalyzes the hydrolysis of 3-hydroxy-L-kynurenine to form L-alanine and 3-hydroxyanthranilate.
References
S Toma, M Nakamura, S TonÃ©, E Okuno, R Kido, J Breton, N Avanzi, L Cozzi, C Speciale, M Mostardini, S Gatti, L Benatti, "Cloning and
recombinant expression of rat and human kynureninase.", FEBS Lett, 408, 1997, 5-10.
Reaction
17.10.5 3-hydroxyanthranilate + O2 => 2-amino-3-carboxymuconate semialdehyde
Description
Cytosolic 3-hydroxyanthranilate oxygenase catalyzes the reaction of 3-hydroxyanthranilate and O2 to form 2-amino-3-carboxymuconate
semialdehyde.
References
P Malherbe, C KÃ¶hler, M Da Prada, G Lang, V Kiefer, R Schwarcz, HW Lahm, AM Cesura, "Molecular cloning and functional expression of
human 3-hydroxyanthranilic-acid dioxygenase.", J Biol Chem, 269, 1994, 13792-7.
Reaction
17.10.6 2-amino-3-carboxymuconate semialdehyde => 2-aminomuconate semialdehyde + CO2
Description
At the beginning of this reaction, 1 molecule of '2-Amino-3-carboxymuconate semialdehyde' is present. At the end of this reaction, 1 molecule of
'CO2', and 1 molecule of '2-Aminomuconate semialdehyde' are present.
This reaction takes place in the 'cytoplasm' and is mediated by the 'carboxy-lyase activity' of '2-amino-3-carboxymuconate-6-semialdehyde
decarboxylase homodimer'.
References
S Fukuoka, K Ishiguro, K Yanagihara, A Tanabe, Y Egashira, H Sanada, K Shibata, "Identification and expression of a cDNA encoding human
alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD). A key enzyme for the tryptophan-niacine pathway and
"quinolinate hypothesis".", J Biol Chem, 277, 2002, 35162-7.
Source reaction
This reaction was inferred from the corresponding reaction "2-amino-3-carboxymuconate semialdehyde => 2-aminomuconate semialdehyde +
CO2 [rat]" in species Rattus norvegicus.
A Tanabe, Y Egashira, S Fukuoka, K Shibata, H Sanada, "Purification and molecular cloning of rat 2-amino-3-carboxymuconate-6-semialdehyde
decarboxylase.", Biochem J, 361, 2002, 567-75.
Reaction
17.10.7 2-aminomuconate semialdehyde + NAD+ => aminomuconate + NADH + H+
Authors
Description
This reaction has been characterized in vitro using enzyme partially purified from cat liver. The human event is inferred from cat one. Neither the
cat nor the human protein has been fully purified or sequenced.
References
A Ichiyama, S Nakamura, H Kawai, T Honjo, Y Nishizuka, O Hayaishi, S Senoh, "Studies on the metabolism of the benzene ring of tryptophan in
mammalian tissues. II. Enzymatic formation of alpha-muconic acid from 2-hydroxyanthranilic acid", J Biol Chem, 240, 1965, 740-9.
Source reaction
This reaction was inferred from the corresponding reaction "2-aminomuconate semialdehyde + NAD+ => aminomuconate + NADH + H+" in
species Felis catus.
A Ichiyama, S Nakamura, H Kawai, T Honjo, Y Nishizuka, O Hayaishi, S Senoh, "Studies on the metabolism of the benzene ring of tryptophan in
mammalian tissues. II. Enzymatic formation of alpha-muconic acid from 2-hydroxyanthranilic acid", J Biol Chem, 240, 1965, 740-9.
Reaction
17.11 Carnitine synthesis
Authors
2003-04-29.
Editors
Description
Carnitine is synthesized in four steps from trimethyllysine (generated in turn by the S-adenosyl-methionine-mediated methylation of lysine
residues in proteins, followed by protein hydrolysis). The enzymes that catalyze the first three steps of carnitine synthesis, converting
trimethyllysine to gamma-butyrobetaine, are widely distributed in human tissues. The enzyme that catalyzes the last reaction, converting
gamma-butyrobetaine to carnitine, is found only in liver and kidney cells, and at very low levels in brain tissues. Other tissues that require
carnitine, such as muscle, are dependent on transport systems that mediate its export from the liver and uptake by other tissues.
References
J Kerner, C Hoppel, "Genetic disorders of carnitine metabolism and their nutritional management.", Annu Rev Nutr, 18, 1998, 179-206.
17.11.1 trimethyllysine + alpha-ketoglutarate + O2 => beta-hydroxy-trimethyllysine + succinate + CO2
Description
At the beginning of this reaction, 1 molecule of 'Oxygen', 1 molecule of '2-Oxoglutarate', and 1 molecule of 'N6,N6,N6-Trimethyl-L-lysine' are
present. At the end of this reaction, 1 molecule of 'CO2', 1 molecule of '3-Hydroxy-N6,N6,N6-trimethyl-L-lysine', and 1 molecule of 'Succinate'
are present.
This reaction takes place in the 'mitochondrion' and is mediated by the 'oxidoreductase activity' of 'molecular oxygen, 2-oxoglutarate as one
donor, and incorporation of one atom each of oxygen into both donors of trimethyllysine dioxygenase holoenzyme homodimer'.
References
FM Vaz, R Ofman, K Westinga, JW Back, RJ Wanders, "Molecular and Biochemical Characterization of Rat epsilon -N-Trimethyllysine
Hydroxylase, the First Enzyme of Carnitine Biosynthesis.", J Biol Chem, 276, 2001, 33512-7.
Reaction
17.11.2 beta-hydroxy-trimethyllysine => gamma-butyrobetaine aldehyde + glycine
Description
At the beginning of this reaction, 1 molecule of '3-Hydroxy-N6,N6,N6-trimethyl-L-lysine' is present. At the end of this reaction, 1 molecule of
'Glycine', and 1 molecule of '4-Trimethylammoniobutanal' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'glycine hydroxymethyltransferase activity' of 'serine hydroxymethyltransferase
holoenzyme tetramer'.
References
JD Hulse, SR Ellis, LM Henderson, "Carnitine biosynthesis. beta-Hydroxylation of trimethyllysine by an alpha-ketoglutarate-dependent

mitochondrial dioxygenase.", J Biol Chem, 253, 1978, 1654-9.
Reaction
17.11.3 gamma-butyrobetaine semialdehyde + NAD+ => gamma-butyrobetaine + NADH + H+
Description
At the beginning of this reaction, 1 molecule of 'NAD+', and 1 molecule of '4-Trimethylammoniobutanal' are present. At the end of this reaction, 1
molecule of 'H+', 1 molecule of '4-Trimethylammoniobutanoate', and 1 molecule of 'NADH' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'aldehyde dehydrogenase activity' of '4-trimethylaminobutyraldehyde
References
FM Vaz, SW Fouchier, R Ofman, M Sommer, RJ Wanders, "Molecular and biochemical characterization of rat
gamma-trimethylaminobutyraldehyde dehydrogenase and evidence for the involvement of human aldehyde dehydrogenase 9 in carnitine
biosynthesis.", J Biol Chem, 275, 2000, 7390-4.
G Kurys, W Ambroziak, R Pietruszko, "Human aldehyde dehydrogenase. Purification and characterization of a third isozyme with low Km for
gamma-aminobutyraldehyde.", J Biol Chem, 264, 1989, 4715-21.
Reaction
17.11.4 gamma-butyrobetaine + alpha-ketoglutarate + O2 => carnitine + succinate + CO2
Description
At the beginning of this reaction, 1 molecule of 'Oxygen', 1 molecule of '4-Trimethylammoniobutanoate', and 1 molecule of '2-Oxoglutarate' are
present. At the end of this reaction, 1 molecule of 'CO2', 1 molecule of 'Succinate', and 1 molecule of 'Carnitine' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'gamma-butyrobetaine dioxygenase activity' of 'gamma-butyrobetaine
hydroxylase holoenzyme dimer'.
References
S Lindstedt, I Nordin, "Multiple forms of gamma-butyrobetaine hydroxylase (EC 1.14.11.1).", Biochem J, 223, 1984, 119-27.
FM Vaz, S van Gool, R Ofman, L Ijlst, RJ Wanders, "Carnitine biosynthesis: identification of the cDNA encoding human gamma-butyrobetaine
hydroxylase.", Biochem Biophys Res Commun, 250, 1998, 506-10.
Reaction
17.12 Creatine metabolism
Authors
2003-04-23.
Editors
Description
In humans, creatine is synthesized primarily in the liver and kidney, from glycine, arginine, and S-adenosylmethionine, in a sequence of two
reactions. From the liver, creatine is exported to tissues such as skeletal muscle and brain, where it undergoes phosphorylation and serves as a
short-term energy store. The mechanism by which creatine leaves producer tissues is unclear, but its uptake by consumer tissues is mediated
by the SLC6A8 transporter.
Once formed, phosphocreatine undergoes a slow spontaneous reaction to form creatinine, which is excreted from the body.
References
J Kerner, C Hoppel, "Genetic disorders of carnitine metabolism and their nutritional management.", Annu Rev Nutr, 18, 1998, 179-206.
M Wyss, R Kaddurah-Daouk, "Creatine and creatinine metabolism", Physiol Rev, 80, 2000, 1107-213.
17.12.1 arginine + glycine => ornithine + guanidoacetate

Description
Glycine amindinotransferase, localized to the mitochondrial intermembrane space, catalyzes the reaction of arginine and glycine to form
guanidinoacetate and ornithine. The active form of the enzyme is a dimer (Humm et al. 1997 {EMBO J]; Humm et al 1997 [Biochem J]). Its
function in vivo has been confirmed by molecular and biochemical studies of patients deficient in the enzyme (Item et al. 2001).
References
A Humm, E Fritsche, K Mann, M Gohl, R Huber, "Recombinant expression and isolation of human L-arginine:glycine amidinotransferase and
identification of its active-site cysteine residue", Biochem J, 322, 1997, 771-6.
CB Item, S Stockler-Ipsiroglu, C Stromberger, A Muhl, MG Alessandri, MC Bianchi, M Tosetti, F Fornai, G Cioni, "Arginine:glycine
amidinotransferase deficiency: the third inborn error of creatine metabolism in humans", Am J Hum Genet, 69, 2001, 1127-33.
A Humm, E Fritsche, S Steinbacher, R Huber, "Crystal structure and mechanism of human L-arginine:glycine amidinotransferase: a
mitochondrial enzyme involved in creatine biosynthesis.", EMBO J, 16, 1997, 3373-85.
Reaction
17.12.2 guanidinoacetate + S-adenosylmethionine => creatine + S-adenosylhomocysteine
Description
Cytosolic guanidinoacetate methyltransferase catalyzes the reaction of S-adenosylmethionine and guanidinoacetate to form
S-adenosylhomocysteine and creatine (Stockler et al. 1996).
References
S Stockler, D Isbrandt, F Hanefeld, B Schmidt, K von Figura, "Guanidinoacetate methyltransferase deficiency: the first inborn error of creatine
metabolism in man.", Am J Hum Genet, 58, 1996, 914-22.
Reaction
17.12.3 creatine + ATP => phosphocreatine + ADP [CKB,CKM]
Authors
Description
Cytosolic creatine kinase catalyzes the reaction of creatine and ATP to form phosphocreatine and ADP. The active form of the enzyme is a
dimer. Monomers of the cytosolic enzyme occur in two isoforms, B and M, so called because of their abundance in brain and muscle
respectively. The enzyme is widely expressed in the body and many tissues express both isoforms. Both homo- (BB, MM) and heterodimers
(BM) are catalytically active.
References
PF Wang, AJ Flynn, MM Naor, JH Jensen, G Cui, Jr Merz KM, GL Kenyon, MJ McLeish, "Exploring the role of the active site cysteine in human
muscle creatine kinase", Biochemistry, 45, 2006, 11464-72.
Reaction
17.12.4 creatine + ATP => phosphocreatine + ADP [CK octamer]
Authors
Description
Creatine kinase octamers associated with the inner mitochondrial membrane catalyze the reaction of creatine and ATP to form phosphocreatine
and ADP. Two mitochondrial creatine kinase proteins have been identified, one encoded by CKMT1A and B that is found in many tissues and
one encoded by CKMT2 that is found in sarcomeres (Haas et al. 1988; Haas and Straus 1990). Studies of sarcomeric creatine kinase octamers
suggest that their organization and association with phospholipids in the inner mitochondrial membrane may facilitate energy transfer from ATP
generated in the mitochondrial matrix to cytosolic phosphocreatine (Khuchua et al. 1998; Schlattner et al. 2004).
References
RC Haas, AW Strauss, "Separate nuclear genes encode sarcomere-specific and ubiquitous human mitochondrial creatine kinase isoenzymes", J
Biol Chem, 265, 1990, 6921-7.
RC Haas, C Korenfeld, Z Zhang, B Perryman, D Roman, AW Strauss, "Isolation and characterization of the gene and cDNA encoding human
mitochondrial creatine kinase", J Biol Chem, 264, 1989, 2890-7.
U Schlattner, F Gehring, N Vernoux, M Tokarska-Schlattner, D Neumann, O Marcillat, C Vial, T Wallimann, "C-terminal lysines determine
phospholipid interaction of sarcomeric mitochondrial creatine kinase", J Biol Chem, 279, 2004, 24334-42.
ZA Khuchua, W Qin, J Boero, J Cheng, RM Payne, VA Saks, AW Strauss, "Octamer formation and coupling of cardiac sarcomeric mitochondrial
creatine kinase are mediated by charged N-terminal residues", J Biol Chem, 273, 1998, 22990-6.
Reaction
17.12.5 phosphocreatine + H2O => creatinine + orthophosphate

Authors
Description
Cytosolic phosphocreatine spontaneously hydrolyzes to yield creatinine and orthophosphate (Borsook and Dubnoff 1947). Creatinine cannot be
metabolized further and is excreted from the body in the urine. Creatinine formation proceeds at a nearly constant rate and the amount produced
by an individual is a function of muscle mass, so urinary creatinine output is clinically useful as a normalization factor in assays of urinary output
of other molecules. Iyengar et al. (1985) have suggested that an alternative reaction sequence, proceeding via phosphocreatinine but also
spontaneous, may contribute to creatinine formation.
References
MR Iyengar, DW Coleman, TM Butler, "Phosphocreatinine, a high-energy phosphate in muscle, spontaneously forms phosphocreatine and
creatinine under physiological conditions", J Biol Chem, 260, 1985, 7562-7.
H Borsook, JW Dubnoff, "The hydrolysis of phosphocreatine and the origin of urinary creatinine", J Biol Chem, 168, 1947, 493-510.
Reaction
18 Metabolism of carbohydrates
Authors
D'Eustachio, P, Schmidt, EE, 2003-11-03.
Editors
D'Eustachio, P, Schmidt, EE, 0000-00-00.
Description
These pathways together are responsible for: 1) the extraction of energy and carbon skeletons for biosyntheses from dietary sugars and related
molecules; 2) the short-term storage of glucose in the body (as glycogen) and its mobilization during a short fast; and 3) the synthesis of glucose
from pyruvate during extended fasts.
18.1 Digestion of dietary carbohydrate
Authors
Reviewers
Nichols, BL, 2007-01-15.
Description
Carbohydrate is a major component of the human diet, and includes starch (amylose and amylopectin) and disaccharides such as sucrose,
lactose, maltose and, in small amounts, trehalose. The digestion of starch begins with the action of amylase enzymes secreted in the saliva and
small intestine, which convert it to maltotriose, maltose, limit dextrins, and some glucose. Digestion of the limit dextrins and disaccharides, both
dietary and starch-derived, to monosaccharides - glucose, galactose, and fructose - is accomplished by enzymes located on the luminal surfaces
of enterocytes lining the microvilli of the small intestine (Semenza et al. 2001).
References
G Semenza, S Auricchio, N Mantei, "Small-intestinal disaccharidases", The Metabolic and Molecular Bases of Inherited Disease, 8th ed (Scriver
CR, et al., editors), 1, 2001, 1623-1650.
18.1.1 Digestion of linear starch (amylose) by extracellular amylase
Authors
Reviewers
Nichols, BL, 2007-01-15.
Description
Extracellular amylose starch, linear polymers of glucose joined by alpha-1,4 linkages, is digested by the endoglucosidase activity of
alpha-amylases, yielding maltose, maltotriose, and longer maltosides (Semenza et al. 2001). The human genome contains five functional
alpha-amylase genes, encoding structurally closely related isoenzymes (Gumucio et al. 1988). Three of these genes encode proteins
synthesized in the parotid glands and released into the saliva (amylase 1A, B, and C), and the other two encode proteins synthesized in the
exocrine pancreas and released into the small intestine (amylase 2A and B). In the human body, starch digestion thus commences in the mouth,
mediated by salivary amylases, and is continued in the small intestine, mediated by the pancreatic ones.
X-ray crystallographic studies of amylase 1A and 2A proteins show them to be monomers, complexed with single calcium and chloride ions
(Ramasubbu et al. 1996; Brayer et al. 2000). Biochemical characterization of amylase 2A indicates that the enzyme efficiently cleaves
poly-glucose chains so as to release maltose - a glucose disaccharide - from the reducing end of the chain (Braun et al. 1993; Brayer et al.
2000).
References
CR, et al., editors), 1, 2001, 1623-1650.
GD Brayer, G Sidhu, R Maurus, EH Rydberg, C Braun, Y Wang, NT Nguyen, CM Overall, SG Withers, "Subsite mapping of the human
pancreatic alpha-amylase active site through structural, kinetic, and mutagenesis techniques", Biochemistry, 39, 2000, 4778-91.
DL Gumucio, K Wiebauer, RM Caldwell, LC Samuelson, MH Meisler, "Concerted evolution of human amylase genes", Mol Cell Biol, 8, 1988,
1197-205.
N Ramasubbu, V Paloth, Y Luo, GD Brayer, MJ Levine, "Structure of human salivary alpha-amylase at 1.6 A resolution: implications for its role in
the oral cavity.", Acta Crystallogr D Biol Crystallogr, 52, 1996, 435-46.
C Braun, A Meinke, L Ziser, SG Withers, "Simultaneous high-performance liquid chromatographic determination of both the cleavage pattern
and the stereochemical outcome of the hydrolysis reactions catalyzed by various glycosidases", Anal Biochem, 212, 1993, 259-62.
Reaction
18.1.2 Digestion of branched starch (amylopectin) by extracellular amylase
Authors
Reviewers
Nichols, BL, 2007-01-15.

Description
The 1-4 linkages of extracellular amylopectin starch, a glucose polymer containing linear segments formed by alpha-1,4 linkages and a smaller
number of alpha-1,6 linkages forming branch points, are digested by the endoglucosidase activity of alpha-amylases, yielding maltose,
maltotriose, and longer maltosides from the alpha-1,4 linear segments and alpha-limit dextrins from the branch points (Semenza et al. 2001).
Alpha-limit dextrins are glucose (G) oligomers linked by 1-4 and 1-6 bonds. 1-6 branch points make up about 5% of all amylopectin glucose
bonds - the exact fraction depends on the source of the starch. Mass spectroscopic analysis of alpha-limit dextrin shows it to be a mixture of
maltosides and isomaltosides containing two to forty G residues, but the most common contain fewer than seven. Maltose (G2) is the shortest
1-4 maltoside produced by alpha-amylase. Isomaltose (G2) is the shortest 1-6 isomaltoside.
The human genome contains five functional alpha-amylase genes, encoding structurally closely related isoenzymes (Gumucio et al. 1988).
Three of these genes encode proteins synthesized in the parotid glands and released into the saliva (amylase 1A, B, and C), and the other two
encode proteins synthesized in the exocrine pancreas and released into the small intestine (amylase 2A and B). In the human body, starch
digestion thus commences in the mouth, mediated by salivary amylases, and is continued in the small intestine, mediated by the pancreatic
ones.
X-ray crystallographic studies of amylase 1A and 2A proteins show them to be monomers, complexed with single calcium and chloride ions
(Ramasubbu et al. 1996; Brayer et al. 2000). Biochemical characterization of amylase 2A indicates that the enzyme efficiently cleaves
poly-glucose chains so as to release maltose - a glucose disaccharide - from the reducing end of the chain (Braun et al. 1993; Brayer et al.
2000).
References
CR, et al., editors), 1, 2001, 1623-1650.
GD Brayer, G Sidhu, R Maurus, EH Rydberg, C Braun, Y Wang, NT Nguyen, CM Overall, SG Withers, "Subsite mapping of the human
pancreatic alpha-amylase active site through structural, kinetic, and mutagenesis techniques", Biochemistry, 39, 2000, 4778-91.
DL Gumucio, K Wiebauer, RM Caldwell, LC Samuelson, MH Meisler, "Concerted evolution of human amylase genes", Mol Cell Biol, 8, 1988,
1197-205.
N Ramasubbu, V Paloth, Y Luo, GD Brayer, MJ Levine, "Structure of human salivary alpha-amylase at 1.6 A resolution: implications for its role in
the oral cavity.", Acta Crystallogr D Biol Crystallogr, 52, 1996, 435-46.
C Braun, A Meinke, L Ziser, SG Withers, "Simultaneous high-performance liquid chromatographic determination of both the cleavage pattern
and the stereochemical outcome of the hydrolysis reactions catalyzed by various glycosidases", Anal Biochem, 212, 1993, 259-62.
Reaction
18.1.3 Digestion of 1-6 linkages of limit dextrins to yield maltose, maltotriose, longer maltosides, and
glucose
Authors
Reviewers
Nichols, BL, 2007-01-15.
Description
The 1-6 linkages in extracellular limit dextrins are hydrolyzed by sucrase-isomaltase to yield maltose, maltotriose, longer maltosides, and
glucose (Conklin et al. 1975; Nichols et al. 1998; Semenza et al. 2001). In the body, this enzyme is found on the external face of enterocytes in
microvilli of the small intestine (Hauri et al. 1985), and acts on limit dextrins generated by the hydrolysis of amylopectin starch.
References
CR, et al., editors), 1, 2001, 1623-1650.
BL Nichols, J Eldering, S Avery, D Hahn, A Quaroni, "Human small intestinal maltase-glucoamylase cDNA cloning. Homology to
sucrase-isomaltase.", J Biol Chem, 273, 1998, 3076-81.
HP Hauri, EE Sterchi, D Bienz, JA Fransen, A Marxer, "Expression and intracellular transport of microvillus membrane hydrolases in human
intestinal epithelial cells", J Cell Biol, 101, 1985, 838-51.
KA Conklin, KM Yamashiro, GM Gray, "Human intestinal sucrase-isomaltase. Identification of free sucrase and isomaltase and cleavage of the
hybrid into active distinct subunits.", J Biol Chem, 250, 1975, 5735-41.
Reaction
18.1.4 maltotriose + H2O => maltose + D-glucose (maltase-glucoamylase)
Authors
Reviewers
Nichols, BL, 2007-01-15.
Description
Maltotriose is representative of linear glucose oligomers containing more than two residues. The 1-4 linkages of extracellular maltotriose are
hydrolyzed to yield maltose and glucose in a reaction catalyzed by the exoglucosidase activity of maltase-glucoamylase (Nichols et al. 1998;
Semenza et al. 2001). In the body, this enzyme is found as a dimer on the external face of enterocytes in microvilli of the small intestine (Hauri et
al. 1985), and acts on maltotriose derived directly from the diet and from the hydrolysis of starch. This reaction can also be catalyzed by
sucrase-isomaltase, but maltase-glucoamylase is about a hundredfold more active.
References
CR, et al., editors), 1, 2001, 1623-1650.
BL Nichols, S Avery, P Sen, DM Swallow, D Hahn, EE Sterchi, "The maltase-glucoamylase gene: common ancestry to sucrase-isomaltase with
complementary starch digestion activities", Proc Natl Acad Sci U S A, 100, 2003, 1432-7.
Reaction
18.1.5 maltotriose + H2O => maltose + D-glucose (sucrase-isomaltase)
Authors
Reviewers
Nichols, BL, 2007-01-15.
Description
Maltotriose is representative of linear glucose oligomers containing more than two residues. The 1-4 linkages of extracellular maltotriose are
hydrolyzed to yield maltose and glucose in a reaction catalyzed by the exoglucosidase activity of sucrase-isomaltase (Nichols et al. 1998;
al. 1985), and acts on maltotriose derived directly from the diet and from the hydrolysis of starch, although with lower activity than
maltase-glucoamylase.
References
CR, et al., editors), 1, 2001, 1623-1650.
Reaction
18.1.6 maltose + H2O => 2 D-glucose (maltase-glucoamylase)
Authors
Reviewers
Nichols, BL, 2007-01-15.
Description
The alpha-1,4 linkages of extracellular maltose are hydrolyzed to yield glucose in a reaction catalyzed by maltase-glucoamylase (Nichols et al.
1998; Semenza et al. 2001). In the body, this enzyme is found as a dimer on the external face of enterocytes in microvilli of the small intestine
(Hauri et al. 1985), and acts on maltose derived directly from the diet and from the hydrolysis of starch.
References
Reaction
18.1.7 maltose + H2O => 2 D-glucose (sucrase-isomaltase)
Authors
Reviewers
Nichols, BL, 2007-01-15.
Description
The alpha-1,4 linkages of extracellular maltose are hydrolyzed to yield glucose in a reaction catalyzed by sucrase-maltase (Nichols et al. 1998;
al. 1985), and acts on maltose derived directly from the diet and from the hydrolysis of starch.
References
Reaction
18.1.8 lactose + H2O => D-glucose + D-galactose
Authors
Reviewers
Nichols, BL, 2007-01-15.
Description
Extracellular lactose is hydrolyzed to yield molecules of glucose and galactose, in a reaction catalyzed by the lactase activity of lactase-phlorizin
hydrolase associated with the plasma membrane. In the body, lactase-phlorzin hydrolase is found on the external face of enterocytes in microvilli
of the small intestine (Hauri et al. 1985). Expression of the enzyme is developmentally regulated and subject to a genetic polymorphism: enzyme
levels fall after weaning but the extent of the fall varies sharply between human populations (Grand et al. 2003; Swallow 2003). The
lactase-phlorizin hydrolase polypeptide undergoes dimerization and two rounds of proteolytic cleavage in the course of its maturation and
transport to the cell surface (Grunberg and Sterchi 1995; Wuthrich et al. 1996).
References
M Wuthrich, J Grunberg, D Hahn, R Jacob, I Radebach, HY Naim, EE Sterchi, "Proteolytic processing of human lactase-phlorizin hydrolase is a
two-step event: identification of the cleavage sites", Arch Biochem Biophys, 336, 1996, 27-34.
DM Swallow, "Genetics of lactase persistence and lactose intolerance", Annu Rev Genet, 37, 2003, 197-219.
J Grunberg, EE Sterchi, "Human lactase-phlorizin hydrolase: evidence of dimerization in the endoplasmic reticulum", Arch Biochem Biophys,
323, 1995, 367-72.
RJ Grand, RK Montgomery, DK Chitkara, JN Hirschhorn, "Changing genes; losing lactase", Gut, 52, 2003, 617-9.
Reaction
18.1.9 sucrose + H2O => glucose + fructose
Authors
Reviewers
Nichols, BL, 2007-01-15.
Description
Extracellular sucrose is hydrolyzed to yield glucose and fructose in a reaction catalyzed by the sucrase domain of sucrase-isomaltase (Conklin et
al. 1975). In the body, this enzyme is found on the external face of enterocytes in microvilli of the small intestine (Hauri et al. 1985). The
sucrase-isomaltase polypeptide is cleaved into its sucrase and isomaltase domains, which remain associated and, by analogy to the
corresponding pig enzyme, are thought to dimerize (Cowell et al. 1986).
References
GM Cowell, J Tranum-Jensen, H Sjostrom, O Noren, "Topology and quaternary structure of pro-sucrase/isomaltase and final-form
sucrase/isomaltase", Biochem J, 237, 1986, 455-61.
KA Conklin, KM Yamashiro, GM Gray, "Human intestinal sucrase-isomaltase. Identification of free sucrase and isomaltase and cleavage of the
hybrid into active distinct subunits.", J Biol Chem, 250, 1975, 5735-41.
Reaction
18.1.10 trehalose + H2O => 2 D-glucose
Authors
Reviewers
Nichols, BL, 2007-01-15.
Description
Extracellular trehalose, a disaccharide, is cleaved by trehalase associated with the plasma membrane to yield two molecules of glucose.
Trehalase has been purified to homogeneity from rabbit intestine and shown to be a monomer attached to the plasma membrane by a GPI
anchor (Galand 1984; Ruf et al. 1990). A human cDNA encoding a closely homologous protein has been cloned and the protein product of the
clone has been shown to have trehalase activity in vitro (Ishihara et al. 1997). The human enzyme has not been characterized further, and so
both the posttranslational modifications of the human enzyme and its activity in vivo have been inferred from the properties of the well studied
rabbit enzyme.
References
J Ruf, H Wacker, P James, M Maffia, P Seiler, G Galand, A von Kieckebusch, G Semenza, N Mantei, "Rabbit small intestinal trehalase.
Purification, cDNA cloning, expression, and verification of glycosylphosphatidylinositol anchoring.", J Biol Chem, 265, 1990, 15034-9.
R Ishihara, S Taketani, M Sasai-Takedatsu, M Kino, R Tokunaga, Y Kobayashi, "Molecular cloning, sequencing and expression of cDNA
encoding human trehalase", Gene, 202, 1997, 69-74.
G Galand, "Purification and characterization of kidney and intestinal brush-border membrane trehalases from the rabbit", Biochim Biophys Acta,
789, 1984, 10-9.
Source reaction
This reaction was inferred from the corresponding reaction "trehalose + H2O => 2 D-glucose" in species Oryctolagus cuniculus.
J Ruf, H Wacker, P James, M Maffia, P Seiler, G Galand, A von Kieckebusch, G Semenza, N Mantei, "Rabbit small intestinal trehalase.
Purification, cDNA cloning, expression, and verification of glycosylphosphatidylinositol anchoring.", J Biol Chem, 265, 1990, 15034-9.
R Ishihara, S Taketani, M Sasai-Takedatsu, M Kino, R Tokunaga, Y Kobayashi, "Molecular cloning, sequencing and expression of cDNA
encoding human trehalase", Gene, 202, 1997, 69-74.
G Galand, "Purification and characterization of kidney and intestinal brush-border membrane trehalases from the rabbit", Biochim Biophys Acta,
789, 1984, 10-9.
Reaction
18.2 Hexose uptake
Authors
Reviewers
Wright, EM, 2007-01-15.
Description
Hexoses, notably fructose, glucose, and galactose, generated in the lumen of the small intestine by breakdown of dietary carbohydrate are taken
up by enterocytes lining the microvilli of the small intestine and released from them into the blood. Uptake into enterocytes is mediated by two
transporters localized on the lumenal surfaces of the cells, SGLT1 (glucose and galactose, together with sodium ions) and GLUT5 (fructose).
GLUT2, localized on the basolateral surfaces of enterocytes, mediates the release of these hexoses into the blood (Wright et al. 2004). GLUT2
may also play a role in hexose uptake from the gut lumen into enterocytes when the lumenal content of monosaccharides is very high (e.g.,
Kellet and Brot-Laroche, 2005).
References
EM Wright, DD Loo, BA Hirayama, E Turk, "Surprising versatility of Na+-glucose cotransporters: SLC5", Physiology (Bethesda), 19, 2004, 370-6.
GL Kellett, E Brot-Laroche, "Apical GLUT2: a major pathway of intestinal sugar absorption", Diabetes, 54, 2005, 3056-62.
18.2.1 Transport (influx) of fructose by GLUT5
Authors
Reviewers
Wright, EM, 2007-01-15.
Description
The reversible transport of extracellular fructose into the cytosol is mediated by GLUT5. In the small intestine, GLUT5 is localized on the lumenal
surfaces of enterocytes (Davidson et al. 1992) and thus mediates the uptake of dietary fructose, which can be released into the circulation in a
separate transport step mediated by basolaterally localized GLUT2. The specificity of GLUT5 has been worked out by studying sugar transport
in Xenopus oocytes expressing recombinant human GLUT5 protein (Burant et al. 1992).
References
CF Burant, J Takeda, E Brot-Laroche, GI Bell, NO Davidson, "Fructose transporter in human spermatozoa and small intestine is GLUT5", J Biol
Chem, 267, 1992, 14523-6.
NO Davidson, AM Hausman, CA Ifkovits, JB Buse, GW Gould, CF Burant, GI Bell, "Human intestinal glucose transporter expression and
localization of GLUT5", Am J Physiol, 262, 1992, C795-800.
Reaction
18.2.2 Transport (influx) of glucose, galactose, and sodium ions by SGLT1
Authors
Reviewers
Wright, EM, 2007-01-15.
Description
The transport of extracellular glucose and galactose into the cytosol, coupled to the uptake of two sodium ions for each hexose transported is
mediated by SGLT1. In the small intestine, SGLT1 is localized on the lumenal surfaces of enterocytes and thus mediates the uptake of dietary
glucose and galactose, which can be released into the circulation in a separate transport step mediated by basolaterally localized GLUT2
(Wright et al. 2004). The specificity of SGLT1 has been worked out by studying sugar transport in plasma membrane vesicles containing
recombinant human SGLT1 protein (Quick et al. 2003). Consistent with these in vitro results, children lacking functional SGLT1 protein fail to
absord dietary glucose and galactose (Martin et al. 1996).
References
EM Wright, DD Loo, BA Hirayama, E Turk, "Surprising versatility of Na+-glucose cotransporters: SLC5", Physiology (Bethesda), 19, 2004, 370-6.
MG Martin, E Turk, MP Lostao, C Kerner, EM Wright, "Defects in Na+/glucose cotransporter (SGLT1) trafficking and function cause
glucose-galactose malabsorption", Nat Genet, 12, 1996, 216-20.
M Quick, J Tomasevic, EM Wright, "Functional asymmetry of the human Na+/glucose transporter (hSGLT1) in bacterial membrane vesicles",
Biochemistry, 42, 2003, 9147-52.
Reaction
18.2.3 Transport (efflux) of fructose, galactose, and glucose by GLUT2
Authors
Reviewers
Wright, EM, 2007-01-15.
Description
The reversible facilitated diffusion of fructose, galactose, and glucose from the cytosol to the extracellular space is mediated by the GLUT2
transporter in the plasma membrane. In the epithelial cells of the small intestine, the basolateral localization of GLUT2 (Thorens et al. 1990)
enables hexose sugars derived from the diet and taken up by the action of the SGLT1 and GLUT5 transporters to be released into the
circulation. The specificity of the GLUT2 transporter has been established directly through biochemical assays of purified recombinant proteins
(Colville et al. 1993; Wu et al. 1998) and indirectly through studies of patients deficient in GLUT2 transporter protein (Santer et al. 1997).
References
R Santer, R Schneppenheim, A Dombrowski, H Gotze, B Steinmann, J Schaub, "Mutations in GLUT2, the gene for the liver-type glucose
transporter, in patients with Fanconi-Bickel syndrome", Nat Genet, 17, 1997, 324-6.
CA Colville, MJ Seatter, TJ Jess, GW Gould, HM Thomas, "Kinetic analysis of the liver-type (GLUT2) and brain-type (GLUT3) glucose
transporters in Xenopus oocytes: substrate specificities and effects of transport inhibitors", Biochem J, 290, 1993, 701-6.
B Thorens, ZQ Cheng, D Brown, HF Lodish, "Liver glucose transporter: a basolateral protein in hepatocytes and intestine and kidney cells", Am
J Physiol, 259, 1990, C279-85.
L Wu, JD Fritz, AC Powers, "Different functional domains of GLUT2 glucose transporter are required for glucose affinity and substrate
specificity", Endocrinology, 139, 1998, 4205-12.
Reaction
18.3 Glucose metabolism
Authors
Schmidt, EE, 2003-02-05.
Editors
Description
Glucose is the major form in which dietary sugars are made available to cells of the human body. Its breakdown is a major source of energy for
all cells, and is essential for the brain and red blood cells. The first stage of glucose utilization consists of its uptake by cells, and its conversion
to a phosphorylated form, glucose-6-phosphate, that cannot traverse the cell membrane. Glucose-6-phosphate is converted to pyruvate through
the pathway of glycolysis. Little energy is obtained from glycolysis. The complete oxidation of glucose to CO2 and H2O requires two additional
pathways, oxidative decarboxylation of pyruvate to acetyl CoA, and the citric acid cycle, that are common to the metabolism of many small
molecules. In some tissues, notably the liver, glucose can be synthesized from pyruvate, by the pathway of gluconeogenesis. Under anaerobic
conditions, e.g. in exercising muscle, glucose is metabolized only to pyruvate, which is then converted to lactate and exported into the blood.
Liver cells can take up this lactate and re-synthesize glucose. This process for re-utilizing glucose is known as the Cori cycle.
18.3.1 Glucose uptake

Authors
2003-02-15.
Description
Glucose is the major chemical form in which dietary carbohydrate absorbed through the intestinal epithelium is presented to cells. Cells take up
glucose by facilitated diffusion, via glucose transporters (GLUTs) associated with the plasma membrane. Four tissue-specific human GLUT
isoforms are known. Once in the cytoplasm, glucose is phosphorylated, to yield glucose-6-phosphate, which cannot cross the plasma membrane
because of its negative charge. The phosphorylation of glucose consumes ATP and is irreversible. In the liver, this reaction is catalyzed by
glucokinase, which has a low affinity for glucose (Km about 10 mM), but is not inhibited by glucose-6-phosphate. In tissues other than the liver,
this reaction is catalyzed by one of the isoforms of hexokinase. These enzymes are feedback-inhibited by glucose-6-phosphate, and have a high
affinity for glucose (Km about 0.1 mM). Liver cells can thus accumulate large amounts of glucose 6-phosphate, but only when blood glucose
concentrations are high, while most other tissues can take up glucose even when blood glucose concentrations are low, but cannot accumulate
much intracellular glucose 6-phosphate. These differences are consistent with the view that that the liver functions to buffer blood glucose
concentrations, while most other tissues take up glucose to meet immediate metabolic needs.
The absence of glucose-6-phosphatase from most tissues (exceptions are liver and kidney) makes glucose uptake by these tissues essentially
irreversible, again consistent with the view that glucose is taken up for local metabolic use.
References
Q Liu, JC Vera, H Peng, DW Golde, "The predicted ATP-binding domains in the hexose transporter GLUT1 critically affect transporter activity.",
Biochemistry, 40, 2001, 7874-81.
HG Joost, GI Bell, JD Best, MJ Birnbaum, MJ Charron, YT Chen, H Doege, DE James, HF Lodish, KH Moley, JF Moley, M Mueckler, S Rogers,
A SchÃ¼rmann, S Seino, B Thorens, "Nomenclature of the GLUT/SLC2A family of sugar/polyol transport facilitators.", Am J Physiol Endocrinol
Metab, 282, 2002, E974-6.
JE Pessin, GI Bell, "Mammalian facilitative glucose transporter family: structure and molecular regulation.", Annu Rev Physiol, 54, 1992, 911-30.
PW Hruz, MM Mueckler, "Structural analysis of the GLUT1 facilitative glucose transporter (review).", Mol Membr Biol, 18, 2001, 183-93.
MJ Seatter, SA De la Rue, LM Porter, GW Gould, "QLS motif in transmembrane helix VII of the glucose transporter family interacts with the C-1
position of D-glucose and is involved in substrate selection at the exofacial binding site.", Biochemistry, 37, 1998, 1322-6.
NJ Bryant, R Govers, DE James, "Regulated transport of the glucose transporter GLUT4.", Nat Rev Mol Cell Biol, 3, 2002, 267-77.
18.3.1.1 Glucose is carried across the plasma membrane by a glucose transport protein (GLUT)
Editors
Description
Glucose crosses the plasma membrane by facilitated diffusion, mediated by four glucose transporter proteins (GLUTs). These proteins are
members of a larger family of transporter proteins. The three dimensional structures of these proteins have not been determined, but conserved
sequence motifs suggest the existence of shared structural features, that have been confirmed by in situ labeling and mutagenesis studies. Each
GLUT protein has twelve membrane-spanning domains, organized to form an aqueous channel. While monomeric protein can form such a
channel and transport glucose, kinetic studies suggest that the functional form of the protein is a homotetramer. Different GLUT proteins are
expressed in different tissues, and differences in their affinities for glucose are correlated with tissue specific differences in glucose uptake. In
addition, GLUT4, found in muscle and adipose tissue, is sequestered inside cells except in the presence of elevated levels of insulin when it is
rapidly translocated to the cell surface. GLUTs transport molecules in addition to glucose: GLUT1 mediates ascorbate uptake, for example.
References
HG Joost, GI Bell, JD Best, MJ Birnbaum, MJ Charron, YT Chen, H Doege, DE James, HF Lodish, KH Moley, JF Moley, M Mueckler, S Rogers,
A SchÃ¼rmann, S Seino, B Thorens, "Nomenclature of the GLUT/SLC2A family of sugar/polyol transport facilitators.", Am J Physiol Endocrinol
Metab, 282, 2002, E974-6.
JC Vera, CI Rivas, FV VelÃ¡squez, RH Zhang, II Concha, DW Golde, "Resolution of the facilitated transport of dehydroascorbic acid from its
intracellular accumulation as ascorbic acid.", J Biol Chem, 270, 1995, 23706-12.
18.3.1.1.1 alpha-D-glucose (extracellular) <=> alpha-D-glucose (cytosol) [GLUT1]
Description
GLUT1 is expressed by many cell types, notably red blood cells and cells of the brain. Its low Km for glucose (~1 mM) relative to normal blood
glucose concentration (~5 mM) allows these cells to take up glucose independent of changes in blood glucose levels. Its abundance in red blood
cells has allowed it to be purified and biochemically characterized (Hruz and Mueckler 2001).
References
Reaction
Description
GLUT2 is present on liver cells and pancreatic beta cells. Its Km for glucose (~15-20 mM) is substantially higher than normal blood glucose
concentrations (~5 mM), so the rate of glucose uptake into these cells is proportional to blood glucose concentration. GLUT2 also mediates
glucose export from liver cells when gluconeogenesis is underway.
References
R Santer, R Schneppenheim, A Dombrowski, H Gotze, B Steinmann, J Schaub, "Mutations in GLUT2, the gene for the liver-type glucose
transporter, in patients with Fanconi-Bickel syndrome", Nat Genet, 17, 1997, 324-6.
L Wu, JD Fritz, AC Powers, "Different functional domains of GLUT2 glucose transporter are required for glucose affinity and substrate
specificity", Endocrinology, 139, 1998, 4205-12.
Reaction
Description
GLUT3 is expressed by cells of the brain and possibly other cell types with a high constitutive requirement for glucose. Its low Km for glucose
(~1.5 mM) relative to normal blood glucose concentrations (~5 mM) allows these cells to import glucose independent of fluctuations in blood
glucose levels.
References
Reaction
Description
Like GLUT1 and GLUT3, GLUT4 has a high affinity for glucose. GLUT4 is abundant in cells of skeletal muscle and adipose tissue. GLUT4
molecules are translocated from an intracellular store to the cell surface in response to increased insulin levels (Bryant et al. 2002; Fukumoto et
al. 1989).
References
H Fukumoto, T Kayano, JB Buse, YH Edwards, PF Pilch, GI Bell, S Seino, "Cloning and characterization of the major insulin-responsive glucose
transporter expressed in human skeletal muscle and other insulin-responsive tissues.", J Biol Chem, 264, 1989, 7776-9.
Reaction
18.3.1.2 Glucose + ATP => glucose-6-phosphate + ADP
Description
Glucose and ATP react to form glucose-6-phosphate and ADP.
18.3.1.2.1 ATP + alpha-D-Glucose => ADP + alpha-D-glucose 6-phosphate [glucokinase]
Description
The first ATP investment - phosphorylation of glucose by hexokinase. This reaction is essentially irreversible.
References
J Takeda, M Gidh-Jain, LZ Xu, P Froguel, G Velho, M Vaxillaire, D Cohen, F Shimada, H Makino, S Nishi, M Stoffel, N Vionnet, R St. Charles,
RW Harrison, IT Weber, GI Bell, SJ Pilkis, "Structure/function studies of human beta-cell glucokinase. Enzymatic properties of a sequence
polymorphism, mutations associated with diabetes, and other site-directed mutants.", J Biol Chem, 268, 1993, 15200-4.
Reaction
18.3.1.2.2 ATP + alpha-D-Glucose => ADP + alpha-D-glucose 6-phosphate [hexokinase 1]
Description
References
AE Aleshin, C Zeng, GP Bourenkov, HD Bartunik, HJ Fromm, RB Honzatko, "The mechanism of regulation of hexokinase: new insights from the
crystal structure of recombinant human brain hexokinase complexed with glucose and glucose-6-phosphate", Structure, 6, 1998, 39-50.
Reaction
Description
References
M Lehto, X Huang, EM Davis, MM Le Beau, E Laurila, KF Eriksson, GI Bell, LC Groop, "Human hexokinase II gene: exon-intron organization,
mutation screening in NIDDM, and its relationship to muscle hexokinase activity", Diabetologia, 38, 1995, 1466-74.
Reaction
Description
References
G Rijksen, GE Staal, PJ Beks, M Streefkerk, JW Akkerman, "Compartmentation of hexokinase in human blood cells. Characterization of soluble
and particulate enzymes.", Biochim Biophys Acta, 719, 1982, 431-7.
Reaction
18.3.1.3 Negative Regulation of Glucokinase by Glucokinase Regulatory Protein
Editors
Description
The activity of glucokinase (GK) is negatively regulated by glucokinase regulatory protein (GKRP), which reversibly binds the enzyme to form an
inactive complex. Binding is stimulated by fructose 6-phosphate and sorbitol 6-phosphate (hence high concentrations of these molecules tend to
reduce GK activity) and inhibited by fructose 1-phosphate (hence a high concentration of this molecule tends to increase GK activity). Once
formed, the complex is translocated to the nucleus. In the presence of high glucose concentrations, the nuclear GK:GKRP complex dissociates,
freeing GK to return to the cytosol. The free GKRP is thought also to return to the cytosol under these conditions, but this return has not been
confirmed experimentally. Possible physiological role for this sequestration process are to decrease futile cycling between glucose and glucose
6-phosphate in hepatocytes under low-glucose conditions, but also to decrease the lag time between a rise in intracellular glucose levels and the
onset of glucose phosphorylation in both hepatocytes and pancreatic beta cells (Brocklehurst et al. 2004; Shiota et al. 1999).
This process has been studied in both rat and human cells, and with purified proteins from both species. Its qualitative features are well
conserved, but there is an intriguing quantitative difference between humans and rats: while fructose 6-phosphate and sorbitol 6-phosphate
stimulate GKRP activity in both species, the purified human protein, but not the rat protein, has a substantial basal binding activity even in the
absence of either molecule. Confirmation of this result in vivo would suggest a major quantitative difference in the response of glucose
metabolism to levels of other sugar phosphates in humans versus rodents (Brocklehurst et al. 2004).
References
C Shiota, J Coffey, J Grimsby, JF Grippo, MA Magnuson, "Nuclear import of hepatic glucokinase depends upon glucokinase regulatory protein,
whereas export is due to a nuclear export signal sequence in glucokinase", J Biol Chem, 274, 1999, 37125-30.
KJ Brocklehurst, RA Davies, L Agius, "Differences in regulatory properties between human and rat glucokinase regulatory protein", Biochem J,
378, 2004, 693-7.
18.3.1.3.1 glucokinase (GK) + glucokinase regulatory protein (GKRP) <=> GK:GKRP complex
Editors
Description
Glucokinase (GK) reversibly binds glucokinase regulatory protein (GKRP) to form an inactive complex. Binding is stimulated by fructose
6-phosphate and sorbitol 6-phosphate (hence high concentrations of these molecules tend to reduce GK activity) and inhibited by fructose
1-phosphate (hence a high concentration of this molecule tends to increase GK activity) (Brocklehurst et al. 2004).
References
378, 2004, 693-7.
Reaction
18.3.1.3.2 cytosolic GK:GKRP complex <=> glucokinase (GK) + glucokinase regulatory protein (GKRP)
Editors
Description
Glucokinase (GK) binds glucokinase regulatory protein (GKRP) to form an inactive complex. Complex formation is reversible in vitro
(Brocklehurst et al. 2004), and so is annotated here. The extent to which cytosolic complexes dissociate under physiological conitions is unclear,
however, and their major fate appears to be transport to the nucleus (Shiota et al. 1999).
References
378, 2004, 693-7.
Reaction
18.3.1.3.3 GK:GKRP [cytosol] => GK:GKRP [nucleoplasm]
Editors
Description
The complex of glucokinase (GK) and glucokinase regulatory protein (GKRP) is translocated to the nucleus via the nuclear pore (Shiota et al.
1999).
References
Reaction
18.3.1.3.4 nucleoplasmic GK:GKRP complex => glucokinase (GK) + glucokinase regulatory protein (GKRP)
Editors
Description
In the presence of high glucose concentrations, the complex of nucleoplasmic glucokinase (GK) and glucokinase regulatory protein (GKRP)
dissociates (Shiota et al. 1999).
References
Reaction
18.3.1.3.5 glucokinase [nucleoplasm] => glucokinase [cytosol]
Editors
Description
Free glucokinase (GK) leaves the nucleus via the nuclear pore. While the GK protein contains a nuclear export sequence motif, the molecular
details of the GK export process remain to be worked out (Shiota et al. 1999).
References
Reaction
18.3.2 Glycolysis
Authors
Schmidt, EE, 2003-01-28.
Description
Glycolysis occurs in the cytosol, yielding 2 ATP, 2 pyruvate, and 2 NADH + 2 H+ from each glucose molecule. The first two steps of glycolysis
convert glucose-6-phosphate into a form that is easily cleaved into phosphorylated three-carbon units. High energy phosphate (as ATP) is
generated from the three-carbon units in the remaining steps.
18.3.2.1 Glucose 6-phosphate is isomerized to form fructose-6-phosphate
Authors
Schmidt, EE, 2003-01-28.
Editors
Description
Glucose-6-phosphate and fructose-6-phosphate are freely interconverted by phosphoglucose isomerase.
A note about stereochemistry -- In the KEGG database of metabolic pathways, the alpha anomer of D-glucose 6-phosphate is said to be
converted to the beta anomer of D-fructose 6-phosphate. While the involvement of the alpha anomer of D-glucose in this reaction is
well-established, the stereochemistry of the fructose moiety is less clear. Noltmann, reviewing available data concerning reaction mechanisms in
1972, argued that the reaction likely involved conversion of alpha-D-glucose 6-phosphate to alpha-D-fructose 6-phosphate, but conceded that
relevant experimental data were limited. In an equally authoritative review the next year, Bloxham and Lardy reached the opposite conclusion,
based on unpublished substrate specificity data. The anomeric conformation of fructose phosphate in this reaction is therefore left unspecified in
GK.
References
DP Bloxham, HA Lardy, "Phosphofructokinase", The Enzymes, 3rd ed (Boyer PD, editor), 8, 1973, 239-278.
EA Noltmann, "Aldose-ketose isomerases", The Enzymes, 3rd ed (Boyer PD, editor), 6, 1972, 271-354.
18.3.2.1.1 alpha-D-glucose 6-phosphate <=> D-fructose 6-phosphate
Description
The second reaction - readily reversible isomerisation of glucose-6-phosphate by phosphoglucoisomerase.
References
KK Tsuboi, J Estrada, PB Hudson, "Enzymes of the human erythrocyte. IV. Phosphoglucose isomerase, purification and properties.", J Biol
Chem, 231, 1958, 19-29.
W Xu, E Beutler, "The characterization of gene mutations for human glucose phosphate isomerase deficiency associated with chronic hemolytic
anemia", J Clin Invest, 94, 1994, 2326-9.
Reaction
18.3.2.2 Fructose 6-phosphate and ATP react to form fructose 2,6-bisphosphate and ADP
Authors
2003-04-29.
Editors
Schmidt, EE, D'Eustachio, P, 0000-00-00.
Description
The formation of fructose 2,6-bisphosphate from fructose 6-phosphate and ATP is the last step in the regulatory pathway initiated when the
hormone insulin binds to its receptor on the cell surface. Fructose 2,6-bisphosphate is not itself on the pathway of glycolysis. Rather, it acts as a
postive allosteric effector of phosphofructokinase 1, greatly increasing the rate of synthesis of fructose 1,6-bisphosphate and hence the overall
rate of glycolysis. A separate regulatory pathway, initiated by glucagon or epinephrine binding, has the opposite effect on fructose
2,6-bisphosphate production, and thus inhibits glycolysis.
References
SJ Pilkis, TH Claus, IJ Kurland, AJ Lange, "6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase: a metabolic signaling enzyme.", Annu Rev
Biochem, 64, 1995, 799-835.
18.3.2.2.1 ATP + D-fructose 6-phosphate => ADP + D-fructose 2,6-bisphosphate [brain + placenta]
Description
This reaction takes place in the 'cytoplasm' and is mediated by the '6-phosphofructokinase activity' of
'6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 homodimer'.
References
NP Manes, MR el-Maghrabi, "The kinase activity of human brain 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is regulated via
inhibition by phosphoenolpyruvate", Arch Biochem Biophys, 438, 2005, 125-36.
Reaction
18.3.2.2.2 ATP + D-fructose 6-phosphate => ADP + D-fructose 2,6-bisphosphate [heart]
Description
This reaction takes place in the 'cytoplasm' and is mediated by the '6-phosphofructo-2-kinase activity' of
References
T Hirata, M Kato, N Okamura, M Fukasawa, R Sakakibara, "Expression of human placental-type 6-phosphofructo-2-kinase/fructose

2,6-bisphosphatase in various cells and cell lines", Biochem Biophys Res Commun, 242, 1998, 680-4.
Reaction
18.3.2.2.3 ATP + D-fructose 6-phosphate => ADP + D-fructose 2,6-bisphosphate [liver + muscle]
Description
References
Reaction
18.3.2.2.4 ATP + D-fructose 6-phosphate => ADP + D-fructose 2,6-bisphosphate [testis]
Description
References
A Manzano, JX Perez, M Nadal, X Estivill, A Lange, R Bartrons, "Cloning, expression and chromosomal localization of a human testis
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene", Gene, 229, 1999, 83-9.
Reaction
18.3.2.3 ATP + D-fructose 6-phosphate => ADP + D-fructose 1,6-bisphosphate
Description
The cytosolic reaction of one molecule each of fructose-6-phosphate and ATP to form one molecule each of fructose-1,6-bisphosphate and ADP
is catalyzed by phosphofructokinase 1. This reaction, irreversible under physiological conditions, is the rate-limiting step of glycolysis.
Phosphofructokinase 1 activity is allosterically regulated by ATP, citrate, and fructose-2,6-bisphosphate.
Phosphofructokinase 1 is active as a tetramer (although higher-order multimers, not annotated here, may form in vivo). Two isoforms of
phosphofructokinase 1 monomer, L and M, are widely expressed in human tissues. Different tissues can contain different tetramers or tetramer
combinations: L4 in liver, M4 in muscle, and all possible tetramers - L4, L3M, L2M2, LM3, and M4 - in red blood cells, for example (Raban et al.
1995; Vora et al. 1980, 1987; Vora 1981). A third isoform, P, is abundant in platelets, where it is found in P4, P3L, P2L2, and PL3 tetramers (Eto
et al. 1994; Vora et al. 1987).
References
S Vora, C Seaman, S Durham, S Piomelli, "Isozymes of human phosphofructokinase: identification and subunit structural characterization of a
new system", Proc Natl Acad Sci U S A, 77, 1980, 62-6.
S Vora, "Isozymes of human phosphofructokinase in blood cells and cultured cell lines: molecular and genetic evidence for a trigenic system",
Blood, 57, 1981, 724-32.
S Vora, S DiMauro, D Spear, D Harker, MJ Danon, "Characterization of the enzymatic defect in late-onset muscle phosphofructokinase
deficiency. New subtype of glycogen storage disease type VII.", J Clin Invest, 80, 1987, 1479-85.
N Raben, R Exelbert, R Spiegel, JB Sherman, H Nakajima, P Plotz, J Heinisch, "Functional expression of human mutant phosphofructokinase in
yeast: genetic defects in French Canadian and Swiss patients with phosphofructokinase deficiency", Am J Hum Genet, 56, 1995, 131-41.
K Eto, H Sakura, K Yasuda, T Hayakawa, E Kawasaki, R Moriuchi, S Nagataki, Y Yazaki, T Kadowaki, "Cloning of a complete protein-coding
sequence of human platelet-type phosphofructokinase isozyme from pancreatic islet.", Biochem Biophys Res Commun, 198, 1994, 990-8.
Reaction
18.3.2.4 D-fructose 1,6-bisphosphate <=> dihydroxyacetone phosphate + D-glyceraldehyde 3-phosphate

Description
In this freely reversible cytosolic reaction, one molecule of D-fructose 1,6-bisphosphate is cleaved to yield single molecules of dihydroxyacetone
phosphate and D-glyceraldehyde 3-phosphate. The active form of aldolase, the enzyme that catalyzes the reaction, is a homotetramer. Three
aldolase isozymes have been identified which differ in their patterns of expression in various adult tissues and during development but are
otherwise functionally indistinguishable (Ali and Cox 1995; Freemont et al. 1988).
References
M Ali, TM Cox, "Diverse mutations in the aldolase B gene that underlie the prevalence of hereditary fructose intolerance", Am J Hum Genet, 56,
1995, 1002-5.
B Dunbar, LA Fothergill-Gilmore, "The complete amino acid sequence of human skeletal-muscle fructose-bisphosphate aldolase", Biochem J,
249, 1988, 779-88.
Reaction
18.3.2.5 Dihydroxyacetone phosphate is isomerized to form glyceraldehyde-3-phosphate
Description
Dihydroxyacetone phosphate, unlike glyceraldehyde-3-phosphate, is not on the direct pathway of glycolysis. Triose phosphate isomerase
catalyzes the freely reversible interconversion of these two molecules.
18.3.2.5.1 dihydroxyacetone phosphate <=> D-glyceraldehyde 3-phosphate
Description
The fifth reaction - isomerisation of the triose phosphates.

References
HS Lu, PM Yuan, RW Gracy, "Primary structure of human triosephosphate isomerase", J Biol Chem, 259, 1984, 11958-68.
Reaction
18.3.2.6 Glyceraldehyde 3-phosphate, NAD+, and orthophosphate react to form 1,3-bisphosphoglycerate, NADH, and H+
Description
Glyceraldehyde-3-phosphate is reversibly oxidized to 1,3-bisphosphoglycerate by glyceraldehyde-3-phosphate dehydrogenase. The energy

released in the oxidation reaction is captured in the form of NADH + H+.
References
JF Taylor, SF Velick, GT Cori, CF Cori, MW Slein, "The prosthetic group of crystalline D-glyceraldehyde-3-phosphate dehydrogenase", J Biol
Chem, 173, 1948, 619-626.
GT Cori, MW Slein, CF Cori, "Crystalline D-glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle", J Biol Chem, 173, 1948, 605-618.
18.3.2.6.1 D-glyceraldehyde 3-phosphate + orthophosphate + NAD+ <=> 1,3-bisphospho-D-glycerate + NADH + H+
Description
Cytosolic glyceraldehyde-3-phosphate dehydrogenase catalyzes the reaction of glyceraldehyde 3-phosphate, orthophosphate, and NAD+ to
form NADH + H+ and 1,3-bisphosphoglycerate, the first energy-rich intermediate of glycoslysis.
While there are multiple human glyceraldehyde 3-phosphate dehydrogenase-like pseudogenes, there is only one glyceraldehyde 3-phosphate
dehydrogenase gene expressed in somatic tissue (Benham and Povey 1989). Consistent with this conclusion, the homogeneous enzymes
purified from various human tissues had indistinguishable physical and immunochemical properties (Heinz and Freimuller 1982), and studies of
human erythrocytes of various ages suggested that variant forms of the enzyme arise as a result of post-translational modifications (Edwards et
al. 1976). There is, however, an authentic second isoform of glyceraldehyde 3-phosphate dehydrogenase whose expression is confined to
spermatogenic cells of the testis (Welch et al. 2000).
References
JE Welch, PL Brown, DA O'Brien, PL Magyar, DO Bunch, C Mori, EM Eddy, "Human glyceraldehyde 3-phosphate dehydrogenase-2 gene is
expressed specifically in spermatogenic cells.", J Androl, 21, 2000, 328-38.
FJ Benham, S Povey, "Members of the human glyceraldehyde-3-phosphate dehydrogenase-related gene family map to dispersed chromosomal
locations.", Genomics, 5, 1989, 209-14.
L Ercolani, B Florence, M Denaro, M Alexander, "Isolation and complete sequence of a functional human glyceraldehyde-3-phosphate
dehydrogenase gene", J Biol Chem, 263, 1988, 15335-41.
YH Edwards, P Clark, H Harris, "Isozymes of glyceraldehyde-3-phosphate dehydrogenase in man and other mammals.", Ann Hum Genet, 40,
1976, 67-77.
F Heinz, B FreimÃ¼ller, "Glyceraldehyde-3-phosphate dehydrogenase from human tissues.", Methods Enzymol, 89, 1983, 301-5.
Reaction
18.3.2.7 1,3-bisphosphoglycerate and ADP react to form 3-phosphoglycerate and ATP
Description
The high-energy phosphate bond from 1,3-bisphosphoglycerate is transferred to form ATP, in a reversible reaction catalyzed by
phosphoglycerate kinase. This reaction is an instance of a substrate-level phosphorylation.
18.3.2.7.1 ADP + 3-Phospho-D-glyceroyl phosphate <=> ATP + 3-Phospho-D-glycerate
Description
Cytosolic phosphoglycerate kinase catalyzes the reaction of ADP and 1,3-bisphosphoglycerate to form D-glyceraldehyde 3-phosphate and ATP.
This is the first substrate-level phosphorylation reaction in glycolysis.
References
IY Huang, CD Welch, A Yoshida, "Complete amino acid sequence of human phosphoglycerate kinase. Cyanogen bromide peptides and
complete amino acid sequence.", J Biol Chem, 255, 1980, 6412-20.
IY Huang, E Rubinfien, A Yoshida, "Complete amino acid sequence of human phosphoglycerate kinase. Isolation and amino acid sequence of
tryptic peptides.", J Biol Chem, 255, 1980, 6408-11.
Reaction
18.3.2.8 3-Phospho-D-glycerate <=> 2-Phospho-D-glycerate
Description
The reversible isomerisation of 3- and 2-phosphoglycerate is catalyzed by cytosolic phosphoglycerate mutase. This is the eighth reaction of
glycolysis. The active form of the enzyme is a dimer. There are two isoforms of this enzyme, PGAM1 (isoform B) and PGAM2 (isoform M). In the
body, erythrocytes express only PGAM1, while skeletal muscle expresses only PGAM2. Other tissues express both isoforms.
References
S Tsujino, S Shanske, S Sakoda, G Fenichel, S DiMauro, "The molecular genetic basis of muscle phosphoglycerate mutase (PGAM)
deficiency", Am J Hum Genet, 52, 1993, 472-477.
Y Blouquit, MC Calvin, R Rosa, D Prome, JC Prome, F Pratbernou, M Cohen-Solal, J Rosa, "Sequence of the human erythrocyte
phosphoglycerate mutase by microsequencer and mass spectrometry", J Biol Chem, 263, 1988, 16906-10.
A Repiso, MJR Baso, J-LV Corrons, J Carreras, F Climent, "Phosphoglycerate mutase BB isoenzyme deficiency in a patient with
non-spherocytic anemia: familial and metabolic studies", Haematologica, 90, 2005, 257-259.
Reaction
18.3.2.9 2-Phospho-D-glycerate <=> Phosphoenolpyruvate + H2O
Authors
Editors
Description
In this freely reversible cytosolic reaction, one molecule of 2-phosphoglycerate reacts to form one molecule each of phosphoenolpyruvate and
water, elevating the transfer potential of the phosphoryl group.
The enzyme that catalyzes the reaction is a homodimer. Three isozymes have been purified and biochemically characterized. The alpha isoform
is expressed in many normal human tissues (Giallongo et al. 1986). The beta isoform is expressed in muscle. Evidence for its function in vivo in
humans comes from analysis of a patient in which a point mutation in the gene encoding the enzyme was associated specifically with reduced
enolase activity in muscle extracts, and with other symptoms consistent with a defect in glycolysis (Comi et al. 2001). The gamma isoform of
human enolase is normally expressed in neural tissue. It is not known to have distinctive biochemical functions, but is of possible clinical interest
as a marker of some types of neuroendocrine and lung tumors (McAleese et al. 1988). Verma and Kurl (1993) identifed a possible fourth
isoform, a "lung-specific" enolase whose expression is increased in response to dexamethasone treatment. The protein has not been
biochemically characterized, however, nor have the levels of mRNA and protein in other tissues been examined. Thus, the observation that this
protein is particularly similar in its predicted amino acid sequence to a duck crystallin (Wistow et al. 1988) raises the possibility that its normal
function is unrelated to glycolysis.
References
GP Comi, F Fortunato, S Lucchiari, A Bordoni, A Prelle, S Jann, A Keller, P Ciscato, S Galbiati, L Chiveri, Y Torrente, G Scarlato, N Bresolin,
"Beta-enolase deficiency, a new metabolic myopathy of distal glycolysis", Ann Neurol, 50, 2001, 202-7.
M Verma, RN Kurl, "Human lung enolase: cloning and sequencing of cDNA and its inducibility with dexamethasone", Biochem Mol Biol Int, 30,
1993, 293-303.
GJ Wistow, T Lietman, LA Williams, SO Stapel, WW de Jong, J Horwitz, J Piatigorsky, "Tau-crystallin/alpha-enolase: one gene encodes both an
enzyme and a lens structural protein", J Cell Biol, 107, 1988, 2729-36.
SM McAleese, B Dunbar, JE Fothergill, LJ Hinks, IN Day, "Complete amino acid sequence of the neurone-specific gamma isozyme of enolase
(NSE) from human brain and comparison with the non-neuronal alpha form (NNE)", Eur J Biochem, 178, 1988, 413-7.
A Giallongo, S Feo, R Moore, CM Croce, LC Showe, "Molecular cloning and nucleotide sequence of a full-length cDNA for human alpha
enolase", Proc Natl Acad Sci U S A, 83, 1986, 6741-5.
Reaction
18.3.2.10 Phosphoenolpyruvate and ADP react to form pyruvate and ATP
Description
The transfer of a high-energy phosphate bond from phosphoenolpyruvate, to form ATP, is catalyzed by pyruvate kinase. This reaction, an
instance of substrate-level phosphorylation, is essentially irreversible. Pyruvate kinase activity is regulated both allosterically and by
glucagon-mediated phosphorylation.
18.3.2.10.1 ADP + Phosphoenolpyruvate => ATP + Pyruvate (pyruvate kinase M2)
Authors
Description
The muscle (M) isoform of pyruvate kinase is expressed both in muscle and in a variety of other tissues in the normal human adult (Takenaka et
al. 1991).
References
M Takenaka, T Noguchi, S Sadahiro, H Hirai, K Yamada, T Matsuda, E Imai, T Tanaka, "Isolation and characterization of the human pyruvate
kinase M gene", Eur J Biochem, 198, 1991, 101-6.
Reaction
18.3.2.10.2 ADP + Phosphoenolpyruvate => ATP + Pyruvate (pyruvate kinase R/L)
Description
The R/L isoform of pyruvate kinase is the major form of the enzyme in human red blood cells (R) and liver (L); deficiencies in the enzyme are
associated with reduced pyruvate kinase activity in red blood cells and with hemolytic anemia (Tani et al. 1988; Miwa et al. 1993).
References
K Tani, H Fujii, S Nagata, S Miwa, "Human liver type pyruvate kinase: complete amino acid sequence and the expression in mammalian cells",
S Miwa, H Kanno, H Fujii, "Concise review: pyruvate kinase deficiency: historical perspective and recent progress of molecular genetics", Am J
Hematol, 42, 1993, 31-5.
Reaction
18.3.3 Gluconeogenesis
Editors
Description
In this series of reactions, two molecules of pyruvate are used to synthesize one molecule of glucose, at a cost of six high-energy bonds. The
glucose produced by these reactions can be exported into the blood from the cells that synthesize it (e.g., from the liver under fasting conditions)
to meet the glucose requirements of the brain and of red blood cells.
18.3.3.1 Pyruvate, CO2, and ATP react to form oxaloacetate, ADP, and orthophosphate
Description
The carboxylation of pyruvate to form oxaloacetate, catalyzed by pyruvate carboxylase, is an irreversible and allosterically regulated reaction
(Jitrapakdee and Wallace 1999). This is the first of four reactions that differ between gluconeogenesis and glycolysis. The reaction takes place in
the mitochondrion, while the rest of the reactions of gluconeogenesis occur in the cytoplasm. Mitochondrial oxaloacetate is reduced to form
malate, which is transferred to the cytosol and reoxidized to oxaloacetate by malate dehydrogenase.
References
MA Carbone, BH Robinson, "Expression and characterization of a human pyruvate carboxylase variant by retroviral gene transfer", Biochem J,
370, 2003, 275-282.
S Jitrapakdee, JC Wallace, "Structure, function and regulation of pyruvate carboxylase", Biochem J, 340, 1999, 1-16.
Reaction
18.3.3.2 Oxaloacetate + NADH + H+ <=> (S)-Malate + NAD+

Description
Mitochondrial malate dehydrogenase catalyzes the reaction of malate and NAD+ to form oxaloacetate and NADH + H+. The dimeric structure of
the human dehydrogenase is inferred from that established for its well-studied pig homolog (Sanchez et al. 1998).
References
SA Sanchez, TL Hazlett, JE Brunet, DM Jameson, "Aggregation states of mitochondrial malate dehydrogenase", Protein Sci, 7, 1998,
2184-2189.
I Morgunov, PA Srere, "Interaction between citrate synthase and malate dehydrogenase. Substrate channeling of oxaloacetate", J Biol Chem,
273, 1998, 29540-29544.
Reaction
18.3.3.3 Exchange of malate and alpha-ketoglutarate (2-oxoglutarate) across the inner mitochondrial membrane
Authors
Description
The SLC25A11 transport protein in the inner mitochondrial membrane mediates the export of mitochondrial malate into the cytosol, coupled to
the import of cyrosolic alpha-ketoglutarate (2-oxoglutarate) into the mitochondrial matrix. Recent studies of SLC25A11, in addition to providing
direct experimental evidence for the malate:alpha-ketoglutarate transport function of the human protein, suggest that it may also be responsible
for the transport of porphyrin intermediates from the cytosol to the mitochondrial matrix in the course of heme biosynthesis (Kabe et al. 2006)
References
Y Kabe, M Ohmori, K Sinouchi, Y Tsuboi, S Hirao, M Azuma, H Watanabe, I Okura, H Handa, "Porphyrin accumulation in mitochondria is
mediated by 2-oxoglutarate carrier", J Biol Chem, 281, 2006, 31729â€“31735.
Reaction
18.3.3.4 malate + NAD+ <=> oxaloacetate + NADH + H+
Authors
Description
Cytosolic malate dehydrogenase catalyzes the reaction of malate and NAD+ to form oxaloacetate and NADH + H+. The dimeric structure of the
human dehydrogenase is inferred from that established for its well-studied pig homolog (Birktoft et al. 1989).
References
AS-Y Lo, C-T Liew, S-M Ngai, SK-W Tsui, K-P Fung, C-Y Lee, MM-Y Waye, "Developmental regulation and cellular distribution of human
cytosolic malate dehydrogenase (MDH1)", J Cell Biochem, 94, 2005, 763-773.
JJ Birktoft, G Rhodes, LJ Banaszak, "Refined crystal structure of cytoplasmic malate dehydrogenase at 2.5-A resolution", Biochemistry, 28,
1989, 6065-6081.
Reaction
18.3.3.5 Oxaloacetate and GTP react to form phosphoenolpyruvate, CO2, and GDP
Description
The transfer of a high-energy phosphate bond from GTP to form phosphoenolpyruvate is catalyzed by cytosolic phosphoenolpyruvate
carboxykinase. This is the second of four reactions that differ between gluconeogenesis and glycolysis.
References
P Dunten, C Belunis, R Crowther, K Hollfelder, U Kammlott, W Levin, H Michel, GB Ramsey, A Swain, D Weber, SJ Wertheimer, "Crystal
structure of human cytosolic phosphoenolpyruvate carboxykinase reveals a new GTP-binding site", J Mol Biol, 316, 2002, 257-264.
Reaction
18.3.3.6 Phosphoenolpyruvate + H2O <=> 2-Phospho-D-glycerate
Description
In this freely reversible cytosolic reaction, one molecule each of phosphoenolpyruvate and water react to form one molecule of
2-phosphoglycerate.
References
1993, 293-303.
Reaction
18.3.3.7 2-Phospho-D-glycerate <=> 3-Phospho-D-glycerate
Description
The reversible isomerisation of 2- and 3-phosphoglycerate is catalyzed by cytosolic phosphoglycerate mutase. The active form of the enzyme is
a dimer. There are two isoforms of this enzyme, PGAM1 (isoform B) and PGAM2 (isoform M). In the body, erythrocytes express only PGAM1,
while skeletal muscle expresses only PGAM2. Other tissues express both isoforms.
References
Reaction
18.3.3.8 ATP + 3-Phospho-D-glycerate <=> ADP + 1,3-bisphospho-D-glycerate
Description
The phosphorylation of 3-phosphoglycerate to form 1,3-bisphosphoglycerate, is catalyzed by cytosolic phosphoglycerate kinase.
References
Reaction
18.3.3.9 1,3-bisphospho-D-glycerate + NADH + H+ <=> D-glyceraldehyde 3-phosphate + Orthophosphate + NAD+
Description
The reduction of 1,3-bisphosphoglycerate to form glyceraldehyde-3-phosphate is catalyzed by cytosolic glyceraldehyde-3-phosphate

dehydrogenase.
References
1976, 67-77.
Reaction
18.3.3.10 D-glyceraldehyde 3-phosphate <=> dihydroxyacetone phosphate
Description
The conversion of glyceraldehyde-3-phosphate to dihydroxyacetone phosphate, catalyzed by triose phosphate isomerase, is simply the reverse
of a reaction of glycolysis.
References
M Watanabe, BC Zingg, HW Mohrenweiser, "Molecular analysis of a series of alleles in humans with reduced activity at the triosephosphate
isomerase locus", Am J Hum Genet, 58, 1996, 308-16.
Reaction
18.3.3.11 dihydroxyacetone phosphate + D-glyceraldehyde 3-phosphate <=> D-fructose 1,6-bisphosphate
Description
In this freely reversible cytosolic reaction, single molecules of dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate react to form one
molecule of D-fructose 1,6-bisphosphate. The active form of aldolase, the enzyme that catalyzes the reaction, is a homotetramer. Three aldolase
isozymes have been identified which differ in their patterns of expression in various adult tissues and during development but are otherwise
functionally indistinguishable (Ali and Cox 1995; Freemont et al. 1988).
References
1995, 1002-5.
249, 1988, 779-88.
Reaction
18.3.3.12 Fructose 1,6-bisphosphate is hydrolyzed to form fructose 6-phosphate and orthophosphate
Description
The hydrolysis of fructose-1,6,-bisphosphate to form fructose-6-phosphate, is catalyzed by fructose-1,6-bisphosphatase. This reaction is

essentially irreversible, and is the third of four reactions that differ between gluconeogenesis and glycolysis.
18.3.3.12.1 D-fructose 1,6-bisphosphate + H2O => D-fructose 6-phosphate + orthophosphate
Description
Cytosolic fructose-1,6-bisphosphatase catalyzes the hydrolysis of fructose-1,6-bisphosphate to form fuctose-6-phosphate and orthophosphate.
References
Y Kikawa, M Inuzuka, BY Jin, S Kaji, J-i Koga, Y Yamamoto, K Fujisawa, I Hata, A Nakai, Y Shigematsu, H Mizunuma, A Taketo, M Mayumi, M
Sudo, "Identification of genetic mutations in Japanese patients with fructose-1,6-bisphosphatase deficiency", Am J Hum Genet, 61, 1997,
852-861.
Reaction
18.3.3.12.2 D-fructose 1,6-bisphosphate + H2O => D-fructose 6-phosphate + orthophosphate
Description
In the body, this isoform of fructose-1,6-bisphosphatase is muscle-specific (Tillma and Eschrich 1998).
References
H Tillmann, K Eschrich, "Isolation and characterization of an allelic cDNA for human muscle fructose-1,6-bisphosphatase", Gene, 212, 1998,
295-304.
Reaction
18.3.3.13 D-fructose 6-phosphate <=> alpha-D-Glucose 6-phosphate
Description
The isomerization of fructose-6-phosphate to form glucose-6-phosphate, catalyzed by phosphoglucose isomerase, is simply the reverse of a
reaction of glycolysis.
References
Chem, 231, 1958, 19-29.
Reaction
18.3.3.14 alpha-D-glucose 6-phosphate [cytosol] => alpha-D-glucose 6-phosphate [endoplasmic reticulum lumen]
Authors
Description
The SLC37A4 transport protein in the endoplasmic reticulum membrane mediates the translocation of glucose-6-phosphate from the cytosol into
the lumen of the endoplasmic reticulum. Defects in this transporter are associated with glycogen storage disease type ib (Gerin et al. 1997).
References
I Gerin, M Veiga-da-Cunha, Y Achouri, J-F Collet, E Van Schaftingen, "Sequence of a putative glucose 6-phosphate translocase, mutated in
glycogen storage disease type Ib", FEBS Lett, 419, 1997, 235-238.
Reaction
18.3.3.15 alpha-D-Glucose 6-phosphate + H2O => alpha-D-Glucose + Orthophosphate
Description
Glucose-6-phosphatase associated with the inner face of the endoplasmic reticulum membrane catalyzes the hydrolysis of glucose-6-phosphate
to glucose and orthophosphate (reviewed by Van Scaftingen and Gerin 2002). This reaction is essentially irreversible. It is the last of the four
reactions that differ between gluconeogenesis and glycolysis. Defects in glucose-6-phosphatase are the cause of glycogen storage disease type
1a (Lei et al. 1995).
References
K-J Lei, Y-T Chen, H Chen, L-JC Wong, J-L Liu, A McConkie-Rosell, JLK Van Hove, HC-Y Ou, NJ Yeh, LY Pan, JY Chou, "Genetic basis of
glycogen storage disease type 1a: Prevalent mutations at the glucose-6-phosphatase locus", Am J Hum Genet, 57, 1995, 766-771.
E Van Schaftingen, I Gerin, "The glucose-6-phosphatase system", Biochem J, 362, 2002, 513-532.
Reaction
18.3.3.16 Efflux of glucose from the endoplasmic reticulum
Authors
Description
Glucose generated within the endoplasmic reticulum is exported from the cell. Several mechansims for this transport process have been
proposed but experimental data remain incomplete and contradictory (e.g., Hosokawa and Thorens 2002; Fehr et al. 2005; Van Schaftingen and
Gerin 2002).
References
M Hosokawa, B Thorens, "Glucose release from GLUT2-null hepatocytes: characterization of a major and a minor pathway", Am J Physiol
Endocrinol Metab, 282, 2002, E794-E801.
M Fehr, H Takanaga, DW Ehrhardt, WB Frommer, "Evidence for high-capacity bidirectional glucose transport across the endoplasmic reticulum
membrane by genetically encoded fluoresence resonance energy transfer nanosensors", Mol Cell Biol, 25, 2005, 11102-11112.
Reaction
18.3.3.17 Fructose 2,6-bisphosphate is hydrolyzed to form fructose-6-phosphate and orthophosphate
Description
At the beginning of this reaction, 1 molecule of 'D-Fructose 2,6-bisphosphate', and 1 molecule of 'H2O' are present. At the end of this reaction, 1
This reaction takes place in the 'cytoplasm' and is mediated by the 'fructose-bisphosphatase activity' of 'unknown fructose-bisphosphatase'.
References
Biochem, 64, 1995, 799-835.
Reaction
18.3.4 Cori Cycle (interconversion of glucose and lactate)
Description
Under conditions in which oxygen supply is limiting, e.g., in exercising muscle, or in the absence of mitochondria, e.g., in red blood cells,
re-oxidation of NADH produced by glycolysis cannot be coupled to generation of ATP. Instead, its re-oxidation is coupled to the reduction of
pyruvate to lactate. This lactate is released into the blood, and is taken up primarily by the liver, where it is oxidized to pyruvate and can be used
for gluconeogenesis.
18.3.4.1 Glycolysis
Authors
Schmidt, EE, 2003-01-28.
Description
Glycolysis occurs in the cytosol, yielding 2 ATP, 2 pyruvate, and 2 NADH + 2 H+ from each glucose molecule. The first two steps of glycolysis
convert glucose-6-phosphate into a form that is easily cleaved into phosphorylated three-carbon units. High energy phosphate (as ATP) is
generated from the three-carbon units in the remaining steps.
18.3.4.1.1 Glucose 6-phosphate is isomerized to form fructose-6-phosphate
Authors
Schmidt, EE, 2003-01-28.
Editors
Description
Glucose-6-phosphate and fructose-6-phosphate are freely interconverted by phosphoglucose isomerase.
A note about stereochemistry -- In the KEGG database of metabolic pathways, the alpha anomer of D-glucose 6-phosphate is said to be
converted to the beta anomer of D-fructose 6-phosphate. While the involvement of the alpha anomer of D-glucose in this reaction is
well-established, the stereochemistry of the fructose moiety is less clear. Noltmann, reviewing available data concerning reaction mechanisms in
1972, argued that the reaction likely involved conversion of alpha-D-glucose 6-phosphate to alpha-D-fructose 6-phosphate, but conceded that
relevant experimental data were limited. In an equally authoritative review the next year, Bloxham and Lardy reached the opposite conclusion,
based on unpublished substrate specificity data. The anomeric conformation of fructose phosphate in this reaction is therefore left unspecified in
GK.
References
DP Bloxham, HA Lardy, "Phosphofructokinase", The Enzymes, 3rd ed (Boyer PD, editor), 8, 1973, 239-278.
18.3.4.1.1.1 alpha-D-glucose 6-phosphate <=> D-fructose 6-phosphate
Description
The second reaction - readily reversible isomerisation of glucose-6-phosphate by phosphoglucoisomerase.
References
Chem, 231, 1958, 19-29.
Reaction
18.3.4.1.2 Fructose 6-phosphate and ATP react to form fructose 2,6-bisphosphate and ADP
Authors
2003-04-29.
Editors
Schmidt, EE, D'Eustachio, P, 0000-00-00.
Description
The formation of fructose 2,6-bisphosphate from fructose 6-phosphate and ATP is the last step in the regulatory pathway initiated when the
hormone insulin binds to its receptor on the cell surface. Fructose 2,6-bisphosphate is not itself on the pathway of glycolysis. Rather, it acts as a
postive allosteric effector of phosphofructokinase 1, greatly increasing the rate of synthesis of fructose 1,6-bisphosphate and hence the overall
rate of glycolysis. A separate regulatory pathway, initiated by glucagon or epinephrine binding, has the opposite effect on fructose
2,6-bisphosphate production, and thus inhibits glycolysis.
References
Biochem, 64, 1995, 799-835.
18.3.4.1.2.1 ATP + D-fructose 6-phosphate => ADP + D-fructose 2,6-bisphosphate [brain + placenta]
Description
This reaction takes place in the 'cytoplasm' and is mediated by the '6-phosphofructokinase activity' of
References
NP Manes, MR el-Maghrabi, "The kinase activity of human brain 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is regulated via
inhibition by phosphoenolpyruvate", Arch Biochem Biophys, 438, 2005, 125-36.
Reaction
18.3.4.1.2.2 ATP + D-fructose 6-phosphate => ADP + D-fructose 2,6-bisphosphate [heart]
Description
References
T Hirata, M Kato, N Okamura, M Fukasawa, R Sakakibara, "Expression of human placental-type 6-phosphofructo-2-kinase/fructose

2,6-bisphosphatase in various cells and cell lines", Biochem Biophys Res Commun, 242, 1998, 680-4.
Reaction
18.3.4.1.2.3 ATP + D-fructose 6-phosphate => ADP + D-fructose 2,6-bisphosphate [liver + muscle]
Description
References
Reaction
18.3.4.1.2.4 ATP + D-fructose 6-phosphate => ADP + D-fructose 2,6-bisphosphate [testis]
Description
References
A Manzano, JX Perez, M Nadal, X Estivill, A Lange, R Bartrons, "Cloning, expression and chromosomal localization of a human testis
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene", Gene, 229, 1999, 83-9.
Reaction
18.3.4.1.3 ATP + D-fructose 6-phosphate => ADP + D-fructose 1,6-bisphosphate
Description
The cytosolic reaction of one molecule each of fructose-6-phosphate and ATP to form one molecule each of fructose-1,6-bisphosphate and ADP
is catalyzed by phosphofructokinase 1. This reaction, irreversible under physiological conditions, is the rate-limiting step of glycolysis.
Phosphofructokinase 1 activity is allosterically regulated by ATP, citrate, and fructose-2,6-bisphosphate.
Phosphofructokinase 1 is active as a tetramer (although higher-order multimers, not annotated here, may form in vivo). Two isoforms of
phosphofructokinase 1 monomer, L and M, are widely expressed in human tissues. Different tissues can contain different tetramers or tetramer
combinations: L4 in liver, M4 in muscle, and all possible tetramers - L4, L3M, L2M2, LM3, and M4 - in red blood cells, for example (Raban et al.
1995; Vora et al. 1980, 1987; Vora 1981). A third isoform, P, is abundant in platelets, where it is found in P4, P3L, P2L2, and PL3 tetramers (Eto
et al. 1994; Vora et al. 1987).
References
S Vora, C Seaman, S Durham, S Piomelli, "Isozymes of human phosphofructokinase: identification and subunit structural characterization of a
new system", Proc Natl Acad Sci U S A, 77, 1980, 62-6.
S Vora, "Isozymes of human phosphofructokinase in blood cells and cultured cell lines: molecular and genetic evidence for a trigenic system",
Blood, 57, 1981, 724-32.
N Raben, R Exelbert, R Spiegel, JB Sherman, H Nakajima, P Plotz, J Heinisch, "Functional expression of human mutant phosphofructokinase in
yeast: genetic defects in French Canadian and Swiss patients with phosphofructokinase deficiency", Am J Hum Genet, 56, 1995, 131-41.
K Eto, H Sakura, K Yasuda, T Hayakawa, E Kawasaki, R Moriuchi, S Nagataki, Y Yazaki, T Kadowaki, "Cloning of a complete protein-coding
sequence of human platelet-type phosphofructokinase isozyme from pancreatic islet.", Biochem Biophys Res Commun, 198, 1994, 990-8.
Reaction
18.3.4.1.4 D-fructose 1,6-bisphosphate <=> dihydroxyacetone phosphate + D-glyceraldehyde 3-phosphate
Description
In this freely reversible cytosolic reaction, one molecule of D-fructose 1,6-bisphosphate is cleaved to yield single molecules of dihydroxyacetone
phosphate and D-glyceraldehyde 3-phosphate. The active form of aldolase, the enzyme that catalyzes the reaction, is a homotetramer. Three
aldolase isozymes have been identified which differ in their patterns of expression in various adult tissues and during development but are
otherwise functionally indistinguishable (Ali and Cox 1995; Freemont et al. 1988).
References
1995, 1002-5.
249, 1988, 779-88.
Reaction
18.3.4.1.5 Dihydroxyacetone phosphate is isomerized to form glyceraldehyde-3-phosphate

Description
Dihydroxyacetone phosphate, unlike glyceraldehyde-3-phosphate, is not on the direct pathway of glycolysis. Triose phosphate isomerase
catalyzes the freely reversible interconversion of these two molecules.
18.3.4.1.5.1 dihydroxyacetone phosphate <=> D-glyceraldehyde 3-phosphate
Description
The fifth reaction - isomerisation of the triose phosphates.
References
Reaction
18.3.4.1.6 Glyceraldehyde 3-phosphate, NAD+, and orthophosphate react to form 1,3-bisphosphoglycerate, NADH, and H+
Description
Glyceraldehyde-3-phosphate is reversibly oxidized to 1,3-bisphosphoglycerate by glyceraldehyde-3-phosphate dehydrogenase. The energy

released in the oxidation reaction is captured in the form of NADH + H+.
References
JF Taylor, SF Velick, GT Cori, CF Cori, MW Slein, "The prosthetic group of crystalline D-glyceraldehyde-3-phosphate dehydrogenase", J Biol
Chem, 173, 1948, 619-626.
GT Cori, MW Slein, CF Cori, "Crystalline D-glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle", J Biol Chem, 173, 1948, 605-618.
18.3.4.1.6.1 D-glyceraldehyde 3-phosphate + orthophosphate + NAD+ <=> 1,3-bisphospho-D-glycerate + NADH + H+
Description
Cytosolic glyceraldehyde-3-phosphate dehydrogenase catalyzes the reaction of glyceraldehyde 3-phosphate, orthophosphate, and NAD+ to
form NADH + H+ and 1,3-bisphosphoglycerate, the first energy-rich intermediate of glycoslysis.
References
FJ Benham, S Povey, "Members of the human glyceraldehyde-3-phosphate dehydrogenase-related gene family map to dispersed chromosomal
locations.", Genomics, 5, 1989, 209-14.
1976, 67-77.
Reaction
18.3.4.1.7 1,3-bisphosphoglycerate and ADP react to form 3-phosphoglycerate and ATP
Description
The high-energy phosphate bond from 1,3-bisphosphoglycerate is transferred to form ATP, in a reversible reaction catalyzed by
phosphoglycerate kinase. This reaction is an instance of a substrate-level phosphorylation.
18.3.4.1.7.1 ADP + 3-Phospho-D-glyceroyl phosphate <=> ATP + 3-Phospho-D-glycerate
Description
Cytosolic phosphoglycerate kinase catalyzes the reaction of ADP and 1,3-bisphosphoglycerate to form D-glyceraldehyde 3-phosphate and ATP.
This is the first substrate-level phosphorylation reaction in glycolysis.
References
Reaction
18.3.4.1.8 3-Phospho-D-glycerate <=> 2-Phospho-D-glycerate
Description
The reversible isomerisation of 3- and 2-phosphoglycerate is catalyzed by cytosolic phosphoglycerate mutase. This is the eighth reaction of
glycolysis. The active form of the enzyme is a dimer. There are two isoforms of this enzyme, PGAM1 (isoform B) and PGAM2 (isoform M). In the
body, erythrocytes express only PGAM1, while skeletal muscle expresses only PGAM2. Other tissues express both isoforms.
References
Y Blouquit, MC Calvin, R Rosa, D Prome, JC Prome, F Pratbernou, M Cohen-Solal, J Rosa, "Sequence of the human erythrocyte
phosphoglycerate mutase by microsequencer and mass spectrometry", J Biol Chem, 263, 1988, 16906-10.
Reaction
18.3.4.1.9 2-Phospho-D-glycerate <=> Phosphoenolpyruvate + H2O
Authors
Editors
Description
In this freely reversible cytosolic reaction, one molecule of 2-phosphoglycerate reacts to form one molecule each of phosphoenolpyruvate and
water, elevating the transfer potential of the phosphoryl group.
References
1993, 293-303.
Reaction
18.3.4.1.10 Phosphoenolpyruvate and ADP react to form pyruvate and ATP
Description
The transfer of a high-energy phosphate bond from phosphoenolpyruvate, to form ATP, is catalyzed by pyruvate kinase. This reaction, an
instance of substrate-level phosphorylation, is essentially irreversible. Pyruvate kinase activity is regulated both allosterically and by
glucagon-mediated phosphorylation.
18.3.4.1.10.1 ADP + Phosphoenolpyruvate => ATP + Pyruvate (pyruvate kinase M2)
Authors
Description
The muscle (M) isoform of pyruvate kinase is expressed both in muscle and in a variety of other tissues in the normal human adult (Takenaka et
al. 1991).
References
M Takenaka, T Noguchi, S Sadahiro, H Hirai, K Yamada, T Matsuda, E Imai, T Tanaka, "Isolation and characterization of the human pyruvate
kinase M gene", Eur J Biochem, 198, 1991, 101-6.
Reaction
18.3.4.1.10.2 ADP + Phosphoenolpyruvate => ATP + Pyruvate (pyruvate kinase R/L)
Description
The R/L isoform of pyruvate kinase is the major form of the enzyme in human red blood cells (R) and liver (L); deficiencies in the enzyme are
associated with reduced pyruvate kinase activity in red blood cells and with hemolytic anemia (Tani et al. 1988; Miwa et al. 1993).
References
K Tani, H Fujii, S Nagata, S Miwa, "Human liver type pyruvate kinase: complete amino acid sequence and the expression in mammalian cells",
S Miwa, H Kanno, H Fujii, "Concise review: pyruvate kinase deficiency: historical perspective and recent progress of molecular genetics", Am J
Hematol, 42, 1993, 31-5.
Reaction
18.3.4.2 Gluconeogenesis
Editors
Description
In this series of reactions, two molecules of pyruvate are used to synthesize one molecule of glucose, at a cost of six high-energy bonds. The
glucose produced by these reactions can be exported into the blood from the cells that synthesize it (e.g., from the liver under fasting conditions)
to meet the glucose requirements of the brain and of red blood cells.
18.3.4.2.1 Pyruvate, CO2, and ATP react to form oxaloacetate, ADP, and orthophosphate
Description
The carboxylation of pyruvate to form oxaloacetate, catalyzed by pyruvate carboxylase, is an irreversible and allosterically regulated reaction
(Jitrapakdee and Wallace 1999). This is the first of four reactions that differ between gluconeogenesis and glycolysis. The reaction takes place in
the mitochondrion, while the rest of the reactions of gluconeogenesis occur in the cytoplasm. Mitochondrial oxaloacetate is reduced to form
malate, which is transferred to the cytosol and reoxidized to oxaloacetate by malate dehydrogenase.
References
MA Carbone, BH Robinson, "Expression and characterization of a human pyruvate carboxylase variant by retroviral gene transfer", Biochem J,
370, 2003, 275-282.
S Jitrapakdee, JC Wallace, "Structure, function and regulation of pyruvate carboxylase", Biochem J, 340, 1999, 1-16.
Reaction
18.3.4.2.2 Oxaloacetate + NADH + H+ <=> (S)-Malate + NAD+
Description
Mitochondrial malate dehydrogenase catalyzes the reaction of malate and NAD+ to form oxaloacetate and NADH + H+. The dimeric structure of
the human dehydrogenase is inferred from that established for its well-studied pig homolog (Sanchez et al. 1998).
References
2184-2189.
273, 1998, 29540-29544.
Reaction
18.3.4.2.3 Exchange of malate and alpha-ketoglutarate (2-oxoglutarate) across the inner mitochondrial membrane
Authors
Description
The SLC25A11 transport protein in the inner mitochondrial membrane mediates the export of mitochondrial malate into the cytosol, coupled to
the import of cyrosolic alpha-ketoglutarate (2-oxoglutarate) into the mitochondrial matrix. Recent studies of SLC25A11, in addition to providing
direct experimental evidence for the malate:alpha-ketoglutarate transport function of the human protein, suggest that it may also be responsible
for the transport of porphyrin intermediates from the cytosol to the mitochondrial matrix in the course of heme biosynthesis (Kabe et al. 2006)
References
Y Kabe, M Ohmori, K Sinouchi, Y Tsuboi, S Hirao, M Azuma, H Watanabe, I Okura, H Handa, "Porphyrin accumulation in mitochondria is
mediated by 2-oxoglutarate carrier", J Biol Chem, 281, 2006, 31729â€“31735.
Reaction
18.3.4.2.4 malate + NAD+ <=> oxaloacetate + NADH + H+
Authors
Description
Cytosolic malate dehydrogenase catalyzes the reaction of malate and NAD+ to form oxaloacetate and NADH + H+. The dimeric structure of the
human dehydrogenase is inferred from that established for its well-studied pig homolog (Birktoft et al. 1989).
References
AS-Y Lo, C-T Liew, S-M Ngai, SK-W Tsui, K-P Fung, C-Y Lee, MM-Y Waye, "Developmental regulation and cellular distribution of human
cytosolic malate dehydrogenase (MDH1)", J Cell Biochem, 94, 2005, 763-773.
JJ Birktoft, G Rhodes, LJ Banaszak, "Refined crystal structure of cytoplasmic malate dehydrogenase at 2.5-A resolution", Biochemistry, 28,
1989, 6065-6081.
Reaction
18.3.4.2.5 Oxaloacetate and GTP react to form phosphoenolpyruvate, CO2, and GDP
Description
The transfer of a high-energy phosphate bond from GTP to form phosphoenolpyruvate is catalyzed by cytosolic phosphoenolpyruvate
carboxykinase. This is the second of four reactions that differ between gluconeogenesis and glycolysis.
References
P Dunten, C Belunis, R Crowther, K Hollfelder, U Kammlott, W Levin, H Michel, GB Ramsey, A Swain, D Weber, SJ Wertheimer, "Crystal
structure of human cytosolic phosphoenolpyruvate carboxykinase reveals a new GTP-binding site", J Mol Biol, 316, 2002, 257-264.
Reaction
18.3.4.2.6 Phosphoenolpyruvate + H2O <=> 2-Phospho-D-glycerate
Description
In this freely reversible cytosolic reaction, one molecule each of phosphoenolpyruvate and water react to form one molecule of
2-phosphoglycerate.
References
1993, 293-303.
Reaction
18.3.4.2.7 2-Phospho-D-glycerate <=> 3-Phospho-D-glycerate
Description
The reversible isomerisation of 2- and 3-phosphoglycerate is catalyzed by cytosolic phosphoglycerate mutase. The active form of the enzyme is
a dimer. There are two isoforms of this enzyme, PGAM1 (isoform B) and PGAM2 (isoform M). In the body, erythrocytes express only PGAM1,
while skeletal muscle expresses only PGAM2. Other tissues express both isoforms.
References
Reaction
18.3.4.2.8 ATP + 3-Phospho-D-glycerate <=> ADP + 1,3-bisphospho-D-glycerate
Description
The phosphorylation of 3-phosphoglycerate to form 1,3-bisphosphoglycerate, is catalyzed by cytosolic phosphoglycerate kinase.
References
Reaction
18.3.4.2.9 1,3-bisphospho-D-glycerate + NADH + H+ <=> D-glyceraldehyde 3-phosphate + Orthophosphate + NAD+
Description
The reduction of 1,3-bisphosphoglycerate to form glyceraldehyde-3-phosphate is catalyzed by cytosolic glyceraldehyde-3-phosphate

dehydrogenase.
References
1976, 67-77.
Reaction
18.3.4.2.10 D-glyceraldehyde 3-phosphate <=> dihydroxyacetone phosphate
Description
The conversion of glyceraldehyde-3-phosphate to dihydroxyacetone phosphate, catalyzed by triose phosphate isomerase, is simply the reverse
of a reaction of glycolysis.
References
M Watanabe, BC Zingg, HW Mohrenweiser, "Molecular analysis of a series of alleles in humans with reduced activity at the triosephosphate
isomerase locus", Am J Hum Genet, 58, 1996, 308-16.
Reaction
18.3.4.2.11 dihydroxyacetone phosphate + D-glyceraldehyde 3-phosphate <=> D-fructose 1,6-bisphosphate
Description
In this freely reversible cytosolic reaction, single molecules of dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate react to form one
molecule of D-fructose 1,6-bisphosphate. The active form of aldolase, the enzyme that catalyzes the reaction, is a homotetramer. Three aldolase
isozymes have been identified which differ in their patterns of expression in various adult tissues and during development but are otherwise
functionally indistinguishable (Ali and Cox 1995; Freemont et al. 1988).
References
1995, 1002-5.
249, 1988, 779-88.
Reaction
18.3.4.2.12 Fructose 1,6-bisphosphate is hydrolyzed to form fructose 6-phosphate and orthophosphate
Description
The hydrolysis of fructose-1,6,-bisphosphate to form fructose-6-phosphate, is catalyzed by fructose-1,6-bisphosphatase. This reaction is

essentially irreversible, and is the third of four reactions that differ between gluconeogenesis and glycolysis.
18.3.4.2.12.1 D-fructose 1,6-bisphosphate + H2O => D-fructose 6-phosphate + orthophosphate
Description
References
Y Kikawa, M Inuzuka, BY Jin, S Kaji, J-i Koga, Y Yamamoto, K Fujisawa, I Hata, A Nakai, Y Shigematsu, H Mizunuma, A Taketo, M Mayumi, M
Sudo, "Identification of genetic mutations in Japanese patients with fructose-1,6-bisphosphatase deficiency", Am J Hum Genet, 61, 1997,
852-861.
Reaction
18.3.4.2.12.2 D-fructose 1,6-bisphosphate + H2O => D-fructose 6-phosphate + orthophosphate
Description
In the body, this isoform of fructose-1,6-bisphosphatase is muscle-specific (Tillma and Eschrich 1998).
References
H Tillmann, K Eschrich, "Isolation and characterization of an allelic cDNA for human muscle fructose-1,6-bisphosphatase", Gene, 212, 1998,
295-304.
Reaction
18.3.4.2.13 D-fructose 6-phosphate <=> alpha-D-Glucose 6-phosphate
Description
The isomerization of fructose-6-phosphate to form glucose-6-phosphate, catalyzed by phosphoglucose isomerase, is simply the reverse of a
reaction of glycolysis.
References
Chem, 231, 1958, 19-29.
Reaction
18.3.4.2.14 alpha-D-glucose 6-phosphate [cytosol] => alpha-D-glucose 6-phosphate [endoplasmic reticulum lumen]
Authors
Description
The SLC37A4 transport protein in the endoplasmic reticulum membrane mediates the translocation of glucose-6-phosphate from the cytosol into
the lumen of the endoplasmic reticulum. Defects in this transporter are associated with glycogen storage disease type ib (Gerin et al. 1997).
References
I Gerin, M Veiga-da-Cunha, Y Achouri, J-F Collet, E Van Schaftingen, "Sequence of a putative glucose 6-phosphate translocase, mutated in
glycogen storage disease type Ib", FEBS Lett, 419, 1997, 235-238.
Reaction
18.3.4.2.15 alpha-D-Glucose 6-phosphate + H2O => alpha-D-Glucose + Orthophosphate
Description
Glucose-6-phosphatase associated with the inner face of the endoplasmic reticulum membrane catalyzes the hydrolysis of glucose-6-phosphate
to glucose and orthophosphate (reviewed by Van Scaftingen and Gerin 2002). This reaction is essentially irreversible. It is the last of the four
reactions that differ between gluconeogenesis and glycolysis. Defects in glucose-6-phosphatase are the cause of glycogen storage disease type
1a (Lei et al. 1995).
References
K-J Lei, Y-T Chen, H Chen, L-JC Wong, J-L Liu, A McConkie-Rosell, JLK Van Hove, HC-Y Ou, NJ Yeh, LY Pan, JY Chou, "Genetic basis of
glycogen storage disease type 1a: Prevalent mutations at the glucose-6-phosphatase locus", Am J Hum Genet, 57, 1995, 766-771.
Reaction
18.3.4.2.16 Efflux of glucose from the endoplasmic reticulum
Authors
Description
Glucose generated within the endoplasmic reticulum is exported from the cell. Several mechansims for this transport process have been
proposed but experimental data remain incomplete and contradictory (e.g., Hosokawa and Thorens 2002; Fehr et al. 2005; Van Schaftingen and
Gerin 2002).
References
M Hosokawa, B Thorens, "Glucose release from GLUT2-null hepatocytes: characterization of a major and a minor pathway", Am J Physiol
Endocrinol Metab, 282, 2002, E794-E801.
M Fehr, H Takanaga, DW Ehrhardt, WB Frommer, "Evidence for high-capacity bidirectional glucose transport across the endoplasmic reticulum
membrane by genetically encoded fluoresence resonance energy transfer nanosensors", Mol Cell Biol, 25, 2005, 11102-11112.
Reaction
18.3.4.2.17 Fructose 2,6-bisphosphate is hydrolyzed to form fructose-6-phosphate and orthophosphate
Description
At the beginning of this reaction, 1 molecule of 'D-Fructose 2,6-bisphosphate', and 1 molecule of 'H2O' are present. At the end of this reaction, 1
This reaction takes place in the 'cytoplasm' and is mediated by the 'fructose-bisphosphatase activity' of 'unknown fructose-bisphosphatase'.
References
Biochem, 64, 1995, 799-835.
Reaction
18.3.5 Glycogen synthesis
Authors
2003-02-15.
Description
Glycogen, a highly branched glucose polymer, is formed and broken down in most human tissues, but is most abundant in liver and muscle,
where it serves as a major stored fuel. Glycogen metabolism has been studied in most detail in muscle, although considerable experimental data
are available concerning these reactions in liver as well. Glycogen metabolism in other tissues has not been studied extensively, and is thought
to resemble the muscle process.
The biochemical steps of the glycogen synthetic pathway are the same in liver and muscle. The first two steps, conversion of glucose
6-phosphate to glucose 1-phosphate and the synthesis of UDP-glucose from glucose 1-phosphate and UTP, are shared with several other
pathways. The next three events, the auto-catalyzed synthesis of a glucose oligomer on glycogenin; the linear extension of the glucose oligomer
catalyzed by glycogen synthase; and the formation of branches catalyzed by glycogen branching enzyme, are unique to glycogen synthesis.
These events are shown in the figure. Repetition of the last two events allows the generation of large, extensively branched glycogen polymers.
The catalysis of several of these steps by distinct isozymes in liver and muscle allows them to be regulated independently in the two tissues.
References
SA Westphal, FQ Nuttall, "Comparative characterization of human and rat liver glycogen synthase.", Arch Biochem Biophys, 292, 1992, 479-86.
Y Bao, P Kishnani, JY Wu, YT Chen, "Hepatic and neuromuscular forms of glycogen storage disease type IV caused by mutations in the same
glycogen-branching enzyme gene.", J Clin Invest, 97, 1996, 941-8.
BJ Gibbons, PJ Roach, TD Hurley, "Crystal structure of the autocatalytic initiator of glycogen biosynthesis, glycogenin.", J Mol Biol, 319, 2002,
463-77.
J Mu, AV Skurat, PJ Roach, "Glycogenin-2, a novel self-glucosylating protein involved in liver glycogen biosynthesis.", J Biol Chem, 272, 1997,
27589-97.
MF Browner, K Nakano, AG Bang, RJ Fletterick, "Human muscle glycogen synthase cDNA sequence: a negatively charged protein with an
asymmetric charge distribution.", Proc Natl Acad Sci U S A, 86, 1989, 1443-7.
YC Chao, JG Huang, HL Peng, HY Chang, "Sequence differences between human muscle and liver cDNAs for UDPglucose pyrophosphorylase
and kinetic properties of the recombinant enzymes expressed in Escherichia coli.", Eur J Biochem, 235, 1996, 173-9.
PJ Roach, KR Warren, DE Atkinson, "Uridine diphosphate glucose synthase from calf liver: determinants of enzyme activity in vitro.",
Biochemistry, 14, 1976, 5445-50.
Y Cao, AM Mahrenholz, AA DePaoli-Roach, PJ Roach, "Characterization of rabbit skeletal muscle glycogenin. Tyrosine 194 is essential for
function.", J Biol Chem, 268, 1993, 14687-93.
F Barbetti, M Rocchi, M Bossolasco, R Cordera, P Sbraccia, P Finelli, GG Consalez, "The human skeletal muscle glycogenin gene: cDNA,
tissue expression and chromosomal localization.", Biochem Biophys Res Commun, 220, 1996, 72-7.
J Mu, PJ Roach, "Characterization of human glycogenin-2, a self-glucosylating initiator of liver glycogen metabolism.", J Biol Chem, 273, 1999,
34850-6.
18.3.5.1 alpha-D-Glucose 6-phosphate <=> D-Glucose 1-phosphate
Description
Glucose 1-phosphate is converted to glucose 6-phosphate by cytosolic phosphoglucomutase I.
References
RE March, W Putt, M Hollyoake, JH Ives, JU Lovegrove, DA Hopkinson, YH Edwards, DB Whitehouse, "The classical human
phosphoglucomutase (PGM1) isozyme polymorphism is generated by intragenic recombination", Proc Natl Acad Sci U S A, 90, 1993, 10730-3.
Reaction
18.3.5.2 UTP and glucose 1-phosphate react to form UDP-glucose and pyrophosphate
Description
UTP and glucose 1-phosphate react to form UDP-glucose and pyrophosphate, in a reaction that can be catalyzed by both liver and muscle forms
of UTP:glucose-1-phosphate uridylyltransferase activity.
References
RL Turnquist, RG Hansen, "Uridine diphosphoryl glucose pyrophosphorylase", The Enzymes, 3rd ed (Boyer PD, editor), 8, 1973, 51-71.
18.3.5.2.1 UTP + D-glucose 1-phosphate <=> pyrophosphate + UDP-glucose [liver]
Reviewers
Description
UDPglucose pyrophosphorylase (UDPGPase) catalyses the transfer of a glucose moiety from glucose-1-phosphate to UTP, forming
UDP-Glucose and PPi. Liver requires UDP-glucose for the formation of UDPglucuronate, which acts as a conjugating source for the formation of
soluble glucuronides of xenobiotic and endobiotic metabolites.
References
FEBS Lett, 329, 1993, 153-8.
Reaction
18.3.5.2.2 UTP + D-glucose 1-phosphate <=> pyrophosphate + UDP-glucose [muscle]
Description
At the beginning of this reaction, 1 molecule of 'D-Glucose 1-phosphate', and 1 molecule of 'UTP' are present. At the end of this reaction, 1
molecule of 'pyrophosphate', and 1 molecule of 'UDPglucose' are present.
This reaction takes place in the 'cytoplasm' and is mediated by the 'UTP:glucose-1-phosphate uridylyltransferase activity' of
'UTP-glucose-1-phosphate uridylyltransferase 2 octamer [muscle]'.
References
FEBS Lett, 329, 1993, 153-8.
Reaction
18.3.5.3 Glycogenin catalyzes the synthesis of an oligo(1,4)glucose moiety covalently attached to itself
Description
Glycogenin1 and 2 can each catalyze the synthesis of a covalently attached oligo(1,4) glucose moiety - a kind of auto-post-translational
modification.
18.3.5.3.1 n UDP-glucose + glycogenin-2 => n UDP + {(1,4)-alpha-D-glucosyl}n glycogenin-2
Description
At the beginning of this reaction, 1 molecule of 'Glycogenin-2 ', and 1 molecule of 'UDPglucose' are present. At the end of this reaction, 1
molecule of 'uridine 5'-diphosphate', and 1 molecule of 'oligo((1,4)-alpha-glycosyl)glycogenin-2' are present.
This reaction takes place in the 'glycogen granule' and is mediated by the 'glycogenin glucosyltransferase activity' of 'glycogenin-2 dimer'.
References
463-77.
Reaction
18.3.5.3.2 n UDP-glucose + glycogenin-1 => n UDP + {(1,4)-alpha-D-glucosyl}n glycogenin-1
Description
At the beginning of this reaction, 1 molecule of 'glycogenin-1', and 1 molecule of 'UDPglucose' are present. At the end of this reaction, 1
molecule of 'uridine 5'-diphosphate', and 1 molecule of 'oligo((1,4)-alpha-glucosyl)glycogenin-1' are present.
This reaction takes place in the 'glycogen granule' and is mediated by the 'glycogenin glucosyltransferase activity' of 'glycogenin-1 dimer'.
References
463-77.
Reaction
18.3.5.4 Glycogen synthase catalyzes the addition of glucose residues to the non-reducing end of a (1,4)-alpha-D-glucose multimer
Description
Glycogen synthase catalyzes the addition of glucose residues to the non-reducing end of a (1,4)-alpha-D-glucose multimer formed on glycogenin
1 or glycogenin 2.
18.3.5.4.1 m UDP-glucose + {(1,4)-alpha-D-glucosyl}n glycogenin-1 => m UDP + {(1,4)-alpha-D-glucosyl}m+n glycogenin-1 [muscle, I

form]
Description
At the beginning of this reaction, 1 molecule of 'UDPglucose', and 1 molecule of 'oligo((1,4)-alpha-glucosyl)glycogenin-1' are present. At the end
of this reaction, 1 molecule of 'uridine 5'-diphosphate', and 1 molecule of 'poly{(1,4)-alpha-glucosyl}glycogenin-1' are present.
This reaction takes place in the 'glycogen granule' and is mediated by the 'glycogen (starch) synthase activity' of 'glycogen synthase 1 tetramer, I
form [muscle]'.
References
MF Browner, K Nakano, AG Bang, RJ Fletterick, "Human muscle glycogen synthase cDNA sequence: a negatively charged protein with an
asymmetric charge distribution.", Proc Natl Acad Sci U S A, 86, 1989, 1443-7.
Reaction
18.3.5.4.2 m UDP-glucose + {(1,4)-alpha-D-glucosyl}n glycogenin-2 => m UDP + {(1,4)-alpha-D-glucosyl}m+n glycogenin-2 [liver, I form]
Description
At the beginning of this reaction, 1 molecule of 'UDPglucose', and 1 molecule of 'oligo((1,4)-alpha-glycosyl)glycogenin-2' are present. At the end
This reaction takes place in the 'glycogen granule' and is mediated by the 'glycogen (starch) synthase activity' of 'glycogen synthase 2 tetramer, I
form [liver]'.
References
M Orho, NU Bosshard, NR Buist, R Gitzelmann, A Aynsley-Green, P Blumel, MC Gannon, FQ Nuttall, LC Groop, "Mutations in the liver glycogen
synthase gene in children with hypoglycemia due to glycogen storage disease type 0", J Clin Invest, 102, 1998, 507-15.
Reaction
18.3.5.5 Phosphorylated glycogen synthase catalyzes the addition of glucose residues to the non-reducing end of a
(1,4)-alpha-D-glucose multimer when activated by glucose-6-phosphate
Description
Phosphorylated glycogen synthase catalyzes the addition of glucose residues to the non-reducing end of a (1,4)-alpha-D-glucose multimer
formed on either glycogenin 1 or glycogenin 2 when activated by glucose-6-phosphate.
18.3.5.5.1 m UDP-glucose + {(1,4)-alpha-D-glucosyl}n glycogenin-1 => m UDP + {(1,4)-alpha-D-glucosyl}m+n glycogenin-1 [muscle, D

form]
Description
At the beginning of this reaction, 1 molecule of 'UDPglucose', and 1 molecule of 'oligo((1,4)-alpha-glucosyl)glycogenin-1' are present. At the end
This reaction takes place in the 'glycogen granule' and is mediated by the 'glycogen (starch) synthase activity' of 'glycogen synthase 1 tetramer,
D form [muscle]'.
References
E Cid, RR Gomis, RA Geremia, JJ Guinovart, JC Ferrer, "Identification of two essential glutamic acid residues in glycogen synthase", J Biol
Chem, 275, 2000, 33614-21.
Reaction
18.3.5.5.2 m UDP-glucose + {(1,4)-alpha-D-glucosyl}n glycogenin-2 => m UDP + {(1,4)-alpha-D-glucosyl}m+n glycogenin-2 [liver, D

form]
Description
At the beginning of this reaction, 1 molecule of 'UDPglucose', and 1 molecule of 'oligo((1,4)-alpha-glycosyl)glycogenin-2' are present. At the end
This reaction takes place in the 'glycogen granule' and is mediated by the 'glycogen (starch) synthase activity' of 'glycogen synthase 2 tetramer,
D form [liver]'.
References
Reaction
18.3.5.6 Glycogen branching enzyme transfers terminal alpha(1,4)glucose blocks to form alpha(1,6) branches
Description
Glycogen branching enzyme transfers terminal alpha(1,4) glucose blocks to form alpha(1,6) branches on growing glycogen molecules formed on
glycogenin 1 or glycogenin 2.
18.3.5.6.1 poly{(1,4)-alpha-D-glucosyl} glycogenin-1 => glycogen-glycogenin-1
Description
At the beginning of this reaction, 1 molecule of 'poly{(1,4)-alpha-glucosyl}glycogenin-1' is present. At the end of this reaction, 1 molecule of
'glycogen-glycogenin-1' is present.
This reaction takes place in the 'glycogen granule' and is mediated by the '1,4-alpha-glucan branching enzyme activity' of 'glycogen branching
enzyme'.
References
Reaction
18.3.5.6.2 poly{(1,4)-alpha-D-glucosyl} glycogenin-2 => glycogen-glycogenin-2
Description
At the beginning of this reaction, 1 molecule of 'poly{(1,4)-alpha-glucosyl}glycogenin-2' is present. At the end of this reaction, 1 molecule of
'glycogen-glycogenin-2' is present.
This reaction takes place in the 'glycogen granule' and is mediated by the '1,4-alpha-glucan branching enzyme activity' of 'glycogen branching
enzyme'.
References
Reaction
18.3.6 Glycogen breakdown (glycogenolysis)
Authors
2003-02-15.
Description
Glycogen breakdown, like glycogen synthesis, occurs via the same biochemical steps in liver and muscle, but is separately regulated via
tissue-specific isozymes and signaling pathways. Glycogen phosphorylase (which can be activated by phosphorylase kinase) catalyzes the
removal of glucose residues, as glucose 1-phosphate, from the ends of glycogen branches. The final four residues of each branch are removed
in two steps catalyzed by debranching enzyme, and further glycogen phosphorylase activity completes the process of glycogen breakdown. The
figure shows the actions of phosphorylase and debranching enzyme. The first glucose residue in each branch is released as free glucose; all
other residues are released as glucose 1-phosphate. The latter molecule can be converted to glucose 6-phosphate in a step shared with other
pathways.
References
NB Madsen, "Glycogen phosphorylase", The Enzymes, 3rd ed (Boyer PD, Krebs EG, editors), 17, 1986, 365-394.
DJ Graves, JH Wang, "Alpha-glycan phosphorylases - chemical and physical basis of catalysis and regulation", The Enzymes, 3rd ed (Boyer
PD, editor), 7, 1972, 435-482.
J Shen, Y Bao, HM Liu, P Lee, JV Leonard, YT Chen, "Mutations in exon 3 of the glycogen debranching enzyme gene are associated with
glycogen storage disease type III that is differentially expressed in liver and muscle.", J Clin Invest, 98, 1996, 352-7.
CA Pickett-Gies, DA Walsh, "Phosphorylase kinase", The Enzymes, 3rd ed (Boyer PD, Krebs EG, editors), 17, 1986, 395-459.
18.3.6.1 Phosphorylase kinase activates glycogen phosphorylase
Description
Phosphorylase kinase catalyzes the phosphorylation of both the liver and muscle forms of glycogen phosphorylase.
18.3.6.1.1 glycogen phosphorylase, liver form, tetramer b + ATP => glycogen phosphorylase, liver form, tetramer a + ADP
Description
At the beginning of this reaction, 1 molecule of 'glycogen phosphorylase, liver form, tetramer b', and 1 molecule of 'ATP' are present. At the end
of this reaction, 1 molecule of 'glycogen phosphorylase, liver form, tetramer a', and 1 molecule of 'ADP' are present.
This reaction takes place in the 'glycogen granule' and is mediated by the 'phosphorylase kinase activity' of 'phosphorylase kinase complex, liver
form'.
References
IE van den Berg, EA van Beurden, HE MalingrÃ©, HK van Amstel, BT Poll-The, JA Smeitink, WH Lamers, R Berger, "X-linked liver
phosphorylase kinase deficiency is associated with mutations in the human liver phosphorylase kinase alpha subunit.", Am J Hum Genet, 56,
1995, 381-7.
Reaction
18.3.6.1.2 glycogen phosphorylase, muscle form, tetramer b + ATP => glycogen phosphorylase, muscle form, tetramer a + ADP
Description
At the beginning of this reaction, 1 molecule of 'glycogen phosphorylase, muscle form, tetramer b', and 1 molecule of 'ATP' are present. At the
end of this reaction, 1 molecule of 'glycogen phosphorylase, muscle form, tetramer a', and 1 molecule of 'ADP' are present.
This reaction takes place in the 'glycogen granule' and is mediated by the 'phosphorylase kinase activity' of 'phosphorylase kinase complex,
muscle form'.
References
Reaction
18.3.6.2 Glycogen-glycogenin reacts with n orthophosphates, yielding n glucose 1-phosphates and limit dextrin-glycogenin
Description
Glycogen-glycogenin 1 and glycogen-glycogenin 2 can each react with n orthophosphates, yielding n glucose 1-phosphates and limit dextrin
attached to glycogenin
18.3.6.2.1 glycogen-glycogenin-1 + n orthophosphate => limit dextrin-glycogenin-1 + n D-glucose 1-phosphate [muscle a form]
Description
At the beginning of this reaction, 1 molecule of 'glycogen-glycogenin-1', and 1 molecule of 'Orthophosphate' are present. At the end of this
reaction, 1 molecule of 'D-Glucose 1-phosphate', and 1 molecule of 'limit dextrin-glycogenin-1' are present.
This reaction takes place in the 'glycogen granule' and is mediated by the 'glycogen phosphorylase activity' of 'glycogen phosphorylase, muscle
form, tetramer a'.
References
PD, editor), 7, 1972, 435-482.
Reaction
18.3.6.2.2 glycogen-glycogenin-1 + n orthophosphate => limit dextrin-glycogenin-1 + n D-glucose 1-phosphate [muscle b form]
Description
form, tetramer b'.
References
PD, editor), 7, 1972, 435-482.
Reaction
18.3.6.2.3 glycogen-glycogenin-2 + n orthophosphate => limit dextrin-glycogenin-2 + n D-glucose 1-phosphate [liver a form]
Description
This reaction takes place in the 'glycogen granule' and is mediated by the 'glycogen phosphorylase activity' of 'glycogen phosphorylase, liver
form, tetramer a'.
References
PD, editor), 7, 1972, 435-482.
Reaction
18.3.6.2.4 glycogen-glycogenin-2 + n orthophosphate => limit dextrin-glycogenin-2 + n D-glucose 1-phosphate [liver b form]
Description
form, tetramer b'.
References
PD, editor), 7, 1972, 435-482.
Reaction
18.3.6.3 Debranching enzyme transfers 3-glucose blocks from branches in limit dextrin
Description
Debranching enzyme transfers 3-glucose blocks from branches in limit dextrin formed on glycogenin 1 or glycogenin 2.
18.3.6.3.1 limit dextrin-glycogenin-1 => {(1,6)-alpha-glucosyl}poly{(1,4)-alpha-glucosyl} glycogenin-1
Description
At the beginning of this reaction, 1 molecule of 'limit dextrin-glycogenin-1' is present. At the end of this reaction, 1 molecule of
'{(1,6)-alpha-glucosyl}poly{(1,4)-alpha-glucosyl}glycogenin-1' is present.
This reaction takes place in the 'glycogen granule' and is mediated by the '4-alpha-glucanotransferase activity' of 'glycogen debranching
enzyme'.
References
YT Chen, JK He, JH Ding, BI Brown, "Glycogen debranching enzyme: purification, antibody characterization, and immunoblot analyses of type III
glycogen storage disease.", Am J Hum Genet, 41, 1988, 1002-15.
Reaction
18.3.6.3.2 limit dextrin-glycogenin-2 => {(1,6)-alpha-glucosyl}poly{(1,4)-alpha-glucosyl} glycogenin-2
Description
At the beginning of this reaction, 1 molecule of 'limit dextrin-glycogenin-2' is present. At the end of this reaction, 1 molecule of
'{(1,6)-alpha-glucosyl}poly{(1,4)-alpha-glucosyl}glycogenin-2' is present.
This reaction takes place in the 'glycogen granule' and is mediated by the '4-alpha-glucanotransferase activity' of 'glycogen debranching
enzyme'.
References
Reaction
18.3.6.4 (1,6)-alpha-glucose residues are removed, as D-glucose, from limit dextrin by debranching enzyme
Description
(1,6)-alpha-glucose residues are removed from limit dextrins formed on glycogenin 1 or glycogenin 2 by debranching enzyme.
18.3.6.4.1 {(1,6)-alpha-glucosyl}poly{(1,4)-alpha-glucosyl}glycogenin-1 => poly{(1,4)-alpha-glucosyl} glycogenin-1 + alpha-D-glucose
Description
At the beginning of this reaction, 1 molecule of '{(1,6)-alpha-glucosyl}poly{(1,4)-alpha-glucosyl}glycogenin-1' is present. At the end of this
reaction, 1 molecule of 'poly{(1,4)-alpha-glucosyl}glycogenin-1', and 1 molecule of 'alpha-D-glucose' are present.
This reaction takes place in the 'cytoplasm' and is mediated by the 'amylo-alpha-1,6-glucosidase activity' of 'glycogen debranching enzyme'.
References
Reaction
18.3.6.4.2 {(1,6)-alpha-glucosyl}poly{(1,4)-alpha-glucosyl}glycogenin-2 => poly{(1,4)-alpha-glucosyl} glycogenin-2 + alpha-D-glucose
Description
At the beginning of this reaction, 1 molecule of '{(1,6)-alpha-glucosyl}poly{(1,4)-alpha-glucosyl}glycogenin-2' is present. At the end of this
reaction, 1 molecule of 'alpha-D-glucose', and 1 molecule of 'poly{(1,4)-alpha-glucosyl}glycogenin-2' are present.
This reaction takes place in the 'cytoplasm' and is mediated by the 'amylo-alpha-1,6-glucosidase activity' of 'glycogen debranching enzyme'.
References
Reaction
18.3.6.5 Poly{(1,4)-alpha-glucosyl} glycogenin reacts with n orthophosphates to form glycogenin and n D-glucose 1-phosphates
Description
Poly{(1,4)-alpha-glucosyl} moieties covalently attached to either glycogenin 1 or glycogenin 2 react with n orthophosphates to form glycogenin 1
or 2 and n D-glucose 1-phosphates. The reaction can be catalyzed by both a and b forms of both liver and muscle glycogen phosphorylases.
18.3.6.5.1 poly{(1,4)-alpha-glucosyl} glycogenin-1 + n orthophosphate => glycogenin-1 + n D-glucose 1-phosphate [muscle a form]
Description
At the beginning of this reaction, 1 molecule of 'poly{(1,4)-alpha-glucosyl}glycogenin-1', and 1 molecule of 'Orthophosphate' are present. At the
end of this reaction, 1 molecule of 'D-Glucose 1-phosphate', and 1 molecule of 'glycogenin-1' are present.
form, tetramer a'.
References
PD, editor), 7, 1972, 435-482.
Reaction
18.3.6.5.2 poly{(1,4)-alpha-glucosyl} glycogenin-1 + n orthophosphate => glycogenin-1 + n D-glucose 1-phosphate [muscle b form]
Description
end of this reaction, 1 molecule of 'D-Glucose 1-phosphate', and 1 molecule of 'glycogenin-1' are present.
form, tetramer b'.
References
PD, editor), 7, 1972, 435-482.
Reaction
18.3.6.5.3 poly{(1,4)-alpha-glucosyl} glycogenin-2 + n orthophosphate => glycogenin-2 + n D-glucose 1-phosphate [liver a form]
Description
end of this reaction, 1 molecule of 'D-Glucose 1-phosphate', and 1 molecule of 'Glycogenin-2 ' are present.
form, tetramer a'.
References
PD, editor), 7, 1972, 435-482.
Reaction
18.3.6.5.4 poly{(1,4)-alpha-glucosyl} glycogenin-2 + n orthophosphate => glycogenin-2 + n D-glucose 1-phosphate [liver b form]
Description
end of this reaction, 1 molecule of 'D-Glucose 1-phosphate', and 1 molecule of 'Glycogenin-2 ' are present.
form, tetramer b'.
References
PD, editor), 7, 1972, 435-482.
Reaction
18.3.6.6 D-Glucose 1-phosphate <=> alpha-D-Glucose 6-phosphate
Description
Glucose 1-phosphate is converted to glucose 6-phosphate by cytosolic phosphoglucomutase I.
References
RE March, W Putt, M Hollyoake, JH Ives, JU Lovegrove, DA Hopkinson, YH Edwards, DB Whitehouse, "The classical human
phosphoglucomutase (PGM1) isozyme polymorphism is generated by intragenic recombination", Proc Natl Acad Sci U S A, 90, 1993, 10730-3.
Reaction
18.4 Fructose catabolism
Authors
2003-03-01.
Description
Fructose occurs naturally in foods as a free monosaccharide and as a component of the disaccharide sucrose. It is also widely used as a
sweetener. In the body, fructose is converted to dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate, two intermediates in the
glycolytic pathway, in a sequence of three reactions: fructose is phosphorylated, then cleaved by aldolase to yield dihydroxyacetone phosphate
and D-glyceraldehyde, and the latter compound is phosphorylated to yield D-glyceraldehyde 3-phosphate. Other pathways exist for the
conversion of D-glyceraldehyde to intermediates of glycolysis, but these appear to play only a minor role in normal fructose metabolism.
The cleavage of fructose 1-phosphate is catalyzed by the same enzyme that catalyzes the reversible cleavage of fructose 1,6-bisphosphate in
glycolysis. The isoform of this enzyme found in liver, kidney, and intestine (B) is approximately equally active with fructose 1-phosphate and
fructose 1,6-bisphosphate as substrates, while the muscle and brain isoforms (A and C, respectively), have little activity with fructose
1-phosphate. Fructose metabolism thus occurs mainly in tissues expressing the B isoform, and mutations that affect the catalytic activity of this
isoform are associated with hereditary fructose intolerance.
References
B Steinmann, R Gitzelmann, G Van den Berghe, "Disorders of fructose metabolism", The Metabolic and Molecular Bases of Inherited Disease,
HG Hers, "Triokinases", The Enzymes, 2nd ed (Boyer PD, Lardy H, Myrback K, editors), 6, 1962, 75-83.
18.4.1 ATP + beta-D-fructose => ADP + D-fructose 1-phosphate
Description
At the beginning of this reaction, 1 molecule of 'D-Fructose', and 1 molecule of 'ATP' are present. At the end of this reaction, 1 molecule of
'D-Fructose 1-phosphate', and 1 molecule of 'ADP' are present.
This reaction takes place in the 'cytoplasm' and is mediated by the 'ketohexokinase activity' of 'Ketohexokinase '.
References
Reaction
18.4.2 D-fructose 1-phosphate <=> D-glyceraldehyde + dihydroxyacetone phosphate
Description
At the beginning of this reaction, 1 molecule of 'D-Fructose 1-phosphate' is present. At the end of this reaction, 1 molecule of 'Dihydroxyacetone
phosphate', and 1 molecule of 'D-Glyceraldehyde' are present.
This reaction takes place in the 'cytoplasm' and is mediated by the 'fructose-bisphosphate aldolase activity' of 'fructose-bisphosphate aldolase B,
class I holoenzyme'.
References
Reaction
18.4.3 ATP + D-glyceraldehyde => ADP + D-glyceraldehyde 3-phosphate
Description
At the beginning of this reaction, 1 molecule of 'D-Glyceraldehyde', and 1 molecule of 'ATP' are present. At the end of this reaction, 1 molecule of
'ADP', and 1 molecule of 'D-Glyceraldehyde 3-phosphate' are present.
This reaction takes place in the 'cytoplasm' and is mediated by the 'triokinase activity' of 'triokinase'.
References
HG Hers, "Triokinases", The Enzymes, 2nd ed (Boyer PD, Lardy H, Myrback K, editors), 6, 1962, 75-83.
Reaction
18.5 Galactose catabolism
Authors
2003-02-25.
Description
The main sources of galactose in the human diet are milk and milk products. The disaccharide lactose, from these sources, is hydrolyzed in the
intestine to its constituent monosaccharides, glucose and galactose. Galactose is metabolized primarily in the liver, in a sequence of three
reactions that yield one molecule of glucose 1-phosphate per molecule of galactose. First, it is phosphorylated to yield galactose 1-phosphate.
Then, galactose 1-phosphate and UDP-glucose react to form UDP-galactose and glucose 1-phosphate, and UDP-galactose undergoes
epimerization to form UDP-glucose. In a reaction shared with other pathways, glucose 1-phosphate can be converted into glucose 6-phosphate.
References
JB Holton, JH Walter, LA Tyfield, "Galactosemia", The Metabolic and Molecular Bases of Inherited Disease, 8th ed (Scriver CR, et al., editors),
1, 2001, 1553-1587.
LJ Elsas, K Lai, "The molecular biology of galactosemia.", Genet Med, 1, 2001, 40-8.
18.5.1 ATP + D-galactose => ADP + D-galactose 1-phosphate
Description
At the beginning of this reaction, 1 molecule of 'D-Galactose', and 1 molecule of 'ATP' are present. At the end of this reaction, 1 molecule of
'ADP', and 1 molecule of 'alpha-D-Galactose 1-phosphate' are present.
This reaction takes place in the 'cytoplasm' and is mediated by the 'galactokinase activity' of 'Galactokinase '.
References
Y Ai, M Basu, DJ Bergsma, D Stambolian, "Comparison of the enzymatic activities of human galactokinase GALK1 and a related human
galactokinase protein GK2", Biochem Biophys Res Commun, 212, 1995, 687-91.
Reaction
18.5.2 D-galactose 1-phosphate + UDP-glucose <=> D-glucose 1-phosphate + UDP-galactose
Authors
2003-02-25.
Description
The reaction of alpha-D-galactose 1-phosphate and UDP glucose to form D-glucose 1-phosphate and UDP galactose is catalyzed by GALT. It
takes place in the cytosol.
References
JB Holton, JH Walter, LA Tyfield, "Galactosemia", The Metabolic and Molecular Bases of Inherited Disease, 8th ed (Scriver CR, et al., editors),
1, 2001, 1553-1587.
JK Reichardt, S Packman, SL Woo, "Molecular characterization of two galactosemia mutations: correlation of mutations with highly conserved
domains in galactose-1-phosphate uridyl transferase", Am J Hum Genet, 49, 1991, 860-7.
LJ Elsas, K Lai, "The molecular biology of galactosemia.", Genet Med, 1, 2001, 40-8.
Reaction
18.5.3 UDP-galactose <=> UDP-glucose
Description
At the beginning of this reaction, 1 molecule of 'UDP-D-galactose' is present. At the end of this reaction, 1 molecule of 'UDPglucose' is present.
This reaction takes place in the 'cytoplasm' and is mediated by the 'UDP-glucose 4-epimerase activity' of 'UDP-galactose 4-epimerase
holoenzyme'.
References
JM Schulz, AL Watson, R Sanders, KL Ross, JB Thoden, HM Holden, JL Fridovich-Keil, "Determinants of function and substrate specificity in
human UDP-galactose 4'-epimerase", J Biol Chem, 279, 2004, 32796-803.
Reaction
18.6 Pentose phosphate pathway (hexose monophosphate shunt)
Authors
2003-02-15.
Description
The pentose phosphate pathway is responsible for the generation of a substantial fraction of the cytoplasmic NADPH required for biosynthetic
reactions, and for the generation of ribose 5-phosphate for nucleotide synthesis. Although the pentose phosphate pathway and glycolysis are
distinct, they involve three common intermediates, glucose 6-phosphate, glyceraldehyde 3-phosphate, and fructose 6-phosphate, so the two
pathways are interconnected. The pentose phosphate pathway consists of eight reactions:1. Conversion glucose 6-phosphate to
D-glucono-1,5-lactone 6-phosphate, with the formation of NADPH; 2. Conversion of D-glucono-1,5-lactone 6-phosphate to
6-phospho-D-gluconate; 3. Conversion of 6-phospho-D-gluconate to ribulose 5-phosphate, with the formation of NADPH; 4. Conversion of
ribulose 5-phosphate to xylulose 5-phosphate; 5. Conversion of ribulose 5-phosphate to ribose 5-phosphate; 6. Rearrangement of ribose
5-phosphate and xylulose 5-phosphate to form sedoheptulose 7-phosphate and glyceraldehyde 3-phosphate; 7. Rearrangement of
sedoheptulose 7-phosphate and glyceraldehyde 3-phosphate to form erythrose 4-phosphate and fructose 6-phosphate; and 8. Rearrangement
of xylulose 5-phosphate and erythrose 4-phosphate to form glyceraldehyde 3-phosphate and fructose-6-phosphate.
The oxidative branch of the pentose phosphate pathway, reactions 1-3, generates NADPH and pentose 5-phosphate. The non-oxidative branch
of the pathway, reactions 4-8, converts pentose 5-phosphate to other sugars.
The overall pathway can operate to generate only NADPH (glucose 6-phosphate is converted to pentose 5-phosphates, which are directed to
the synthesis of fructose 6-phosphate and glyceraldehyde 3-phosphate, which in turn are converted back to glucose 6-phosphate). The reactions
of the non-oxidative branch can operate to generate net amounts of ribose 5-phosphate with no production of NADPH. Net flux through this
network of reactions appears to depend on the metabolic state of the cell and the nature of the biosynthetic reactions underway.
References
JP Casazza, RL Veech, "The content of pentose-cycle intermediates in liver in starved, fed ad libitum and meal-fed rats.", Biochem J, 236, 1987,
635-41.
C Bublitz, S Steavenson, "The pentose phosphate pathway in the endoplasmic reticulum.", J Biol Chem, 263, 1988, 12849-53.
18.6.1 alpha-D-glucose 6-phosphate + NADP+ => D-glucono-1,5-lactone 6-phosphate + NADPH + H+ [G6PD

dimer]
Description
At the beginning of this reaction, 1 molecule of 'NADP+', and 1 molecule of 'alpha-D-Glucose 6-phosphate' are present. At the end of this
reaction, 1 molecule of 'H+', 1 molecule of 'D-Glucono-1,5-lactone 6-phosphate', and 1 molecule of 'NADPH' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'glucose-6-phosphate 1-dehydrogenase activity' of 'G6PD dimer'.
References
L Luzzatto, A Afolayan, "Enzymic properties of different types of human erythrocyte glucose-6-phosphate dehydrogenase, with characterization
of two new genetic variants", J Clin Invest, 47, 1968, 1833-42.
Reaction
18.6.2 alpha-D-glucose 6-phosphate + NADP+ => D-glucono-1,5-lactone 6-phosphate + NADPH + H+ [G6PD

tetramer]
Description
At the beginning of this reaction, 1 molecule of 'NADP+', and 1 molecule of 'alpha-D-Glucose 6-phosphate' are present. At the end of this
reaction, 1 molecule of 'H+', 1 molecule of 'D-Glucono-1,5-lactone 6-phosphate', and 1 molecule of 'NADPH' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'glucose-6-phosphate 1-dehydrogenase activity' of 'G6PD tetramer'.
References
L Luzzatto, A Afolayan, "Enzymic properties of different types of human erythrocyte glucose-6-phosphate dehydrogenase, with characterization
of two new genetic variants", J Clin Invest, 47, 1968, 1833-42.
Reaction
18.6.3 D-glucono-1,5-lactone 6-phosphate + H2O => 6-phospho-D-gluconate
Description
At the beginning of this reaction, 1 molecule of 'D-Glucono-1,5-lactone 6-phosphate', and 1 molecule of 'H2O' are present. At the end of this
reaction, 1 molecule of '6-Phospho-D-gluconate' is present.
This reaction takes place in the 'cytosol' and is mediated by the '6-phosphogluconolactonase activity' of '6-phosphogluconolactonase '.
References
E Beutler, W Kuhl, "Limiting role of 6-phosphogluconolactonase in erythrocyte hexose monophosphate pathway metabolism", J Lab Clin Med,
106, 1985, 573-7.
F Collard, JF Collet, I Gerin, M Veiga-da-Cunha, E Van Schaftingen, "Identification of the cDNA encoding human 6-phosphogluconolactonase,
the enzyme catalyzing the second step of the pentose phosphate pathway(1)", FEBS Lett, 459, 1999, 223-6.
Reaction
18.6.4 6-phospho-D-gluconate + NADP+ => D-ribulose 5-phosphate + CO2 + NADPH + H+
Description
At the beginning of this reaction, 1 molecule of 'NADP+', and 1 molecule of '6-Phospho-D-gluconate' are present. At the end of this reaction, 1
molecule of 'CO2', 1 molecule of 'D-Ribulose 5-phosphate', 1 molecule of 'H+', and 1 molecule of 'NADPH' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'phosphogluconate 2-dehydrogenase activity' of '6-phosphogluconate
dehydrogenase, decarboxylating'.
References
E Beutler, W Kuhl, "Limiting role of 6-phosphogluconolactonase in erythrocyte hexose monophosphate pathway metabolism", J Lab Clin Med,
106, 1985, 573-7.
M Rippa, PP Giovannini, MP Barrett, F Dallocchio, S Hanau, "6-Phosphogluconate dehydrogenase: the mechanism of action investigated by a
comparison of the enzyme from different species", Biochim Biophys Acta, 1429, 1998, 83-92.
Reaction
18.6.5 D-ribulose 5-phosphate <=> xylulose 5-phosphate
Description
At the beginning of this reaction, 1 molecule of 'D-Ribulose 5-phosphate' is present. At the end of this reaction, 1 molecule of 'D-Xylulose
5-phosphate' is present.
This reaction takes place in the 'cytosol' and is mediated by the 'ribulose-phosphate 3-epimerase activity' of 'ribulose-5-phosphate epimerase'.
References
GR Boss, RB Pilz, "Phosphoribosylpyrophosphate synthesis from glucose decreases during amino acid starvation of human lymphoblasts", J
Biol Chem, 260, 1985, 6054-9.
Reaction
18.6.6 xylulose 5-phosphate <=> D-ribulose 5-phosphate
Description
At the beginning of this reaction, 1 molecule of 'D-Xylulose 5-phosphate' is present. At the end of this reaction, 1 molecule of 'D-Ribulose
5-phosphate' is present.
This reaction takes place in the 'cytosol' and is mediated by the 'ribulose-phosphate 3-epimerase activity' of 'ribulose-5-phosphate epimerase'.
References
GR Boss, RB Pilz, "Phosphoribosylpyrophosphate synthesis from glucose decreases during amino acid starvation of human lymphoblasts", J
Biol Chem, 260, 1985, 6054-9.
Reaction
18.6.7 ribose 5-phosphate + xylulose 5-phosphate <=> sedoheptulose 7-phosphate + D-glyceraldehyde

3-phosphate
Description
At the beginning of this reaction, 1 molecule of 'D-Xylulose 5-phosphate', and 1 molecule of 'D-ribose 5-phosphate' are present. At the end of this
reaction, 1 molecule of 'D-Glyceraldehyde 3-phosphate', and 1 molecule of 'Sedoheptulose 7-phosphate' are present.
This reaction takes place in the 'cytoplasm' and is mediated by the 'transketolase activity' of 'transketolase dimer'.
References
Reaction
18.6.8 D-glyceraldehyde 3-phosphate + sedoheptulose 7-phosphate<=> xylulose 5-phosphate+ribose

5-phosphate
Description
At the beginning of this reaction, 1 molecule of 'Sedoheptulose 7-phosphate', and 1 molecule of 'D-Glyceraldehyde 3-phosphate' are present. At
the end of this reaction, 1 molecule of 'D-ribose 5-phosphate', and 1 molecule of 'D-Xylulose 5-phosphate' are present.
References
Reaction
18.6.9 sedoheptulose 7-phosphate + D-glyceraldehyde 3-phosphate <=> D-erythrose 4-phosphate +

D-fructose 6-phosphate
Description
At the beginning of this reaction, 1 molecule of 'D-Glyceraldehyde 3-phosphate', and 1 molecule of 'Sedoheptulose 7-phosphate' are present. At
the end of this reaction, 1 molecule of 'D-Erythrose 4-phosphate', and 1 molecule of 'D-Fructose 6-phosphate' are present.
References
Reaction
18.6.10 D-fructose 6-phosphate + D-erythrose 4-phosphate <=> sedoheptulose 7-phosphate +

D-glyceraldehyde 3-phosphate
Description
At the beginning of this reaction, 1 molecule of 'D-Erythrose 4-phosphate', and 1 molecule of 'D-Fructose 6-phosphate' are present. At the end of
this reaction, 1 molecule of 'Sedoheptulose 7-phosphate', and 1 molecule of 'D-Glyceraldehyde 3-phosphate' are present.
References
Reaction
18.6.11 xylulose 5-phosphate + D-erythrose 4-phosphate <=> D-glyceraldehyde 3-phosphate + D-fructose

6-phosphate
Description
At the beginning of this reaction, 1 molecule of 'D-Erythrose 4-phosphate', and 1 molecule of 'D-Xylulose 5-phosphate' are present. At the end of
this reaction, 1 molecule of 'D-Glyceraldehyde 3-phosphate', and 1 molecule of 'D-Fructose 6-phosphate' are present.
References
Reaction
18.6.12 D-glyceraldehyde 3-phosphate + D-fructose 6-phosphate <=> xylulose 5-phosphate + D-erythrose

4-phosphate
Description
At the beginning of this reaction, 1 molecule of 'D-Glyceraldehyde 3-phosphate', and 1 molecule of 'D-Fructose 6-phosphate' are present. At the
end of this reaction, 1 molecule of 'D-Erythrose 4-phosphate', and 1 molecule of 'D-Xylulose 5-phosphate' are present.
References
Reaction
18.6.13 D-ribulose 5-phosphate <=> ribose 5-phosphate
Editors
Description
The reversible interconversion of ribose 5-phosphate and ribulose 5-phosphate is catalyzed by ribose 5-phosphate isomerase (Huck et al. 2004).
References
JH Huck, NM Verhoeven, EA Struys, GS Salomons, C Jakobs, MS van der Knaap, "Ribose-5-phosphate isomerase deficiency: new inborn error
in the pentose phosphate pathway associated with a slowly progressive leukoencephalopathy", Am J Hum Genet, 74, 2004, 745-51.
Reaction
18.6.14 ribose 5-phosphate <=> D-ribulose 5-phosphate
Editors
Description
The reversible interconversion of ribose 5-phosphate and ribulose 5-phosphate is catalyzed by ribose 5-phosphate isomerase (Huck et al. 2004).
References
JH Huck, NM Verhoeven, EA Struys, GS Salomons, C Jakobs, MS van der Knaap, "Ribose-5-phosphate isomerase deficiency: new inborn error
in the pentose phosphate pathway associated with a slowly progressive leukoencephalopathy", Am J Hum Genet, 74, 2004, 745-51.
Reaction
18.7 5-Phosphoribose 1-diphosphate biosynthesis
Authors
Editors
Description
5-Phospho-alpha-D-ribose 1-diphosphate (PRPP) is a key intermediate in both the de novo and salvage pathways of purine and pyrimidine
synthesis. PRPP and the enzymatic activity responsible for its synthesis were first described by Kornberg et al. (1955). The enzyme,
phosphoribosyl pyrophosphate synthetase 1, has been purified from human erythrocytes and characterized biochemically. The purified enzyme
readily forms multimers; its smallest active form appears to be a dimer and for simplicity it is annotated as a dimer here. It specifically catalyzes
the transfer of pyrophosphate from ATP or dATP to D-ribose 5-phosphate, and has an absolute requirement for Mg++ and orthophosphate (Fox
and Kelley 1971; Roth et al. 1974). (The significance of the reaction with dATP in vivo is unclear, as the concentration of cytosolic dATP is
normally much lower than that of ATP.) The importance of this enzyme for purine synthesis in vivo has been established by demonstrating
excess phosphoribosyl pyrophosphate synthetase activity, correlated with elevated enzyme levels or altered enzyme properties, in individuals
whose rates of uric acid production are constitutively abnormally high (Becker and Kim 1987; Roessler et al. 1993).
Molecular cloning studies have revealed the existence of two additional genes that encode phosphoribosyl pyrophosphate synthetase-like
proteins, one widely expressed (phosphoribosyl pyrophosphate synthetase 2) and one whose expression appears to be confined to the testis
(phosphoribosyl pyrophosphate synthetase 1-like 1) (Taira et al. 1989; 1991). Neither of these proteins has been purified and characterized
enzymatically, nor have variations in the abundance or sequence of either protein been associated with alterations in human nucleotide
metabolism (Roessler et al. 1993; Becker et al. 1996), so their dimerization and ability to catalyze the synthesis of PRPP from D-ribose
5-phosphate are inferred here on the basis of their predicted amino acid sequence similarity to phosphoribosyl pyrophosphate synthetase 1.
References
M Taira, T Iizasa, H Shimada, J Kudoh, N Shimizu, M Tatibana, "A human testis-specific mRNA for phosphoribosylpyrophosphate synthetase
that initiates from a non-AUG codon", J Biol Chem, 265, 1990, 16491-16497.
A Kornberg, I Lieberman, ES Simms, "Enzymatic synthesis and properties of 5-phosphoribosylpyrophosphate", J Biol Chem, 215, 1955,
389-402.
IH Fox, WN Kelley, "Human phosphoribosylpyrophosphate synthetase", J Biol Chem, 246, 1971, 5739-5748.
BJ Roessler, JM Nosal, PR Smith, SA Heidler, TD Palella, RL Switzer, MA Becker, "Human X-linked phosphoribosylpyrophosphate synthetase
superactivity is associated with distinct point mutations in the PRPS1 gene", J Biol Chem, 268, 1993, 26476-26481.
M Taira, T Iizasa, K Yamada, H Shimada, M Tatibana, "Tissue-differential expression of two distinct genes for phosphoribosyl pyrophosphate
synthetase and existence of the testic-specific transcript", Biochim Biophys Acta, 1007, 1989, 203-208.
MA Becker, W Taylor, PR Smith, M Ahmed, "Overexpression of the normal phosphoribosylpyrophosphate synthetase 1 isoform undelies
catalytic superactivity of human phosphoribosylpyrophosphate synthetase", J Biol Chem, 271, 1996, 19894-19899.
DG Roth, E Shelton, TF Deuel, "Purification and properties of phosphoribosyl pyrophosphate synthetase from rat liver", J Biol Chem, 249, 1974,
291-296.
MA Becker, M Kim, "Regulation of purine synthesis de novo in human fibroblasts by purine nucleotides and phosphoribosylpyrophosphate", J
Biol Chem, 262, 1987, 14531-14537.
18.7.1 D-ribose 5-phosphate + 2'-deoxyadenosine 5'-triphosphate (dATP) => 5-Phospho-alpha-D-ribose

1-diphosphate (PRPP) + 2'-deoxyadenosine 5'-monophosphate
Description
Cytosolic phosphoribosyl pyrophosphate synthetase 1 catalyzes the reaction of D-ribose 5-phosphate and dATP to form
5-phospho-alpha-D-ribose 1-diphosphate and 2'-deoxyadenosine 5'-monophosphate. While phosphoribosyl pyrophosphate synthetase 1 works
well with either ATP or dATP as a substrate in vitro, the extent of the dATP reaction in vivo is unclear, as cellular dATP concentrations are
normally very low (Fox and Kelley 1971).
References
Reaction
18.7.2 D-ribose 5-phosphate + ATP => 5-phospho-alpha-D-ribose 1-diphosphate (PRPP) + adenosine

5'-monophosphate
Description
Cytosolic phophoribosyl pyrophosphate synthetase catalyzes the reaction of D-ribose 5-phosphate and ATP to form 5-phospho-alpha-D-ribose
and AMP. Three isoforms of the enzyme have been described. The first has been purified and characterized biochemically (Fox and Kelley
1971). The others are known only as inferred protein products of cloned genes; their catalytic properties have not been determined.
References
Reaction
19 Metabolism of nitric oxide
Authors
Hemish, J, 2007-10-19.
Reviewers
Enikolopov, G, 2008-02-28.
Description
Nitric oxide (NO), a diffusible multifunctional second messenger, is implicated in numerous physiological functions in mammals, ranging from
immune response and potentiation of synaptic transmission, to dilation of blood vessels and muscle relaxation. NO is synthesized from
L-arginine by a family of nitric oxide synthases (NOS). Three NOS isoforms have been characterized: neuronal NOS (nNOS, NOS1) primarily
found in neuronal tissue and skeletal muscle; inducible NOS (iNOS, NOS2) originally isolated from macrophages and later discovered in many
other cells types; and endothelial NOS (eNOS, NOS3) present in vascular endothelial cells, cardiac myocytes, and in blood platelets. The
enzymatic activity of all three isoforms is dependent on calmodulin, which binds to nNOS and eNOS at elevated intracellular calcium levels,
while it is tightly associated with iNOS (even at basal calcium levels). As a result, the enzymatic activity of nNOS and eNOS is modulated by
changes in intracellular calcium levels, leading to transient NO production, while iNOS continuously releases NO independent of fluctuations in
intracellular calcium levels and is mainly regulated at the gene expression level.
The NOS enzymes share a common basic structural organization and requirement for substrate cofactors for enzymatic activity. A central
calmodulin-binding motif separates an oxygenase (NH2-terminal) domain from a reductase (COOH-terminal) domain. Binding sites for cofactors
NADPH, FAD, and FMN are located within the reductase domain, while binding sites for tetrahydrobiopterin (BH4) and heme are located within
the oxygenase domain. Once calmodulin binds, it facilitates electron transfer from the cofactors in the reductase domain to heme enabling nitric
oxide production. Both nNOS and eNOS contain an additional insert (40-50 amino acids) in the middle of the FMN-binding subdomain that
serves as autoinhibitory loop, destabilizing calmodulin binding at low calcium levels and inhibiting electron transfer from FMN to the heme in the
absence of calmodulin. iNOS does not contain this insert.
Because NOS enzymatic activity is modulated by the presence of its substrates and cofactors within the cell, under certain conditions, NOS may
generate superoxide instead of NO, a process referred to as uncoupling (uncoupling of NADPH oxidation and NO synthesis).
NO is a highly active molecule that diffuses across cell membranes and cannot be stored inside the producing cell. Its signaling capacity must be
controlled at the levels of biosynthesis and local availability. Indeed, NO production by NO synthases is under complex and tight control, being
regulated at transcriptional and translational levels, through co- and posttranslational modifications, and by subcellular localization.
19.1 eNOS activation and regulation
Reviewers
Description
Originally identified as endothelium-derived relaxing factor, eNOS derived NO is a critical signaling molecule in vascular homeostasis. It
regulates blood pressure and vascular tone, and is involved in vascular smooth muscle cell proliferation, platelet aggregation, and leukocyte
adhesion. Loss of the bioavailability of endothelium derived NO is a key feature of endothelial dysfunction and is implicated in the pathogenesis
of cardiovascular disease such as hypertension and atherosclerosis. The endothelial isoform eNOS is unique among the nitric oxide synthase
(NOS) family in that it is co-translationally modified at its amino terminus by myristoylation and is further acylated by palmitoylation (two residues
next to the myristoylation site). These modifications target eNOS to the plasma membrane caveolae and lipid rafts.
eNOS activation and subsequent nitric oxide (NO) production is stimulated by a variety of stimuli, such as fluid shear stress generated by blood
flow, vascular endothelial growth factor (VEGF), bradykinin, estrogen, insulin, and angiopoietin. The activity of eNOS is further regulated by
numerous post-translational modifications, including protein-protein interactions, phosphorylation, and subcellular localization.
Following activation, eNOS shuttles between caveolae and other subcellular compartments such as the noncaveolar plasma membrane
portions, Golgi apparatus, and perinuclear structures. This subcellular distribution is variable depending upon cell type and mode of activation.
Subcellular localization of eNOS has a profound effect on its ability to produce NO as the availability of its substrates and cofactors will vary with
location. eNOS is primarily particulate, and depending on the cell type, eNOS can be found in several membrane compartments: plasma
membrane caveolae, lipid rafts, and intracellular membranes such as the Golgi complex. In addition, it has been reported that eNOS can also be
detected in the nucleus, however, the conditions associated with nuclear localization of eNOS and its precise role in this cell compartment
remains to be determined.
Several stimuli can trigger a transient displacement of eNOS from the plasma membrane to other subcellular locations. This process can be
mediated through various protein-protein interactions and/or changes in post-translational modifications. Knowledge of the precise molecular
mechanisms governing the intracellular redistribution process is still rather limited.
References
S Oess, A Icking, D Fulton, R Govers, W Muller-Esterl, "Subcellular targeting and trafficking of nitric oxide synthases", Biochem J, 396, 2006,
401-9.
BC Kone, T Kuncewicz, W Zhang, ZY Yu, "Protein interactions with nitric oxide synthases: controlling the right time, the right place, and the right
amount of nitric oxide", Am J Physiol Renal Physiol, 285, 2003, F178-90.
19.1.1 eNOS activation
Reviewers
Description
eNOS activity is regulated by numerous post-translational modifications, including protein-protein interactions, phosphorylation, and subcellular
localization.
In general, following myristoylation and palmitoylation, eNOS localizes to caveolae in the plasma membrane, where in resting cells, it is bound to
caveolin and remains inactive. Several agonists that raise intracellular calcium concentrations promote calmodulin binding to eNOS and the
dissociation of caveolin from the enzyme, leading to an activated eNOS-calmodulin complex.
Phosphorylation plays a significant role in regulating eNOS activity, especially the phosphorylation of Ser1177, located within the reductase
domain, which increases enzyme activity by enhancing reductase activity and calcium sensitivity. In unstimulated, cultured endothelial cells,
Ser1177 is rapidly phosphorylated following a variety of stimuli: fluid shear stress, insulin, estrogen, VEGF, or bradykinin. The kinases involved
in this process depend upon the stimuli applied. For instance, shear stress phosphorylates Ser1177 by activating Akt and PKA; insulin activates
both Akt and the AMP-activated protein kinase (AMPK); estrogen and VEGF mainly phosphorylate eNOS via Akt; whereas the
bradykinin-induced phosphorylation of Ser1177 is mediated by CaMKII. When Ser1177 is phosphorylated, NO production is increased
several-fold above basal levels.
The phosphorylation of a threonine residue (Thr 495), located in the CaM binding domain, is associated with a decrease in eNOS activity. When
this residue is dephosphorylated, substantially more CaM binds to eNOS and elevates enzyme activity. Stimuli associated with
dephosphorylation of Thr495 (e.g., bradykinin, histamine, and Ca2+ ionophores) also increase Ca2+ levels resulting in the phosphorylation of
Ser1177.
Additional phosphorylation sites, such as Ser114 and Ser633, and tyrosine phosphorylation have all been detected, but their functional
relevance remains unclear. It is speculated that the tyrosine phosphorylation of eNOS is unlikely to affect enzyme activity directly, but more likely
to impact the protein-protein interactions with associated scaffolding and regulatory proteins.
References
I Fleming, R Busse, "Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase", Am J Physiol Regul Integr Comp
Physiol, 284, 2003, R1-12.
R Govers, TJ Rabelink, "Cellular regulation of endothelial nitric oxide synthase", Am J Physiol Renal Physiol, 280, 2001, F193-206.
19.1.1.1 N-myristoylation of eNOS
Authors
Hemish, J, 2007-10-19.
Reviewers
Description
A glycine residue (Gly2) at the N-terminus of eNOS is myristoylated, providing membrane localization.
References
J Liu, WC Sessa, "Identification of covalently bound amino-terminal myristic acid in endothelial nitric oxide synthase", J Biol Chem, 269, 1994,
11691-4.
Reaction
19.1.1.2 palmitoylation of eNOS
Authors
Hemish, J, 2007-10-19.
Reviewers
Description
DHHC-21 is a Golgi-localized acyl transferase that palmitoylates eNOS, which targets eNOS to plasmalemmal caveolae. Localization to this
microdomain is likely to optimize eNOS activation and the extracellular release of nitric oxide.
References
G Garcia-Cardena, P Oh, J Liu, JE Schnitzer, WC Sessa, "Targeting of nitric oxide synthase to endothelial cell caveolae via palmitoylation:
implications for nitric oxide signaling", Proc Natl Acad Sci U S A, 93, 1996, 6448-53.
PW Shaul, EJ Smart, LJ Robinson, Z German, IS Yuhanna, Y Ying, RG Anderson, T Michel, "Acylation targets emdothelial nitric-oxide synthase
to plasmalemmal caveolae", J Biol Chem, 271, 1996, 6518-22.
C Fernandez-Hernando, M Fukata, PN Bernatchez, Y Fukata, MI Lin, DS Bredt, WC Sessa, "Identification of Golgi-localized acyl transferases
that palmitoylate and regulate endothelial nitric oxide synthase", J Cell Biol, 174, 2006, 369-77.
Reaction
19.1.1.3 eNOS translocation from Golgi to Caveolae
Reviewers
Description
The transport of myristoylated and palmitoylated eNOS from the Golgi to the plasma membrane is thought to be vesicular, but the details remain
unknown.
References
401-9.
Reaction
19.1.1.4 Calcium binds calmodulin
Authors
Schmidt, EE, 2003-07-11.
Reviewers
Description
Calmodulin gets activated upon binding of at least two, and up to four, calcium ions. For our purposes, we assume 4 calcium ions bound
produces full activation of calmodulin.
Reaction
19.1.1.5 eNOS associates with Caveolin-1
Authors
Hemish, J, 2007-10-19.
Reviewers
Description
Caveolin-1 is the primary negative regulatory protein for eNOS. Caveolin-1 binding to eNOS compromises its ability to bind Calmodulin (CaM),
thereby inhibiting enzyme activity. The major binding region of caveolin-1 for eNOS is within amino acids 60-101 and to a lesser extent, amino
acids 135-178.
References
M Drab, P Verkade, M Elger, M Kasper, M Lohn, B Lauterbach, J Menne, C Lindschau, F Mende, FC Luft, A Schedl, H Haller, TV Kurzchalia,
"Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice", Science, 293, 2001, 2449-52.
O Feron, L Belhassen, L Kobzik, TW Smith, RA Kelly, T Michel, "Endothelial nitric oxide synthase targeting to caveolae. Specific interactions
with caveolin isoforms in cardiac myocytes and endothelial cells.", J Biol Chem, 271, 1996, 22810-4.
S Ghosh, R Gachhui, C Crooks, C Wu, MP Lisanti, DJ Stuehr, "Interaction between caveolin-1 and the reductase domain of endothelial
nitric-oxide synthase. Consequences for catalysis.", J Biol Chem, 273, 1998, 22267-71.
G Garcia-Cardena, P Martasek, BS Masters, PM Skidd, J Couet, S Li, MP Lisanti, WC Sessa, "Dissecting the interaction between nitric oxide
synthase (NOS) and caveolin. Functional significance of the nos caveolin binding domain in vivo.", J Biol Chem, 272, 1997, 25437-40.
Reaction
19.1.1.6 eNOS:Caveolin-1 complex binds to CaM

Authors
Hemish, J, 2007-10-19.
Reviewers
Description
Caveolin inhibition of eNOS is relieved by calmodulin, which causes dissociation of eNOS from caveolin.
References
JB Michel, O Feron, D Sacks, T Michel, "Reciprocal regulation of endothelial nitric-oxide synthase by Ca2+-calmodulin and caveolin", J Biol
Chem, 272, 1997, 15583-6.
Reaction
19.1.1.7 HSP90 binds eNOS:Caveolin-1:CaM complex
Authors
Hemish, J, 2007-10-19.
Reviewers
Description
HSP90 interacts with the amino terminus of eNOS (amino acids 442-600) and facilitates displacement of caveolin by calmodulin (CaM).
References
I Fleming, R Busse, "Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase", Am J Physiol Regul Integr Comp
Physiol, 284, 2003, R1-12.
J Fontana, D Fulton, Y Chen, TA Fairchild, TJ McCabe, N Fujita, T Tsuruo, WC Sessa, "Domain mapping studies reveal that the M domain of
hsp90 serves as a molecular scaffold to regulate Akt-dependent phosphorylation of endothelial nitric oxide synthase and NO release", Circ Res,
90, 2002, 866-73.
G Garcia-Cardena, R Fan, V Shah, R Sorrentino, G Cirino, A Papapetropoulos, WC Sessa, "Dynamic activation of endothelial nitric oxide
synthase by Hsp90", Nature, 392, 1998, 821-4.
Reaction
19.1.1.8 Caveolin-1 dissociates from eNOS:CaM:HSP90 complex
Authors
Hemish, J, 2007-10-19.
Reviewers
Description
HSP90 facilitates the CaM-induced displacement of caveolin from eNOS.
References
JP Gratton, J Fontana, DS O'Connor, G Garcia-Cardena, TJ McCabe, WC Sessa, "Reconstitution of an endothelial nitric-oxide synthase
(eNOS), hsp90, and caveolin-1 complex in vitro. Evidence that hsp90 facilitates calmodulin stimulated displacement of eNOS from caveolin-1.", J
Biol Chem, 275, 2000, 22268-72.
Reaction
19.1.1.9 Akt binds eNOS complex via HSP90
Authors
Hemish, J, 2007-10-19.
Reviewers
Description
Akt is recruited to the M domain of HSP90.
References
S Takahashi, ME Mendelsohn, "Synergistic activation of endothelial nitric-oxide synthase (eNOS) by HSP90 and Akt: calcium-independent
eNOS activation involves formation of an HSP90-Akt-CaM-bound eNOS complex", J Biol Chem, 278, 2003, 30821-7.
J Fontana, D Fulton, Y Chen, TA Fairchild, TJ McCabe, N Fujita, T Tsuruo, WC Sessa, "Domain mapping studies reveal that the M domain of
hsp90 serves as a molecular scaffold to regulate Akt-dependent phosphorylation of endothelial nitric oxide synthase and NO release", Circ Res,
90, 2002, 866-73.
Reaction
19.1.1.10 Akt phosphorylates eNOS
Authors
Hemish, J, 2007-10-19.
Reviewers
Description
HSP90 serves as a scaffold to promote productive interaction between Akt and eNOS. Due to the proximity of these proteins once complexed
with HSP90, Akt phosphorylates eNOS at Ser1177. When Ser1177 is phosphorylated, the level of NO production is elevated two- to three-fold
above basal level.
References
D Fulton, JP Gratton, TJ McCabe, J Fontana, Y Fujio, K Walsh, TF Franke, A Papapetropoulos, WC Sessa, "Regulation of endothelium-derived
nitric oxide production by the protein kinase Akt", Nature, 399, 1999, 597-601.
BJ Michell, JE Griffiths, KI Mitchelhill, I Rodriguez-Crespo, T Tiganis, S Bozinovski, PR de Montellano, BE Kemp, RB Pearson, "The Akt kinase
signals directly to endothelial nitric oxide synthase", Curr Biol, 9, 1999, 845-8.
S Dimmeler, I Fleming, B Fisslthaler, C Hermann, R Busse, AM Zeiher, "Activation of nitric oxide synthase in endothelial cells by Akt-dependent
phosphorylation", Nature, 399, 1999, 601-5.
Reaction
19.1.1.11 NO biosynthesis
Authors
Hemish, J, 2007-10-19.
Reviewers
Description
Nitric oxide (NO) is produced from L-arginine by the family of nitric oxide synthases (NOS) enzymes, forming the free radical NO and citrulline as
byproduct.
References
P Klatt, K Schmidt, ER Werner, B Mayer, "Determination of nitric oxide synthase cofactors: heme, FAD, FMN, and tetrahydrobiopterin", Methods
Enzymol, 268, 1996, 358-65.
BM List, B Klosch, C Volker, AC Gorren, WC Sessa, ER Werner, WR Kukovetz, K Schmidt, B Mayer, "Characterization of bovine endothelial
nitric oxide synthase as a homodimer with down-regulated uncoupled NADPH oxidase activity: tetrahydrobiopterin binding kinetics and role of
haem in dimerization", Biochem J, 323, 1997, 159-65.
N Tuteja, M Chandra, R Tuteja, MK Misra, "Nitric Oxide as a Unique Bioactive Signaling Messenger in Physiology and Pathophysiology", J
Biomed Biotechnol, 2004, 2004, 227-237.
DS Bredt, SH Snyder, "Nitric oxide: a physiologic messenger molecule", Annu Rev Biochem, 63, 1994, 175-95.
Reaction
19.1.2 eNOS acylation cycle
Reviewers
Description
It is thought that eNOS may undergo a cycle of depalmitoylation and repalmitoylation, resulting in a shuttling of the enzyme between the Golgi
and the plasma membrane.
References
401-9.
DC Yeh, JA Duncan, S Yamashita, T Michel, "Depalmitoylation of endothelial nitric-oxide synthase by acyl-protein thioesterase 1 is potentiated
by Ca(2+)-calmodulin", J Biol Chem, 274, 1999, 33148-54.
19.1.2.1 palmitoylation of eNOS
Authors
Hemish, J, 2007-10-19.
Reviewers
Description
DHHC-21 is a Golgi-localized acyl transferase that palmitoylates eNOS, which targets eNOS to plasmalemmal caveolae. Localization to this
microdomain is likely to optimize eNOS activation and the extracellular release of nitric oxide.
References
G Garcia-Cardena, P Oh, J Liu, JE Schnitzer, WC Sessa, "Targeting of nitric oxide synthase to endothelial cell caveolae via palmitoylation:
implications for nitric oxide signaling", Proc Natl Acad Sci U S A, 93, 1996, 6448-53.
PW Shaul, EJ Smart, LJ Robinson, Z German, IS Yuhanna, Y Ying, RG Anderson, T Michel, "Acylation targets emdothelial nitric-oxide synthase
to plasmalemmal caveolae", J Biol Chem, 271, 1996, 6518-22.
C Fernandez-Hernando, M Fukata, PN Bernatchez, Y Fukata, MI Lin, DS Bredt, WC Sessa, "Identification of Golgi-localized acyl transferases
that palmitoylate and regulate endothelial nitric oxide synthase", J Cell Biol, 174, 2006, 369-77.
Reaction
19.1.2.2 eNOS translocation from Golgi to Caveolae

Reviewers
Description
The transport of myristoylated and palmitoylated eNOS from the Golgi to the plasma membrane is thought to be vesicular, but the details remain
unknown.
References
401-9.
Reaction
19.1.2.3 depalmitoylated eNOS translocates from plasma membrane
Authors
Hemish, J, 2007-10-19.
Reviewers
Description
Once depalmitoylated, it's proposed that eNOS is displaced from the plasma membrane and redistributed to other intracellular membranes,
including the Golgi, where re-palmitoylation occurs. The mechanism of transport from the plasma membrane is still unknown.
References
T Michel, "Targeting and translocation of endothelial nitric oxide synthase", Braz J Med Biol Res, 32, 1999, 1361-6.
Reaction
19.1.2.4 depalmitoylation of eNOS
Authors
Hemish, J, 2007-10-19.
Reviewers
Description
Increases in intracellular calcium and calmodulin stimulate depalmitoylation of eNOS by acyl protein thioesterase 1, which displaces eNOS from
the membrane. This might be a mechanism to downregulate NO production following intense stimuli.
References
DC Yeh, JA Duncan, S Yamashita, T Michel, "Depalmitoylation of endothelial nitric-oxide synthase by acyl-protein thioesterase 1 is potentiated
by Ca(2+)-calmodulin", J Biol Chem, 274, 1999, 33148-54.
Reaction
19.1.3 NOSIP mediated eNOS trafficking
Reviewers
Description
eNOS-interacting protein (NOSIP) is a 34-kDa nucleocytoplasmic shuttling protein that binds to the COOH-terminal region (amino acids
366-486) of the eNOS oxygenase domain. This protein association promotes translocation of eNOS from the plasma membrane caveolae to the
cytoskeleton and inhibits eNOS activity. Studies have found that NOSIP accumulates in the cytoplasm specifically during the G2 phase of the
cell cycle.
References
J Dedio, P Konig, P Wohlfart, C Schroeder, W Kummer, W Muller-Esterl, "NOSIP, a novel modulator of endothelial nitric oxide synthase activity",
FASEB J, 15, 2001, 79-89.
401-9.
M Schleicher, F Brundin, S Gross, W Muller-Esterl, S Oess, "Cell cycle-regulated inactivation of endothelial NO synthase through
NOSIP-dependent targeting to the cytoskeleton", Mol Cell Biol, 25, 2005, 8251-8.
A Icking, S Matt, N Opitz, A Wiesenthal, W Muller-Esterl, K Schilling, "NOSTRIN functions as a homotrimeric adaptor protein facilitating
internalization of eNOS", J Cell Sci, 118, 2005, 5059-69.
19.1.3.1 eNOS binds NOSIP
Reviewers
Description
NOSIP (eNOS interacting protein) binds to the carboxyl-terminal region of the eNOS oxygenase domain. Note that the eNOS binding sites for
caveolin and NOSIP overlap.
References
FASEB J, 15, 2001, 79-89.
Reaction
19.1.3.2 eNOS:NOSIP translocation from caveolae to intracellular compartments
Reviewers
Description
NOSIP promotes translocation of eNOS from the plasma membrane to intracellular sites, thereby uncoupling eNOS from plasma membrane
caveolae and inhibiting NO synthesis. eNOS appears to be shifted to intracellular sites that colocalize with Golgi and/or cytoskeletal marker
proteins.
References
FASEB J, 15, 2001, 79-89.
K Zimmermann, N Opitz, J Dedio, C Renne, W Muller-Esterl, S Oess, "NOSTRIN: a protein modulating nitric oxide release and subcellular
distribution of endothelial nitric oxide synthase", Proc Natl Acad Sci U S A, 99, 2002, 17167-72.
Reaction
19.1.4 NOSTRIN mediated eNOS trafficking
Reviewers
Description
eNOS traffic inducer (NOSTRIN) is a novel 506-amino acid eNOS-interacting protein. Along with a decrease in eNOS activity, NOSTRIN causes
translocation of eNOS from the plasma membrane to intracellular vesicular structures. NOSTRIN functions as an adaptor protein through
homotrimerization and recruitment of eNOS, dynamin-2, and N-WASP to its SH3 domain. Studies indicated that NOSTRIN may facilitate vesicle
fission and endocytosis of eNOS by coordinating the function of dynamin and N-WASP, which in turn, recruits the Arp2/3 complex, initiating actin
filament polymerization. Overall, this process is thought to occur via caveolar endocytosis.
References
401-9.
19.1.4.1 eNOS associates with Caveolin-1
Authors
Hemish, J, 2007-10-19.
Reviewers
Description
Caveolin-1 is the primary negative regulatory protein for eNOS. Caveolin-1 binding to eNOS compromises its ability to bind Calmodulin (CaM),
thereby inhibiting enzyme activity. The major binding region of caveolin-1 for eNOS is within amino acids 60-101 and to a lesser extent, amino
acids 135-178.
References
M Drab, P Verkade, M Elger, M Kasper, M Lohn, B Lauterbach, J Menne, C Lindschau, F Mende, FC Luft, A Schedl, H Haller, TV Kurzchalia,
"Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice", Science, 293, 2001, 2449-52.
O Feron, L Belhassen, L Kobzik, TW Smith, RA Kelly, T Michel, "Endothelial nitric oxide synthase targeting to caveolae. Specific interactions
with caveolin isoforms in cardiac myocytes and endothelial cells.", J Biol Chem, 271, 1996, 22810-4.
S Ghosh, R Gachhui, C Crooks, C Wu, MP Lisanti, DJ Stuehr, "Interaction between caveolin-1 and the reductase domain of endothelial
nitric-oxide synthase. Consequences for catalysis.", J Biol Chem, 273, 1998, 22267-71.
G Garcia-Cardena, P Martasek, BS Masters, PM Skidd, J Couet, S Li, MP Lisanti, WC Sessa, "Dissecting the interaction between nitric oxide
synthase (NOS) and caveolin. Functional significance of the nos caveolin binding domain in vivo.", J Biol Chem, 272, 1997, 25437-40.
Reaction
19.1.4.2 eNOS:Caveolin-1 complex binds to Nostrin
Authors
Hemish, J, 2007-10-19.
Reviewers
Description
eNOS interacts with the SH3 domain of NOSTRIN (positions 434-506). Caveolin-1 also binds directly to NOSTRIN (residues 323-434), thus
allowing formation of a ternary complex.
References
K Schilling, N Opitz, A Wiesenthal, S Oess, R Tikkanen, W Muller-Esterl, A Icking, "Translocation of endothelial nitric-oxide synthase involves a
ternary complex with caveolin-1 and NOSTRIN", Mol Biol Cell, 17, 2006, 3870-80.
Reaction
19.1.4.3 eNOS:Caveolin-1:NOSTRIN complex binds dynamin-2
Reviewers
Description
NOSTRIN binds to dynamin via its SH3 domain.
References
401-9.
Reaction
19.1.4.4 eNOS:Caveolin-1:NOSTRIN:dynamin-2 complex binds N-WASP
Reviewers
Description
NOSTRIN interacts with the actin nucleation promoting factor N-WASP by means of its SH3 domain.
References
401-9.
Reaction
19.1.4.5 NOSTRIN mediated translocation of eNOS from plasma membrane
Reviewers
Description
NOSTRIN translocates eNOS from the plasma membrane to intracellular vesicular structures. NOSTRIN internalization of eNOS is proposed to
occur via vesicle fission and caveolar transport through cooperation with dynamin and N-WASP.
References
Reaction
20 Metabolism of non-coding RNA
Description
The term non-coding is commonly employed for RNA that does not encode a protein, but this does not mean that such RNAs do not contain
information nor have function. There is considerable evidence that the majority of mammalian and other complex organism's genomes is
transcribed into non-coding RNAs, many of which are alternatively spliced and/or processed into smaller products. Around 98% of all
transcriptional output in humans is non-coding RNA. RNA-mediated gene regulation is widespread in higher eukaryotes and complex genetic
phenomena like RNA interference are mediated by such RNAs. These non-coding RNAs are a growing list and include rRNAs, tRNAs, snRNAs,
snoRNAs siRNAs, 7SL RNA, 7SK RNA, the RNA component of RNase P RNA, the RNA component of RNase MRP, and the RNA component of
telomerase.
20.1 snRNP Assembly
Authors

Reviewers
Luhrmann, R, 2007-04-30.
Description
Small nuclear ribonucleoproteins (snRNPs) are crucial for pre-mRNA processing to mRNAs. Each snRNP contains a small nuclear RNA
(snRNA) and an extremely stable core of seven Sm proteins. The U6 snRNA differs from the other snRNAs; it binds seven Sm-like proteins and
its assembly does not involve a cytoplasmic phase. The snRNP biogenesis pathway for all of the other snRNAs is complex, involving nuclear
export of snRNA, Sm-core assembly in the cytoplasm and re-import of the mature snRNP. The assembly of the snRNA:Sm-core is carried out by
the survival of motor neurons (SMN) complex. The SMN complex stringently scrutinizes RNAs for specific features that define them as snRNAs
and binds the RNA-binding Sm proteins.
References
J Yong, L Wan, G Dreyfuss, "Why do cells need an assembly machine for RNA-protein complexes?", Trends Cell Biol, 14, 2004, 226-32.
20.1.1 Nuclear export of snRNA transcripts
Authors
Reviewers
Luhrmann, R, 2007-04-30.
Description
The snRNAs, except U6 snRNA, are transcribed by RNA polymerase II, co-transcriptionally capped and exported rapidly to the cytoplasm in
association with a cap-binding complex and the export factor PHAX.
References
A Segref, IW Mattaj, M Ohno, "The evolutionarily conserved region of the U snRNA export mediator PHAX is a novel RNA-binding domain that is
essential for U snRNA export", RNA, 7, 2001, 351-60.
Reaction
20.1.2 Loading and methylation of Sm proteins onto SMN Complexes
Authors
Reviewers
Luhrmann, R, 2007-04-30.
Description
The survival of motor neurons (SMN) complex binds to Sm proteins and small nuclear RNAs (snRNAs) in the cytoplasm. Sm is part the SMN
multiprotein complex that contains Gemins 2 â€" 7, including the DEAD-box RNA helicase Gemin3. The binding of the SMN complex to the
snRNAs depends on the presence of specific, high-affinity (nanomolar) binding domains in the snRNAs. The SMN complex binds the Sm
proteins through the Sm domains interaction with the Gemins, the TUDOR domain, and through unique arginine- and glycine-rich (RG) domains
found in three of these, SmB, SmD1 and SmD3. The association with RG domains is strongly enhanced by the post-translational symmetric
dimethylation of specific arginines in these domains, a process that is carried out by the methylosome (JBP1 or PRMT5) complex.
References
Q Liu, U Fischer, F Wang, G Dreyfuss, "The spinal muscular atrophy disease gene product, SMN, and its associated protein SIP1 are in a
complex with spliceosomal snRNP proteins", Cell, 90, 1997, 1013-21.
AK Gubitz, Z Mourelatos, L Abel, J Rappsilber, M Mann, G Dreyfuss, "Gemin5, a novel WD repeat protein component of the SMN complex that
binds Sm proteins", J Biol Chem, 277, 2002, 5631-6.
J Baccon, L Pellizzoni, J Rappsilber, M Mann, G Dreyfuss, "Identification and characterization of Gemin7, a novel component of the survival of
motor neuron complex", J Biol Chem, 277, 2002, 31957-62.
H Brahms, L Meheus, V de Brabandere, U Fischer, R Luhrmann, "Symmetrical dimethylation of arginine residues in spliceosomal Sm protein
B/B' and the Sm-like protein LSm4, and their interaction with the SMN protein", RNA, 7, 2001, 1531-42.
WJ Friesen, G Dreyfuss, "Specific sequences of the Sm and Sm-like (Lsm) proteins mediate their interaction with the spinal muscular atrophy
disease gene product (SMN)", J Biol Chem, 275, 2000, 26370-5.
L Pellizzoni, J Baccon, J Rappsilber, M Mann, G Dreyfuss, "Purification of native survival of motor neurons complexes and identification of
Gemin6 as a novel component", J Biol Chem, 277, 2002, 7540-5.
U Fischer, Q Liu, G Dreyfuss, "The SMN-SIP1 complex has an essential role in spliceosomal snRNP biogenesis", Cell, 90, 1997, 1023-9.
B Charroux, L Pellizzoni, RA Perkinson, A Shevchenko, M Mann, G Dreyfuss, "Gemin3: A novel DEAD box protein that interacts with SMN, the
spinal muscular atrophy gene product, and is a component of gems", J Cell Biol, 147, 1999, 1181-94.
H Brahms, J Raymackers, A Union, F de Keyser, L Meheus, R Luhrmann, "The C-terminal RG dipeptide repeats of the spliceosomal Sm
proteins D1 and D3 contain symmetrical dimethylarginines, which form a major B-cell epitope for anti-Sm autoantibodies", J Biol Chem, 275,
2000, 17122-9.
B Charroux, L Pellizzoni, RA Perkinson, J Yong, A Shevchenko, M Mann, G Dreyfuss, "Gemin4. A novel component of the SMN complex that is
found in both gems and nucleoli.", J Cell Biol, 148, 2000, 1177-86.
G Meister, D Buhler, R Pillai, F Lottspeich, U Fischer, "A multiprotein complex mediates the ATP-dependent assembly of spliceosomal U
snRNPs", Nat Cell Biol, 3, 2001, 945-9.
Reaction
20.1.3 snRNP complex assembly
Authors
Reviewers
Luhrmann, R, 2007-04-30.
Description
To facilitate snRNP assembly, the SMN complex must bring together the Sm proteins and an Sm-site-containing snRNA. The SMN:Sm protein
complex binds to the m7G capped snRNAs in the cytoplasm.
References
G Meister, C Eggert, U Fischer, "SMN-mediated assembly of RNPs: a complex story", Trends Cell Biol, 12, 2002, 472-8.
D Buhler, V Raker, R Luhrmann, U Fischer, "Essential role for the tudor domain of SMN in spliceosomal U snRNP assembly: implications for
spinal muscular atrophy", Hum Mol Genet, 8, 1999, 2351-7.
L Pellizzoni, N Kataoka, B Charroux, G Dreyfuss, "A novel function for SMN, the spinal muscular atrophy disease gene product, in pre-mRNA
splicing", Cell, 95, 1998, 615-24.
Reaction
20.1.4 snRNA Cap hypermethylation
Authors
Reviewers
Luhrmann, R, 2007-04-30.
Description
The snRNA:SMN:SM protein complex is engaged by a hypermethylase that hypermethylates the snRNA cap from m7G (7-methylguanosine) to
m3G (2,2,7-trimethylguanosine).
References
IW Mattaj, "Cap trimethylation of U snRNA is cytoplasmic and dependent on U snRNP protein binding", Cell, 46, 1986, 905-11.
J Mouaikel, U Narayanan, C Verheggen, AG Matera, E Bertrand, J Tazi, R Bordonne, "Interaction between the small-nuclear-RNA cap
hypermethylase and the spinal muscular atrophy protein, survival of motor neuron", EMBO Rep, 4, 2003, 616-22.
G Plessel, U Fischer, R Luhrmann, "m3G cap hypermethylation of U1 small nuclear ribonucleoprotein (snRNP) in vitro: evidence that the U1
small nuclear RNA-(guanosine-N2)-methyltransferase is a non-snRNP cytoplasmic protein that requires a binding site on the Sm core domain",
Mol Cell Biol, 14, 1994, 4160-72.
J Mouaikel, C Verheggen, E Bertrand, J Tazi, R Bordonne, "Hypermethylation of the cap structure of both yeast snRNAs and snoRNAs requires
a conserved methyltransferase that is localized to the nucleolus", Mol Cell, 9, 2002, 891-901.
Reaction
20.1.5 snRNP:Snurportin complex formation
Authors
Reviewers
Luhrmann, R, 2007-04-30.
Description
The nuclear import signal has two parts; Cap hypermethylation triggers nuclear import via snurportin1 binding and by receptor recognition of the
Sm proteins. Snurportin1 (SPN) is an adaptor that links the assembled snRNP to the nuclear transport machinery, recruiting importin beta for
nuclear import. The import receptor that recognizes the Sm proteins is not yet known.
References
U Narayanan, JK Ospina, MR Frey, MD Hebert, AG Matera, "SMN, the spinal muscular atrophy protein, forms a pre-import snRNP complex with
snurportin1 and importin beta", Hum Mol Genet, 11, 2002, 1785-95.
J Mouaikel, U Narayanan, C Verheggen, AG Matera, E Bertrand, J Tazi, R Bordonne, "Interaction between the small-nuclear-RNA cap
hypermethylase and the spinal muscular atrophy protein, survival of motor neuron", EMBO Rep, 4, 2003, 616-22.
J Huber, U Cronshagen, M Kadokura, C Marshallsay, T Wada, M Sekine, R Luhrmann, "Snurportin1, an m3G-cap-specific nuclear import
receptor with a novel domain structure", EMBO J, 17, 1998, 4114-26.
Reaction
20.1.6 snRNP nuclear import and release
Authors

Reviewers
Luhrmann, R, 2007-04-30.
Description
A properly assembled Sm core and the m3G cap structure are prerequisites for small nuclear ribonucleoprotein (snRNP) import into the nucleus.
Once imported into the nucleus, the snRNPs are initially concentrated in Cajal bodies (CBs), where there is further processing of the snRNAs
plus binding of additional proteins, from CRBs they transit to "speckles", from where they are engaged for pre-mRNA splicing. The SMN
complexes in the nucleus are found throughout the nucleoplasm but are particularly concentrated in Gems, the "twins" of the snRNP-rich CBs.
References
JE Sleeman, AI Lamond, "Newly assembled snRNPs associate with coiled bodies before speckles, suggesting a nuclear snRNP maturation
pathway", Curr Biol, 9, 1999, 1065-74.
Reaction
21 Metabolism of vitamins and cofactors
Authors
Jassal, B, 2007-04-24.
Description
Vitamins are a diverse group of organic compounds, required in small amounts in the diet. They have distinct biochemical roles, often as
coenzymes, and are either not synthesized or synthesized only in limited amounts by human cells. Vitamins are classified according to their
solubility, either fat-soluble or water-soluble. The physiological processes dependent on vitamin-requiring reactions include many aspects of
intermediary metabolism, vision, bone formation, and blood coagulation, and vitamin deficiencies are associated with a correspondingly diverse
and severe group of diseases.
21.1 Metabolism of water-soluble vitamins and cofactors
Authors
Jassal, B, 2007-04-24.
Description
Water-soluble vitamins are a structurally and functionally diverse group of molecules, including ascorbate (vitamin C) and the members of the B
group: thiamin (vitamin B1), riboflavin (B2), niacin (B3), pantothenate (B5), pyridoxine (B6), biotin (B7), folate (B9), and cobalamin (B12).
Metabolic processes annotated in Reactome that involve these molecules are the synthesis of thiamin pyrophosphate (TPP) from thiamin (B1),
the synthesis of FMN and FAD from riboflavin (B2), the synthesis of nicotinic acid (niacin - B3) from tryptophan, the synthesis of Coenzyme A
from pantothenate (B5), and features of the metabolism of folate (B9).
21.1.1 Vitamin C (ascorbate) metabolism
Authors
Jassal, B, 2007-04-24.
Description
Vitamin C (ascorbate) is an antioxidant and a cofactor in reactions catalyzed by Cu+-dependent monooxygenases and Fe++-dependent
dioxygenases. Many mammals can synthesize ascorbate de novo; humans and other primates cannot due to an evolutionarily recent mutation in
the gene catalyzing the last step of the biosynthetic pathway. Reactions annotated here mediate the uptake of ascorbate and its fully oxidized
form, dehydroascorbate (DHA) by cells, and the reduction of DHA and monodehydroascorbate to regenerate ascorbate (Linster and Van
Schaftingen 2007).
References
CL Linster, E Van Schaftingen, "Vitamin C: biosynthesis, recycling and degradation in mammals", FEBS J, 274, 2007, 1-22.
21.1.1.1 Dehydroascorbate transport across the plasma membrane
Authors
Description
The uptake of extracellular dehydroascorbate into the cytosol is mediated by GLUT1, GLUT3, and GLUT4, associated with the plasma
membrane (Rumsey et al. 1997). This process may play a significant role in ascorbate utilization in the central nervous system (Agus et al.
1997). The process is efficiently competitively inhibited by glucose, leading to the suggestion that inhibited dehydroascorbate uptake may
contribute to the pathology of diabetes (Liang et al. 2001; Rumsey et al. 2000).
References
SC Rumsey, O Kwon, GW Xu, CF Burant, I Simpson, M Levine, "Glucose transporter isoforms GLUT1 and GLUT3 transport dehydroascorbic
acid", J Biol Chem, 272, 1997, 18982-18989.
DB Agus, SS Gambhir, WM Pardridge, C Spielholz, J Baselga, JC Vera, DW Golde, "Vitamin C crosses the blood-brain barrier in the oxidized
form through glucose transporters", J Clin Invest, 100, 1997, 2842-2848.
SC Rumsey, R Daruwala, H Al-Hasani, MJ Zarnowski, I Simpson, M Levine, "Dehydroascorbic acid transport by GLUT4 in Xenopus oocytes and
isolated rat adipocytes", J Biol Chem, 275, 2000, 28246-28253.
W-J Liang, D Johnson, SM Jarvis, "Vitamin C transport systems of mammalian cells", Mol Membr Biol, 18, 2001, 87-95.
Reaction
21.1.1.2 Ascorbate transport across the plasma membrane
Authors
Description
The plasma membrane-associated transport proteins SVCT1 and SVCT2 are each capable of mediating the uptake of one molecule of
ascorbate and two sodium ions from the extracellular space to the cytosol (Daruwala et al. 1999; Rajan et al. 1999; Wang et al. 1999). In the
body SVCT2 is expressed in most tissues, while SVCT1 is largely confined to epithelial cells (Liang et al. 2001). SVCT2 may mediate fetal
uptake of ascorbate from the maternal circulation (Rajan et al. 1999). The transporters responsible for its uptake from the small intestine and for
its release from enterocytes into the circulation have not been identified, although both SVCT1 and 2 are expressed in intestinal cells.
References
R Daruwala, J Song, WS Koh, SC Rumsey, M Levine, "Cloning and functional characterization of the human sodium-dependent vitamin C
transporters hSVCT1 and hSVCT2", FEBS Lett, 460, 1999, 480-484.
H Wang, B Dutta, W Huang, LD Devoe, FH Leibach, V Ganapathy, PD Prasad, "Human Na+-dependent vitamin C transporter 1 (hSVCT1):
primary structure, functional characteristics and evidence for a non-functional splice variant", Biochim Biophys Acta, 1461, 1999, 1-9.
DP Rajan, W Huang, B Dutta, LD Devoe, FH Leibach, V Ganapathy, PD Prasad, "Human placental sodium-dependent vitamin C transporter
(SVCT2): molecular cloning and transport function", Biochem Biophys Res Commun, 262, 1999, 762-768.
W-J Liang, D Johnson, SM Jarvis, "Vitamin C transport systems of mammalian cells", Mol Membr Biol, 18, 2001, 87-95.
Reaction
21.1.1.3 Reduction of semidehydroascorbate to ascorbate

Authors
Description
The reduction of cytosolic semidehydroascorbate to ascorbate is catalyzed by cytochrome B5 (CYB5A) associated with the mitochondrial outer
membrane. In the course of the reaction, the heme iron of the cytochrome is oxidized (Linster and Van Schaftingen 2007; Shirabe et al. 1995).
References
K Shirabe, MT Landi, M Takeshita, G Uziel, E Fedrizzi, N Borgese, "A novel point mutation in a 3' splice site of the NADH-cytochrome b5
reductase gene results in immunologically undetectable enzyme and impaired NADH-dependent ascorbate regeneration in cultured fibroblasts of
a patient with type II hereditary methemoglobinemia", Am J Hum Genet, 57, 1995, 302-310.
CL Linster, E Van Schaftingen, "Vitamin C: biosynthesis, recycling and degradation in mammals", FEBS J, 274, 2007, 1-22.
Reaction
21.1.1.4 Reduction of dehydroascorbate to ascorbate
Authors
Description
Cytosolic omega class glutathione transferases (GSTO1 and GSTO2) catalyze the reaction of dehydroascorbate (DHA) and glutathione (GSH)
to form ascorbate and oxidized glutathione (GSSG). The GSTO enzymes occur as homodimers (Board et al. 2000), and while both have
dehydroascorbate reductase activity in vitro, that of GSTO2 is much greater than that of GSTO1 (Schmuck et al. 2005). Polymorphisms affecting
the activities of the two enzymes have been described (Whitbread et al. 2005).
References
EM Schmuck, PG Board, AK Whitbread, N Tetlow, JA Cavanaugh, AC Blackburn, A Masoumi, "Characterization of the monomethylarsonate
reductase and dehydroascorbate reductase activities of Omega class glutathione transferase variants: implications for arsenic metabolism and
the age-at-onset of Alzheimer's and Parkinson's diseases", Pharmacogenet Genomics, 15, 2005, 493-501.
PG Board, M Coggan, G Chelvanayagam, S Easteal, LS Jermiin, GK Schulte, DE Danley, LR Hoth, MC Griffor, AV Kamath, MH Rosner, BA
Chrunyk, DE Perregaux, CA Gabel, KF Geoghegan, J Pandit, "Identification, characterization, and crystal structure of the Omega class
glutathione transferases", J Biol Chem, 275, 2000, 24798-806.
AK Whitbread, A Masoumi, N Tetlow, E Schmuck, M Coggan, PG Board, "Characterization of the omega class of glutathione transferases",
Methods Enzymol, 401, 2005, 78-99.
Reaction
21.1.2 Vitamin B1 (thiamin) metabolism
Authors
Jassal, B, 2007-04-24.
Description
Vitamin B1 (thiamin) is found naturally in certain foodstuffs such as green peas, spinach, liver, bananas, whole grains and legumes. Human
diseases associated with thiamin deficiency include beriberi, due to a thiamin-deficient diet, TMRA, due to defects in the SLC19A2 transport
protein, and Wernicke-Korsakoff Syndrome, associated with thiamin deficiency in alcoholism (Haas 1988). Thiamin is water-soluble so is not
stored in the body. When pyrophosphorylated, thiamin is converted into the coenzyme thiamin pyrophosphate (ThPP, codecarboxylase) which
plays an essential role in oxidative decarboxylation and group transfer reactions.
References
RH Haas, "Thiamin and the brain", Annu Rev Nutr, 8, 1988, 483-515.
21.1.2.1 Thiamin transport across the plasma membrane
Authors
Description
Two transport proteins, SLC19A2 (THTR1) and SLC19A3 (THTR2), associated with the plasma membrane, are each able to mediate the
transport of extracellular thiamin into the cytosol. In the body, both transporters are widely distributed, and both are abundant in kidney and
intestinal epithelia, consistent with their involvement in thiamin uptake under physiological conditions (Ashokkumar et al. 2006; Said et al. 2004;
Subramanian et al. 2006 - J Biol Chem). The observation that mutations in SLC19A2 (THTR1) cause a progressive disorder that can be partially
reversed by treatment with high doses of thiamin likewise suggests a role for this protein in thiamin uptake under normal conditions (Diaz et al.
1999; Fleming et al. 1999; Labay et al. 1999).
Two features of this transport process remain incompletely understood, however. First, mutations in SLC19A3 cause a progressive disorder that
is responsive to biotin treatment (Zhou et al. 2005), although studies of cultured cells indicate that the protein has no affinity for biotin
(Subramanian et al. 2006 - Am J Physiol). Also, studies to date provide little information about the mechanism by which thiamin, once taken up
by epithelial cells in the intestine and kidney, is released from these cells into the blood.
References
VS Subramanian, JS Marchant, HM Said, "Biotin-responsive basal ganglia disease-linked mutations inhibit thiamine transport via hTHTR2: biotin
is not a substrate for hTHTR2", Am J Physiol Cell Physiol, 291, 2006, C851-9.
HM Said, K Balamurugan, VS Subramanian, JS Marchant, "Expression and functional contribution of hTHTR-2 in thiamin absorption in human
intestine", Am J Physiol Gastrointest Liver Physiol, 286, 2004, G491-8.
B Ashokkumar, ND Vaziri, HM Said, "Thiamin uptake by the human-derived renal epithelial (HEK-293) cells: cellular and molecular
mechanisms", Am J Physiol Renal Physiol, 291, 2006, F796-805.
JC Fleming, E Tartaglini, MP Steinkamp, DF Schorderet, N Cohen, EJ Neufeld, "The gene mutated in thiamine-responsive anaemia with
diabetes and deafness (TRMA) encodes a functional thiamine transporter", Nat Genet, 22, 1999, 305-8.
V Labay, T Raz, D Baron, H Mandel, H Williams, T Barrett, R Szargel, L McDonald, A Shalata, K Nosaka, S Gregory, N Cohen, "Mutations in
SLC19A2 cause thiamine-responsive megaloblastic anaemia associated with diabetes mellitus and deafness", Nat Genet, 22, 1999, 300-4.
VS Subramanian, JS Marchant, HM Said, "Targeting and trafficking of the human thiamine transporter-2 in epithelial cells", J Biol Chem, 281,
2006, 5233-45.
GA Diaz, M Banikazemi, K Oishi, RJ Desnick, BD Gelb, "Mutations in a new gene encoding a thiamine transporter cause thiamine-responsive
megaloblastic anaemia syndrome", Nat Genet, 22, 1999, 309-12.
Reaction
21.1.2.2 Thiamin is pyrophosphorylated
Description
Cytosolic thiamin pyrophosphokinase (TPK) catalyzes the reaction of thiamin and ATP to form thiamin pyrophosphate (TPP) (thiamin
diphosphate) and ADP. TPP is an active cofactor for transketolase, pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase, enzymes
involved in glycolysis and energy production. The gene encoding the human enzyme has been cloned and its protein product has been shown to
have TPK activity (Nosaka et al. 2001; Zhao et al. 2001). Its homodomeric structure and association with Mg++ are inferred from properties of
the homologous yeast enzyme (Baker et al. 2001).
References
R Zhao, F Gao, ID Goldman, "Molecular cloning of human thiamin pyrophosphokinase", Biochim Biophys Acta, 1517, 2001, 320-2.
K Nosaka, M Onozuka, N Kakazu, S Hibi, H Nishimura, H Nishino, T Abe, "Isolation and characterization of a human thiamine
pyrophosphokinase cDNA", Biochim Biophys Acta, 1517, 2001, 293-7.
LJ Baker, JA Dorocke, RA Harris, DE Timm, "The crystal structure of yeast thiamin pyrophosphokinase", Structure, 9, 2001, 539-46.
Reaction
21.1.3 Vitamin B2 (riboflavin) metabolism
Description
Riboflavin (vitamin B2, E101) is an essential component for the cofactors FAD (flavin-adenine dinucleotide) and FMN (flavin mononucleotide).
Together with NAD+ and NADP+, FAD and FMN are important hydrogen carriers and take part in more than 100 redox reactions involved in
energy metabolism. Riboflavin is present in many vegetables and meat and during digestion, various flavoproteins from food are degraded and
riboflavin is resorbed. The major degradation and excretion product in humans is riboflavin.
21.1.3.1 Riboflavin is phosphorylated to FMN
Description
Phosphorylation of riboflavin results in the formation of the first cofactor, FMN (flavin mononucleotide). This reaction is catalyzed by riboflavin
kinase, a cytosolic enzyme existing as a monomer. It utilizes either zinc or magnesium ions in the reaction.
References
S Karthikeyan, Q Zhou, F Mseeh, NV Grishin, AL Osterman, H Zhang, "Crystal structure of human riboflavin kinase reveals a beta barrel fold
and a novel active site arch", Structure, 11, 2003, 265-73.
Reaction
21.1.3.2 FMN is futher phosphorylated to FAD
Description
FMN can be phosphorylated and adenylated to produce the second cofactor from riboflavin origins, FAD (flavin adenine dinucleotide). The
enzyme responsible , FMN adenylyltransferase (FAD synthase), is cytosolic and transfers a phosphate and an adenyl group from ATP to form
FAD.
References
C Brizio, M Galluccio, R Wait, EM Torchetti, V Bafunno, R Accardi, E Gianazza, C Indiveri, M Barile, "Over-expression in Escherichia coli and
characterization of two recombinant isoforms of human FAD synthetase", Biochem Biophys Res Commun, 344, 2006, 1008-16.
Reaction
21.1.3.3 FAD can be hydrolyzed back to FMN

Description
Phosphatase action on FAD can reform FMN. The enzyme performing the reaction is nucleotide pyrophosphatase and it exists as a homodimer
on the plasma membrane.
References
SI Belli, JW Goding, "Biochemical characterization of human PC-1, an enzyme possessing alkaline phosphodiesterase I and nucleotide
pyrophosphatase activities", Eur J Biochem, 226, 1994, 433-43.
Reaction
21.1.3.4 FMN can be hydrolyzed back to riboflavin
Description
Cytosolic TRAP (tartrate-resistant acid phosphatase type 5) catalyzes the hydrolysis of FMN to yield riboflavin and orthophosphate.
References
AR Hayman, MJ Warburton, JA Pringle, B Coles, TJ Chambers, "Purification and characterization of a tartrate-resistant acid phosphatase from
human osteoclastomas", Biochem J, 261, 1989, 601-9.
Reaction
21.1.4 Vitamin B5 (pantothenate) metabolism
Authors
Description
Panthothenate (vitamin B5) is the precursor of coenzyme A and is the prosthetic group of acyl carrier protein (ACP). Its name is derived from the
Greek pantothen meaning "from everywhere" and small quantities of pantothenic acid are found in nearly every foodstuff.
21.1.4.1 Pantothenate transport across the plasma membrane
Authors
Description
The plasma membrane-associated transport protein SLC5A6 (SMVT) mediates the uptake of one molecule of pantothenate and two sodium ions
from the extracellular space to the cytosol. Limited Northern blotting studies suggest that SLC5A6 is widely expressed, and abundant in placenta
and small intestine. SLC5A6 may thus play a central role in pantothenate uptake. SLC5A6 also mediates the uptake of biotin and lipoate (Prasad
et al. 1999; Wang et al. 1999).
References
H Wang, W Huang, YJ Fei, H Xia, TL Yang-Feng, FH Leibach, LD Devoe, V Ganapathy, PD Prasad, "Human placental Na+-dependent
multivitamin transporter. Cloning, functional expression, gene structure, and chromosomal localization.", J Biol Chem, 274, 1999, 14875-83.
PD Prasad, H Wang, W Huang, YJ Fei, FH Leibach, LD Devoe, V Ganapathy, "Molecular and functional characterization of the intestinal
Na+-dependent multivitamin transporter", Arch Biochem Biophys, 366, 1999, 95-106.
Reaction
21.1.4.2 Coenzyme A biosynthesis
Authors
Jassal, B, 2007-04-24.
Description
Coenzyme A (CoA) is a ubiquitous cofactor that functions as an acyl group carrier in diverse processes including fatty acid metabolism and the
TCA cycle (Lipmann 1953). It is synthesized from the vitamin pantothenate in a sequence of five reactions (Daugherty et al. 2002; Leonardi et al.
2005; Robishaw and Neely 1985). These reactions all occur in the cytosol or the mitochondrial intermembrane space (Leonardi et al. 2005). A
recently described transport protein appears to mediate the uptake of Coenzyme A into the mitochondrial matrix (Prohl et al. 2001).
References
JD Robishaw, JR Neely, "Coenzyme A metabolism", Am J Physiol, 248, 1985, E1-9.
F Lipmann, "On chemistry and function of coenzyme A", Bacteriol Rev, 17, 1953, 1-16.
M Daugherty, B Polanuyer, M Farrell, M Scholle, A Lykidis, V de Crecy-Lagard, A Osterman, "Complete reconstitution of the human coenzyme A
biosynthetic pathway via comparative genomics", J Biol Chem, 277, 2002, 21431-9.
C Prohl, W Pelzer, K Diekert, H Kmita, T Bedekovics, G Kispal, R Lill, "The yeast mitochondrial carrier Leu5p and its human homologue Graves'
disease protein are required for accumulation of coenzyme A in the matrix", Mol Cell Biol, 21, 2001, 1089-97.
R Leonardi, YM Zhang, CO Rock, S Jackowski, "Coenzyme A: back in action", Prog Lipid Res, 44, 2005, 125-53.
21.1.4.2.1 Pantothenate is phosphorylated [PANK2]
Authors
Jassal, B, 2007-04-24.
Description
Pantothenate kinase 2 catalyzes the reaction of ATP and pantothenate to form ADP and phosphopantothenate. While pantothenate kinase 2
co-purifies with mitocondria, its precise location within the mitochondrion has not been established (Hortnagel et al. 2003; Johnson et al. 2004).
Recent work by Leonardi et al. (2007) supports a model in which the enzyme is located in the intermembrane space, hence freely accessible to
small molecules from the cytosol.Pantothenate is phosphorylated by pantothenate kinase (PANK). Deficiencies in PANK2 cause a progressive
neurodegenerative disorder associated with iron accumulation in the brain, but the relationship between disease symptoms and pantothenate
metabolism remains unclear (Zhou et al. 2001; Zhang et al. 2006).
References
K Hortnagel, H Prokisch, T Meitinger, "An isoform of hPANK2, deficient in pantothenate kinase-associated neurodegeneration, localizes to
mitochondria", Hum Mol Genet, 12, 2003, 321-7.
R Leonardi, CO Rock, S Jackowski, YM Zhang, "Activation of human mitochondrial pantothenate kinase 2 by palmitoylcarnitine", Proc Natl Acad
Sci U S A, 104, 2007, 1494-9.
YM Zhang, CO Rock, S Jackowski, "Biochemical properties of human pantothenate kinase 2 isoforms and mutations linked to pantothenate
kinase-associated neurodegeneration", J Biol Chem, 281, 2006, 107-14.
MA Johnson, YM Kuo, SK Westaway, SM Parker, KH Ching, J Gitschier, SJ Hayflick, "Mitochondrial localization of human PANK2 and
hypotheses of secondary iron accumulation in pantothenate kinase-associated neurodegeneration", Ann N Y Acad Sci, 1012, 2004, 282-98.
B Zhou, SK Westaway, B Levinson, MA Johnson, J Gitschier, SJ Hayflick, "A novel pantothenate kinase gene (PANK2) is defective in
Hallervorden-Spatz syndrome", Nat Genet, 28, 2001, 345-9.
Reaction
21.1.4.2.2 Pantothenate is phosphorylated [PANK1;3;4]
Authors
Description
Cytosolic pantothenate kinases catalyze the reaction of ATP and pantothenate to form ADP and phosphopantothenate. This enzymatic activity
has been demonstrated in crude cell extracts for two isoforms of mouse pantothenate kinase 1 (Rock et al. 2002) and for their human
homologues (Ramaswamy and 2004). Two additional human genes, PANK3 and PANK4, encode closely related proteins but pantothenate
kinase activity has not been demonstrated experimentally for them (Leonardi et al. 2005; Zhou et al. 2001).
References
R Leonardi, YM Zhang, CO Rock, S Jackowski, "Coenzyme A: back in action", Prog Lipid Res, 44, 2005, 125-53.
G Ramaswamy, MA Karim, KG Murti, S Jackowski, "PPARalpha controls the intracellular coenzyme A concentration via regulation of
PANK1alpha gene expression", J Lipid Res, 45, 2004, 17-31.
CO Rock, MA Karim, YM Zhang, S Jackowski, "The murine pantothenate kinase (Pank1) gene encodes two differentially regulated pantothenate
kinase isozymes", Gene, 291, 2002, 35-43.
B Zhou, SK Westaway, B Levinson, MA Johnson, J Gitschier, SJ Hayflick, "A novel pantothenate kinase gene (PANK2) is defective in
Hallervorden-Spatz syndrome", Nat Genet, 28, 2001, 345-9.
Reaction
21.1.4.2.3 Condensation of phosphopantothenate with cysteine
Authors
Jassal, B, 2007-04-24.
Description
The conjugation of cysteine and 4'- phosphopantothenate to form 4-phosphopantothenoylcysteine, coupled to the conversion of ATP to AMP
and pyrophosphate, is catalyzed by cytosolic phosphopantothenate-cysteine ligase (Phosphopantothenoylcysteine synthase, PPC synthase).
Mammalian PPC synthase prefers ATP to CTP, unlike the E. coli ortholog (Daughtery et al. 2002; Manoj et al. 2003).
References
N Manoj, E Strauss, TP Begley, SE Ealick, "Structure of human phosphopantothenoylcysteine synthetase at 2.3 A resolution.", Structure, 11,
2003, 927-36.
Reaction
21.1.4.2.4 Phosphopantothenoylcysteine is decarboxylated
Authors
Jassal, B, 2007-04-24.
Description
The decarboxylation of phosphopantothenoylcysteine is carried out by phosphopantothenoylcysteine decarboxylase (PPCDC). PPCDC is

cytosolic and exists as a homotrimer, binding one FMN cofactor per subunit. While a second isoform has been inferred from large-scale
sequnceing studies, it lacks the protein's FMN-binding domain and would thus appear to be nonfunctional if it is expressed.
References
E Strauss, H Zhai, LA Brand, FW McLafferty, TP Begley, "Mechanistic studies on phosphopantothenoylcysteine decarboxylase: trapping of an
enethiolate intermediate with a mechanism-based inactivating agent", Biochemistry, 43, 2004, 15520-33.
Reaction
21.1.4.2.5 Adenylation of phosphopantetheine
Authors
Jassal, B, 2007-04-24.
Description
The adenylate transferase activity of bifunctional coenzyme A synthase catalyzes the reaction of pantetheine phosphate and ATP to form
dephospho-Coenzyme A and orthophosphate (Daugherty et al. 2002). The enzyme is associated with the mitochondrial outer membrane
(Zhyvoloup et al. 2003).
References
S Aghajanian, DM Worrall, "Identification and characterization of the gene encoding the human phosphopantetheine adenylyltransferase and
dephospho-CoA kinase bifunctional enzyme (CoA synthase)", Biochem J, 365, 2002, 13-8.
A Zhyvoloup, I Nemazanyy, G Panasyuk, T Valovka, T Fenton, H Rebholz, ML Wang, R Foxon, V Lyzogubov, V Usenko, R Kyyamova, O
Gorbenko, G Matsuka, V Filonenko, IT Gout, "Subcellular localization and regulation of coenzyme A synthase", J Biol Chem, 278, 2003,
50316-21.
Reaction
21.1.4.2.6 Phosphorylation of dephospho-CoA to produce CoA
Authors
Jassal, B, 2007-04-24.
Description
The kinase activity of CoA synthase catalyzes the reaction of Dephospho-CoA and ATP to form Coenzyme A and ADP. The enzyme is located
in the mitochondrial outer membrane (Daugherty et al. 2002; Zhyvoloup et al. 2003).
References
S Aghajanian, DM Worrall, "Identification and characterization of the gene encoding the human phosphopantetheine adenylyltransferase and
dephospho-CoA kinase bifunctional enzyme (CoA synthase)", Biochem J, 365, 2002, 13-8.
A Zhyvoloup, I Nemazanyy, G Panasyuk, T Valovka, T Fenton, H Rebholz, ML Wang, R Foxon, V Lyzogubov, V Usenko, R Kyyamova, O
Gorbenko, G Matsuka, V Filonenko, IT Gout, "Subcellular localization and regulation of coenzyme A synthase", J Biol Chem, 278, 2003,
50316-21.
Reaction
21.1.4.2.7 CoA transport across the inner mitochondrial membrane
Authors
Description
SLC25A16, associated with the inner mitochondrial membrane, mediates the transport of CoA (coenzyme A) into the mitochondrial matrix.
Evidence for this event is indirect. The protein has the sequence motifs expected for a transport protein, and yeast cells deficient in its
homologue, Leu5p, fail to accumulate mitochondrial CoA and can be rescued by expression of SLC25A16. At the same time, neither the yeast
nor the human protein has been shown directly to function as a transporter (Prohl et al. 2001; Leonardi et al. 2007).
References
R Leonardi, CO Rock, S Jackowski, YM Zhang, "Activation of human mitochondrial pantothenate kinase 2 by palmitoylcarnitine", Proc Natl Acad
Sci U S A, 104, 2007, 1494-9.
C Prohl, W Pelzer, K Diekert, H Kmita, T Bedekovics, G Kispal, R Lill, "The yeast mitochondrial carrier Leu5p and its human homologue Graves'
disease protein are required for accumulation of coenzyme A in the matrix", Mol Cell Biol, 21, 2001, 1089-97.
Reaction
21.1.4.3 Phosphopantetheine conjugation of the ACP domain of FAS
Authors
Description
Cytosolic AASDHPPT (alpha-aminoadipic semialdehyde dehydrogenase-phosphopantetheinyl transferase) catalyzes the transfer of a

phosphopantetheine moiety from coenzyme A to serine 2156 within the ACP domain of FAS (fatty acyl synthase). Only a single human enzyme
with phosphopantetheinyl transferase activity has been identified, and its broad substrate specificity suggests that it may be responsible as well
for the postranslational modification of enzymes of lysine catabolism (Joshi et al. 2003; Praphanphoj et al. 2001).
References
AK Joshi, L Zhang, VS Rangan, S Smith, "Cloning, expression, and characterization of a human 4'-phosphopantetheinyl transferase with broad
substrate specificity", J Biol Chem, 278, 2003, 33142-9.
V Praphanphoj, KA Sacksteder, SJ Gould, GH Thomas, MT Geraghty, "Identification of the alpha-aminoadipic semialdehyde

dehydrogenase-phosphopantetheinyl transferase gene, the human ortholog of the yeast LYS5 gene", Mol Genet Metab, 72, 2001, 336-42.
Reaction
21.1.5 Vitamin B12 (cobalamin) metabolism
Description
Under construction - the process by which vitamin B12 is metabolized.

21.1.6 Biotin metabolism
Authors
Jassal, B, 2007-04-24.
Description
Biotin is an essential cofactor in a variety of carboxylation reactions. It is abundant in the human diet, and can be taken up from the intestinal
lumen by the SLC5A6 transporter. Its covalent attachment to carboxylase enzymes is catalyzed by the HLCS enzyme in a reaction to be
annotated in a future release of Reactome.
21.1.6.1 Biotin transport across the plasma membrane
Authors
Description
The plasma membrane-associated transport protein SLC5A6 (SMVT) mediates the uptake of one molecule of biotin and two sodium ions from
the extracellular space to the cytosol. Limited Northern blotting studies suggest that SLC5A6 is widely expressed, and abundant in placenta and
small intestine. SLC5A6 may thus play a central role in biotin uptake. SLC5A6 also mediates the uptake of pantothenate and lipoate (Prasad et
al. 1999; Wang et al. 1999).
References
H Wang, W Huang, YJ Fei, H Xia, TL Yang-Feng, FH Leibach, LD Devoe, V Ganapathy, PD Prasad, "Human placental Na+-dependent
multivitamin transporter. Cloning, functional expression, gene structure, and chromosomal localization.", J Biol Chem, 274, 1999, 14875-83.
PD Prasad, H Wang, W Huang, YJ Fei, FH Leibach, LD Devoe, V Ganapathy, "Molecular and functional characterization of the intestinal
Na+-dependent multivitamin transporter", Arch Biochem Biophys, 366, 1999, 95-106.
Reaction
21.1.7 Nicotinate metabolism
Description
Nicotinate (niacin) and nicotinamide are precursors of the coenzymes nicotinamide-adenine dinucleotide (NAD+) and nicotinamide-adenine
dinucleotide phosphate (NADP+). When NAD+ and NADP+ are interchanged in a reaction with their reduced forms, NADH and NADPH
respectively, they are important cofactors in several hundred redox reactions. Nicotinate is synthesized from tryptophan, which is an essential
amino acid in humans.
References
G Magni, A Amici, M Emanuelli, G Orsomando, N Raffaelli, S Ruggieri, "Enzymology of NAD+ homeostasis in man", Cell Mol Life Sci, 61, 2004,
19-34.
21.1.7.1 tryptophan + O2 => N-formylkynurenine [TDO]
Description
Cytosolic tryptophan 2,3-dioxygenase (TDO) tetramer catalyzes the conversion of L-tryptophan and oxygen to formylkynurenine. The structure
and catalytic properties of the human enzyme are inferred from the close similarity of its predicted amino acid sequence (Comings et al. 1995) to
that of the well-studied rat enzyme (Dick et al. 2001). In the body, TDO is found predominantly in the liver and is induced by metabolites such as
tryptophan and histidine, and by glucocorticoids. These properties, together with TDO's narrow substrate specificity, are consistent with the
hypothesis that the enzyme functions functions primarily in tryptophan catabolism and NAD biosynthesis (Taylor and Feng 1991).
References
2516-2522.
DE Comings, G Dietz, GL Forest, D Muhleman, M Sherman, "Sequence of human tryptophan 2,3-dioxygenase (TDO2): presence of a
glucocorticoid response-like element composed of a GTT repeat and an intronic CCCCT repeat", Genomics, 29, 1995, 390-396.
R Dick, BP Murray, MJ Reid, MA Correia, "Structure-function relationships of rat hepatic tryptophan 2,3-dioxygenase: identification of the
putative heme-ligating histidine residues", Arch Biochem Biophys, 392, 2001, 71-78.
Reaction
21.1.7.2 tryptophan + O2 => N-formylkynurenine [IDO]
Description
Cytosolic indoleamine 2,3-dioxygenase (IDO) catalyzes the conversion of L-tryptophan and oxygen to formylkynurenine. The structure and
catalytic properties of the human enzyme have been analyzed directly (Sugimoto et al. 2006); the subcellular location and monomeric state of
the active form of the enzyme are inferred from the properties of its rabbit ortholog (Shimizu et al. 1976). In the body, IDO is ubiquitously
expressed and is induced by interferon. These properties, together with IDO's broad substrate specificity, are consistent with the hypothesis that
the enzyme functions functions in anti bacterial and inflammatory processes (Taylor and Feng 1991).
References
2516-2522.
H Sugimoto, SI Oda, T Otsuki, T Hino, T Yoshida, Y Shiro, "Crystal structure of human indoleamine 2,3-dioxygenase: catalytic mechanism of O2
incorporation by a heme-containing dioxygenase", Proc Natl Acad Sci USA, 103, 2006, 2611-2616.
T Shimizu, S Nomiyama, F Hirata, O Hayaishi, "Indoleamine 2,3-dioxygenase. Purification and some properties", J Biol Chem, 253, 1978,
4700-4706.
Reaction
21.1.7.3 N-formylkynurenine + H2O => kynurenine + formate
Description
In this cytosolic reaction, arylformamidase catalyzes the hydrolysis of formylkynurenine to yield formate and L-kynurenine.
Source reaction
This reaction was inferred from the corresponding reaction "N-formylkynurenine + H2O => kynurenine + formate [mouse]" in species Mus
musculus.
MK Pabarcus, JE Casida, "Kynurenine formamidase: determination of primary structure and modeling-based prediction of tertiary structure and
catalytic triad.", Biochim Biophys Acta, 1596, 2002, 201-11.
Reaction
21.1.7.4 kynurenine + O2 + NADPH + H+ => 3-hydroxykynurenine + NADP+ + H2O
Description
Cytosolic kynurenine 3-monooxygenase catalyzes the reaction of L-kynurenine, NADPH + H+, and O2 to form 3-hydroxy-L-kynurenine, NADP+,
and H2O.
References
J Breton, N Avanzi, S Magagnin, N Covini, G Magistrelli, L Cozzi, A Isacchi, "Functional characterization and mechanism of action of
recombinant human kynurenine 3-hydroxylase", Eur J Biochem, 267, 2000, 1092-1099.
Reaction
21.1.7.5 3-hydroxykynurenine + H2O => 3-hydroxyanthranilate + alanine
Description
Cytosolic kynureninase catalyzes the hydrolysis of 3-hydroxy-L-kynurenine to form L-alanine and 3-hydroxyanthranilate.
References
S Toma, M Nakamura, S TonÃ©, E Okuno, R Kido, J Breton, N Avanzi, L Cozzi, C Speciale, M Mostardini, S Gatti, L Benatti, "Cloning and
recombinant expression of rat and human kynureninase.", FEBS Lett, 408, 1997, 5-10.
Reaction
21.1.7.6 3-hydroxyanthranilate + O2 => 2-amino-3-carboxymuconate semialdehyde
Description
Cytosolic 3-hydroxyanthranilate oxygenase catalyzes the reaction of 3-hydroxyanthranilate and O2 to form 2-amino-3-carboxymuconate
semialdehyde.
References
P Malherbe, C KÃ¶hler, M Da Prada, G Lang, V Kiefer, R Schwarcz, HW Lahm, AM Cesura, "Molecular cloning and functional expression of
human 3-hydroxyanthranilic-acid dioxygenase.", J Biol Chem, 269, 1994, 13792-7.
Reaction
21.1.7.7 2-amino 3-carboxymuconate semialdehyde transforms non-enzymatically to quinolinate
Description
Cytosolic 2-amino 3-carboxymuconate semialdehyde reacts non-enzymatically to form quinolinate and water (Fukuoka et al. 1998).
References
SI Fukuoka, CM Nyaruhucha, K Shibata, "Characterization and functional expression of the cDNA encoding human brain quinolinate
phosphoribosyltransferase", Biochim Biophys Acta, 1395, 1998, 192-201.
Reaction
21.1.7.8 A phospho-ribosyl group is added to quinolinate
Description
The enzyme, nicotinate nucleotide pyrophosphorylase, is specific for quinolinate. Its activity is strictly dependent on Mg2+ ions being present. A
phosphoribosyl group is transferred to quinolinate to form nicotinate D-ribonucleotide. This reaction represents another rate-limiting step of the
pathway from tryptophan to NAD+.
References
SI Fukuoka, CM Nyaruhucha, K Shibata, "Characterization and functional expression of the cDNA encoding human brain quinolinate
phosphoribosyltransferase", Biochim Biophys Acta, 1395, 1998, 192-201.
Reaction
21.1.7.9 Nicotinate D-ribonucleotide + ATP => deamino-NAD+ + pyrophosphate [NMNAT1]
Description
NMNAT1 catalyzes the reaction of nicotinate D-ribonucleotide and ATP to form deamino-NAD+ (nicotinate adenine dinucleotide) and
pyrophosphate (Schweiger et al. 2001). The active form of the enzyme in vitro is a hexamer (Zhou et al. 2002), and its activity is substantially
greater in the presence of Zn++ than of Mg++ (Sorci et al. 2007). The predicted amino acid sequence of the enzyme contains a nuclear
localization domain and the protein is observed to localize to the nucleus (Schweiger et al. 2001; Berger et al. 2005).
References
T Zhou, OV Kurnasov, DR Tomchick, DD Binns, NV Grishin, VE Marquez, AL Osterman, H Zhang, "Structure of human nicotinamide/nicotinic
acid mononucleotide adenylyltransferase. Basis for the dual substrate specificity and activation of the oncolytic agent tiazofurin.", J Biol Chem,
277, 2002, 13148-54.
M Schweiger, K Hennig, F Lerner, M Niere, M Hirsch-Kauffmann, T Specht, C Weise, SL Oei, M Ziegler, "Characterization of recombinant
human nicotinamide mononucleotide adenylyl transferase (NMNAT), a nuclear enzyme essential for NAD synthesis", FEBS Lett, 492, 2001,
95-100.
F Berger, C Lau, M Dahlmann, M Ziegler, "Subcellular compartmentation and differential catalytic properties of the three human nicotinamide
mononucleotide adenylyltransferase isoforms", J Biol Chem, 280, 2005, 36334-41.
L Sorci, F Cimadamore, S Scotti, R Petrelli, L Cappellacci, P Franchetti, G Orsomando, G Magni, "Initial-rate kinetics of human
NMN-adenylyltransferases: substrate and metal ion specificity, inhibition by products and multisubstrate analogues, and isozyme contributions to
NAD+ biosynthesis", Biochemistry, 46, 2007, 4912-22.
Reaction
Description
pyrophosphate (Sorci et al. 2007). The active form of the enzyme is a monomer in vitro (Raffaelli et al. 2002). Although the predicted amino acid
sequence of the enzyme lacks an obvious signal sequence or transmembrane domain (Yalowitz et al. 2004), recombinant FLAG-tagged protein
expressed in HeLa cells localizes predominantly to the Golgi apparatus (Berger et al. 2005). Its localization within the Golgi apparatus is
unknown and the annotation here is based on the plausible but speculative assumption that the enzyme is associated with the Gogi membrane
and accessible from the cytosol. Immunostaining studies indicate that the protein is abundant in Islets of Langerhans and in several regions of
the brain (Yalowitz et al. 2004).
References
JA Yalowitz, S Xiao, MP Biju, AC Antony, OW Cummings, MA Deeg, HN Jayaram, "Characterization of human brain nicotinamide
5'-mononucleotide adenylyltransferase-2 and expression in human pancreas", Biochem J, 377, 2004, 317-26.
N Raffaelli, L Sorci, A Amici, M Emanuelli, F Mazzola, G Magni, "Identification of a novel human nicotinamide mononucleotide
adenylyltransferase", Biochem Biophys Res Commun, 297, 2002, 835-40.
Reaction
Description
pyrophosphate (Sorci et al. 2007). The active form of the enzyme is a tetramer in vitro (Zhang et al. 2003). Recombinant FLAG-tagged protein
expressed in HeLa cells localizes both to the cytosol and to mitochondria (Berger et al. 2005). The cytosolic protein is annotated here.
References
X Zhang, OV Kurnasov, S Karthikeyan, NV Grishin, AL Osterman, H Zhang, "Structural characterization of a human cytosolic NMN/NaMN
adenylyltransferase and implication in human NAD biosynthesis", J Biol Chem, 278, 2003, 13503-11.
Reaction
21.1.7.12 Amidation of deamino-NAD+ to NAD+
Description
NAD synthase catalyzes the final step in the biosynthesis of NAD+, both in the de novo synthesis and in the salvage pathways. The enzyme
makes use of glutamine as an amide donor in the reaction. NAD synthase exists as a homohexamer in the cytosol. There are two forms of NAD
synthase in humans, NADsyn1 and NADsyn2. The major difference between the two forms is that NADsyn1 appears to be glutamine-dependent
whereas NADsyn2 is strictly ammonia-dependent.
References
N Hara, K Yamada, M Terashima, H Osago, M Shimoyama, M Tsuchiya, "Molecular identification of human glutamine- and ammonia-dependent
NAD synthetases. Carbon-nitrogen hydrolase domain confers glutamine dependency.", J Biol Chem, 278, 2003, 10914-21.
Reaction
21.1.7.13 NAD+ is phosphorylated to NADP+
Description
NAD+ kinase catalyzes the transfer of a phosphate group from ATP to NAD+, forming NADP+. This is the only way to generate NADP+ in all
living organisms. The enzyme requires a divalent metal to be effective. Zn2+ is the best metal for this purpose.
References
F Lerner, M Niere, A Ludwig, M Ziegler, "Structural and functional characterization of human NAD kinase", Biochem Biophys Res Commun, 288,
2001, 69-74.
Reaction
21.1.7.14 Nicotinamide salvaging
Description
Nicotinamide levels are modulated by the action of three enzymes involved in nicotinamide salvaging. They are nicotinamide deaminase,
nicotinamide phosphoribosyltransferase and nicotinate phosphoribosyltransferase. These enzymes are poorly characterized in humans, depsite
their importance in nicotinamide utilization.
References
G Magni, A Amici, M Emanuelli, G Orsomando, N Raffaelli, S Ruggieri, "Enzymology of NAD+ homeostasis in man", Cell Mol Life Sci, 61, 2004,
19-34.
21.1.7.14.1 Deamination of nicotinamide to nicotinate

Description
Nicotinamide deaminase deaminates nicotinamide to nicotinate. There is no literature on the human enzyme but there is evidence showing a
marked nicotinamide deaminase activity when red blood cells are infected with Plasmodium falciparum (Zerez C. et al, 1990). What is not clear
is whether this activity is stimulated by the parasite or encoded by its genome.
References
CR Zerez, Jr Roth EF, S Schulman, KR Tanaka, "Increased nicotinamide adenine dinucleotide content and synthesis in Plasmodium
falciparum-infected human erythrocytes", Blood, 75, 1990, 1705-10.
Source reaction
This reaction was inferred from the corresponding reaction "E.Coli deamination of nicotinamide" in species Escherichia coli.
R Frothingham, WA Meeker-O'Connell, EA Talbot, JW George, KN Kreuzer, "Identification, cloning, and expression of the Escherichia coli
pyrazinamidase and nicotinamidase gene, pncA", Antimicrob Agents Chemother, 40, 1996, 1426-31.
Reaction
21.1.7.14.2 Condensation of nicotinamide to nicotinamide D-ribonucleotide (NMN)
Description
Nicotinamide phosphoribosyltransferase (NamPRT) catalyzes the condensation of nicotinamide with 5- phosphoribosyl-1-pyrophosphate to yield
nicotinamide D-ribonucleotide (NMN), an intermediate in the biosynthesis of NAD. It is the rate limiting component in the mammalian NAD
biosynthesis pathway.
References
B Samal, Y Sun, G Stearns, C Xie, S Suggs, I McNiece, "Cloning and characterization of the cDNA encoding a novel human pre-B-cell
colony-enhancing factor", Mol Cell Biol, 14, 1994, 1431-7.
Reaction
21.1.7.14.3 A phosphoribosyl group is added to nicotinate to form nicotinate mononucleotide (NaMN)
Description
Nicotinate phosphoribosyltransferase (NaPRT) catalyzes the conversion of nicotinate to nicotinate mononucleotide (NaMN, nicotinate
D-ribonucleotide). This reaction was first demonstrated in human erythrocytes (Preiss J. and Handler P., 1958) and the enzyme purified and
characterized in 1973 (Niedel J. and Dietrich L.S., 1973). There is no sequence available for this protein.
References
J PREISS, P Handler, "Biosynthesis of diphosphopyridine nucleotide. II. Enzymatic aspects.", J Biol Chem, 233, 1958, 493-500.
J Niedel, LS Dietrich, "Nicotinate phosphoribosyltransferase of human erythrocytes. Purification and properties.", J Biol Chem, 248, 1973,
3500-5.
Reaction
21.1.8 Metabolism of folate and pterines
Description
Folates are essential cofactors that provide one-carbon moieties in various states of reduction for biosynthetic reactions. Processes annotated
here include transport reactions by which folates are taken up by cells and moved intracellularly, folate conjugation with glutamate (required for
folate retention within a cell), and some of the key reactions in the generation of reduced folates and one-carbon derivatives of folate.
References
JM Scott, "Folate and vitamin B12", Proc Nutr Soc, 58, 1999, 441-8.
M Lucock, "Folic acid: nutritional biochemistry, molecular biology, and role in disease processes", Mol Genet Metab, 71, 2000, 121-38.
21.1.8.1 Extracellular folate import across the plasma membrane
Description
SLC46A1 protein in the plasma membrane mediates the reversible transport of folate between the extracellular space and the cytosol. Retention
of folate within the cell is dependent on polyglutamate addition. Although the SLC46A1 gene is expressed in several tissues in the body, this
transporter appears to be primarily needed for absorption of dietary folate from the intestinal lumen (Qiu et al. 2006; Chen et al. 1996).
References
A Qiu, M Jansen, A Sakaris, SH Min, S Chattopadhyay, E Tsai, C Sandoval, R Zhao, MH Akabas, ID Goldman, "Identification of an intestinal
folate transporter and the molecular basis for hereditary folate malabsorption", Cell, 127, 2006, 917-28.
L Chen, H Qi, J Korenberg, TA Garrow, YJ Choi, B Shane, "Purification and properties of human cytosolic folylpoly-gamma-glutamate
synthetase and organization, localization, and differential splicing of its gene", J Biol Chem, 271, 1996, 13077-87.
Reaction
21.1.8.2 Cytosolic folate export across the plasma membrane
Description
SLC46A1 protein in the plasma membrane mediates the reversible transport of folate between the extracellular space and the cytosol. Retention
of folate within the cell is dependent on polyglutamate addition (Qiu et al. 2006; Chen et al. 1996).
References
A Qiu, M Jansen, A Sakaris, SH Min, S Chattopadhyay, E Tsai, C Sandoval, R Zhao, MH Akabas, ID Goldman, "Identification of an intestinal
folate transporter and the molecular basis for hereditary folate malabsorption", Cell, 127, 2006, 917-28.
Reaction
21.1.8.3 Extracellular 5-methyltetrahydrofolate import across the plasma membrane
Description
SLC19A1 protein, associated with the plasma membrane, mediates the uptake of extracellular 5-methyltetrahydrofolate and other reduced
folates (Williams and Flintoff 1995; Ferguson and Flintoff 1999).
References
FM Williams, WF Flintoff, "Isolation of a human cDNA that complements a mutant hamster cell defective in methotrexate uptake", J Biol Chem,
270, 1995, 2987-92.
PL Ferguson, WF Flintoff, "Topological and functional analysis of the human reduced folate carrier by hemagglutinin epitope insertion", J Biol
Chem, 274, 1999, 16269-78.
Reaction
21.1.8.4 Folate is reduced to dihydrofolate (DHF)
Description
Cytosolic dihydrofolate reductase catalyzes the reaction of folate, NADPH, and H+ to form dihydrofolate and NADP+ (Chen et al. 1984; Davies
et al. 1990).
References
MJ Chen, T Shimada, AD Moulton, A Cline, RK Humphries, J Maizel, AW Nienhuis, "The functional human dihydrofolate reductase gene", J Biol
Chem, 259, 1984, 3933-43.
2nd Davies JF, TJ Delcamp, NJ Prendergast, VA Ashford, JH Freisheim, J Kraut, "Crystal structures of recombinant human dihydrofolate
reductase complexed with folate and 5-deazafolate", Biochemistry, 29, 1990, 9467-79.
Reaction
21.1.8.5 DHF is reduced to tetrahydrofolate (THF)

Description
Cytosolic dihydrofolate reductase catalyzes the reaction of dihydrofolate, NADPH, and H+ to form tetrahydrofolate (THF) and NADP+ (Chen et
al. 1984; Davies et al. 1990).
References
MJ Chen, T Shimada, AD Moulton, A Cline, RK Humphries, J Maizel, AW Nienhuis, "The functional human dihydrofolate reductase gene", J Biol
Chem, 259, 1984, 3933-43.
2nd Davies JF, TJ Delcamp, NJ Prendergast, VA Ashford, JH Freisheim, J Kraut, "Crystal structures of recombinant human dihydrofolate
reductase complexed with folate and 5-deazafolate", Biochemistry, 29, 1990, 9467-79.
Reaction
21.1.8.6 Conversion of cytosolic THF to THF-polyglutamate
Description
Cytosolic folylpolyglutamate synthase catalyzes the reaction of THF-glutamate(n), L-glutamate, and ATP to form THF-glutamate(n+1), ADP, and
orthophosphate. (The first glutamate residue is attached to the glutamate moiety of THF itself; for convenience the process is annotated here as
if it proceeded in a single concerted step.) The extent of conjugation is variable, but the commonest cytosolic form of THF has five added
glutamate residues. Although its properties as a donor of one-carbon units are not affected by glutamate addition, THF that lacks added
glutamate residues cannot be retained in the cytosol so this reaction is needed for normal THF function under physiological conditions (Garrow
et al. 1992; Chen et al. 1996).
References
TA Garrow, A Admon, B Shane, "Expression cloning of a human cDNA encoding folylpoly(gamma-glutamate) synthetase and determination of
its primary structure", Proc Natl Acad Sci U S A, 89, 1992, 9151-5.
Reaction
21.1.8.7 Conversion of cytosolic 5-methyltetrahydrofolate (5-methylTHF) to 5-methylTHF-polyglutamate
Description
Cytosolic folylpolyglutamate synthase catalyzes the reaction of 5-methylTHF-glutamate(n), L-glutamate, and ATP to form
5-methylTHF-glutamate(n+1), ADP, and orthophosphate. (The first glutamate residue is attached to the glutamate moiety of 5-methylTHF itself;
for convenience the process is annotated here as if it proceeded in a single concerted step.) The extent of conjugation is variable, but the
commonest cytosolic form of 5-methylTHF has five added glutamate residues. Although its properties as a donor of one-carbon units are not
affected by glutamate addition, 5-methylTHF that lacks added glutamate residues cannot be retained in the cytosol so this reaction is needed for
normal 5-methylTHF function under physiological conditions (Garrow et al. 1992; Chen et al. 1996).
References
Reaction
21.1.8.8 Cytosolic tetrahydrofolate import across the inner mitochondrial membrane

Description
SLC25A32 protein in the inner mitochondrial membrane mediates the reversible transport of tetrahydrofolate between the cytosol and the
mitochondrial matrix. Retention of tetrahydrofolate within the mitochondrial matrix is dependent on mitochondrial polyglutamate addition (Titus
and Moran 2000; Chen et al. 1996).
References
SA Titus, RG Moran, "Retrovirally mediated complementation of the glyB phenotype. Cloning of a human gene encoding the carrier for entry of
folates into mitochondria.", J Biol Chem, 275, 2000, 36811-7.
Reaction
21.1.8.9 Mitochondrial tetrahydrofolate export across the inner mitochondrial membrane
Description
SLC25A32 protein in the inner mitochondrial membrane mediates the reversible transport of tetrahydrofolate between the cytosol and the
mitochondrial matrix. Retention of tetrahydrofolate within the mitochondrial matrix is dependent on mitochondrial polyglutamate addition (Titus
and Moran 2000; Chen et al. 1996).
References
SA Titus, RG Moran, "Retrovirally mediated complementation of the glyB phenotype. Cloning of a human gene encoding the carrier for entry of
folates into mitochondria.", J Biol Chem, 275, 2000, 36811-7.
Reaction
21.1.8.10 Conversion of mitochondrial THF to THF-polyglutamate
Description
Mitochondrial folylpolyglutamate synthase catalyzes the reaction of THF-glutamate(n), L-glutamate, and ATP to form THF-glutamate(n+1), ADP,
and orthophosphate. (The first glutamate residue is attached to the glutamate moiety of THF itself; for convenience the process is annotated
here as if it proceeded in a single concerted step.) The extent of conjugation is variable, but the commonest mitochondrial form of THF has six
added glutamate residues. Although its properties as a donor of one-carbon units are not affected by glutamate addition, THF that lacks added
glutamate residues cannot be retained in the mitochondrial matrix so this reaction is needed for normal THF function under physiological
conditions. The mitochondrial and cytosolic forms of folylpolyglutamate synthase are encoded by the same gene - alternative splicing generates
mRNA with or without an initial exon encoding a mitochondrial targeting sequence (Garrow et al. 1992; Chen et al. 1996).
References
Reaction
21.1.8.11 THF polyglutamate + formate + ATP => 10-formylTHF polyglutamate + ADP + orthophosphate
Description
The formate-tetrahydrofolate ligase activity of the trifunctional MTHFD1 enzyme catalyzes the reaction of THF polyglutamate, formate, and ATP
to form 10-formylTHF polyglutamate, ADP, and orthophosphate. MTHFD1 is cytosolic and occurs as a dimer. The human enzyme has been
identified and partially characterized biochemically (Hum et al. 1988); additional reaction details can be inferred from the properties of the
well-studied homologous rabbit enzyme (Villar et al. 1985).
References
E Villar, B Schuster, D Peterson, V Schirch, "C1-Tetrahydrofolate synthase from rabbit liver. Structural and kinetic properties of the enzyme and
its two domains.", J Biol Chem, 260, 1985, 2245-52.
DW Hum, AW Bell, R Rozen, RE MacKenzie, "Primary structure of a human trifunctional enzyme. Isolation of a cDNA encoding
methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase.", J Biol Chem, 263, 1988,
15946-50.
Reaction
21.1.8.12 10-formylTHF polyglutamate <=> 5,10-methenylTHF polyglutamate + H2O
Description
The methenyltetrahydrofolate cyclohydrolase activity of the trifunctional MTHFD1 enzyme catalyzes the reversible reaction of 10-formylTHF
polyglutamate to form 5,10-methenylTHF polyglutamate and H2O. MTHFD1 is cytosolic and occurs as a dimer. The human enzyme has been
References
15946-50.
Reaction
21.1.8.13 5,10-methenylTHF polyglutamate + NADPH + H+ <=> 5,10-methyleneTHF polyglutamate + NADP+
Description
The methylenetetrahydrofolate dehydrogenase activity of the trifunctional MTHFD1 enzyme catalyzes the reversible reaction of
5,10-methenylTHF polyglutamate, NADPH, and H+ to form 5,10-methyleneTHF polyglutamate and NADP+. MTHFD1 is cytosolic and occurs as
a dimer. The human enzyme has been identified and partially characterized biochemically (Hum et al. 1988); additional reaction details can be
inferred from the properties of the well-studied homologous rabbit enzyme (Villar et al. 1985).
References
15946-50.
Reaction
21.1.8.14 Tetrahydrofolate polyglutamate (THF polyglutamate) + serine <=> 5,10-methyleneTHF polyglutamate + glycine
Description
Cytosolic serine hydroxymethyltransferase catalyzes the reversible reaction of tetrahydrofolate polyglutamate (THF polyglutamate) and serine to
form 5,10-methyleneTHF polyglutamate and glycine. The active form of the enzyme is a tetramer (Renwick et al. 1998). In the body, this reaction
is a major source of 5,10-methyleneTHF, which in turn is a critical precursor in the synthesis of dTMP.
References
SB Renwick, K Snell, U Baumann, "The crystal structure of human cytosolic serine hydroxymethyltransferase: a target for cancer
chemotherapy", Structure, 6, 1998, 1105-16.
Reaction
21.1.8.15 5,10-methyleneTHF polyglutamate + NADPH + H+ => 5-methylTHF polyglutamate + NADP+
Description
Cytosolic MTHFR dimer catalyzes the reaction of 5,10-methyleneTHF polyglutamate, NADPH, and H+ to form 5-methylTHF polyglutamate and
NADP+. The specificity and importance of this reaction in vivo have been established through the study of patients deficient in the enzyme
(Goyette et al. 1995).
References
P Goyette, P Frosst, DS Rosenblatt, R Rozen, "Seven novel mutations in the methylenetetrahydrofolate reductase gene and
genotype/phenotype correlations in severe methylenetetrahydrofolate reductase deficiency", Am J Hum Genet, 56, 1995, 1052-9.
Reaction
21.1.8.16 5,10-methyleneTHF polyglutamate + glycine <=> tetrahydrofolate polyglutamate (THF polyglutamate) + serine
Description
Cytosolic serine hydroxymethyltransferase catalyzes the reversible reaction of 5,10-methyleneTHF polyglutamate and glycine to form
tetrahydrofolate polyglutamate (THF polyglutamate) and serine. The active form of the enzyme is a tetramer (Renwick et al. 1998).
References
SB Renwick, K Snell, U Baumann, "The crystal structure of human cytosolic serine hydroxymethyltransferase: a target for cancer
chemotherapy", Structure, 6, 1998, 1105-16.
Reaction
21.1.8.17 5,10-methyleneTHF polyglutamate + NADP+ <=> 5,10-methenylTHF polyglutamate + NADPH + H+
Description
The methylenetetrahydrofolate dehydrogenase activity of the trifunctional MTHFD1 enzyme catalyzes the reversible reaction of
5,10-methyleneTHF polyglutamate and NADP+ to form 5,10-methenylTHF polyglutamate, NADPH, and H+. MTHFD1 is cytosolic and occurs as
a dimer. The human enzyme has been identified and partially characterized biochemically (Hum et al. 1988); additional reaction details can be
inferred from the properties of the well-studied homologous rabbit enzyme (Villar et al. 1985).
References
15946-50.
Reaction
21.1.8.18 5,10-methenylTHF polyglutamate + H2O <=> 10-formylTHF polyglutamate
Description
The methenyltetrahydrofolate cyclohydrolase activity of the trifunctional MTHFD1 enzyme catalyzes the reversible reaction of 5,10-methenylTHF
polyglutamate and H2O to form 10-formylTHF polyglutamate. MTHFD1 is cytosolic and occurs as a dimer. The human enzyme has been
References
15946-50.
Reaction
22 Nucleotide metabolism
Authors
Jassal, B, 2003-06-26.
Editors
D'Eustachio, P, Gillespie, ME, Joshi-Tope, G, 0000-00-00.
Reviewers
Graves, L, Rush, MG, 0000-00-00.
Description
Nucleotides and their derivatives are used for short-term energy storage (ATP, GTP), for intra- and extra-cellular signaling (cAMP; adenosine),
as enzyme cofactors (NAD, FAD), and for the synthesis of DNA and RNA. Most dietary nucleotides are consumed by gut flora; the human body's
own supply of these molecules is synthesized de novo. Additional metabolic pathways allow the interconversion of nucleotides, the salvage and
reutilization of nucleotides released by degradation of DNA and RNA, and the catabolism of excess nucleotides. These pathways are regulated
to control the total size of the intracellular nucleotide pool, to balance the relative amounts of individual nucleotides, and to couple the synthesis
of deoxyribonucleotides to the onset of DNA replication (S phase of the cell cycle).
These pathways are also of major clinical interest as they are the means by which nucleotide analogues used as anti-viral and anti-tumor drugs
are taken up by cells, activated, and catabolized.
22.1 Transport of nucleosides and free purine and pyrimidine bases across
the plasma membrane
Authors
Editors
Description
Two families of transport proteins mediate the movement of nucleosides and free purine and pyrimidine bases across the plasma membrane.
Equilibrative nucleoside transporters allow the movement of these molecules along concentration gradients into or out of cells (Baldwin et al.
2003); concentrative nucleoside transporters actively transport nucleosides into cells by coupling their transport to the inward movement of
sodium ions (Gray et al. 2003).
Of the four human equilibrative nucleoside transporters, two are well characterized. SLC29A1 (solute carrier family 29 (nucleoside transporters),
member 1) mediates the transport of nucleosides across the plasma membrane. SLC29A2 (solute carrier family 29 (nucleoside transporters),
member 2) mediates the transport of both nucleosides and free bases. Transporter specificities were determined by expressing cloned human
genes in Xenopus oocytes or in mammalian cultured cell lines whose own nucleotide transporters had been disrupted by mutation. These
studies establish that the transport processes are specific and saturable, and that the multiple nucleotides and bases compete for a single
binding site on each transporter. Some features of SLC29A2 specificity are complex. For example, in the Xenopus oocyte system, radiolabeled
uracil and adenine are taken up, and an excess of either molecule inhibits uptake of radiolabeled hypoxanthine, while in the cultured mammalian
cell system, neither adenine nor uracil can inhibit uptake of radiolabeled uridine. If these results reflect ENT2 function in vivo, they indicate that
the net movement of a nucleoside or base across the cell membrane is determined not only by its own concentrations in the extracellular space
and the cytosol, but also by the concentrations of the other nucleosides and bases competing for access to the transporter.
The human genome encodes three concentrative transporters, SLC28A1, 2, and 3 (solute carrier family 28 (sodium-coupled nucleoside
transporter), member 1, 2, and 3). All three genes have been cloned, and expression of the human proteins in Xenopus oocytes has allowed
their transport properties to be determined. SLC28A1 mediates the uptake of pyrimidine nucleosides and adenosine (Ritzel et al. 1997);
SLC28A2 the uptake of purine nucleosides and uridine (Wang et al. 1997); and SLC28A3 the uptake of purine and pyrimidine nucleosides
(Ritzel et al. 2001). Amino acid sequence motifs that determine the specificities of these transporters have been identified in studies of chimeric
and mutant proteins (Loewen et al. 1999). SLC28A3 protein co-transports two sodium ions per nucleoside; SLC28A1 and 2 transport one
sodium per nucleoside (Ritzel et al. 2001).
Physiological roles for nucleoside and base transport include provision of nucleosides to cells with little capacity to synthesize these molecules
de novo, and regulation of extracellular levels of adenosine, which is released from muscle during intense exercise and has signaling properties.
In kidney and intestinal epithelia, the combination of apically localized CNT transporters and basolaterally localized ENT transporters provides a
mechanism for net transport of nucleosides (Mangravite et al. 2003). These transporters also mediate the uptake of nucleoside analogs used
clinically as anti-viral and anti-tumor drugs.
Orthologs of human concentrative and equilibrative transporter proteins have been identified in many eukaryotes, but functional studies of
transporters even from organisms closely related to humans (e.g. rat, Gerstin et al. 2002) have revealed differences in substrate specificities.
Prediction of drug uptake and other functions of these molecules by human - model organism orthology is thus risky.
References
LM Mangravite, G Xiao, KM Giacomini, "Localization of human equilibrative nucleoside transporters, hENT1 and hENT2, in renal epithelial cells",
Am J Physiol Renal Physiol, 284, 2003, 902-910.
SK Loewen, AM Ng, SY Yao, CE Cass, SA Baldwin, JD Young, "Identification of amino acid residues responsible for the pyrimidine and purine
nucleoside specificities of human concentrative Na+ nucleoside cotransporters hCNT1 and hCNT2", J Biol Chem, 274, 1999, 24475-24484.
MW Ritzel, SY Yao, MY Huang, JF Elliott, CE Cass, JD Young, "Molecular cloning and functional expression of cDNAs encoding a human
Na+-nucleoside cotransporter (hCNT1)", Am J Physiol Cell Physiol, 272, 1997, 707-714.
MW Ritzel, AM Ng, SY Yao, K Graham, SK Loewen, KM Smith, RG Ritzel, DA Mowles, P Carpenter, XZ Chen, E Karpinski, RJ Hyde, SA
Baldwin, CE Cass, JD Young, "Molecular identification and characterization of novel human and mouse concentrative Na+-nucleoside
cotransporter proteins (hCNT3 and mCNT3) broadly selective for purine and pyrimidine nucleosides (system cib)", J Biol Chem, 276, 2001,
2914-2927.
SA Baldwin, PR Beal, SY Yao, AE King, CE Cass, JD Young, "The equilibrative nucleoside transporter family, SLC29", Pflugers Arch, 2003,
preprint.
J Wang, SF Su, MJ Dresser, ME Schaner, CB Washington, KM Giacomini, "Na+-dependent purine nucleoside transporter from human kidney:
cloning and functional characterization", Am J Physiol Renal Physiol, 273, 1997, 1058-1065.
JH Gray, RP Owen, KM Giacomini, "The concentrative nucleoside transporter family, SLC28", Pflugers Arch, 2003, preprint.
KM Gerstin, MJ Dresser, KM Giacomini, "Specificty of human and rat orthologs of the concentrative nucleoside transporter, SPNT", Am J Physiol
Renal Physiol, 283, 2002, 344-349.
22.1.1 Concentrative transport (import) of a nucleoside and a sodium ions by solute carrier family 28
(sodium-coupled nucleoside transporter), member 1
Description
The plasma membrane-associated protein SLC28A1 mediates the transport of one molecule of 2'-deoxyadenosine, adenosine, cytidine,
thymidine, or uridine, and one sodium ion, from the extracellular space to the cytosol.
References
MW Ritzel, SY Yao, MY Huang, JF Elliott, CE Cass, JD Young, "Molecular cloning and functional expression of cDNAs encoding a human
Na+-nucleoside cotransporter (hCNT1)", Am J Physiol Cell Physiol, 272, 1997, 707-714.
Reaction
22.1.2 Concentrative transport (import) of a nucleoside and two sodium ions by solute carrier family 28
Description
The plasma membrane-associated protein SLC28A3 mediates the transport of one molecule of adenosine, cytidine, guanosine, inosine,
thymidine, or uridine, and two sodium ions, from the extracellular space to the cytosol.
References
MW Ritzel, AM Ng, SY Yao, K Graham, SK Loewen, KM Smith, RG Ritzel, DA Mowles, P Carpenter, XZ Chen, E Karpinski, RJ Hyde, SA
Baldwin, CE Cass, JD Young, "Molecular identification and characterization of novel human and mouse concentrative Na+-nucleoside
cotransporter proteins (hCNT3 and mCNT3) broadly selective for purine and pyrimidine nucleosides (system cib)", J Biol Chem, 276, 2001,
2914-2927.
Reaction
22.1.3 Concentrative transport (import) of nucleosides plus sodium ions by solute carrier family 28
Description
The plasma membrane-associated protein SLC28A2 mediates the transport of one molecule of adenosine, guanosine, inosine, or uridine, and
one sodium ion, from the extracellular space to the cytosol.
References
SK Loewen, AM Ng, SY Yao, CE Cass, SA Baldwin, JD Young, "Identification of amino acid residues responsible for the pyrimidine and purine
nucleoside specificities of human concentrative Na+ nucleoside cotransporters hCNT1 and hCNT2", J Biol Chem, 274, 1999, 24475-24484.
J Wang, SF Su, MJ Dresser, ME Schaner, CB Washington, KM Giacomini, "Na+-dependent purine nucleoside transporter from human kidney:
cloning and functional characterization", Am J Physiol Renal Physiol, 273, 1997, 1058-1065.
Reaction
22.1.4 Equilibrative transport (export) of nucleosides and free bases by solute carrier family 29 (nucleoside
transporters), member 1
Description
The plasma membrane-associated protein SLC29A1 mediates the reversible transport of one molecule of adenosine, cytosine, guanosine,
inosine, thymidine, or uridine from the extracellular space to the cytosol.
References
M Griffiths, N Beaumont, SY Yao, M Sundaram, CE Boumah, A Davies, FY Kwong, I Coe, CE Cass, JD Young, SA Baldwin, "Cloning of a
human nucleoside transporter implicated in the cellular uptake of adenosine and chemotherapeutic drugs", Nature Medicine, 1997, 89-93.
Reaction
22.1.5 Equilibrative transport (export) of nucleosides and free bases by solute carrier family 29 (nucleoside
Description
The plasma membrane-associated protein SLC29A2 mediates the reversible transport of one molecule of adenine, adenosine, cytidine,
cytosine, guanine, guanosine, hypoxanthine, inosine, thymidine, thymine, uracil, or uridine from the cytosol to the extracellular space.
References
CR Crawford, DH Patel, C Naeve, JA Belt, "Cloning of the human equilibrative, nitrobenzylmercaptopurine riboside (NBMPR)-insensitive
nucleoside transporter ei by functional expression in a transport-deficient cell line", J Biol Chem, 273, 1998, 5288-5293.
Reaction
22.1.6 Equilibrative transport (import) of nucleosides and free bases by solute carrier family 29 (nucleoside
Description
The plasma membrane-associated protein SLC29A1 mediates the reversible transport of one molecule of adenosine, guanosine, inosine, or
uridine from the cytosol to the extracellular space.
References
M Griffiths, N Beaumont, SY Yao, M Sundaram, CE Boumah, A Davies, FY Kwong, I Coe, CE Cass, JD Young, SA Baldwin, "Cloning of a
human nucleoside transporter implicated in the cellular uptake of adenosine and chemotherapeutic drugs", Nature Medicine, 1997, 89-93.
Reaction
22.1.7 Equilibrative transport (import) of nucleosides and free bases by solute carrier family 29 (nucleoside
Description
The plasma membrane-associated protein SLC29A2 mediates the reversible transport of one molecule of adenine, adenosine, cytidine,
cytosine, guanine, guanosine, hypoxanthine, inosine, thymidine, thymine, uracil, or uridine from the extracellular space to the cytosol.
References
CR Crawford, DH Patel, C Naeve, JA Belt, "Cloning of the human equilibrative, nitrobenzylmercaptopurine riboside (NBMPR)-insensitive
nucleoside transporter ei by functional expression in a transport-deficient cell line", J Biol Chem, 273, 1998, 5288-5293.
Reaction
22.2 Purine metabolism
Authors
Jassal, B, 2003-06-26.
Editors
Reviewers
Rush, MG, 2008-01-11.

Description
The events of human purine metabolism are conveniently, if somewhat arbitrarily, grouped into four pathways: de novo synthesis of inosine
5'-monophosphate (IMP), the biosynthesis of other purine ribo- and deoxyribonucleotides, purine salvage reactions, and purine catabolism.
De novo synthesis of inosine 5'-monophosphate (IMP). The purine ribonucleotide IMP is assembled on 5-phospho-alpha-D-ribose 1-diphosphate
(PRPP).
Purine biosynthesis [interconversion]. Purine ribo- and deoxyribonucleotide di- and triphosphates are synthesized, both from IMP and from
guanosine and adenosine ribo- and deoxyribonucleotide monophosphates generated in salvage reactions.
Purine salvage reactions. Purine nucleosides and free bases generated by DNA and RNA breakdown are converted to nucleotide
monophosphates.
Purine catabolism. The purine bases guanine and hypoxanthine are degraded to uric acid, which is excreted from the body.
22.2.1 De novo synthesis of IMP
Authors
Jassal, B, 2003-06-26.
Editors
Description
The purine ribonucleotide inosine 5'-monophosphate (IMP) is assembled on 5-phospho-alpha-D-ribose 1-diphosphate (PRPP), with atoms
derived from aspartate, glutamine, glycine, N10-formyl-tetrahydrofolate, and carbon dioxide. Although several of the individual reactions in this
sequence are reversible, as indicated by the double-headed arrows in the diagram, other irreversible steps drive the pathway in the direction of
IMP synthesis in the normal cell. All of these reactions are thus annotated here only in the direction of IMP synthesis. IMP is the starting point for
the biosynthesis of other purine nucleotides.
References
H Zalkin, JE Dixon, "De novo purine nucleotide biosynthesis", Prog Nucleic Acid Res Mol Biol, 42, 1992, 259-287.
22.2.1.1 5-phospho-alpha-D-ribose 1-diphosphate (PRPP) + H2O + L-glutamine <=> 5-phosphoribosylamine + L-glutamate

+pyrophosphate
Authors
Jassal, B, 2003-06-26.
Editors
Jassal, B, D'Eustachio, P, 0000-00-00.
Description
This event is the committed step in de novo purine synthesis. The reaction itself is reversible, but it is pulled strongly in the direction of
5'-phosphoribosylamine synthesis by the irreversible hydrolysis of the pyrophosphate that is also formed in the reaction. The enzyme that
catalyzes it, phosphoribosyl pyrophosphate amidotransferase, is inferred to be an iron-sulfur protein, like its well-characterized B. subtilis
homologue, because incubation of purified enzyme with molecular oxygen or chelating agents inactivates it, and activity can be restored by
incubation with ferrous iron and inorganic sulfide. The stoichiometry of the iron-sulfur moiety and its role in enzyme activity remain unknown
(Itakura and Holmes 1979). The fully active form of the enzyme is a dimer, which can associate further to form a tetramer with sharply reduced
activity (Holmes et al. 1973b; Iwahana et al. 1993). Interaction of the enzyme with inosine 5'-monophosphate (IMP), guanosine
5'-monophosphate (GMP), and adenosine 5'-monophosphate (AMP), end products of de novo purine biosynthesis, favors tetramer formation,
while interaction with 5-phospho-alpha-D-ribose 1-diphosphate (PRPP), a required substrate, favors formation of the active dimer. Kinetic
studies suggest that the enzyme's binding site for GMP and IMP is separate from its AMP binding site (Holmes et al. 1973a).
References
EW Holmes, JB Wyngaarden, WN Kelley, "Human glutamine phosphoribosylpyrophosphate amidotransferase. Two molecular forms
interconvertible by purine ribonucleotides and phosphoribosylpyrophosphate", J Biol Chem, 248, 1973, 6035-6040.
EW Holmes, JA McDonald, JM McCord, JB Wyngaarden, WN Kelley, "Human glutamine phosphoribosylpyrophosphate amidotransferase.

Kinetic and regulatory properties", J Biol Chem, 248, 1973, 144-150.
M Itakura, EW Holmes, "Human amidophosphoribosyltransferase. An oxygen-sensitive iron-sulfur protein", J Biol Chem, 254, 1979, 333-338.
H Iwahana, J Oka, N Mizusawa, E Kudo, S Ii, K Yoshimoto, EW Holmes, M Itakura, "Molecular cloning of human
amidophosphoribosyltransferase", Biochem Biophys Res Commun, 190, 1993, 192-200.
Reaction
22.2.1.2 5-phosphoribosylamine + glycine + ATP <=> 5-phosphoribosylglycinamide (GAR) + adenosine 5'-diphosphate +

orthophosphate
Authors
Editors
D'Eustachio, P, Jassal, B, 0000-00-00.
Description
The reversible synthesis of cytosolic 5-phosphoribosylglycinamide from 5-phosphoribosylamine and glycine, accompanied by the conversion of
ATP to ADP and orthophosphate, is catalyzed by the phosphoribosylglycinamide synthetase domain of the trifunctional protein,
"phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazole synthetase" (Aimi et al.
1990; Daubner et al. 1985). The active form of the protein is annotated here as a cytosolic monomer, as the limited data available do not suggest
its association into larger complexes (Gooljarsingh et al. 2001).
References
LT Gooljarsingh, J Ramcharan, S Gilroy, SJ Benkovic, "Localization of GAR transformylase in Escherichia coli and mammalian cells", Proc Natl
Acad Sci USA, 98, 2001, 6565-6570.
J Aimi, H Qiu, J Williams, H Zalkin, JE Dixon, "De novo purine nucleotide biosynthesis: cloning of human and avian cDNAs encoding the
trifunctional glycinamide ribonucleotide synthetase-aminoimidazole ribonucleotide synthetase-glycinamide ribonucleotide transformylase by
functional complementation in E. coli", Nucleic Acids Res, 18, 1990, 6665-6672.
SC Daubner, JL Schrimsher, FJ Schendel, M Young, S Henikoff, D Patterson, J Stubbe, SJ Benkovic, "A multifunctional protein possessing
glycinamide ribonucleotide synthetase, glycinamide ribonucleotide transformylase, and aminoimidazole ribonucleotide synthetase activities in de
novo purine biosynthesis", Biochemistry, 24, 1985, 7059-7062.
Reaction
22.2.1.3 5-phosphoribosylglycinamide (GAR) + 10-formyl-tetrahydrofolate => 5'-phosphoribosylformylglycinamide (FGAR) +

tetrahydrofolate
Authors
Editors
Description
The irreversible transfer of a formyl group to cytosolic 5-phosphoribosylglycinamide (GAR) to form 5'-phosphoribosylformylglycinamide (FGAR)
is catalyzed by the phosphoribosylglycinamide formyltransferase domain of the trifunctional protein, "phosphoribosylglycinamide
formyltransferase, phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazole synthetase" (Aimi et al. 1990; Daubner et al. 1985;
Zhang et al. 2002). The active form of the protein is annotated here as a cytosolic monomer, as the limited data available do not suggest its
association into larger complexes (Gooljarsingh et al. 2001).
References
Acad Sci USA, 98, 2001, 6565-6570.
Y Zhang, J Desharnais, SE Greasley, GP Beardsley, DL Boger, IA Wilson, "Crystal structures of human GAR Tfase at low and high pH and with
substrate beta-GAR", Biochemistry, 41, 2002, 14206-14215.
Reaction
22.2.1.4 5'-phosphoribosylformylglycinamide (FGAR) + L-glutamine + ATP + H2O => 5'-phosphoribosylformylglycinamidine (FGAM) +

L-glutamate + adenosine 5'-diphosphate + orthophosphate
Authors
Editors
Description
The irreversible transfer of an amino group from L-glutamine to 5'-phosphoribosylformylglycinamide (FGAR), forming
5'-phosphoribosylformylglycinamidine (FGAM), accompanied by the conversion of ATP to ADP and orthophosphate, is catalyzed by
phosphoribosylformylglycinamidine synthetase. Although the human enzyme has been purified and characterized biochemically (Barnes et al.
1994), its subunit structure is unknown. It is annotated here as a monomer by analogy to the well-characterized chicken enzyme (Frere et al.
1971).
References
JM Frere, DD Schroeder, JM Buchanan, "Biosynthesis of the purines. XXXV. Reversible polymerization of formylglycinamide ribonucleotide
amidotransferase", J Biol Chem, 246, 1971, 4727-4730.
TS Barnes, JH Bleskan, IM Hart, KA Walton, JW Barton, D Patterson, "Purification of, generation of monoclonal antibodies to, and mapping of
phosphoribosyl N-formylglycinamide amidotransferase", Biochemistry, 33, 1994, 1850-1860.
Reaction
22.2.1.5 5'-phosphoribosylformylglycinamidine (FGAM) + ATP => 5'-phosphoribosyl-5-aminoimidazole (AIR) + ADP + orthophosphate
Authors
Editors
Description
The irreversible synthesis of cytosolic 5'-phosphoribosyl-5-aminoimidazole (AIR) from 5'-phosphoribosylformylglycinamidine (FGAM),

accompanied by the conversion of ATP to ADP and orthophosphate, is catalyzed by the phosphoribosylaminoimidazole synthetase domain of
the trifunctional protein, "phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazole
synthetase" (Aimi et al. 1990; Daubner et al. 1985). The active form of the protein is annotated here as a cytosolic monomer, as the limited data
available do not suggest its association into larger complexes (Gooljarsingh et al. 2001).
References
Acad Sci USA, 98, 2001, 6565-6570.
Reaction
22.2.1.6 5'-phosphoribosyl-5-aminoimidazole (AIR) + CO2 <=> 5'-phosphoribosyl-5-aminoimidazole-4-carboxylate (CAIR)
Authors
Editors
Description
The reversible carboxylation of 5'-phosphoribosyl-5-aminoimidazole (AIR) to form 5'-phosphoribosyl-5-aminoimidazole-4-carboxylate (CAIR) is

catalyzed by the phosphoribosylaminoimidazole carboxylase domain of the bifunctional protein "phosphoribosylaminoimidazole carboxylase,
phosphoribosylaminoimidazole succinocarboxamide synthetase" (Schild et al. 1990; Zalkin and Dixon 1992). The subunit structure of the
enzyme is unknown; it is annotated here as a monomer by default.
References
D Schild, AJ Brake, MC Kiefer, D Young, PJ Barr, "Cloning of three human multifunctional de novo purine biosynthetic genes by functional
complementation of yeast mutations", Proc Natl Acad Sci USA, 87, 1990, 2916-2920.
Reaction
22.2.1.7 5'-phosphoribosyl-5-aminoimidazole-4-carboxylate (CAIR) + L-aspartate + ATP <=>

5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide (SAICAR) + adenosine 5'-diphosphate + orthophosphate
Authors
Editors
Description
The reversible conversion of 5'-phosphoribosyl-5-aminoimidazole-4-carboxylate (CAIR) and aspartate to

5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide (SAICAR), accompanied by the conversion of ATP to adenosine 5'-diphosphate
and orthophosphate, is catalyzed by the phosphoribosylaminoimidazole succinocarboxamide synthetase domain of the bifunctional protein
"phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase" (Schild et al. 1990; Zalkin and
Dixon 1992). The subunit structure of the enzyme is unknown; it is annotated here as a monomer by default.
References
D Schild, AJ Brake, MC Kiefer, D Young, PJ Barr, "Cloning of three human multifunctional de novo purine biosynthetic genes by functional
complementation of yeast mutations", Proc Natl Acad Sci USA, 87, 1990, 2916-2920.
Reaction
22.2.1.8 5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide (SAICAR) <=>

5'-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR) + fumarate
Authors
D'Eustachio, P, 2004--0-2-.
Editors

Description
The reversible conversion of 5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide (SAICAR) to

5'-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR) and fumarate is catalyzed by adenylosuccinate lyase. The active form of this
enzyme is a cytosolic tetramer (Stone et al. 1993). The enzyme also catalyzes the conversion of adenylosuccinate to adenosine
5'-monophosphate and fumarate. Humans lacking the enzyme accumulate dephosphorylated forms of both substrates, indicating that the
enzyme mediates both reactions in vivo as well (Race et al. 2000).
References
V Race, S Marie, MF Vincent, G Van den Berghe, "Clinical, biochemical and molecular genetic correlations in adenylosuccinate lyase
deficiency", Hum Mol Genet, 9, 2000, 2159-2165.
RL Stone, H Zalkin, JE Dixon, "Expression, purification, and kinetic characterization of recombinant human adenylosuccinate lyase", J Biol
Chem, 268, 1993, 19710-19716.
Reaction
22.2.1.9 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR) + 10-formyltetrahydrofolate =>

5'-phosphoribosyl-5-formaminoimidazole-4-carboxamide (FAICAR) + tetrahydrofolate
Authors
2007-03-03.
Editors
Description
The irreversible transfer of a formyl group to 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR), to yield

5'-phosphoribosyl-5-formaminoimidazole-4-carboxamide (FAICAR), is catalyzed by the phosphoribosylaminoimidazolecarboxamide
formyltransferase activity of the bifunctional 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase enzyme. This
cytosolic protein occurs in two forms: as a monomer it catalyzes the conversion of FAICAR to inosine 5'-monophosphate; dimerization is
necessary for FAICAR synthetic activity (Rayl et al. 1996; Vergis et al. 2001). The catalyst for this reaction is thus annotated as a dimer.
References
JM Vergis, KG Bulock, KG Fleming, GP Beardsley, "Human 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine

5'-monophosphate cyclohydrolase. A bifunctional protein requiring dimerization for transformylase activity but not for cyclohydrolase activity", J
Biol Chem, 276, 2001, 7727-7733.
EA Rayl, BA Moroson, GP Beardsley, "The human purH gene product, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP
cyclohydrolase", J Biol Chem, 271, 1996, 2225-2233.
Reaction
22.2.1.10 5'-phosphoribosyl-5-formaminoimidazole-4-carboxamide (FAICAR) <=> inosine 5'-monophosphate + H2O
Authors
2007-03-03.
Description
The reversible synthesis of inosine 5'-monophosphate from 5'-phosphoribosyl-5-formaminoimidazole-4-carboxamide (FAICAR) is catalyzed by

the IMP cyclohydrolase activity of the bifunctional 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase
enzyme. This cytosolic protein occurs in two forms: dimerization is necessary for FAICAR synthetic activity, but the monomer has IMP
cyclohydrolase activity (Rayl et al. 1996; Vergis et al. 2001). The catalyst for this reaction is thus annotated as a monomer.
References
JM Vergis, KG Bulock, KG Fleming, GP Beardsley, "Human 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine

5'-monophosphate cyclohydrolase. A bifunctional protein requiring dimerization for transformylase activity but not for cyclohydrolase activity", J
Biol Chem, 276, 2001, 7727-7733.
EA Rayl, BA Moroson, GP Beardsley, "The human purH gene product, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP
cyclohydrolase", J Biol Chem, 271, 1996, 2225-2233.
Reaction
22.2.2 Purine biosynthesis
Authors
Jassal, B, 2003-07-17.
Editors
Description
Inosine 5'-monophosphate (IMP) from de novo biosynthesis, as well as purine ribo- and deoxyribonucleotide monophosphates from purine
salvage, provide the starting materials for these reactions. Guanosine 5'-monophosphate (GMP) and adenosine 5'-monophosphate (AMP) are
synthesized from IMP. Further phosphorylation of GMP and AMP yields GDP, GTP, ADP, and ATP. The synthesis of ADP from ATP occurs as
part of the pathways of electron transport and oxidative phosphorylation. All of the other phosphorylation events ultimately derive their
high-energy phosphate groups from ATP.
Deoxyguanosine 5'-diphosphate (dGDP) and deoxyadenosine 5'-diphosphate (dADP) are generated from GDP and ADP, respectively. These
molecules in turn can be phosphorylated to yield dGTP and dATP, precursors for DNA synthesis. Their production is tightly regulated and linked
to the entry of the cell into S phase of the cell cycle.
22.2.2.1 dGTP formation
Description
GDP is reduced by ribonucleotide reductase and then phosphorylated to yield dGTP.
22.2.2.1.1 guanosine 5'-diphosphate + thioredoxin (reduced) => 2'-deoxyguanosine 5'-diphosphate + thioredoxin (oxidized) + H2O
Description
At the beginning of this reaction, 1 molecule of 'Thioredoxin', and 1 molecule of 'GDP' are present. At the end of this reaction, 1 molecule of
'thioredoxin, oxidized', 1 molecule of 'H2O', and 1 molecule of '2'-deoxyguanosine 5'-diphosphate' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'class I ribonucleotide reductase activity' of 'ribonucleotide reductase'.
References
O Guittet, P Hakansson, N Voevodskaya, S Fridd, A Graslund, Y Nakamura, L Thelander, "Mammalian p53R2 protein forms an active
ribonucleotide reductase in vitro with the R1 protein, which is expressed both in resting cells in response to DNA damage and in proliferating
cells", J Biol Chem, 276, 2001, 40647-40651.
Reaction
22.2.2.1.2 thioredoxin, oxidized + NADPH + H+ => thioredoxin, reduced + NADP+
Description
At the beginning of this reaction, 1 molecule of 'H+', 1 molecule of 'thioredoxin, oxidized', and 1 molecule of 'NADPH' are present. At the end of
this reaction, 1 molecule of 'Thioredoxin', and 1 molecule of 'NADP+' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'thioredoxin-disulfide reductase activity' of 'thioredoxin reductase holoenzyme'.
References
S Urig, J Lieske, K Fritz-Wolf, A Irmler, K Becker, "Truncated mutants of human thioredoxin reductase 1 do not exhibit glutathione reductase
activity", FEBS Lett, 580, 2006, 3595-600.
Reaction
22.2.2.1.3 2'-deoxyguanosine 5'-diphosphate (dGDP) + ATP <=> dGTP + adenosine 5'-diphosphate (ADP)
Description
At the beginning of this reaction, 1 molecule of '2'-deoxyguanosine 5'-diphosphate', and 1 molecule of 'ATP' are present. At the end of this
reaction, 1 molecule of 'ADP', and 1 molecule of 'dGTP' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'nucleoside diphosphate kinase activity' of 'nucleoside diphosphate kinase A,B
heterohexamer'.
References
AM Gilles, E Presecan, A Vonica, I Lascu, "Nucleoside diphosphate kinase from human erythrocytes. Structural characterization of the two
polypeptide chains responsible for heterogeneity of the hexameric enzyme", J Biol Chem, 266, 1991, 8784-8789.
S Schaertl, M Konrad, MA Geeves, "Substrate specificity of human nucleoside-diphosphate kinase revealed by transient kinetic analysis", J Biol
Chem, 273, 1998, 5662-5669.
Reaction
22.2.2.2 ATP formation
Description
The events annotated here trace the pathway of ATP synthesis: the cytosolic conversion of inosine-5'-monophosphate to AMP, the
phosphorylation of AMP to ADP, the transport of ADP into the mitochondrial matrix, and its conversion there to ATP coupled to electron
transport.
22.2.2.2.1 Conversion of cytosolic inosine 5'-monophosphate, L-aspartate, and GTP to adenylosuccinate, guanosine 5'-diphosphate,
and orthophosphate
Authors
Editors
Description
In E. coli, the synthesis of adenylosuccinate from inosine 5'-monophosphate, aspartate, and GTP is catalyzed by a single enzyme,
adenylosuccinate synthetase (Lieberman 1956). Mice have two isoforms of this enzyme, encoded by separate genes, one that is ubiquitously
expressed at low levels, and one that is expressed at high levels in muscle cells. The non-muscle enzyme is thought to be responsible for de
novo synthesis of adenine nucleotides from inosine 5'-monophosphate. Consistent with the function of maintaining adenine levels in balance
with those of other intracellular nucleotides, this isozyme is feedback-inhibited by adenosine 5'-monophosphate. The muscle form is thought to
participate in a substrate cycle in which the large amount of IMP generated during vigorous exercise by the deamination of adenosine
5'-monophosphate is used to resynthesize adenine nucleotides. Consistent with this role, this isozyme is not feedback-inhibited by adenosine
5'-monophosphate. While the physiological function of this substrate cycle remains unclear, it is required for normal muscle function and has
been shown to operate in human skeletal muscle (Bangsbo et al. 1992; Sabine and Holmes 2001).
Both mouse enzymes have been purified and biochemically characterized. The active forms of both enzymes appear to be dimers, with one
Mg++ ion bound to each monomer. Both enzymes are strongly inhibited competitively by adenylosuccinate, guanosine 5'-monophosphate, and
guanosine 5'-diphosphate, and non-competitively by fructose 1,6-bisphosphate (Borza et al. 2003; Guicherit et al. 1994; Lewis et al. 1996).
A human cDNA homologous to the mouse non-muscle adenylosuccinate synthetase has been cloned, and its translation product has been
shown to have enzymatic activity (Powell et al. 1992). A human homologue of the muscle-specific enzyme is known only as a cDNA; there is not
yet direct experimental evidence for its expression and function. The properties of two human reactions in which adenylosuccinate is synthesized
from inosine 5'-monophosphate are therefore inferred here from the homologous mouse reactions.
References
T Borza, CV Iancu, E Pike, RB Honzatko, HJ Fromm, "Variations in the response of mouse isozymes of adenylosuccinate synthetase to
inhibitors of physiological relevance", J Biol Chem, 278, 2003, 6673-6679.
OM Guicherit, BF Cooper, FB Rudolph, RE Kellems, "Amplification of an adenylosuccinate synthetase gene in alanosine-resistant murine
T-lymphoma cells", J Biol Chem, 269, 1994, 4488-4496.
RL Sabine, EW Holmes, "Myoadenylate deaminase deficiency", The Metabolic and Molecular Bases of Inherited Disease, 8th ed (Scriver CR, et
al., editors), 2, 2001, 2627-2638.
SM Powell, H Zalkin, JE Dixon, "Cloning and characterization of the cDNA encoding human adenylosuccinate synthetase", FEBS Lett, 303,
1992, 4-20.
I Lieberman, "Enzymatic synthesis of adenosine-5'-phosphate from inosine-5'-phosphate", J Biol Chem, 223, 1956, 327-329.
J Bangsbo, T Graham, L Johansen, S Strange, C Christensen, B Saltin, "Elevated muscle acidity and energy production during exhaustive
exercise in humans", Am J Physiol, 263, 1992, R891-R899.
AL Lewis, OM Guicherit, SK Datta, GR Hanten, RE Kellems, "Structure and expression of the murine muscle adenylosuccinate synthetase
gene", J Biol Chem, 271, 1996, 22647-22456.
22.2.2.2.1.1 inosine 5'-monophosphate + L-aspartate + GTP => adenylosuccinate + guanosine 5'-diphosphate + orthophosphate
Authors
Editors
Description
This event is thought to be part of the pathway of de novo adenine nucleotide biosynthesis throughout the body.
References
SM Powell, H Zalkin, JE Dixon, "Cloning and characterization of the cDNA encoding human adenylosuccinate synthetase", FEBS Lett, 303,
1992, 4-20.
Source reaction
This reaction was inferred from the corresponding reaction "inosine 5'-monophosphate + L-aspartate + GTP => adenylosuccinate + guanosine
5'-diphosphate + orthophosphate" in species Mus musculus.
Reaction
22.2.2.2.1.2 inosine 5'-monophosphate + L-aspartate + GTP => adenylosuccinate + guanosine 5'-diphosphate + orthophosphate
Authors
Editors
Description
This event is thought to be part of the purine nucleotide cycle in muscle, in which adenosine 5'-monophosphate (AMP) is converted to inosine
5'-monophosphate (IMP) during periods of intense exercise, and regenerated during periods of rest.
Source reaction
This reaction was inferred from the corresponding reaction "inosine 5'-monophosphate + L-aspartate + GTP => adenylosuccinate + guanosine
5'-diphosphate + orthophosphate" in species Mus musculus.
Reaction
22.2.2.2.2 adenylosuccinate => adenosine 5'-monophosphate + fumarate
Authors
Editors
Description
The irreversible conversion of adenylosuccinate to adenosine 5'-monophosphate and fumarate is catalyzed by adenylosuccinate lyase. The
active form of this enzyme is a cytosolic tetramer (Stone et al. 1993). The enzyme also catalyzes the conversion of
5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide (SAICAR) to 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR) and
fumarate. Humans lacking the enzyme accumulate dephosphorylated forms of both substrates, indicating that the enzyme mediates both
reactions in vivo as well (Race et al. 2000).
References
V Race, S Marie, MF Vincent, G Van den Berghe, "Clinical, biochemical and molecular genetic correlations in adenylosuccinate lyase
deficiency", Hum Mol Genet, 9, 2000, 2159-2165.
RL Stone, H Zalkin, JE Dixon, "Expression, purification, and kinetic characterization of recombinant human adenylosuccinate lyase", J Biol
Chem, 268, 1993, 19710-19716.
Reaction
22.2.2.2.3 adenosine 5'-monophosphate (AMP) + ATP <=> adenosine 5'-diphosphate (ADP) + ADP
Description
Cytosolic adenylate kinase 1 catalyzes the reaction of ATP and AMP to form two molecules of ADP.
References
S Matsuura, M Igarashi, Y Tanizawa, M Yamada, F Kishi, T Kajii, H Fujii, S Miwa, M Sakurai, A Nakazawa, "Human adenylate kinase deficiency
associated with hemolytic anemia. A single base substitution affecting solubility and catalytic activity of the cytosolic adenylate kinase.", J Biol
Chem, 264, 1989, 10148-55.
Reaction
22.2.2.2.4 ADP-ATP translocase maintains a high ADP:ATP ratio in the matrix
Authors
Jassal, B, 2005-06-30.
Description
A family of antiport, ATP-ADP translocases, preferentially export ATP from the matrix while importing ADP from the cytosol, thereby maintaining
a high ADP:ATP ratio in the matrix. When there are increased energy demands on the body, such as under heavy exercise, cytosolic ADP rises
and is exchanged with mitochondrial matrix ATP via the transmembrane ADP:ATP translocase. Increased ADP causes the proton-motive force
to be discharged and protons enter via ATPase, thereby regenerating the ATP pool.
There are 3 isoforms of translocases in humans; isoform 1 is the heart/skeletal muscle form, isoform 2 is the fibroblast form and isoform 3 is the
liver form. All isoforms exist as homodimers. The translocase can adopt 2 different conformations, called the CATR (carboxyatractyloside) and
BA (bongkrekic acid) conformations. Amongst the endogenous nucleotides, only ADP and ATP can trigger the rapid conversion between the
CATR and BA conformations.
The reaction can be summed as below:
ADPout + ATPin ADPin + ATPout

References
E Pfaff, M Klingenberg, HW Heldt, "Unspecific permeation and specific exchange of adenine nucleotides in", Biochim Biophys Acta, 104, 1965,
312-5.
C Fiore, V Trezeguet, A Le Saux, P Roux, C Schwimmer, AC Dianoux, F Noel, GJ Lauquin, G Brandolin, PV Vignais, "The mitochondrial
ADP/ATP carrier: structural, physiological and", Biochimie, 80, 1998, 137-50.
M Klingenberg, "Molecular aspects of the adenine nucleotide carrier from mitochondria", Arch Biochem Biophys, 270, 1989, 1-14.
ED Duee, PV Vignais, "[Exchange between extra- and intramitochondrial adenine nucleotides]", Biochim Biophys Acta, 107, 1965, 184-8.
Reaction
22.2.2.2.5 Formation of ATP by chemiosmotic coupling
Authors
Jassal, B, 2005-06-29.
Description
The re-entry of protons into the mitochondrial matrix through Complex V causes conformational changes which result in ATP synthesis. Complex
V (ATP synthase) is composed of 3 parts; an F1 catalytic core (approx 5 subunits), an F0 membrane proton channel (approx 9 subunits) and two
stalks linking F1 to F0. F1 contains three alpha subunits, three beta subunits, and one each of gamma, delta, and epsilon subunits. Each beta
subunit contains an active site for ATP synthesis. F0 has at least 9 subunits (a-g, A6L and F6), with one copy each of subunits b, d and F6.
The mechanism of ATP synthesis by Complex V was predicted by Boyer et al in 1973: ADP and Pi bind to the enzyme resulting in a
conformational change. ATP is then synthesized, still bound to the enzyme. Another change in the active site results in the release of free ATP
into the matrix. The overall reaction is:
ADP + Pi + H+ + nH+memb. space -> ATP + H2O + nH+matrix

References
PD Boyer, RL Cross, W Momsen, "A new concept for energy coupling in oxidative phosphorylation based on a", Proc Natl Acad Sci U S A, 70,
1973, 2837-9.
22.2.2.2.5.1 ADP and Pi bind to ATPase
Authors
Jassal, B, 2005-06-30.
Description
The beta subunit has 3 conformations; tight, open and loose. ADP and Pi bind to the subunit in the loose form. On binding, this subunit is
converted to the tight configuration.
References
1973, 2837-9.
Reaction
22.2.2.2.5.2 ATP is synthesized from ADP and Pi by ATPase
Authors
Jassal, B, 2005-06-30.
Description
In the tight configuration, the beta subunit catalyzes the reaction of ADP + Pi to ATP + water. ATP is still tightly bound to the subunit at this
stage.
References
1973, 2837-9.
Reaction
22.2.2.2.5.3 Enzyme-bound ATP is released
Authors
Jassal, B, 2005-06-30.
Description
In the last step, the beta subunit is converted to the open form and ATP is released. Passage of protons through the Fo part causes a ring of
approximately 10 subunits to rotate. This rotation in turn drives the rotation of the gamma subunits, which forms part of one of the stalks. The
gamma subunit moves between the three beta subunits which are held in place by the second stalk which can be regarded as a stator. The
polypeptide called OSCP connects the stator stalk to the assembly of alpha and beta subunits. It is this step that is coupled to proton
translocation as energy is required to break the strong bond between ATP and the protein.
References
1973, 2837-9.
Reaction
22.2.2.3 dATP formation
Description
ADP is reduced by ribonucleotide reductase and then phosphorylated to yield dATP.
22.2.2.3.1 adenosine 5'-diphosphate + thioredoxin (reduced) => 2'-deoxyadenosine 5'-diphosphate + thioredoxin (oxidized) + H2O
Description
At the beginning of this reaction, 1 molecule of 'Thioredoxin', and 1 molecule of 'ADP' are present. At the end of this reaction, 1 molecule of
'thioredoxin, oxidized', 1 molecule of '2'-deoxyadenosine 5'-diphosphate', and 1 molecule of 'H2O' are present.
References
cells", J Biol Chem, 276, 2001, 40647-40651.
Reaction
22.2.2.3.2 thioredoxin, oxidized + NADPH + H+ => thioredoxin, reduced + NADP+
Description
References
Reaction
22.2.2.3.3 2'-deoxyadenosine 5'-diphosphate (dADP) + ATP <=> dATP + adenosine 5'-diphosphate (ADP)
Description
At the beginning of this reaction, 1 molecule of '2'-deoxyadenosine 5'-diphosphate', and 1 molecule of 'ATP' are present. At the end of this
reaction, 1 molecule of 'ADP', and 1 molecule of 'dATP' are present.
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
22.2.2.4 2'-deoxyadenosine 5'-diphosphate (dADP) + ATP <=> dATP + adenosine 5'-diphosphate (ADP)
Description
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
22.2.2.5 dAMP + ATP <=> dADP + ADP
Description
At the beginning of this reaction, 1 molecule of '2'-deoxyadenosine 5'-monophosphate', and 1 molecule of 'ATP' are present. At the end of this
reaction, 1 molecule of 'ADP', and 1 molecule of '2'-deoxyadenosine 5'-diphosphate' are present.

Reaction
22.2.2.6 2'-deoxyguanosine 5'-diphosphate (dGDP) + ATP <=> dGTP + adenosine 5'-diphosphate (ADP)
Description
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
22.2.2.7 2'-deoxyguanosine 5'-monophosphate (dGMP) + ATP <=> 2'-deoxyguanosine 5'-diphosphate (dGDP) + ADP
Description
At the beginning of this reaction, 1 molecule of '2'-deoxyguanosine 5'-monophosphate', and 1 molecule of 'ATP' are present. At the end of this
reaction, 1 molecule of 'ADP', and 1 molecule of '2'-deoxyguanosine 5'-diphosphate' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'nucleoside kinase activity' of 'guanylate kinase 1'.
References
KC Agarwal, RP Miech, RE Parks, Jr, "Guanylate kinases from human erythrocytes, hog brain, and rat liver", Methods Enzymol, 51, 1978,
483-491.
Reaction
22.2.2.8 guanosine 5'-monophosphate (GMP) + ATP <=> guanosine 5'-diphosphate (GDP) + ADP
Description
At the beginning of this reaction, 1 molecule of 'ATP', and 1 molecule of 'GMP' are present. At the end of this reaction, 1 molecule of 'GDP', and
1 molecule of 'ADP' are present.
References
483-491.
Reaction
22.2.2.9 Conversion of cytosolic inosine 5'-monophosphate (IMP), NAD+, and H2O to xanthosine 5'-monophosphate (XMP) and NADH
+ H+
Authors
Editors
Description
Two human isoenzymes catalyze the irreversible dehydrogenation of inosine 5'-monophosphate (IMP) to form xanthosine 5'-monophosphate
(XMP). The active forms of both isoenzymes are homotetramers, and they are nearly identical in their catalytic efficiencies and their susceptibility
to inhibition by XMP (Carr et al. 1993; Colby et al. 1999; Hager et al. 1995). Distinct functions for the two isozymes have not been established.
However, a variety of experiments suggest that distinct functions exist. While one isoenzyme, IMP (inosine monophosphate dehydrogenase) 1,
is expressed at constant levels; the other, IMP (inosine monophosphate dehydrogenase) 2, is expressed at elevated levels in tumor cells and in
mitotic normal cells. In humans, heterozygosity for mutant forms of isoenzyme 1 is associated with a form of retinitis pigmentosa (Bowne et al.
2002). In laboratory mice, mutations that disrupt the homologue of isoenzyme 1 have no obvious effect at the level of the whole organism, while
ones that disrupt isoenzyme 2 are lethal (Gu et al. 2003).
This reaction is the rate limiting step in the synthesis of guanosine 5'-monophosphate (GMP) from IMP, and GMP competitively inhibits the
well-characterized bacterial IMP dehydrogenase enzyme. Evidence for an inhibitory effect of GMP on the human isoenzymes has not been
reported. Rather, they appear to be inhibited by XMP; in addition, transcription of one or both IMP dehydrogenase mRNAs may be inhibited by
high cellular GMP concentrations (Glesne et al. 1991).
References
DA Glesne, FR Collart, E Huberman, "Regulation of IMP dehydrogenase gene expression by its end products, guanine nucleotides", Mol Cell
Biol, 11, 5417-5425.
JJ Gu, AK Tolin, J Jain, H Huang, L Santiago, BS Mitchell, "Targeted disruption of the inosine 5'-monophosphate dehydrogenase type I gene in
mice", Mol Cell Biol, 23, 2003, 6702-6712.
SJ Bowne, LS Sullivan, SH Blanton, CL Cepko, S Blackshaw, DG Birch, D Hughbanks-Wheaton, JR Heckenlively, SP Daiger, "Mutations in the
inosine monophosphate dehydrogenase 1 gene (IMPDH1) cause the RP10 form of autosomal dominant retinitis pigmentosa", Hum Mol Genet,
11, 2002, 559-568.
PW Hager, FR Collart, E Huberman, BS Mitchell, "Recombinant human inosine monophosphate dehydrogenase type I and type II proteins.
Purification and characterization of inhibitor binding", Biochem Pharmacol, 49, 1995, 1323-1329.
TD Colby, K Vanderveen, MD Strickler, GD Markham, BM Goldstein, "Crystal structure of human type II inosine monophosphate
dehydrogenase: implications for ligand binding and drug design", Proc Natl Acad Sci USA, 96, 1999, 3531-3536.
SF Carr, E Papp, JC Wu, Y Natsumeda, "Characterization of human type I and type II IMP dehydrogenases", J Biol Chem, 268, 1993,
27286-27290.
22.2.2.9.1 inosine 5'-monophosphate (IMP) + NAD+ + H2O => xanthosine 5'-monophosphate (XMP) + NADH + H+
Authors
Editors
Description
Reaction catalyzed by isoenzyme 1
References
27286-27290.
Reaction
22.2.2.9.2 inosine 5'-monophosphate (IMP) + NAD+ + H2O => xanthosine 5'-monophosphate (XMP) + NADH + H+
Authors
Editors
Description
Reaction catalyzed by isoenzyme 2
References
27286-27290.
Reaction
22.2.2.10 xanthosine 5'-monophosphate (XMP) + L-glutamine + ATP + H2O => guanosine 5'-monophosphate (GMP) + L-glutamate +
adenosine 5'-monophosphate (AMP) + pyrophosphate
Authors
Editors
Description
The conversion of cytosolic xanthosine 5'-monophosphate (XMP) to guanosine 5'-monophosphate (GMP), accompanied by the hydrolysis of
ATP to adenosine 5'-monophosphate (AMP) and pyrophosphate, is catalyzed by guanine monophosphate synthetase. The reaction has an
absolute requirement for Mg++. The enzyme from E. coli is a dimer and uses ammonia and glutamine equally efficiently as sources of the amino
group transferred to XMP (Patel et al. 1975). The human enzyme in contrast is monomeric and, while it can utilize ammonia, does so with low
efficiency so that ammonia is unlikely to play a significant role in GMP synthesis under normal conditions in vivo (Hirst et al. 1994; Nakamura
and Lou 1995).
References
M Hirst, E Haliday, J Nakamura, L Lou, "Human GMP synthetase. Protein purification, cloning, and functional expression of cDNA", J Biol Chem,
269, 1994, 23830-23837.
N Patel, HS Moyed, JF Kane, "Xanthosine-5'-phosphate amidotransferase from Escherichia coli", J Biol Chem, 250, 1975, 2609-2613.
J Nakamura, L Lou, "Biochemical characterization of human GMP synthetase", J Biol Chem, 270, 1995, 7347-7353.
Reaction
22.2.3 Purine salvage reactions
Authors
Jassal, B, 2003-07-17.
Editors
Description
Nucleosides and free bases generated by DNA and RNA breakdown are converted back to nucleotide monophosphates, allowing them to
re-enter the pathway of purine biosynthesis. To emphasize the limited salvage events possible for purine deoxyribonucleotides, the pathways of
ribonucleotide salvage and deoxyribonucleotide salvage are shown separately.
22.2.3.1 Deoxyribonucleotide salvage

Description
Nucleosides and free bases generated by DNA breakdown are converted back to nucleotide monophosphates, allowing them to re-enter the
pathway of purine biosynthesis. Under normal conditions, DNA turnover is limited and deoxyribonucleotide salvage operates at a
correspondingly low level.
22.2.3.1.1 2'-deoxyguanosine 5'-monophosphate (dGMP) + H2O => 2'-deoxyguanosine + orthophosphate
Description
At the beginning of this reaction, 1 molecule of '2'-deoxyguanosine 5'-monophosphate', and 1 molecule of 'H2O' are present. At the end of this
reaction, 1 molecule of '2'-deoxyguanosine', and 1 molecule of 'Orthophosphate' are present.
This reaction takes place in the 'cytosol' and is mediated by the '5'-nucleotidase activity' of '5'-nucleotidase cytosolic II holoenzyme'.
References
J Spychala, V Madrid-Marina, IH Fox, "High Km soluble 5'-nucleotidase from human placenta", J Biol Chem, 263, 1988, 18759-18765.
Reaction
22.2.3.1.2 2'-deoxyguanosine + orthophosphate <=> guanine + 2-deoxy-D-ribose 1-phosphate
Description
At the beginning of this reaction, 1 molecule of '2'-deoxyguanosine', and 1 molecule of 'Orthophosphate' are present. At the end of this reaction,
1 molecule of 'guanine', and 1 molecule of '2-deoxy-D-ribose 1-phosphate' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'purine-nucleoside phosphorylase activity' of 'nucleoside phosphorylase
homotrimer'.
References
DA Wiginton, MS Coleman, JJ Hutton, "Characterization of purine nucleoside phosphorylase from human granulocytes and its metabolism of
deoxyribonucleosides", J Biol Chem, 255, 1980, 6663-6669.
Reaction
22.2.3.1.3 2'-deoxyguanosine + ATP => 2'-deoxyguanosine 5'-monophosphate + ADP
Description
At the beginning of this reaction, 1 molecule of '2'-deoxyguanosine', and 1 molecule of 'ATP' are present. At the end of this reaction, 1 molecule
of 'ADP', and 1 molecule of '2'-deoxyguanosine 5'-monophosphate' are present.
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'nucleoside kinase activity' of 'deoxyguanosine kinase holoenzyme'.
References
L Wang, U Hellman, S Eriksson, "Cloning and expression of human mitochondrial deoxyguanosine kinase cDNA", FEBS Lett, 390, 1996, 39-43.
M Johansson, A Karlsson, "Cloning and expression of human deoxyguanosine kinase cDNA", Proc Natl Acad Sci USA, 93, 1996, 7258-7262.
Reaction
22.2.3.1.4 2'-Deoxyadenosine + H2O => 2'-Deoxyinosine + NH3
Description
At the beginning of this reaction, 1 molecule of '2'-deoxyadenosine', and 1 molecule of 'H2O' are present. At the end of this reaction, 1 molecule
of 'NH3', and 1 molecule of '2'-deoxyinosine' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'adenosine deaminase activity' of 'Adenosine deaminase '.
References
AL Akeson, DA Wiginton, MR Dusing, JC States, JJ Hutton, "Mutant human adenosine deaminase alleles and their expression by transfection
into fibroblasts", J Biol Chem, 263, 1988, 16291-6.
Reaction
22.2.3.1.5 2'-deoxyinosine + orthophosphate <=> hypoxanthine + 2-deoxy-D-ribose 1-phosphate
Description
At the beginning of this reaction, 1 molecule of 'Orthophosphate', and 1 molecule of '2'-deoxyinosine' are present. At the end of this reaction, 1
molecule of 'Hypoxanthine', and 1 molecule of '2-deoxy-D-ribose 1-phosphate' are present.
homotrimer'.
References
Reaction
22.2.3.1.6 2'-deoxyadenosine + ATP => 2'-deoxyadenosine 5'-monophosphate + ADP
Description
At the beginning of this reaction, 1 molecule of '2'-deoxyadenosine', and 1 molecule of 'ATP' are present. At the end of this reaction, 1 molecule
of '2'-deoxyadenosine 5'-monophosphate', and 1 molecule of 'ADP' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'adenosine kinase activity' of 'adenosine kinase holoenzyme'.
References
MS Hershfield, JE Fetter, WC Small, AS Bagnara, SR Williams, B Ullman, DW Martin, Jr, DB Wasson, DA Carson, "Effects of mutational loss of
adenosine kinase and deoxycytidine kinase on deoxyATP accumulation and deoxyadenosine toxicity in cultured CEM human T-lymphoblastoid
cells", J Biol Chem, 257, 1982, 6380-6386.
MC Hurley, B Lin, IH Fox, "Regulation of deoxyadenosine and nucleoside analog phosphorylation by human placental adenosine kinase", J Biol
Chem, 260, 1985, 15675-15681.
Reaction
22.2.3.2 Ribonucleotide salvage
Description
Nucleosides and free bases generated by RNA breakdown are converted back to nucleotide monophosphates, allowing them to re-enter the
pathways of purine biosynthesis.
22.2.3.2.1 guanosine 5'-monophosphate (GMP) + H2O => guanosine + orthophosphate
Description
At the beginning of this reaction, 1 molecule of 'H2O', and 1 molecule of 'GMP' are present. At the end of this reaction, 1 molecule of 'guanosine',
and 1 molecule of 'Orthophosphate' are present.
References
Reaction
22.2.3.2.2 guanosine + orthophosphate <=> guanine + D-ribose 1-phosphate
Description
At the beginning of this reaction, 1 molecule of 'guanosine', and 1 molecule of 'Orthophosphate' are present. At the end of this reaction, 1
molecule of 'guanine', and 1 molecule of 'D-ribose 1-phosphate' are present.
homotrimer'.
References
Reaction
22.2.3.2.3 Guanine + PRPP => GMP + PPi
Description
At the beginning of this reaction, 1 molecule of '5-phospho-alpha-D-ribose 1-diphosphate', and 1 molecule of 'guanine' are present. At the end of
this reaction, 1 molecule of 'pyrophosphate', and 1 molecule of 'GMP' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'hypoxanthine phosphoribosyltransferase activity' of 'HGPRT homotetramer'.
References
DJ Jolly, H Okayama, P Berg, AC Esty, D Filpula, P Bohlen, GG Johnson, JE Shively, T Hunkapillar, T Friedmann, "Isolation and
characterization of a full-length expressible cDNA for human hypoxanthine phosphoribosyl transferase", Proc Natl Acad Sci U S A, 80, 1983,
477-81.
Reaction
22.2.3.2.4 Adenine + PRPP => AMP + PPi
Description
At the beginning of this reaction, 1 molecule of 'adenine', and 1 molecule of '5-phospho-alpha-D-ribose 1-diphosphate' are present. At the end of
this reaction, 1 molecule of 'pyrophosphate', and 1 molecule of 'AMP' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'adenine phosphoribosyltransferase activity' of 'APRT homodimer'.
References
JA Holden, GS Meredith, WN Kelley, "Human adenine phosphoribosyltransferase. Affinity purification, subunit structure, amino acid composition,
and peptide mapping.", J Biol Chem, 254, 1979, 6951-5.
Reaction
22.2.3.2.5 adenosine + ATP => adenosine 5'-monophosphate (AMP) + ADP
Description
At the beginning of this reaction, 1 molecule of 'adenosine', and 1 molecule of 'ATP' are present. At the end of this reaction, 1 molecule of 'ADP',
and 1 molecule of 'AMP' are present.
References
II Mathews, MD Erion, SE Ealick, "Structure of human adenosine kinase at 1.5 A resolution.", Biochemistry, 37, 1998, 15607-20.
CM Andres, IH Fox, "Purification and properties of human placental adenosine kinase", J Biol Chem, 254, 1979, 11388-11393.
J Spychala, NS Datta, K Takabayashi, M Datta, IH Fox, T Gribbin, BS Mitchell, "Cloning of human adenosine kinase cDNA: sequence similarity
to microbial ribokinases and fructokinases", Proc Natl Acad Sci USA, 93, 1996, 1232-1237.
Reaction
22.2.3.2.6 adenosine 5'-monophosphate (AMP) + H2O => inosine 5'-monophosphate (IMP) + NH4+ (L isoform)
Description
At the beginning of this reaction, 1 molecule of 'H2O', and 1 molecule of 'AMP' are present. At the end of this reaction, 1 molecule of 'NH4+', and
1 molecule of 'IMP' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'AMP deaminase activity' of 'AMP deaminase isoform L tetramer'.
References
al., editors), 2, 2001, 2627-2638.
H Holmsen, "Platelet AMP deaminase. Purification and kinetic studies.", J Biol Chem, 256, 1981, 10519-23.
Reaction
22.2.3.2.7 adenosine 5'-monophosphate (AMP) + H2O => inosine 5'-monophosphate (IMP) + NH4+ (M isoform)
Description
This reaction takes place in the 'cytosol' and is mediated by the 'AMP deaminase activity' of 'AMP deaminase isoform M monomer'.
References
al., editors), 2, 2001, 2627-2638.
C Frieden, "Adenylate deaminase. Kinetic and binding studies on the rabbit muscle enzyme.", J Biol Chem, 253, 1979, 8728-35.
YP Lee, "5'-Adenylic acid deaminase. III. Properties and kinetic studies.", J Biol Chem, 227, 1958, 999-1007.
Reaction
22.2.3.2.8 adenosine 5'-monophosphate (AMP) + H2O => inosine 5'-monophosphate (IMP) + NH4+ (E isoform)
Description
This reaction takes place in the 'cytosol' and is mediated by the 'AMP deaminase activity' of 'AMP deaminase isoform E tetramer'.
References
al., editors), 2, 2001, 2627-2638.
S Yun, CH Suelter, "Human erythrocyte 5'-AMP aminohydrolase. Purification and characterization.", J Biol Chem, 253, 1978, 404-8.
Reaction
22.2.3.2.9 Adenosine + H2O => Inosine + NH3
Description
At the beginning of this reaction, 1 molecule of 'adenosine', and 1 molecule of 'H2O' are present. At the end of this reaction, 1 molecule of 'NH3',
and 1 molecule of 'inosine' are present.
References
Reaction
22.2.3.2.10 inosine 5'-monophosphate (IMP) + H2O => inosine + orthophosphate
Description
At the beginning of this reaction, 1 molecule of 'IMP', and 1 molecule of 'H2O' are present. At the end of this reaction, 1 molecule of
'Orthophosphate', and 1 molecule of 'inosine' are present.
References
Reaction
22.2.3.2.11 inosine + orthophosphate <=> hypoxanthine + D-ribose 1-phosphate
Description
At the beginning of this reaction, 1 molecule of 'Orthophosphate', and 1 molecule of 'inosine' are present. At the end of this reaction, 1 molecule
of 'Hypoxanthine', and 1 molecule of 'D-ribose 1-phosphate' are present.
homotrimer'.
References
Reaction
22.2.3.2.12 Hypoxanthine + PRPP => IMP + PPi
Description
At the beginning of this reaction, 1 molecule of '5-phospho-alpha-D-ribose 1-diphosphate', and 1 molecule of 'Hypoxanthine' are present. At the
end of this reaction, 1 molecule of 'pyrophosphate', and 1 molecule of 'IMP' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'hypoxanthine phosphoribosyltransferase activity' of 'HGPRT homotetramer'.
References
DJ Jolly, H Okayama, P Berg, AC Esty, D Filpula, P Bohlen, GG Johnson, JE Shively, T Hunkapillar, T Friedmann, "Isolation and
characterization of a full-length expressible cDNA for human hypoxanthine phosphoribosyl transferase", Proc Natl Acad Sci U S A, 80, 1983,
477-81.
Reaction
22.2.4 Purine catabolism
Authors
Jassal, B, 2003-07-17.
Editors
Description
The purine bases guanine and hypoxanthine (derived from adenine by events in the purine salvage pathways) are converted to xanthine and
then to uric acid, which is excreted from the body. The end-point of this pathway in humans and hominoid primates is unusual. Most other
mammals metabolize uric acid further to yield more soluble end products, and much speculation has centered on possible roles for high uric acid
levels in normal human physiology.
22.2.4.1 Guanine + H2O => Xanthine + NH3
Description
At the beginning of this reaction, 1 molecule of 'guanine', and 1 molecule of 'H2O' are present. At the end of this reaction, 1 molecule of 'NH3',
and 1 molecule of 'Xanthine' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'guanine deaminase activity' of 'Guanine deaminase homodimer'.
References
G Yuan, JC Bin, DJ McKay, FF Snyder, "Cloning and characterization of human guanine deaminase. Purification and partial amino acid
sequence of the mouse protein.", J Biol Chem, 274, 1999, 8175-80.
Reaction
22.2.4.2 Hypoxanthine + H2O + O2 => Xanthine + H2O2
Description
At the beginning of this reaction, 1 molecule of 'Oxygen', 1 molecule of 'H2O', and 1 molecule of 'Hypoxanthine' are present. At the end of this
reaction, 1 molecule of 'Xanthine', and 1 molecule of 'H2O2' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'xanthine oxidase activity' of 'Xanthine oxidase homodimer-(FAD-Mo-[2Fe-2S])
complex'.
References
M Saksela, KO Raivio, "Cloning and expression in vitro of human xanthine dehydrogenase/oxidase", Biochem J, 315, 1996, 235-9.
Reaction
22.2.4.3 Xanthine + H2O + O2 => Urate + H2O2

Description
At the beginning of this reaction, 1 molecule of 'Oxygen', 1 molecule of 'Xanthine', and 1 molecule of 'H2O' are present. At the end of this
reaction, 1 molecule of 'H2O2', and 1 molecule of 'Urate' are present.
complex'.
References
Reaction
22.2.4.4 Xanthine formation
Description
The steps which ultimately form xanthine are described.
22.2.4.4.1 Inosine formation
Description
Inosine is generated either by the hydrolytic cleavage of an amino group from adenosine or of a phosphate group from inosine monophosphate.
22.2.4.4.1.1 Adenosine + H2O => Inosine + NH3
Description
At the beginning of this reaction, 1 molecule of 'adenosine', and 1 molecule of 'H2O' are present. At the end of this reaction, 1 molecule of 'NH3',
and 1 molecule of 'inosine' are present.
References
Reaction
22.2.4.4.1.2 inosine 5'-monophosphate (IMP) + H2O => inosine + orthophosphate
Description
References
Reaction
22.2.4.4.2 inosine + orthophosphate <=> hypoxanthine + D-ribose 1-phosphate
Description
At the beginning of this reaction, 1 molecule of 'Orthophosphate', and 1 molecule of 'inosine' are present. At the end of this reaction, 1 molecule
of 'Hypoxanthine', and 1 molecule of 'D-ribose 1-phosphate' are present.
homotrimer'.
References
Reaction
22.2.4.4.3 Hypoxanthine formation
Description
dAMP is converted to hypoxanthine by successive hydrolysis and phosphorolysis reactions.
22.2.4.4.3.1 2'-Deoxyadenosine + H2O => 2'-Deoxyinosine + NH3
Description
At the beginning of this reaction, 1 molecule of '2'-deoxyadenosine', and 1 molecule of 'H2O' are present. At the end of this reaction, 1 molecule
of 'NH3', and 1 molecule of '2'-deoxyinosine' are present.
References
Reaction
22.2.4.4.3.2 2'-deoxyinosine + orthophosphate <=> hypoxanthine + 2-deoxy-D-ribose 1-phosphate
Description
At the beginning of this reaction, 1 molecule of 'Orthophosphate', and 1 molecule of '2'-deoxyinosine' are present. At the end of this reaction, 1
molecule of 'Hypoxanthine', and 1 molecule of '2-deoxy-D-ribose 1-phosphate' are present.
homotrimer'.
References
Reaction
22.2.4.4.4 Hypoxanthine + H2O + O2 => Xanthine + H2O2
Description
At the beginning of this reaction, 1 molecule of 'Oxygen', 1 molecule of 'H2O', and 1 molecule of 'Hypoxanthine' are present. At the end of this
reaction, 1 molecule of 'Xanthine', and 1 molecule of 'H2O2' are present.
complex'.
References
Reaction
22.2.4.4.5 Guanine formation
Description
GMP and dGMP are converted to guanine by successive hydrolysis and phosphorolysis reactions.
22.2.4.4.5.1 2'-deoxyguanosine 5'-monophosphate (dGMP) + H2O => 2'-deoxyguanosine + orthophosphate
Description
References
Reaction
22.2.4.4.5.2 2'-deoxyguanosine + orthophosphate <=> guanine + 2-deoxy-D-ribose 1-phosphate
Description
At the beginning of this reaction, 1 molecule of '2'-deoxyguanosine', and 1 molecule of 'Orthophosphate' are present. At the end of this reaction,
1 molecule of 'guanine', and 1 molecule of '2-deoxy-D-ribose 1-phosphate' are present.
homotrimer'.
References
Reaction
22.2.4.4.5.3 guanosine 5'-monophosphate (GMP) + H2O => guanosine + orthophosphate
Description
References
Reaction
22.2.4.4.5.4 guanosine + orthophosphate <=> guanine + D-ribose 1-phosphate
Description
At the beginning of this reaction, 1 molecule of 'guanosine', and 1 molecule of 'Orthophosphate' are present. At the end of this reaction, 1
molecule of 'guanine', and 1 molecule of 'D-ribose 1-phosphate' are present.
homotrimer'.
References
Reaction
22.2.4.4.6 Guanine + H2O => Xanthine + NH3
Description
At the beginning of this reaction, 1 molecule of 'guanine', and 1 molecule of 'H2O' are present. At the end of this reaction, 1 molecule of 'NH3',
and 1 molecule of 'Xanthine' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'guanine deaminase activity' of 'Guanine deaminase homodimer'.
References
G Yuan, JC Bin, DJ McKay, FF Snyder, "Cloning and characterization of human guanine deaminase. Purification and partial amino acid
sequence of the mouse protein.", J Biol Chem, 274, 1999, 8175-80.
Reaction
22.3 Pyrimidine metabolism
Authors
Editors
Description
The events of human pyrimidine metabolism are conveniently, if somewhat arbitrarily, grouped into four pathways: de novo synthesis of the
pyrimidine ring and its conversion to uridine 5'-monophosphate (UMP), the biosynthesis of other pyrimidine ribo- and deoxyribonucleotides,
pyrimidine salvage reactions, and pyrimidine catabolism.
De novo synthesis of the pyrimidine ring and its conversion to UMP. The pyrimidine base orotate is synthesized from glutamine, bicarbonate,
and aspartate. Orotate reacts with 5-phospho-alpha-D-ribose 1-diphosphate (PRPP) and is then decarboxylated to form the pyrimidine
nucleotide UMP.
Pyrimidine biosynthesis [interconversion]. Pyrimidine ribo- and deoxyribonucleotide di- and triphosphates are synthesized, both from UMP and
from pyrimidine ribonucleotide monophosphates generated in salvage reactions.
Pyrimidine salvage reactions. Pyrimidine nucleosides and free bases generated by DNA and RNA breakdown are converted to nucleotide
monophosphates.
Pyrimidine catabolism. The pyrimidine bases thymine and uracil are degraded to beta-aminoisobutyrate and beta-alanine, respectively, which
are excreted from the body.
22.3.1 De novo synthesis of the pyrimidine ring and conversion to UMP
Authors
Jassal, B, 2003-06-17.
Editors
Description
The pyrimidine orotate (orotic acid) is synthesized in a sequence of four reactions, deriving its atoms from glutamine, bicarbonate, and aspartate.
A single multifunctional cytosolic enzyme catalyzes the first three of these reactions, while the last one is catalyzed by a mitochondrial enzyme.
In two further reactions, catalyzed by a bifunctional cytosolic enzyme, orotate reacts with 1-phosphoribosyl 5-pyrophosphate (PRPP) to yield
orotidine 5'-monophosphate, which is decarboxylated to yield uridine 5'-monophosphate (UMP). All other pyrimidines are synthesized from UMP.
While several individual reactions in this pathway are reversible, as indicated by the double-headed arrows in the diagram of the pathway, other
irreversible reactions drive the pathway in the direction of UMP biosynthesis in the normal cell. All reactions are thus annotated here only in the
forward direction.
This pathway has been most extensively analyzed at the genetic and biochemical level in cell lines derived from Chinese and Syrian hamsters.
All three enzymes have also been purified from human sources, however, and the key features of these reactions have been confirmed from
studies of this human material (Jones 1980; Webster et al. 2001).
References
ME Jones, "Pyrimidine nucleotide biosynthesis in animals: genes, enzymes, and regulation of UMP biosynthesis.", Annu Rev Biochem, 49,
1980, 253-79.
DR Webster, DM Becroft, AH van Gennip, AB van Kuilenberg, "Hereditary orotic aciduria and other disorders of pyrimidine metabolism", The
Metabolic and Molecular Bases of Inherited Disease, 8th ed (Scriver CR, et al., editors), 2, 2001, 2663-2704.
22.3.1.1 L-glutamine + 2 ATP + HCO3- + H2O => carbamoyl phosphate + L-glutamate + 2 adenosine 5'-diphosphate + orthophosphate
Authors
2007-03-03.
Editors
Description
The irreversible synthesis of carbamoyl phosphate from glutamine, bicarbonate, and ATP is catalyzed by the carbamoyl-phosphate synthase
(glutamine-hydrolyzing) activity of the trifunctional CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase)
protein (Ito and Uchino 1973; Iwahana et al. 1996). The purified human protein is active in several different oligomerization states, as is its
Syrian hamster homologue. The most abundant form of the latter is a hexamer, and the active human protein is annotated as a hexamer for this
reason (Ito and Uchino 1973; Lee et al. 1985).
References
L Lee, RE Kelly, SC Pastra-Landis, DR Evans, "Oligomeric structure of the multifunctional protein CAD that initiates pyrimidine biosynthesis in
mammalian cells", Proc Natl Acad Sci USA, 82, 1985, 6802-6806.
H Iwahana, H Fujimura, S Ii, M Kondo, M Moritani, Y Takahashi, T Yamaoka, K Yoshimoto, M Itakura, "Molecular cloning of a human cDNA
encoding a trifunctional enzyme of carbamoyl-phosphate synthetase-aspartate transcarbamoylase-dihydroorotase in de Novo pyrimidine
synthesis", Biochem Biophys Res Commun, 219, 1996, 249-255.
K Ito, H Uchino, "Control of pyrimidine biosynthesis in human lymphocytes. Simultaneous increase in activities of glutamine-utilizing carbamyl
phosphate synthetase and aspartate transcarbamylase in phytohemagglutinin-stimulated human peripheral lymphocytes and their enzyme
co-purification", J Biol Chem, 248, 1973, 389-392.
Reaction
22.3.1.2 carbamoyl phosphate + L-aspartate <=> N-carbamoyl L-aspartate + orthophosphate
Authors
2007-03-03.
Editors
Description
The reversible (but strongly favored) synthesis of N-carbamoyl L-aspartate from carbamoyl phosphate and L-aspartate is catalyzed by the
aspartate carbamoyltansferase activity of the trifunctional CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and
dihydroorotase) protein (Ito and Uchino 1973; Iwahana et al. 1996). The purified human protein is active in several different oligomerization
states, as is its Syrian hamster homologue. The most abundant form of the latter is a hexamer, and the active human protein is annotated as a
hexamer for this reason (Ito and Uchino 1973; Lee et al. 1985).
References
K Ito, H Uchino, "Control of pyrimidine biosynthesis in human lymphocytes. Simultaneous increase in activities of glutamine-utilizing carbamyl
phosphate synthetase and aspartate transcarbamylase in phytohemagglutinin-stimulated human peripheral lymphocytes and their enzyme
co-purification", J Biol Chem, 248, 1973, 389-392.
Reaction
22.3.1.3 N-carbamoyl L-aspartate + H+ <=> (S)-dihydroorotate + H2O
Authors
2007-03-03.
Editors
Description
The reversible synthesis of dihydroorotate from N-carbamoyl L-aspartate is catalyzed by the dihydroorotase activity of the trifunctional CAD
(carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) protein. This activity has not been directly demonstrated
in experimental studies of the purified human protein, but has been inferred from the behavior of the purified hamster protein and the high
degree of sequence similarity between the cloned hamster and human genes (Iwahana et al. 1996). Also on the basis of this similarity, the active
human protein is annotated as a hexamer (Lee et al. 1985).
References
Reaction
22.3.1.4 (S)-dihydroorotate + ubiquinone => orotate + ubiquinol

Authors
Editors
Description
Dihydroorotate dehydrogenase catalyzes the essentially irreversible oxidation of dihydroorotate to orotate (orotic acid). The enzyme is located in
the inner mitochondrial membrane, oriented so that cytosolic dihydroorotate molecules have access to it, and orotate is released into the cytosol.
The reducing equivalents generated by the reaction are transferred by ubiquinone (Coenzyme Q) to the electron transport chain within the inner
mitochondrial membrane. Despite statements to the contrary in several standard textbooks of biochemistry, there is no evidence for the
involvement of NAD+ as an acceptor of reducing equivalents in this reaction in mammalian cells (Jones 1980). In contrast, the reaction catalyzed
by the enzyme isolated from the anaerobic bacterium Clostridium oroticum requires NAD+ as a cofactor but also, consistent with the greater
electronegativity of NAD+, proceeds strongly in the direction of dihydroorotate synthesis (Lieberman and Kornberg 1953).
Early studies of purified rat liver enzyme by Forman and Kennedy suggested the presence of flavin mononucleotide and iron-sulfur complexes
as cofactors. More recent work by Bader, Beuneu, and their colleagues with recombinant human protein expressed in insect cells has confirmed
the presence of flavin mononucleotide, at a stoichiometry of one molecule per molecule of apoenzyme, but suggests that iron-sulfur complexes,
if indeed they are involved in the oxidation and electron transport process, are not an integral part of the dihydroorotate dehydrogenase
holoenzyme.
References
JH Forman, J Kennedy, "Superoxide production and electron transport in mitochondrial oxidation of dihydroorotic acid.", J Biol Chem, 250, 1975,
4322-6.
B Bader, W Knecht, M Fries, M LÃ¶ffler, "Expression, purification, and characterization of histidine-tagged rat and human flavoenzyme
dihydroorotate dehydrogenase.", Protein Expr Purif, 13, 1998, 414-22.
C Beuneu, R Auger, M LÃ¶ffler, A Guissani, G Lemaire, M Lepoivre, "Indirect inhibition of mitochondrial dihydroorotate dehydrogenase activity
by nitric oxide.", Free Radic Biol Med, 28, 2000, 1206-13.
I Lieberman, A Kornberg, "Enzymic synthesis and breakdown of a pyrimidine, orotic acid. I. Dihydro-orotic dehydrogenase", Biochim Biophys
Acta, 12, 1953, 223-234.
ME Jones, "Pyrimidine nucleotide biosynthesis in animals: genes, enzymes, and regulation of UMP biosynthesis.", Annu Rev Biochem, 49,
1980, 253-79.
Reaction
22.3.1.5 orotate + 5-phospho-alpha-D-ribose 1-diphosphate (PRPP) <=> orotidine 5'-monophosphate (OMP) + pyrophosphate
Authors
2007-03-03.
Editors
Description
The synthesis of orotidine 5'-monophosphate (OMP) from orotate and 5-phospho-alpha-D-ribose 1-diphosphate (PRPP) is catalyzed by the
orotate phosphoribosyltransferase activity of the bifunctional "uridine monophosphate synthetase (orotate phosphoribosyl transferase and
orotidine 5'-decarboxylase)" protein. The reaction itself is freely reversible, but is pulled in the forward direction in vivo by the irreversible
hydrolysis of pyrophosphate. While purified human protein has not been characterized in detail, the close similarity of the human gene to that
encoding the well-studied hamster protein, and the demonstration that mutations in the human gene are associated with failure to convert
orotate to UMP in vivo, provide convincing evidence that the human uridine monophosphate synthetase protein indeed catalyzes these two
reactions (McClard et al. 1980; Suchi et al. 1997). By analogy to its hamster homologue, the active form of the human protein is inferred to be a
monomer.
References
M Suchi, H Mizuno, Y Kawai, T Tsuboi, S Sumi, K Okajima, ME Hodgson, H Ogawa, Y Wada, "Molecular cloning of the human UMP synthase
gene and characterization of point mutations in two hereditary orotic aciduria families", Am J Hum Genet, 60, 1997, 525-539.
RW McClard, MJ Black, LR Livingstone, ME Jones, "Isolation and initial characterization of the single polypeptide that synthesizes uridine
5'-monophosphate from orotate in Ehrlich ascites carcinoma. Purification by tandem affinity chromatography of uridine-5'-monophosphate
synthase", Biochemistry, 19, 1980, 4699-4706.
Reaction
22.3.1.6 orotidine 5'-monophosphate => uridine 5'-monophosphate + CO2
Authors
2007-03-03.
Editors
Description
The decarboxylation of orotidine 5'-monophosphate (OMP) to form uridine 5'-monophosphate (UMP) is catalyzed by the orotidine 5'-phosphate
decarboxylase activity of the bifunctional "uridine monophosphate synthetase (orotate phosphoribosyl transferase and orotidine
5'-decarboxylase)" protein. While purified human protein has not been characterized in detail, the close similarity of the human gene to that
encoding the well-studied hamster protein, and the demonstration that mutations in the human gene are associated with failure to convert
orotate to UMP in vivo, provide convincing evidence that the human uridine monophosphate synthetase protein indeed catalyzes these two
reactions (McClard et al. 1980; Suchi et al. 1997). By analogy to its hamster homologue, the active form of the human protein is inferred to be a
monomer.
References
M Suchi, H Mizuno, Y Kawai, T Tsuboi, S Sumi, K Okajima, ME Hodgson, H Ogawa, Y Wada, "Molecular cloning of the human UMP synthase
gene and characterization of point mutations in two hereditary orotic aciduria families", Am J Hum Genet, 60, 1997, 525-539.
RW McClard, MJ Black, LR Livingstone, ME Jones, "Isolation and initial characterization of the single polypeptide that synthesizes uridine
5'-monophosphate from orotate in Ehrlich ascites carcinoma. Purification by tandem affinity chromatography of uridine-5'-monophosphate
synthase", Biochemistry, 19, 1980, 4699-4706.
Reaction
22.3.2 Pyrimidine biosynthesis (interconversion)
Authors
Jassal, B, 2003-06-17.
Description
Uridine 5'-monophosphate (UMP) is the starting material for the biosynthesis of the ribonucleotide cytidine and the deoxyribonucleotides
thymidine and deoxycytidine. Kinases convert all of these molecules to nucleotide 5'-triphosphates, to serve as substrates for RNA and DNA
biosynthesis. These kinase reactions are reversible (so that, e.g., UDP and ADP can react to form UMP and ATP). Under normal physiological
conditions (high concentrations of adenine nucleotides relative to other nucleotides, and higher concentrations of ATP than ADP), however, they
proceed in the direction of ATP consumption, not ATP synthesis.
References
22.3.2.1 uridine 5'-monophosphate (UMP) + ATP <=> uridine 5'-diphosphate (UDP) + ADP
Description
At the beginning of this reaction, 1 molecule of 'ATP', and 1 molecule of 'uridine 5'-monophosphate' are present. At the end of this reaction, 1
molecule of 'uridine 5'-diphosphate', and 1 molecule of 'ADP' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'nucleoside kinase activity' of 'UMP-CMP kinase '.
References
JY Liou, GE Dutschman, W Lam, Z Jiang, YC Cheng, "Characterization of human UMP/CMP kinase and its phosphorylation of D- and L-form
deoxycytidine analogue monophosphates.", Cancer Res, 62, 2002, 1624-31.
EM Scott, RC Wright, "Kinetics and equilibria of pyrimidine nucleoside monophosphate kinase from human erythrocytes", Biochim Biophys Acta,
571, 1979, 45-54.
Reaction
22.3.2.2 uridine 5'-diphosphate (UDP) + ATP <=> uridine 5'-triphosphate (UTP) + ADP
Description
At the beginning of this reaction, 1 molecule of 'ATP', and 1 molecule of 'uridine 5'-diphosphate' are present. At the end of this reaction, 1
molecule of 'ADP', and 1 molecule of 'UTP' are present.
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
22.3.2.3 uridine 5'-triphosphate + glutamine + ATP + H2O => cytidine 5'-triphosphate + glutamate + ADP + orthophosphate
Description
At the beginning of this reaction, 1 molecule of 'ATP', 1 molecule of 'Glutamine', 1 molecule of 'H2O', and 1 molecule of 'UTP' are present. At the
end of this reaction, 1 molecule of 'Glutamate', 1 molecule of 'CTP', 1 molecule of 'ADP', and 1 molecule of 'Orthophosphate' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'CTP synthase activity' of 'CTP synthase '.
References
GS Han, A Sreenivas, MG Choi, YF Chang, SS Martin, EP Baldwin, GM Carman, "Expression of Human CTP synthetase in Saccharomyces
cerevisiae reveals phosphorylation by protein kinase A", J Biol Chem, 280, 2005, 38328-36.
Reaction
22.3.2.4 adenosine 5'-diphosphate (ADP) + CTP <=> ATP + cytidine 5'-diphosphate (CDP)
Description
At the beginning of this reaction, 1 molecule of 'ADP', and 1 molecule of 'CTP' are present. At the end of this reaction, 1 molecule of 'cytidine
5'-diphosphate', and 1 molecule of 'ATP' are present.
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
22.3.2.5 cytidine 5'-diphosphate + thioredoxin (reduced) => 2'-deoxycytidine 5'-diphosphate + thioredoxin (oxidized) + H2O
Description
At the beginning of this reaction, 1 molecule of 'Thioredoxin', and 1 molecule of 'cytidine 5'-diphosphate' are present. At the end of this reaction,
1 molecule of 'thioredoxin, oxidized', 1 molecule of 'H2O', and 1 molecule of '2'-deoxycytidine 5'-diphosphate' are present.
References
cells", J Biol Chem, 276, 2001, 40647-40651.
Reaction
22.3.2.6 2'-deoxycytidine 5'-diphosphate (dCDP) + ATP <=> dCTP + adenosine 5'-diphosphate (ADP)
Description
At the beginning of this reaction, 1 molecule of '2'-deoxycytidine 5'-diphosphate', and 1 molecule of 'ATP' are present. At the end of this reaction,
1 molecule of 'ADP', and 1 molecule of 'dCTP' are present.
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
22.3.2.7 adenosine 5'-diphosphate (ADP) + dCTP <=> ATP + 2'-deoxycytidine 5'-diphosphate (dCDP)
Description
At the beginning of this reaction, 1 molecule of 'ADP', and 1 molecule of 'dCTP' are present. At the end of this reaction, 1 molecule of
'2'-deoxycytidine 5'-diphosphate', and 1 molecule of 'ATP' are present.
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
22.3.2.8 2'-deoxycytidine 5'-diphosphate (dCDP) + ADP <=> deoxycytidine 5'-monophosphate (dCMP) + ATP
Description
At the beginning of this reaction, 1 molecule of 'ADP', and 1 molecule of '2'-deoxycytidine 5'-diphosphate' are present. At the end of this reaction,
1 molecule of '2'-deoxycytidine 5'-monophosphate', and 1 molecule of 'ATP' are present.
References
571, 1979, 45-54.
Reaction
22.3.2.9 2'-deoxycytidine 5'-monophosphate (dCMP) + H2O => 2'-deoxyuridine 5'-monophosphate (dUMP) + NH4+
Description
At the beginning of this reaction, 1 molecule of 'H2O', and 1 molecule of '2'-deoxycytidine 5'-monophosphate' are present. At the end of this
reaction, 1 molecule of 'NH3', and 1 molecule of '2'-deoxyuridine 5'-monophosphate' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'dCMP deaminase activity' of 'Deoxycytidylate deaminase '.
References
KX Weiner, RS Weiner, F Maley, GF Maley, "Primary structure of human deoxycytidylate deaminase and overexpression of its functional protein
in Escherichia coli", J Biol Chem, 268, 1993, 12983-9.
Reaction
22.3.2.10 2'-deoxycytidine 5'-monophosphate (dCMP) + ATP <=> deoxycytidine 5'-diphosphate (dCDP) + ADP
Description
At the beginning of this reaction, 1 molecule of '2'-deoxycytidine 5'-monophosphate', and 1 molecule of 'ATP' are present. At the end of this
reaction, 1 molecule of 'ADP', and 1 molecule of '2'-deoxycytidine 5'-diphosphate' are present.
References
571, 1979, 45-54.
Reaction
22.3.2.11 cytidine 5'-diphosphate (CDP) + ADP <=> cytidine 5'-monophosphate (CMP) + ATP
Description
At the beginning of this reaction, 1 molecule of 'cytidine 5'-diphosphate', and 1 molecule of 'ADP' are present. At the end of this reaction, 1
molecule of 'cytidine 5'-monophosphate', and 1 molecule of 'ATP' are present.
References
571, 1979, 45-54.
Reaction
22.3.2.12 cytidine 5'-monophosphate (CMP) + ATP <=> cytidine 5'-diphosphate (CDP) + ADP
Description
At the beginning of this reaction, 1 molecule of 'cytidine 5'-monophosphate', and 1 molecule of 'ATP' are present. At the end of this reaction, 1
molecule of 'cytidine 5'-diphosphate', and 1 molecule of 'ADP' are present.
References
571, 1979, 45-54.
Reaction
22.3.2.13 cytidine 5'-diphosphate + ATP <=> cytidine 5'-triphosphate + ADP
Description
At the beginning of this reaction, 1 molecule of 'cytidine 5'-diphosphate', and 1 molecule of 'ATP' are present. At the end of this reaction, 1
molecule of 'ADP', and 1 molecule of 'CTP' are present.
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
22.3.2.14 adenosine 5'-diphosphate (ADP) + UTP <=> ATP + uridine 5'-diphosphate (UDP)
Description
At the beginning of this reaction, 1 molecule of 'ADP', and 1 molecule of 'UTP' are present. At the end of this reaction, 1 molecule of 'uridine
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
22.3.2.15 uridine 5'-diphosphate (UDP) + ADP <=> uridine 5'-monophosphate (UMP) + ATP
Description
At the beginning of this reaction, 1 molecule of 'uridine 5'-diphosphate', and 1 molecule of 'ADP' are present. At the end of this reaction, 1
molecule of 'ATP', and 1 molecule of 'uridine 5'-monophosphate' are present.
References
571, 1979, 45-54.
Reaction
22.3.2.16 uridine 5'-diphosphate + thioredoxin (reduced) => 2'-deoxyuridine 5'-diphosphate + thioredoxin (oxidized) + H2O
Description
At the beginning of this reaction, 1 molecule of 'uridine 5'-diphosphate', and 1 molecule of 'Thioredoxin' are present. At the end of this reaction, 1
molecule of 'thioredoxin, oxidized', 1 molecule of 'H2O', and 1 molecule of '2'-deoxyuridine 5'-diphosphate' are present.
References
cells", J Biol Chem, 276, 2001, 40647-40651.
Reaction
22.3.2.17 2'-deoxyuridine 5'-monophosphate (dUMP) + ATP <=> 2'-deoxyuridine 5'-diphosphate (dUDP) + ADP
Description
At the beginning of this reaction, 1 molecule of 'ATP', and 1 molecule of '2'-deoxyuridine 5'-monophosphate' are present. At the end of this
reaction, 1 molecule of 'ADP', and 1 molecule of '2'-deoxyuridine 5'-diphosphate' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'nucleoside kinase activity' of 'deoxythymidylate kinase (thymidylate kinase)
holoenzyme'.
References
LS Lee, YC Cheng, "Human thymidylate kinase. Purification, characterization, and kinetic behavior of the thymidylate kinase derived from
chronic myelocytic leukemia", J Biol Chem, 252, 1977, 5686-5691.
Reaction
22.3.2.18 2'-deoxyuridine 5'-diphosphate (dUDP) + ATP <=> deoxyuridine 5'-triphosphate (dUTP) + ADP
Description
At the beginning of this reaction, 1 molecule of '2'-deoxyuridine 5'-diphosphate', and 1 molecule of 'ATP' are present. At the end of this reaction,
1 molecule of 'ADP', and 1 molecule of 'dUTP' are present.
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
22.3.2.19 2'-deoxyuridine 5'-triphosphate (dUTP) + H2O <=> deoxyuridine 5'-monophosphate (dUMP) + pyrophosphate
Description
At the beginning of this reaction, 1 molecule of 'H2O', and 1 molecule of 'dUTP' are present. At the end of this reaction, 1 molecule of
'pyrophosphate', and 1 molecule of '2'-deoxyuridine 5'-monophosphate' are present.
References
Chem, 273, 1998, 5662-5669.
Reaction
22.3.2.20 2'-deoxyuridine 5'-monophosphate (dUMP) + N5,N10-methylene tetrahydrofolate => thymidine 5'-monophosphate (TMP) +
dihydrofolate
Description
At the beginning of this reaction, 1 molecule of 'tetrahydrofolate', and 1 molecule of '2'-deoxyuridine 5'-monophosphate' are present. At the end
of this reaction, 1 molecule of 'Dihydrofolate', and 1 molecule of 'thymidine 5'-monophosphate' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'thymidylate synthase activity' of 'TSase-Mg complex'.
References
VJ Davisson, W Sirawaraporn, DV Santi, "Expression of human thymidylate synthase in Escherichia coli", J Biol Chem, 264, 1989, 9145-8.
Reaction
22.3.2.21 thymidine 5'-monophosphate (TMP) + ATP <=> thymidine 5'-diphosphate (TDP) + ADP
Description
At the beginning of this reaction, 1 molecule of 'thymidine 5'-monophosphate', and 1 molecule of 'ATP' are present. At the end of this reaction, 1
molecule of 'thymidine 5'-diphosphate', and 1 molecule of 'ADP' are present.
holoenzyme'.
References
Reaction
22.3.2.22 thymidine 5'-diphosphate (TDP) + ATP <=> thymidine 5'-triphosphate (TTP) + ADP
Description
At the beginning of this reaction, 1 molecule of 'ATP', and 1 molecule of 'thymidine 5'-diphosphate' are present. At the end of this reaction, 1
molecule of 'ADP', and 1 molecule of 'TTP' are present.
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
22.3.2.23 adenosine 5'-diphosphate (ADP) + TTP <=> ATP + thymidine 5'-diphosphate (TDP)
Description
At the beginning of this reaction, 1 molecule of 'TTP', and 1 molecule of 'ADP' are present. At the end of this reaction, 1 molecule of 'thymidine
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
22.3.2.24 thymidine 5'-diphosphate (TDP) + ADP <=> thymidine 5'-monophosphate (TMP) + ATP
Description
At the beginning of this reaction, 1 molecule of 'thymidine 5'-diphosphate', and 1 molecule of 'ADP' are present. At the end of this reaction, 1
molecule of 'thymidine 5'-monophosphate', and 1 molecule of 'ATP' are present.
holoenzyme'.
References
Reaction
22.3.2.25 2'-deoxyuridine 5'-triphosphate (dUTP) + ADP <=> 2'-deoxyuridine 5'-diphosphate (dUDP) + ATP
Description
At the beginning of this reaction, 1 molecule of 'ADP', and 1 molecule of 'dUTP' are present. At the end of this reaction, 1 molecule of
'2'-deoxyuridine 5'-diphosphate', and 1 molecule of 'ATP' are present.
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
22.3.2.26 2'-deoxyuridine 5'-diphosphate (dUDP) + ADP <=> 2'deoxyuridine 5'-monophosphate (dUMP) + ATP
Description
At the beginning of this reaction, 1 molecule of 'ADP', and 1 molecule of '2'-deoxyuridine 5'-diphosphate' are present. At the end of this reaction,
1 molecule of 'ATP', and 1 molecule of '2'-deoxyuridine 5'-monophosphate' are present.
holoenzyme'.
References
Reaction
22.3.3 Pyrimidine salvage reactions
Authors
Jassal, B, 2003-06-17.
Description
In pyrimidine salvage reactions, nucleosides and free bases generated by DNA and RNA breakdown are converted back to nucleotide
monophosphates, allowing them to re-enter the pathways of pyrimidine biosynthesis (interconversion).
References
22.3.3.1 uracil + D-ribose 1-phosphate <=> uridine + orthophosphate
Description
At the beginning of this reaction, 1 molecule of 'uracil', and 1 molecule of 'D-ribose 1-phosphate' are present. At the end of this reaction, 1
molecule of 'uridine', and 1 molecule of 'Orthophosphate' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'uridine phosphorylase activity' of 'Uridine phosphorylase 1 '.
References
SI Watanabe, T Uchida, "Cloning and expression of human uridine phosphorylase", Biochem Biophys Res Commun, 216, 1995, 265-272.
Reaction
22.3.3.2 uridine + orthophosphate <=> uracil + D-ribose 1-phosphate
Description
At the beginning of this reaction, 1 molecule of 'uridine', and 1 molecule of 'Orthophosphate' are present. At the end of this reaction, 1 molecule
of 'uracil', and 1 molecule of 'D-ribose 1-phosphate' are present.
References
Reaction
22.3.3.3 cytidine + H2O => uridine + NH4+
Description
At the beginning of this reaction, 1 molecule of 'cytidine', and 1 molecule of 'H2O' are present. At the end of this reaction, 1 molecule of 'NH3',
and 1 molecule of 'uridine' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'cytidine deaminase activity' of 'cytidine deaminase-Zn complex'.
References
J Laliberte, RL Momparler, "Human cytidine deaminase: purification of enzyme, cloning, and expression of its complementary DNA", Cancer
Res, 54, 1994, 5401-7.
Reaction
22.3.3.4 uridine + ATP => uridine 5'-monophosphate (UMP) + ADP
Description
At the beginning of this reaction, 1 molecule of 'ATP', and 1 molecule of 'uridine' are present. At the end of this reaction, 1 molecule of 'ADP', and
1 molecule of 'uridine 5'-monophosphate' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'nucleoside kinase activity' of 'uridine-cytidine kinase 1 holoenzyme'.
References
AR Van Rompay, A Norda, K Linden, M Johansson, A Karlsson, "Phosphorylation of uridine and cytidine nucleoside analogs by two human
uridine-cytidine kinases", Mol Pharmacol, 59, 2001, 1181-1186.
Reaction
22.3.3.5 uridine 5'-monophosphate + H2O => uridine + orthophosphate
Description
At the beginning of this reaction, 1 molecule of 'H2O', and 1 molecule of 'uridine 5'-monophosphate' are present. At the end of this reaction, 1
This reaction takes place in the 'cytosol' and is mediated by the '5'-nucleotidase activity' of '5'-nucleotidase cytosolic 1A'.
References
SA Hunsucker, J Spychala, BS Mitchell, "Human cytosolic 5'-nucleotidase I. Characterization and role in nucleoside analog resistance", J Biol
Chem, 276, 2001, 10498-10504.
Reaction
22.3.3.6 uracil + 5-phospho-alpha-D-ribose 1-diphosphate (PRPP) <=> uridine 5'-monophosphate (UMP) + pyrophosphate
Description
At the beginning of this reaction, 1 molecule of '5-phospho-alpha-D-ribose 1-diphosphate', and 1 molecule of 'uracil' are present. At the end of
this reaction, 1 molecule of 'pyrophosphate', and 1 molecule of 'uridine 5'-monophosphate' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'uracil phosphoribosyltransferase activity' of 'uracil phosphoribosyltransferase'.
References
Reaction
22.3.3.7 cytidine + ATP => cytidine 5'-monophosphate (CMP) + ADP
Description
At the beginning of this reaction, 1 molecule of 'cytidine', and 1 molecule of 'ATP' are present. At the end of this reaction, 1 molecule of 'ADP',
and 1 molecule of 'cytidine 5'-monophosphate' are present.
References
Reaction
22.3.3.8 cytidine 5'-monophosphate + H2O => cytidine + orthophosphate

Description
At the beginning of this reaction, 1 molecule of 'cytidine 5'-monophosphate', and 1 molecule of 'H2O' are present. At the end of this reaction, 1
molecule of 'cytidine', and 1 molecule of 'Orthophosphate' are present.
References
Chem, 276, 2001, 10498-10504.
Reaction
22.3.3.9 2'-deoxycytidine + ATP => 2'-deoxycytidine 5'-monophosphate (dCMP) + ADP
Description
At the beginning of this reaction, 1 molecule of 'ATP', and 1 molecule of '2'-deoxycytidine' are present. At the end of this reaction, 1 molecule of
'ADP', and 1 molecule of '2'-deoxycytidine 5'-monophosphate' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'nucleoside kinase activity' of 'deoxycytidine kinase holoenzyme'.
References
JC Sarup, MA Johnson, V Verhoef, A Fridland, "Regulation of purine deoxynucleoside phosphorylation by deoxycytidine kinase from human
leukemic blast cells", Biochem Pharmacol, 38, 1989, 2601-2607.
EV Usova, S Eriksson, "Identification of residues involved in the substrate specificity of human and murine dCK", Biochem Pharmacol, 64, 2002,
1559-1567.
NS Datta, DS Shewach, MC Hurley, BS Mitchell, IH Fox, "Human T-lymphoblast deoxycytidine kinase: purification and properties", Biochemistry,
28, 1989, 114-123.
C Bohman, S Eriksson, "Deoxycytidine kinase from human leukemic spleen: preparation and characterization of the homogeneous enzyme",
Biochemistry, 27, 1988, 4258-4265.
Reaction
22.3.3.10 2'-deoxycytidine 5'-monophosphate + H2O => 2'-deoxycytidine + orthophosphate
Description
reaction, 1 molecule of 'Orthophosphate', and 1 molecule of '2'-deoxycytidine' are present.
References
Chem, 276, 2001, 10498-10504.
Reaction
22.3.3.11 2'-deoxycytidine + H2O => 2'-deoxyuridine + NH4+
Description
At the beginning of this reaction, 1 molecule of 'H2O', and 1 molecule of '2'-deoxycytidine' are present. At the end of this reaction, 1 molecule of
'NH3', and 1 molecule of '2'-deoxyuridine' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'cytidine deaminase activity' of 'cytidine deaminase-Zn complex'.
References
J Laliberte, RL Momparler, "Human cytidine deaminase: purification of enzyme, cloning, and expression of its complementary DNA", Cancer
Res, 54, 1994, 5401-7.
Reaction
22.3.3.12 2'-deoxyuridine 5'-monophosphate + H2O => 2'-deoxyuridine + orthophosphate
Description
At the beginning of this reaction, 1 molecule of 'H2O', and 1 molecule of '2'-deoxyuridine 5'-monophosphate' are present. At the end of this
reaction, 1 molecule of 'Orthophosphate', and 1 molecule of '2'-deoxyuridine' are present.
References
Reaction
22.3.3.13 2'-deoxyuridine + orthophosphate <=> uracil + 2-deoxy-D-ribose 1-phosphate
Description
At the beginning of this reaction, 1 molecule of 'Orthophosphate', and 1 molecule of '2'-deoxyuridine' are present. At the end of this reaction, 1
molecule of '2-deoxy-D-ribose 1-phosphate', and 1 molecule of 'uracil' are present.
References
TA Krenitsky, JW Mellors, RK Barclay, "Pyrimidine nucleosidases. Their classification and relationship to uric acid ribonucleoside
phosphorylase", J Biol Chem, 240, 1965, 1281-1286.
Reaction
22.3.3.14 uracil + 2-deoxy-D-ribose 1-phosphate <=> 2'-deoxyuridine + orthophosphate
Description
At the beginning of this reaction, 1 molecule of 'uracil', and 1 molecule of '2-deoxy-D-ribose 1-phosphate' are present. At the end of this reaction,
1 molecule of 'uridine', and 1 molecule of 'Orthophosphate' are present.
References
Reaction
22.3.3.15 thymine + 2-deoxy-D-ribose 1-phosphate <=> thymidine + orthophosphate
Description
At the beginning of this reaction, 1 molecule of '2-deoxy-D-ribose 1-phosphate', and 1 molecule of 'thymine' are present. At the end of this
reaction, 1 molecule of 'thymidine', and 1 molecule of 'Orthophosphate' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'transferase activity' of 'thymidine phosphorylase dimer'.
References
A Yoshimura, Y Kuwazuru, T Furukawa, H Yoshida, K Yamada, S Akiyama, "Purification and tissue distribution of human thymidine
phosphorylase; high expression in lymphocytes, reticulocytes and tumors", Biochim Biophys Acta, 1034, 1990, 107-113.
Reaction
22.3.3.16 thymidine + orthophosphate <=> thymine + 2-deoxy-D-ribose 1-phosphate
Description
At the beginning of this reaction, 1 molecule of 'thymidine', and 1 molecule of 'Orthophosphate' are present. At the end of this reaction, 1
molecule of '2-deoxy-D-ribose 1-phosphate', and 1 molecule of 'thymine' are present.
References
Reaction
22.3.3.17 thymidine + ATP => thymidine 5'-monophosphate (dTMP) + ADP
Description
At the beginning of this reaction, 1 molecule of 'ATP', and 1 molecule of 'thymidine' are present. At the end of this reaction, 1 molecule of
'thymidine 5'-monophosphate', and 1 molecule of 'ADP' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'nucleoside kinase activity' of 'thymidine kinase 1, soluble holoenzyme'.
References
B Munch-Petersen, L Cloos, G Tyrsted, S Eriksson, "Diverging substrate specificity of pure human thymidine kinases 1 and 2 against antiviral
dideoxynucleosides", J Biol Chem, 266, 1991, 9032-9038.
Reaction
22.3.3.18 thymidine 5'-monophosphate + H2O => thymidine + orthophosphate
Description
At the beginning of this reaction, 1 molecule of 'thymidine 5'-monophosphate', and 1 molecule of 'H2O' are present. At the end of this reaction, 1
molecule of 'thymidine', and 1 molecule of 'Orthophosphate' are present.
Reaction
22.3.4 Reversible phosphorolysis of pyrimidine nucleosides
Authors
Editors
Description
The phosphorolysis of pyrimidine nucleosides is catalyzed by two cytosolic enzymes, uridine phosphorylase 1 and thymidine phosphorylase
(endothelial cell growth factor (platelet-derived)). Studies of purified enzymes from a variety of mammals indicate that uridine is a substrate only
for uridine phosphorylase and thymidine is a substrate only for thymidine phosphorylase, but both enzymes act on 2'-deoxyuridine (Friedkin and
Roberts 1954; Kraut and Yamada 1971; Krenitsky et al. 1964, 1965; Zimmerman 1964). These reactions are reversible in vitro (Bose and
Yamada 1974; Friedkin and Roberts 1954), but clinical data suggest that they proceed largely in the direction of formation of free pyrimidine
bases in vivo, and do not contribute significantly to pyrimidine salvage (Pizzorno et al. 2002; Spinazzola et al. 2002).
Despite the name given to thymidine phosphorylase - endothelial cell growth factor (platelet-derived) - recent studies have shown that this
protein is not a conventional growth factor, and addition of the protein to culture media does not stimulate DNA replication. Rather, the
2-deoxy-D-ribose released by thymidine phosphorolysis acts as an angiogenic factor (Brown and Bicknell 1998; Moghaddam and Bicknell 1992).
The human enzymes have been purified and shown to have enzymatic activity in vitro (uridine phosphorylase 1 - Watanabe and Uchida 1995;
thymidine phosphorylase - Yoshimura et al. 1990), but they have not been extensively characterized. Their subunit structures and exact
substrate specificities have been inferred from the consistent results obtained in earlier studies of human cell extracts and purified rodent
enzymes.
References
A Moghaddam, R Bicknell, "Expression of platelet-derived endothelial cell growth factor in Escherichia coli and confirmation of its thymidine
phosphorylase activity", Biochemistry, 31, 1992, 12141-12146.
R Bose, EW Yamada, "Uridine phosphorylase, molecular properties and mechanism of catalysis", Biochemistry, 13, 1974, 2051-2056.
M Zimmerman, "Deoxyribosyl transfer. II. Nucleoside:pyrimidine deoxyribosyltransferase activity of three partially purified thymidine
phosphorylases", J Biol Chem, 239, 1964, 2622-2627.
G Pizzorno, D Cao, JJ Leffert, RL Russell, D Zhang, RE Handschumacher, "Homeostatic control of uridine and the role of uridine phosphorylase:
a biological and clinical update", Biochim Biophys Acta, 1587, 2002, 133-144.
A Kraut, EW Yamada, "Cytoplasmic uridine phosphorylase of rat liver. Characterization and kinetics", J Biol Chem, 246, 1971, 2021-2030.
NS Brown, R Bicknell, "Thymidine phosphorylase, 2-deoxy-D-ribose and angiogenesis", Biochem J, 334, 1998, 1-8.
A Spinazzola, R Marti, I Nishino, AL Andreu, A Naini, S Tadesse, I Pela, E Zammarchi, MA Donati, JA Oliver, M Hirano, "Altered thymidine
metabolism due to defects of thymidine phosphorylase", J Biol Chem, 277, 2002, 4128-4133.
M Friedkin, D Roberts, "The enzymatic synthesis of nucleosides. II. Thymidine and related pyrimidine nucleosides", J Biol Chem, 207, 1954,
257-266.
TA Krenitsky, M Barclay, JA Jacquez, "Specificity of mouse uridine phosphorylase. Chromatography, purification, and properties", J Biol Chem,
239, 1964, 805-812.
22.3.4.1 Reversible phosphorolysis of pyrimidine nucleosides by thymidine phosphorylase
Description
The reversible phosphorolysis of thymidine and 2'-deoxyuridine is catalyzed by thymidine phosphorylase.
22.3.4.1.1 thymidine + orthophosphate <=> thymine + 2-deoxy-D-ribose 1-phosphate
Description
At the beginning of this reaction, 1 molecule of 'thymidine', and 1 molecule of 'Orthophosphate' are present. At the end of this reaction, 1
molecule of '2-deoxy-D-ribose 1-phosphate', and 1 molecule of 'thymine' are present.
References
Reaction
22.3.4.1.2 thymine + 2-deoxy-D-ribose 1-phosphate <=> thymidine + orthophosphate
Description
At the beginning of this reaction, 1 molecule of '2-deoxy-D-ribose 1-phosphate', and 1 molecule of 'thymine' are present. At the end of this
reaction, 1 molecule of 'thymidine', and 1 molecule of 'Orthophosphate' are present.
References
Reaction
22.3.4.1.3 2'-deoxyuridine + orthophosphate <=> uracil + 2-deoxy-D-ribose 1-phosphate
Description
References
Reaction
22.3.4.1.4 uracil + 2-deoxy-D-ribose 1-phosphate <=> 2'-deoxyuridine + orthophosphate
Description
1 molecule of '2'-deoxyuridine', and 1 molecule of 'Orthophosphate' are present.
References
Reaction
22.3.4.2 Reversible phosphorolysis of pyrimidine nucleosides by uridine phosphorylase 1
Description
The reversible phosphorolysis of uridine and 2'-deoxyuridine is catalyzed by uridine phosphorylase.
22.3.4.2.1 uridine + orthophosphate <=> uracil + D-ribose 1-phosphate
Description
At the beginning of this reaction, 1 molecule of 'uridine', and 1 molecule of 'Orthophosphate' are present. At the end of this reaction, 1 molecule
of 'uracil', and 1 molecule of 'D-ribose 1-phosphate' are present.
References
Reaction
22.3.4.2.2 uracil + D-ribose 1-phosphate <=> uridine + orthophosphate
Description
At the beginning of this reaction, 1 molecule of 'uracil', and 1 molecule of 'D-ribose 1-phosphate' are present. At the end of this reaction, 1
References
Reaction
22.3.4.2.3 uracil + 2-deoxy-D-ribose 1-phosphate <=> 2'-deoxyuridine + orthophosphate
Description
1 molecule of 'uridine', and 1 molecule of 'Orthophosphate' are present.
References
Reaction
22.3.4.2.4 2'-deoxyuridine + orthophosphate <=> uracil + 2-deoxy-D-ribose 1-phosphate
Description
References
Reaction
22.3.5 Pyrimidine catabolism
Authors
Jassal, B, 2003-06-17.
Description
In parallel sequences of three reactions each, thymine is converted to beta-aminoisobutyrate and uracil is converted to beta-alanine. Both of
these molecules are excreted in human urine and may be the normal end products of pyrimidine catabolism. Reaction sequences that convert
them to propionyl-CoA and acetyl-CoA, respectively, have been described but their quantitative importance in human metabolism is unknown.
References
OW Griffith, "Beta-amino acids: mammalian metabolism and utility as alpha-amino acid analogues", Annu Rev Biochem, 55, 1986, 855-878.
22.3.5.1 uracil + NADPH + H+ => 5,6-dihydrouracil + NADP+
Description
Cytosolic dihydropyrimidine dehydrogenase catalyzes the reaction of uracil and NADPH + H+ to form 5,6-dihydrouracil and NADP+. The
mechanism of the human reaction is inferred from that of the well-characterized pig enzyme (Yokota et al. 1994).
References
H Yokota, P Fernandez-Salguero, H Furuya, K Lin, OW McBride, B Podschun, KD Schnackerz, FJ Gonzalez, "cDNA cloning and chromosome
mapping of human dihydropyrimidine dehydrogenase, an enzyme associated with 5-fluorouracil toxicity and congenital thymine uraciluria", J Biol
Chem, 269, 1994, 23192-6.
Reaction
22.3.5.2 5,6-dihydrouracil + H2O => beta-ureidopropionate
Description
At the beginning of this reaction, 1 molecule of 'H2O', and 1 molecule of '5,6-Dihydrouracil' are present. At the end of this reaction, 1 molecule of
'3-Ureidopropionate' is present.
This reaction takes place in the 'cytosol' and is mediated by the 'dihydropyrimidinase activity' of 'Dihydropyriminidase-Zn complex'.
References
N Hamajima, M Kouwaki, P Vreken, K Matsuda, S Sumi, M Imaeda, S Ohba, K Kidouchi, M Nonaka, M Sasaki, N Tamaki, Y Endo, R De Abreu,
J Rotteveel, A van Kuilenburg, A van Gennip, H Togari, Y Wada, "Dihydropyrimidinase deficiency: structural organization, chromosomal
localization, and mutation analysis of the human dihydropyrimidinase gene", Am J Hum Genet, 63, 1998, 717-26.
Reaction
22.3.5.3 beta-ureidopropionate + H2O => beta-alanine + NH4+ + CO2
Description
At the beginning of this reaction, 1 molecule of '3-Ureidopropionate', and 1 molecule of 'H2O' are present. At the end of this reaction, 1 molecule
of 'CO2', 1 molecule of 'beta-Alanine', and 1 molecule of 'NH3' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'beta-ureidopropionase activity' of '3-ureidopropionase-Zn complex'.
References
AB van Kuilenburg, R Meinsma, E Beke, B Assmann, A Ribes, I Lorente, R Busch, E Mayatepek, NG Abeling, A van Cruchten, AE Stroomer, H
van Lenthe, L Zoetekouw, W Kulik, GF Hoffmann, T Voit, RA Wevers, F Rutsch, AH van Gennip, "beta-Ureidopropionase deficiency: an inborn
error of pyrimidine degradation associated with neurological abnormalities", Hum Mol Genet, 13, 2004, 2793-801.
Reaction
22.3.5.4 thymine + NADPH + H+ => 5,6-dihydrothymine + NADP+
Description
Cytosolic dihydropyrimidine dehydrogenase catalyzes the reaction of thymine and NADPH + H+ to form 5,6-dihydrothymine and NADP+. The
mechanism of the human reaction is inferred from that of the well-characterized pig enzyme (Yokota et al. 1994).
References
H Yokota, P Fernandez-Salguero, H Furuya, K Lin, OW McBride, B Podschun, KD Schnackerz, FJ Gonzalez, "cDNA cloning and chromosome
mapping of human dihydropyrimidine dehydrogenase, an enzyme associated with 5-fluorouracil toxicity and congenital thymine uraciluria", J Biol
Chem, 269, 1994, 23192-6.
Reaction
22.3.5.5 5,6-dihydrothymine + H2O => beta-ureidoisobutyrate
Description
At the beginning of this reaction, 1 molecule of 'H2O', and 1 molecule of '5,6-Dihydrothymine' are present. At the end of this reaction, 1 molecule
of '3-Ureidoisobutyrate' is present.
This reaction takes place in the 'cytosol' and is mediated by the 'dihydropyrimidinase activity' of 'Dihydropyriminidase-Zn complex'.
References
N Hamajima, M Kouwaki, P Vreken, K Matsuda, S Sumi, M Imaeda, S Ohba, K Kidouchi, M Nonaka, M Sasaki, N Tamaki, Y Endo, R De Abreu,
J Rotteveel, A van Kuilenburg, A van Gennip, H Togari, Y Wada, "Dihydropyrimidinase deficiency: structural organization, chromosomal
localization, and mutation analysis of the human dihydropyrimidinase gene", Am J Hum Genet, 63, 1998, 717-26.
Reaction
22.3.5.6 beta-ureidoisobutyrate + H2O => beta-aminoisobutyrate + NH4+ + CO2
Description
At the beginning of this reaction, 1 molecule of '3-Ureidoisobutyrate', and 1 molecule of 'H2O' are present. At the end of this reaction, 1 molecule
of 'L-3-Amino-isobutanoate', 1 molecule of 'CO2', and 1 molecule of 'NH3' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'beta-ureidopropionase activity' of '3-ureidopropionase-Zn complex'.
References
AB van Kuilenburg, R Meinsma, E Beke, B Assmann, A Ribes, I Lorente, R Busch, E Mayatepek, NG Abeling, A van Cruchten, AE Stroomer, H
van Lenthe, L Zoetekouw, W Kulik, GF Hoffmann, T Voit, RA Wevers, F Rutsch, AH van Gennip, "beta-Ureidopropionase deficiency: an inborn
error of pyrimidine degradation associated with neurological abnormalities", Hum Mol Genet, 13, 2004, 2793-801.
Reaction
22.4 Synthesis of deoxyribonucleoside 5'-diphosphates from ribonucleoside

5'-diphosphates
Authors
Editors
Description
Deoxyribonucleotides are synthesized de novo by the reduction of ribonucleoside 5'-diphosphates (Eklund et al. 2001). This reaction is catalyzed
by ribonucleotide reductase, a tetrameric enzyme containing two large and two small subunits. The enzyme exists in two forms, a cytosolic form
active during S phase of the cell cycle that produces deoxyribonucleotides for DNA replication, and a nuclear form active throughout the cell
cycle that produces deoxyribonucleotides for DNA repair. The two forms have identical large subunits but distinct (though closely similar) small
subunits (Guittet et al. 2001; Shao et al. 2004).
The cytosolic enzyme is active on adenine, uridine, guanidine, and cytidine nucleotides. Its activity is regulated to produce balanced amounts of
its four deoxyribonucleotide products. This regulation involves two allosteric binding sites, an activity site and a specificity site. The activity site
binds either ATP, which greatly increases the enzymeÃ¢â‚¬â„¢s activity, or dATP, which inhibits it. The specificity site binds various
deoxynucleoside triphosphates, which increase or decrease the enzymeÃ¢â‚¬â„¢s activity on specific ribonucleoside 5Ã¢â‚¬â„¢-diphosphates,
illustrated in the table below. Thus, the enzymeÃ¢â‚¬â„¢s activity on ADP is stimulated strongly by dGTP while its activity on CDP is inhibited by
TTP and, to a lesser extent, by dGTP.
The rationale for annotating the specificity and allosteric regulation of human cytosolic ribonucleotide reductase is indirect but compelling. These
features were first determined in studies of partially purified ribonucleotide reductase from calf thymus (Eriksson et al. 1979). Purified
recombinant mouse ribonucleotide reductase was subsequently shown to have the same specificity and regulatory features, suggesting that
these features are very well conserved among mammalian enzymes (Chimploy and Mathews 2001; Reichard et al. 2000). The recent
demonstration that mouse, human, and mouse:human chimeric ribonucleotide reductases have indistinguishable activities, tested with a limited
set of substrates and effectors (Guittet et al. 2001; Shao et al. 2004) is consistent with this suggestion, as is the near-perfect conservation
between these species of amino acid sequence motifs critical in determining substrate and effector specificity (Eklund et al. 2001).
The reducing equivalents needed for the reaction are provided directly by a small protein, glutaredoxin or thioredoxin (Holmgren 1989). Initial
studies in E. coli suggested that thioredoxin plays this role, but studies of E. coli lacking functional thioredoxin established that glutaredoxin was
also effective as a source of reducing equivalents. Human ribonucleotide reductase is active in vitro with either human thioredoxin or human
glutaredoxin proteins (Sun et al. 1998). The relative contributions of glutaredoxin and thioredoxin to deoxyribonucleotide synthesis in normal
cells in vivo are unknown in either the human or the E. coli system. The oxidized small proteins are re-reduced with NADPH as the donor of
reducing equivalents, allowing deoxyribonucleotide synthesis to proceed in the presence of catalytic quantities of glutaredoxin or thioredoxin.
(Chimploy and Mathews 2001, Eklund et al 2001, Eriksson et al 1979, Guittet et al 2001, Holmgren 1989, Reichard et al 2000, Shao et al 2004,
Sun et al 1998).
References
K Chimploy, CK Mathews, "Mouse ribonucleotide reductase control. Influence of substrate binding upon interactions with allosteric effectors", J
Biol Chem, 276, 2001, 7093-7100.
A Holmgren, "Thioredoxin and glutaredoxin systems", J Biol Chem, 264, 1989, 13963-13966.
S Eriksson, L Thelander, M Akerman, "Allosteric regulation of calf thymus ribonucleotide diphosphate reductase", Biochemistry, 18, 1979,
2948-2952.
P Reichard, R Eliasson, R Ingemarson, L Thelander, "Cross-talk between the allosteric effector-binding sites in mouse ribonucleotide
reductase", J Biol Chem, 275, 2000, 33021-33026.
H Eklund, U Uhlin, M Farnegardh, DT Logan, P Nordlund, "Structure and function of the radical enzyme ribonucleotide reductase", Prog Biophys
Mol Biol, 77, 2001, 177-268.
cells", J Biol Chem, 276, 2001, 40647-40651.
C Sun, MJ Berardi, JH Bushweller, "The NMR solution structure of human glutaredoxin in the fully reduced form", J Mol Biol, 280, 1998,
687-701.
J Shao, B Zhou, L Zhu, W Qiu, YC Yuan, B Xi, Y Yen, "In vitro characterization of enzymatic properties and inhibition of the p53R2 subunit of
human ribonucleotide reductase", Cancer Res, 64, 2004, 1-6.
22.4.1 Reduction of cytosolic ribonucleoside 5'-diphosphates to deoxyribonucleoside 5'-diphosphates by

ribonucleotide reductase and glutaredoxin
Description
Deoxyribonucleotides required during S phase for DNA synthesis are synthesized de novo in the cytosol by the reduction of ribonucleoside
5'-diphosphates. The reducing equivalents needed for these reactions can be provided directly by glutaredoxin.
22.4.1.1 Reduction of cytosolic ribonucleoside 5'-diphosphates to deoxyribonucleoside 5'-diphosphates (glutaredoxin)
Description
5'-diphosphates.
References
cells", J Biol Chem, 276, 2001, 40647-40651.
687-701.
22.4.1.1.1 adenosine 5'-diphosphate + glutaredoxin (reduced) => 2'-deoxyadenosine 5'-diphosphate + glutaredoxin (oxidized) + H2O
Description
At the beginning of this reaction, 1 molecule of 'Glutaredoxin', and 1 molecule of 'ADP' are present. At the end of this reaction, 1 molecule of
'glutaredoxin (thioltransferase), oxidized', 1 molecule of '2'-deoxyadenosine 5'-diphosphate', and 1 molecule of 'H2O' are present.
References
cells", J Biol Chem, 276, 2001, 40647-40651.
687-701.
Reaction
22.4.1.1.2 cytidine 5'-diphosphate + glutaredoxin (reduced) => 2'-deoxycytidine 5'-diphosphate + glutaredoxin (oxidized) + H2O
Description
At the beginning of this reaction, 1 molecule of 'Glutaredoxin', and 1 molecule of 'cytidine 5'-diphosphate' are present. At the end of this reaction,
1 molecule of 'glutaredoxin (thioltransferase), oxidized', 1 molecule of 'H2O', and 1 molecule of '2'-deoxycytidine 5'-diphosphate' are present.
References
cells", J Biol Chem, 276, 2001, 40647-40651.
687-701.
Reaction
22.4.1.1.3 guanosine 5'-diphosphate + glutaredoxin (reduced) => 2'-deoxyguanosine 5'-diphosphate + glutaredoxin (oxidized) + H2O
Description
At the beginning of this reaction, 1 molecule of 'Glutaredoxin', and 1 molecule of 'GDP' are present. At the end of this reaction, 1 molecule of
'glutaredoxin (thioltransferase), oxidized', 1 molecule of 'H2O', and 1 molecule of '2'-deoxyguanosine 5'-diphosphate' are present.
References
cells", J Biol Chem, 276, 2001, 40647-40651.
687-701.
Reaction
22.4.1.1.4 uridine 5'-diphosphate + glutaredoxin (reduced) => 2'-deoxyuridine 5'-diphosphate + glutaredoxin (oxidized) + H2O
Description
At the beginning of this reaction, 1 molecule of 'Glutaredoxin', and 1 molecule of 'uridine 5'-diphosphate' are present. At the end of this reaction,
1 molecule of 'glutaredoxin (thioltransferase), oxidized', 1 molecule of 'H2O', and 1 molecule of '2'-deoxyuridine 5'-diphosphate' are present.
References
cells", J Biol Chem, 276, 2001, 40647-40651.
687-701.
Reaction
22.4.1.2 glutaredoxin (oxidized) + glutathione (reduced) => glutaredoxin (reduced) + glutathione (oxidized)
Description
At the beginning of this reaction, 1 molecule of 'glutaredoxin (thioltransferase), oxidized', and 1 molecule of 'Glutathione' are present. At the end
of this reaction, 1 molecule of 'Glutaredoxin', and 1 molecule of 'oxidized glutathione' are present.
Reaction
22.4.1.3 glutathione (oxidized) + NADPH + H+ => 2 glutathione (reduced) + NADP+

Description
At the beginning of this reaction, 1 molecule of 'H+', 1 molecule of 'NADPH', and 1 molecule of 'oxidized glutathione' are present. At the end of
this reaction, 2 molecules of 'Glutathione', and 1 molecule of 'NADP+' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'glutathione-disulfide reductase activity' of 'glutathione reductase holoenzyme'.
References
H Loos, D Roos, R Weening, J Houwerzijl, "Familial deficiency of glutathione reductase in human blood cells", Blood, 48, 1976, 53-62.
Reaction
22.4.2 Reduction of cytosolic ribonucleoside 5'-diphosphates to deoxyribonucleoside 5'-diphosphates by

ribonucleotide reductase and thioredoxin
Description
5'-diphosphates. The reducing equivalents needed for these reactions can be provided directly by thioredoxin.
22.4.2.1 Reduction of cytosolic ribonucleoside 5'-diphosphates to deoxyribonucleoside 5'-diphosphates (thioredoxin)
Description
5'-diphosphates.
References
cells", J Biol Chem, 276, 2001, 40647-40651.
22.4.2.1.1 uridine 5'-diphosphate + thioredoxin (reduced) => 2'-deoxyuridine 5'-diphosphate + thioredoxin (oxidized) + H2O
Description
References
cells", J Biol Chem, 276, 2001, 40647-40651.
Reaction
22.4.2.1.2 cytidine 5'-diphosphate + thioredoxin (reduced) => 2'-deoxycytidine 5'-diphosphate + thioredoxin (oxidized) + H2O
Description
References
cells", J Biol Chem, 276, 2001, 40647-40651.
Reaction
Description
References
cells", J Biol Chem, 276, 2001, 40647-40651.
Reaction
Description
References
cells", J Biol Chem, 276, 2001, 40647-40651.
Reaction
22.4.2.2 thioredoxin, oxidized + NADPH + H+ => thioredoxin, reduced + NADP+
Description
References
Reaction
22.4.3 Reduction of nuclear ribonucleoside 5'-diphosphates to deoxyribonucleoside 5'-diphosphates by

ribonucleotide reductase and thioredoxin
Authors
Editors
Description
Ribonucleotide reductase M2B (TP53 inducible), "p53R2", first identified as the product of a gene expressed in response to p53, can replace the
small subunit of ribonucleotide reductase. The resulting complex localizes to the cell nucleus and has ATP-dependent ribonucleotide reductase
activity in vitro. These features support the hypothesis that this complex is responsible specifically for the synthesis of deoxyribonucleotides used
in DNA repair, especially outside the S phase of the cell cycle (Guittet et al. 2001; Shao et al. 2004; Tanaka et al. 2000). This model would imply
the existence of a nuclear kinase to convert the nuclear repair-specific deoxyribonucleoside diphosphates to triphosphates. Whether this kinase
is a novel enzyme, or whether one of the known cytosolic kinases can also localize to the nucleus, is unknown.
Mechanistic studies in vitro indicate that p53R2-containing ribonucleotide reductase acts on cytidine 5'-diphosphate in the same way as
conventional ribonucleotide reductase, suggesting that either thioredoxin or glutaredoxin directly provides the reducing equivalents for the
reaction, and that activity on each ribonucleoside diphosphate is allosterically regulated by specific ribo- and deoxyribonucleoside triphosphates.
The reduction of other ribonucleoside diphosphates has not yet been studied experimentally, nor have the requirements of the reactions for
cofactors an allosteric regulators been determined. The reduction of adenine, guanine, and uridine ribonucleotides is therefore inferred,
plausibly, from the enzyme's activity on cytidine ribonucleotide, and all reactions are arbitrarily annotated here with thioredoxin as the cofactor,
and without regulatory features.
References
cells", J Biol Chem, 276, 2001, 40647-40651.
H Tanaka, T Yamaguchi, J Shiraishi, S Fukuda, K Matsui, Y Takei, Y Nakamura, "A ribonucleotide reductase gene involved in a p53-dependent
cell-cycle checkpoint for DNA damage", Nature, 404, 2000, 42-49.
22.4.3.1 thioredoxin, reduced (cytosol) <=> thioredoxin, reduced (nucleus)
Description
In this reaction, 1 molecule of 'Thioredoxin' is translocated from cytosol to nucleoplasm.
Reaction
22.4.3.2 Reduction of nuclear ribonucleoside 5'-diphosphates to deoxyribonucleoside 5'-diphosphates (thioredoxin)
Description
Deoxyribonucleotides needed for DNA repair are synthesized de novo by the reduction of ribonucleoside 5'-diphosphates. This reaction is
catalyzed by a nuclear form of ribonucleotide reductase active throughout the cell cycle. The reducing equivalents needed for these reactions
can be provided directly by thioredoxin.
Description
This reaction takes place in the 'nucleus' and is mediated by the 'class I ribonucleotide reductase activity' of 'ribonucleotide reductase M2B
(TP53 inducible) variant'.
Source reaction
This reaction was inferred from the corresponding reaction "cytidine 5'-diphosphate + thioredoxin (reduced) => 2'-deoxycytidine 5'-diphosphate +
thioredoxin (oxidized) + H2O" in species Homo sapiens.
cells", J Biol Chem, 276, 2001, 40647-40651.
Reaction
22.4.3.2.2 cytidine 5'-diphosphate + thioredoxin (reduced) => 2'-deoxycytidine 5'-diphosphate + thioredoxin (oxidized) + H2O
Description
References
cells", J Biol Chem, 276, 2001, 40647-40651.
Reaction
Description
Source reaction
cells", J Biol Chem, 276, 2001, 40647-40651.
Reaction
22.4.3.2.4 uridine 5'-diphosphate + thioredoxin (reduced) => 2'-deoxyuridine 5'-diphosphate + thioredoxin (oxidized) + H2O
Description
Source reaction
cells", J Biol Chem, 276, 2001, 40647-40651.
Reaction
22.4.3.3 thioredoxin, oxidized (nucleus) <=> thioredoxin, oxidized (cytosol)
Description
In this reaction, 1 molecule of 'thioredoxin, oxidized' is translocated from nucleoplasm to cytosol.
Reaction
22.4.3.4 thioredoxin, oxidized + NADPH + H+ => thioredoxin, reduced + NADP+
Description
References
Reaction
22.5 Hydrolysis of nucleoside 5'-monophosphates to nucleosides plus

orthophosphate
Authors
Editors
Reviewers
Rush, MG, 2008-01-11.
Description
Seven human enzymes catalyze the hydrolysis of 5'-nucleoside monophosphates to yield the corresponding nucleosides plus orthophosphate.
One is attached to the external surface of the plasma membrane by a glycolipid anchor and acts on extracellular nucleotides; one, active against
thymidine, uridine, and deoxyuridine monophosphates, is located in the mitochondrial matrix; and five are located in the cytosol. All of the
cytosolic enzymes play a role in regulating the intracellular nucleotide pool, as shown by studies in which inhibiting or overexpressing individual
enzymes alters intra- and extra-cellular nucleotide and nucleoside levels (e.g., Page et al. 1997). Determining the normal contribution of any one
enzyme to the hydrolysis of any specific nucleotide is difficult, however. These enzymes have overlapping substrate specificities, as outlined in
the table below: "+" indicates a reaction observed in studies with purified enzymes in vitro, "-" indicates a reaction not observed, and "?"
indicates an enzyme-substrate pair that has not been characterized. Two of the enzymes have phosphotransferase activity, so that they can not
only hydrolyze substrates, but transfer phosphate groups from one substrate to another. A final source of complexity is the allosteric regulation of
three of the nucleotidases by adenine nucleotides or orthophosphate.
These enzymes have diverse functions in normal humans, some of which are tissue-specific. One general and shared function appears to be
involvement in regulatory substrate cycles, as illustrated in the schematic pathway below (Reichard 1988; Gallinaro et al. 2002; Bianchi and
Spychala 2003). Studies of the responses of cultured mammalian cells to alterations in levels of 5'-nucleotidases have established the ability of
5'-nucleoside phosphatases to hydrolyze excess nucleoside monophosphates to nucleosides, and of nucleoside kinases to convert nucleosides
to nucleoside monophosphates. These cycles, coupled to the transport of nucleosides (but not nucleotides) across the plasma membrane,
appear to allow fine-tuning of cytosolic nucleotide pools.
References
P Reichard, "Interactions between deoxyribonucleotide and DNA synthesis", Annu Rev Biochem, 57, 1988, 349-374.
V Bianchi, J Spychala, "Mammalian 5'-nucleotidases", J Biol Chem, 278, 2003, 46195-46198.
T Page, A Yu, J Fontanesi, WL Nyhan, "Developmental disorder associated with increased cellular nucleotidase activity", Proc Natl Acad Sci
USA, 94, 1997, 11601-11606.
L Gallinaro, K Crovatto, C Rampazzo, G Pontarin, P Ferraro, E Milanese, P Reichard, V Bianchi, "Human mitochondrial 5'-deoxynucleotidase.
Overproduction in cultured cells and functional aspects", J Biol Chem, 277, 2002, 35080-35087.
22.5.1 Hydrolysis of extracellular nucleoside 5'-monophosphates by plasma membrane-bound

5'-nucleotidase
Authors
Editors
Description
5'-nucleotidase, ecto (CD73) catalyzes the hydrolysis of 5'-nucleotides to the corresponding nucleosides plus orthophosphate. The active
enzyme is a homodimer, requires Zn++ as a cofactor (Fini et al. 1990), and is anchored to the external face of the plasma membrane via a
glycophosphatidylinositol moiety added post-translationally. The rat enzyme has been purified and characterized biochemically. It can hydrolyze
a range of 5'-ribonucleotides in vitro, and shows limited activity against 5'thymidine monophosphate and 5'-deoxyriboadenosine monophosphate,
but is most active against 5'-adenosine monophosphate (Le Hir et al. 1989). The human enzyme has been purified and shown to be active
against 5'-adenosine monophosphate (Thompson et al. 1987). Its activity against other nucleotide monophosphates is inferred from its structural
similarity to the rat enzyme (Zimmerman 1992). The function of the enzyme in humans is unknown, but its localization and substrate specificity
suggest that it could modulate signaling by extracellular adenosine. It may also mediate cell-cell interactions, and act as a component of the
antigen receptor complex on T lymphocytes (Bianchi and Spychala 2003; Zimmerman 1992).
References
LF Thompson, JM Ruedi, MG Low, "Purification of 5'-nucleotidase from human placenta after release from plasma membranes by
phosphatidylinositol-specific phospholipase C", Biochem Biophys Res Commun, 145, 1987, 118-125.
M Le Hir, R Gandhi, UC Dubach, "Purification and properties of a 5'-nucleotidase from rat renal membranes", Enzyme, 41, 1989, 87-93.
C Fini, CA Palmerini, P Damiani, U Stochaj, HG Mannherz, A Floridi, "5'-nucleotidase from bull seminal plasma, chicken gizzard and snake
venom is a zinc metalloprotein", Biochim Biophys Acta, 1038, 1990, 18-22.
H Zimmermann, "5'-nucleotidase: molecular structure and functional aspects", Biochem J, 285, 1992, 345-365.
22.5.1.1 adenosine 5'-monophosphate (AMP) + H2O => adenosine + orthophosphate
Description
At the beginning of this reaction, 1 molecule of 'H2O', and 1 molecule of 'adenosine 5'-monophosphate' are present. At the end of this reaction, 1
molecule of 'Orthophosphate', and 1 molecule of 'adenosine' are present.
This reaction takes place on the 'plasma membrane' and is mediated by the '5'-nucleotidase activity' of '5'-nucleotidase, ecto (CD73)
holoenzyme'.
References
LF Thompson, JM Ruedi, MG Low, "Purification of 5'-nucleotidase from human placenta after release from plasma membranes by
phosphatidylinositol-specific phospholipase C", Biochem Biophys Res Commun, 145, 1987, 118-125.
Reaction
22.5.1.2 uridine 5'-monophosphate (UMP) + H2O => uridine + orthophosphate
Description
holoenzyme'.
Source reaction
This reaction was inferred from the corresponding reaction "uridine 5'-monophosphate (UMP) + H2O => uridine + orthophosphate" in species
Rattus norvegicus.
Reaction
22.5.1.3 guanosine 5'-monophosphate (GMP) + H2O => guanosine + orthophosphate
Description
holoenzyme'.
Source reaction
This reaction was inferred from the corresponding reaction "guanosine 5'-monophosphate (GMP) + H2O => guanosine + orthophosphate" in
Reaction
22.5.1.4 inosine 5'-monophosphate (IMP) + H2O => inosine + orthophosphate
Description
At the beginning of this reaction, 1 molecule of 'inosine 5'-monophosphate', and 1 molecule of 'H2O' are present. At the end of this reaction, 1
molecule of 'Orthophosphate', and 1 molecule of 'inosine' are present.
holoenzyme'.
Source reaction
This reaction was inferred from the corresponding reaction "inosine 5'-monophosphate (IMP) + H2O => inosine + orthophosphate" in species
Rattus norvegicus.
Reaction
22.5.1.5 cytidine 5'-monophosphate (CMP) + H2O => cytidine + orthophosphate
Description
holoenzyme'.
Source reaction
This reaction was inferred from the corresponding reaction "cytidine 5'-monophosphate (CMP) + H2O => cytidine + orthophosphate" in species
Rattus norvegicus.
Reaction
22.5.1.6 thymidine 5'-monophosphate (TMP) + H2O => thymidine + orthophosphate
Description
holoenzyme'.
Source reaction
This reaction was inferred from the corresponding reaction "thymidine 5'-monophosphate (TMP) + H2O => thymidine + orthophosphate" in
Reaction
22.5.1.7 2'-deoxyadenosine 5'-monophosphate (dAMP) + H2O => deoxyadenosine + orthophosphate
Description
At the beginning of this reaction, 1 molecule of '2'-deoxyadenosine 5'-monophosphate', and 1 molecule of 'H2O' are present. At the end of this
reaction, 1 molecule of '2'-deoxyadenosine', and 1 molecule of 'Orthophosphate' are present.
holoenzyme'.
Source reaction
This reaction was inferred from the corresponding reaction "2'-deoxyadenosine 5'-monophosphate (dAMP) + H2O => deoxyadenosine +
orthophosphate" in species Rattus norvegicus.
Reaction
22.5.2 Hydrolysis of cytosolic nucleoside 5'-monophosphates by 5'-nucleotidase cytosolic II
Authors
Editors
Description
5'-nucleotidase cytosolic II is one of five human cytosolic enzymes that catalyze the hydrolysis of nucleoside monophosphates to nucleosides
plus orthophosphate. The active form of the enzyme is a tetramer and has an absolute requirement for magnesium ions. In vitro, the purified
enzyme efficiently hydrolyzes inosine, deoxyinosine, guanosine, and deoxyguanosine monophosphates, while showing only limited activity
against adenosine, deoxyadenosine, xanthine, and pyrimidine nucleoside monophosphates (Bianchi and Spychala 2003; Spychala et al. 1988).
Consistent with these results, cultured cells overexpressing the enzyme from a recombinant DNA clone showed enhanced activity against
inosine and guanosine monophosphates, but not adenosine monophosphate (Gazziola et al. 2001; Sala-Newby et al. 2000).
References
GB Sala-Newby, NV Freeman, A Skladanowski, AC Newby, "Distinct roles for recombinant cytosolic 5'-nucleotidase I and -II in AMP and IMP
catabolism in COS-7 and H9c2 rat myoblast cell lines", J Biol Chem, 275, 2000, 11666-11671.
C Gazziola, P Ferraro, M Moras, P Reichard, V Bianchi, "Cytosolic high Km 5'-nucleotidase and 5'(3')-deoxyribonucleotidase in substrate cycles
involved in nucleotide metabolism", J Biol Chem, 276, 2001, 6185-6190.
Description
References
Reaction
22.5.2.2 2'-deoxyguanosine 5'-monophosphate (dGMP) + H2O => 2'-deoxyguanosine + orthophosphate
Description
References
Reaction
Description
References
Reaction
22.5.2.4 2'-deoxyinosine 5'-monophosphate (dIMP) + H2O => 2'-deoxyinosine + orthophosphate
Description
At the beginning of this reaction, 1 molecule of 'H2O', and 1 molecule of '2'-deoxyinosine 5'-monophosphate' are present. At the end of this
reaction, 1 molecule of 'Orthophosphate', and 1 molecule of '2'-deoxyinosine' are present.
References
Reaction
22.5.3 Hydrolysis of cytosolic nucleoside 5'-monophosphates by 5'-nucleotidase cytosolic IA
Authors
Editors
Description
5'-nucleotidase cytosolic IA is one of five human cytosolic enzymes that catalyze the hydrolysis of nucleoside monophosphates to nucleosides
plus orthophosphate. The subunit structure of the human enzyme has not been determined experimentally, but is inferred to be a homotetramer
with one Mg++ ion bound per subunit, based on its similarity to the pigeon heart enzyme (Bianchi and Spychala 2003; Sala-Newby et al. 1999;
Skladanowski and Newby 1990). The human (Hunsucker et al. 2001) and rabbit (Garvey et al. 1998; Yamazaki et al. 1991) enzymes have been
purified and biochemically characterized in vitro. Both enzymes are very active against AMP but also show significant activities against other
purine and pyrimidine ribo- and deoxyribonucleoside monophosphates. Both enzymes are strongly allosterically activated by ADP. Although the
human and rabbit enzymes are qualitatively alike, their quantitative properties can differ significantly. Both enzymes hydrolyze dCMP more
efficiently than AMP, for example, but while the rabbit enzyme is 3.6 times more efficient, the human enzyme is 21.4 times more efficient.
Whether such differences reflect real differences in the function of 5'-nucleotidase cytosolic IA between humans and other mammals, or only
differences in the methods used to purify and characterize the enzymes, remains to be determined.
References
EP Garvey, GT Lowen, MR Almond, "Nucleotide and nucleoside analogues as inhibitors of cytosolic 5'-nucleotidase I from heart", Biochemistry,
37, 1998, 9043-9051.
Y Yamazaki, VL Truong, JM Lowenstein, "5'-nucleotidase I from rabbit heart", Biochemistry, 30, 1991, 1503-1509.
A Skladanowski, AC Newby, "Partial purification and properties of an AMP-specific soluble 5'-nucleotidase from pigeon heart", Biochem J, 268,
1990, 117-122.
GB Sala-Newby, A Skladanowski, AC Newby, "The mechanism of adenosine formation in cells. Cloning of cytosolic 5'-nucleotidase-I", J Biol
Chem, 274, 1999, 17789-17793.
Chem, 276, 2001, 10498-10504.
Description
This reaction takes place in the 'cytosol' and is mediated by the '5'-nucleotidase activity' of '5'-nucleotidase cytosolic 1A holoenzyme'.
References
Chem, 276, 2001, 10498-10504.
Reaction
22.5.3.2 2'-deoxycytidine 5'-monophosphate (dCMP) + H2O => 2'-deoxycytidine + orthophosphate
Description
References
Chem, 276, 2001, 10498-10504.
Reaction
22.5.3.3 2'-deoxyuridine 5'-monophosphate (dUMP) + H2O => 2'-deoxyuridine + orthophosphate
Description
References
Chem, 276, 2001, 10498-10504.
Reaction
22.5.3.4 2'-deoxyadenosine 5'-monophosphate (dAMP) + H2O => 2'-deoxyadenosine + orthophosphate
Description
At the beginning of this reaction, 1 molecule of '2'-deoxyadenosine 5'-monophosphate', and 1 molecule of 'H2O' are present. At the end of this
reaction, 1 molecule of '2'-deoxyadenosine', and 1 molecule of 'Orthophosphate' are present.
References
Chem, 276, 2001, 10498-10504.
Reaction
Description
References
Chem, 276, 2001, 10498-10504.
Reaction
22.5.3.6 2'-deoxyinosine 5'-monophosphate (dIMP) + H2O => 2'deoxyinosine + orthophosphate
Description
References
Chem, 276, 2001, 10498-10504.
Reaction
Description
At the beginning of this reaction, 1 molecule of 'H2O', and 1 molecule of 'AMP' are present. At the end of this reaction, 1 molecule of
'Orthophosphate', and 1 molecule of 'adenosine' are present.
References
Chem, 276, 2001, 10498-10504.
Reaction
Description
References
Chem, 276, 2001, 10498-10504.
Reaction
Description
References
Chem, 276, 2001, 10498-10504.
Reaction
Description
References
Chem, 276, 2001, 10498-10504.
Reaction
Description
References
Chem, 276, 2001, 10498-10504.
Reaction
22.5.4 Hydrolysis of cytosolic nucleoside 5'-monophosphates by 5'-nucleotidase cytosolic IB
Authors
Editors
Description
5'-nucleotidase cytosolic IB is one of five human cytosolic enzymes that catalyze the hydrolysis of nucleoside monophosphates to nucleosides
plus orthophosphate. cDNA clones encoding human and mouse forms of the enzyme were identified on the basis of their close similarity to one
another, and their lower but significant similarity to clones of human and pigeon 5'-nucleotidase cytosolic IA. Like 5'-nucleotidase cytosolic IA,
5'-nucleotidase cytosolic IB catalyzes the hydrolysis of AMP in a reaction activated by ADP. The enzyme's subcellular localization is inferred to
be cytosolic based on its localization in COS cells overexpressing recombinant protein, but neither its subunit structure, its divalent cation
requirements, nor the range of nucleoside monophosphates on which it is active have been determined experimentally. Its physiological function
is unknown (Sala-Newby and Newby 2001; Sala-Newby et al. 2003).
References
GB Sala-Newby, NV Freeman, MA Curto, AC Newby, "Metabolic and functional consequences of cytosolic 5'-nucleotidase-IA overexpression in
neonatal rat cardiomyocytes", Am J Physiol Heart Circ Physiol, 285, 2003, H991-H998.
GB Sala-Newby, AC Newby, "Cloning of a mouse cytosolic 5'-nucleotidase-I identifies a new gene related to human autoimmune
infertility-related protein", Biochim Biophys Acta, 1521, 2001, 12-18.
Description
At the beginning of this reaction, 1 molecule of 'H2O', and 1 molecule of 'AMP' are present. At the end of this reaction, 1 molecule of
'Orthophosphate', and 1 molecule of 'adenosine' are present.
This reaction takes place in the 'cytosol' and is mediated by the '5'-nucleotidase activity' of '5'-nucleotidase cytosolic IB'.
References
GB Sala-Newby, NV Freeman, MA Curto, AC Newby, "Metabolic and functional consequences of cytosolic 5'-nucleotidase-IA overexpression in
neonatal rat cardiomyocytes", Am J Physiol Heart Circ Physiol, 285, 2003, H991-H998.
GB Sala-Newby, AC Newby, "Cloning of a mouse cytosolic 5'-nucleotidase-I identifies a new gene related to human autoimmune
infertility-related protein", Biochim Biophys Acta, 1521, 2001, 12-18.
Reaction
22.5.5 Hydrolysis of cytosolic nucleoside 5'-monophosphates by 5'-nucleotidase, cytosolic III
Authors
Editors
Description
5'-nucleotidase, cytosolic III is one of five human cytosolic enzymes that catalyze the hydrolysis of nucleoside monophosphates to nucleosides
plus orthophosphate. While it appears to be present in many tissues, it is especially abundant in erythrocytes, where it may function to remove
excess pyrimidine nucleotides generated by nucleic acid breakdown, while sparing purine nucleotides needed for red cell energy metabolism.
Deficiencies in the enzyme are associated with a form of hemolytic anemia, and its inactivation by heavy metals may be responsible for the
hematological abnormalities associated with lead poisoning (Marinaki et al. 2001; Rees et al. 2003). The active form of the enzyme is a
monomer. It has an absolute requirement for Mg++, and catalyzes the hydrolysis of pyrimidine monophosphates (UMP, CMP, dCMP, TMP, and
dUMP). It is inactive against purine nucleotides (Amici et al. 1997; Amici and Magni 2002).
References
A Amici, G Magni, "Human erythrocyte pyrimidine 5'-nucleotidase, PN-I", Arch Biochem Biophys, 397, 2002, 184-190.
DC Rees, JA Duley, AM Marinaki, "Pyrimidine 5' nucleotidase deficiency", Br J Haematol, 120, 2003, 375-383.
A Amici, M Emmanuelli, G Magni, N Raffaelli, S Ruggieri, "Pyrimidine nucleotidases from human erythrocyte possess phosphotransferase
activities specific for pyrimidine nucleotides", FEBS Lett, 1997, 263-267.
AM Marinaki, E Escuredo, JA Duley, A Simmonds, A Amici, V Naponelli, G Magni, M Seip, I Ben-Bassat, EH Harley, SL Thein, DC Rees,
"Genetic basis of hemolytic anemia caused by pyrimidine 5' nucleotidase deficiency", Blood, 97, 2001, 3327-3332.
Description
This reaction takes place in the 'cytosol' and is mediated by the '5'-nucleotidase activity' of '5'-nucleotidase, cytosolic III holoenzyme'.
References
Reaction
Description
References
Reaction
22.5.5.3 2'-deoxycytidine 5'-monophosphate (dCMP) + H2O => 2'-deoxycytidine + orthophosphate
Description
References
Reaction
Description
References
Reaction
Description
References
Reaction
22.5.6 Hydrolysis of cytosolic nucleoside 5'- and 3'-monophosphates by 5',3'-nucleotidase, cytosolic
Authors
Editors
Description
5'3'-nucleotidase, cytosolic is the major deoxyribonucleotidase of human cells, and thus appears to play a central role in the "substrate cycles"
that regulate cytosolic deoxynucleotide levels (Gazziola et al. 2001). The active form of the enzyme is a homodimer, with an absolute
requirement for Mg++ (Hoglund and Reichard 1990; Rampazzo et al. 2000).
References
L Hoglund, P Reichard, "Cytoplasmic 5'(3')-nucleotidase from human placenta", J Biol Chem, 265, 1990, 6589-6595.
C Gazziola, P Ferraro, M Moras, P Reichard, V Bianchi, "Cytosolic high Km 5'-nucleotidase and 5'(3')-deoxyribonucleotidase in substrate cycles
involved in nucleotide metabolism", J Biol Chem, 276, 2001, 6185-6190.
C Rampazzo, M Johansson, L Gallinaro, P Ferraro, U Hellman, A Karlsson, P Reichard, V Bianchi, "Mammalian 5'(3')-deoxyribonucleotidase,
cDNA cloning, and overexpression of the enzyme in Escherichia coli and mammalian cells", J Biol Chem, 275, 2000, 5409-5415.
Description
This reaction takes place in the 'cytosol' and is mediated by the '5'-nucleotidase activity' of '5',3'-nucleotidase, cytosolic holoenzyme'.
References
Reaction
22.5.6.2 2'-deoxyinosine 5'-monophosphate (dIMP) + H2O => 2'-deoxyinosine + orthophosphate
Description
References
Reaction
22.5.6.3 2'-deoxyuridine 3'-monophosphate + H2O => 2'-deoxyuridine + orthophosphate
Description
References
Reaction
Description
References
Reaction
Description
References
Reaction
Description
References
Reaction
Description
References
Reaction
Description
References
Reaction
Description
At the beginning of this reaction, 1 molecule of 'uridine 3'-monophosphate', and 1 molecule of 'H2O' are present. At the end of this reaction, 1
References
Reaction
Description
References
Reaction
22.5.7 Hydrolysis of mitochondrial nucleoside 5'- and 3'-monophosphates by 5',3'-nucleotidase,

mitochondrial
Authors
Editors
Description
5'3'-nucleotidase, mitochondrial is the major deoxyribonucleotidase of human mitochondria, active against dUMP, TMP, and also uridine 2'-, 3'-,
and 5'-monophosphates. It may play a central role in "substrate cycles" to regulate mitochondrial deoxynucleotide levels, especially in
non-dividing cells (Rampazzo et al. 2000; Gallinaro et al. 2002). The active form of the enzyme is a homodimer, with an absolute requirement for
Mg++ (Rampazzo et al. 2000; Rinaldo-Matthis et al. 2002).
References
A Rinaldo-Matthis, C Rampazzo, P Reichard, V Bianchi, P Nordlund, "Crystal structure of a human mitochondrial deoxyribonuclease", Nt Struct
Biol, 9, 2002, 779-787.
C Rampazzo, L Gallinaro, E Milanese, E Frigimelica, P Reichard, V Bianchi, "A deoxynucleotidase in mitochondria: involvement in regulation of
dNTP pools and possible link to genetic disease", Proc Natl Acad Sci USA, 97, 2000, 8239-8444.
Description
This reaction takes place in the 'mitochondrial matrix' and is mediated by the '5'-nucleotidase activity' of '5',3'-nucleotidase, mitochondrial
holoenzyme'.
References
Reaction
Description
holoenzyme'.
References
Reaction
Description
holoenzyme'.
References
Reaction
Description
holoenzyme'.
References
Reaction
Description
At the beginning of this reaction, 1 molecule of 'uridine 3'-monophosphate', and 1 molecule of 'H2O' are present. At the end of this reaction, 1
holoenzyme'.
References
Reaction
22.6 Phosphorylation of nucleosides to form nucleoside 5'-monophosphates
Authors
Editors
Reviewers
Rush, MG, 2008-01-11.
Description
Seven human enzymes, five cytosolic and two mitochondrial, catalyze the phosphorylation of nucleosides to yield the corresponding nucleoside
5'-monophosphates (Arner and Eriksson 1995; Van Rompay et al. 2003). These reactions are part of the nucleotide salvage process by which
nucleosides taken up by the cell, or generated internally by nucleic acid breakdown, are returned to the cell's nucleoside triphosphate pools for
use in nucleic acid synthesis.
There is evidence for both functional and geographical compartmentalization of these pools. Functionally, nuclear DNA repair may draw
preferentially on nucleotides generated via salvage while nuclear DNA replication may draw preferentially on nucleotides generated via de novo
synthesis (e.g., Xu et al. 1995). Geographically, cytosolic kinases may phosphorylate most nucleotides incorporated into nuclear DNA while
mitochondrial kinases may phosphorylate most nucleotides incorporated into mitochondrial DNA (e.g., Zhu et al. 2000).
While all seven enzymes are likely to play some role in regulating intracellular nucleotide pools, determining the normal contribution of any one
enzyme to the synthesis of any one nucleoside is difficult. These enzymes have overlapping substrate specificities, as outlined in the table
below: "+" indicates a reaction observed in studies with purified enzymes in vitro, "-" indicates a reaction not observed, and "?" indicates an
enzyme-substrate pair that has not been characterized. Most of the enzymes can use nucleoside triphosphates in addition to ATP as phosphate
donors, and several are regulated positively or negatively by nucleoside triphosphates, so that the activity and specificity of each of these
enzymes in vivo is likely to be modulated by many features of the cellular nucleotide pools in addition to the abundance of its own nominal
substrate.
References
C Zhu, M Johansson, A Karlsson, "Incorporation of nucleoside analogs into nuclear or mitochondrial DNA is determined by the intracellular
phosphorylation site", J Biol Chem, 275, 2000, 26727-26731.
YZ Xu, P Huang, W Plunkett, "Functional compartmentation of dCTP pools. Preferential utilization of salvaged deoxycytidine for DNA repair in
human lymphoblasts", J Biol Chem, 270, 1995, 631-637.
AR Van Rompay, M Johansson, A Karlsson, "Substrate specificity and phosphorylation of antiviral and anticancer nucleoside analogues by
human deoxyribonucleoside kinases and ribonucleoside kinases", Pharmacol Ther, 100, 2003, 119-139.
ES Arner, S Eriksson, "Mammalian deoxyribonucleotide kinases", Pharmacol Ther, 67, 1995, 155-186.
22.6.1 Phosphorylation of cytosolic nucleosides by adenosine kinase
Authors
Editors
Description
Adenosine kinase holoenzyme is a complex of one molecule of adenosine kinase apoprotein and Mg++ (Andres and Fox 1979; Mathews et al.
1998). It is localized in the cytosol and catalyzes the phosphorylation of adenosine as well as a variety of clinically important nucleoside analogs
(Andres and Fox 1979; Miller et al. 1979; Spychala et al. 1996). Studies of drug resistance in cultured human cells lacking adenosine kinase
suggest that this enzyme may also play a role in phosphorylation of 2'-deoxyadenosine (Hershfield et al. 1982). Kinetic analyses of the purified
human placental enzyme in vitro suggest that this activity is likely to be significant at physiological Mg++ concentrations and when
2'-deoxyadenosine concentrations are elevated (Hurley et al. 1985).
References
RL Miller, DL Adamczyk, WH Miller, GW Koszalka, JL Rideout, LM Beacham, III, EY Chao, JJ Haggerty, TA Krenitsky, GB Elion, "Adenosine
kinase from rabbit liver. II. Substrate and inhibitor specificity", J Biol Chem, 254, 1979, 2346-2352.
cells", J Biol Chem, 257, 1982, 6380-6386.
Chem, 260, 1985, 15675-15681.
22.6.1.1 adenosine + ATP => adenosine 5'-monophosphate (AMP) + ADP
Description
At the beginning of this reaction, 1 molecule of 'adenosine', and 1 molecule of 'ATP' are present. At the end of this reaction, 1 molecule of 'ADP',
and 1 molecule of 'AMP' are present.
References
Reaction
22.6.1.2 2'-deoxyadenosine + ATP => 2'-deoxyadenosine 5'-monophosphate + ADP
Description
References
cells", J Biol Chem, 257, 1982, 6380-6386.
Chem, 260, 1985, 15675-15681.
Reaction
22.6.2 Phosphorylation of cytosolic nucleosides by deoxycytidine kinase
Authors
Editors
Description
Deoxycytidine kinase holoenzyme is a homodimer (Bohman and Eriksson 1988; Chottiner et al. 1991; Datta et al. 1989 [Biochemistry]). Although
a chimeric deoxycytidine kinase - green fluorescent protein expressed at high levels in cultured cells localized to nuclei (Johansson et al. 1997),
endogenous protein is primarily cytosolic (Hatzis et al. 1998). Despite its name, the enzyme has a broad substrate specificity. Several groups,
studying human enzymes purified from multiple sources, have demonstrated it to be active with deoxycytidine, deoxyguanosine, and
deoxyadenosine as substrates (Bohman and Eriksson 1988; Datta et al. 1989 [Biochemistry]; Sarup et al. 1989; Usova and Eriksson 2002). The
enzyme likewise appears to be active with cytidine as a substrate, as shown in one study with human enzyme (Datta et al. 1989 [J Biol Chem])
and in an exhaustive analysis of the bovine enzyme (Krenitsky et al. 1976). While ATP functions efficiently as a phosphate donor, other
nucleoside triphosphates, notably UTP, function efficiently as phosphate donors in vitro and may function in this way in vivo as well (Shewach et
al. 1992).
References
NS Datta, DS Shewach, BS Mitchell, IH Fox, "Kinetic properties and inhibition of human T lymphoblast deoxycytidine kinase", J Biol Chem, 264,
1989, 9359-9364.
TA Krenitsky, JV Tuttle, GW Koszalka, IS Chen, LM Beacham, III, JL Rideout, GB Elion, "Deoxycytidine kinase from calf thymus: substrate and
inhibitor specificity", J Biol Chem, 251, 1976, 4055-4061.
EG Chottiner, DS Shewach, NS Datta, E Ashcraft, D Gribbin, D Ginsberg, IH Fox, BS Mitchell, "Cloning and expression of human deoxycytidine
kinase cDNA", Proc Natl Acad Sci USA, 88, 1991, 1531-1535.
E Sabini, S Ort, C Monnerjahn, M Konrad, A Lavie, "Structure of human dCK suggests strategies to improve anticancer and antiviral therapy",
Nat Struct Biol, 10, 2003, 513-519.
P Hatzis, AS Al-Madhoon, M Jullig, TG Petrakis, S Eriksson, I Talianidis, "The intracellular localization of deoxycytidine kinase", J Biol Chem,
273, 1998, 30239-30243.
1559-1567.
28, 1989, 114-123.
Biochemistry, 27, 1988, 4258-4265.
DS Shewach, KK Reynolds, L Hertel, "Nucleotide specificity of human deoxycytidine kinase", Mol Pharmacol, 42, 1992, 518-524.
L Wang, A Karlsson, ES Arner, S Eriksson, "Substrate specificity of mitochondrial 2'-deoxyguanosine kinase", J Biol Chem, 268, 1993,
22847-22852.
M Johansson, S Brismar, A Karlsson, "Human deoxycytidine kinase is located in the cell nucleus", Proc Natl Acad Sci USA, 94, 1997,
11941-11945.
22.6.2.1 2'-deoxycytidine + ATP => 2'-deoxycytidine 5'-monophosphate (dCMP) + ADP
Description
References
1559-1567.
28, 1989, 114-123.
Biochemistry, 27, 1988, 4258-4265.
Reaction
Description
References
1559-1567.
28, 1989, 114-123.
Biochemistry, 27, 1988, 4258-4265.
Reaction
22.6.2.3 2'-deoxyguanosine + ATP => 2'-deoxyguanosine 5'-monophosphate + ADP
Description
References
1559-1567.
28, 1989, 114-123.
Biochemistry, 27, 1988, 4258-4265.
Reaction
Description
References
NS Datta, DS Shewach, BS Mitchell, IH Fox, "Kinetic properties and inhibition of human T lymphoblast deoxycytidine kinase", J Biol Chem, 264,
1989, 9359-9364.
Reaction
22.6.3 Phosphorylation of mitochondrial nucleosides by deoxyguanosine kinase
Authors
Editors
Description
Biochemical studies have established that the bovine form of deoxyguanosine kinase holoenzyme is a homodimer, localized to mitochondria,
that efficiently phosphorylates 2'-deoxypurine nucleosides (deoxyadenosine, deoxyguanosine, and deoxyinosine), with ATP and UTP both
serving as efficient phosphate donors in vitro (Park and Ives 1988; Wang et al. 1993). A divalent cation requirement for the enzyme has not been
demonstrated experimentally. The enzyme has not been purified from human tissues, but studies of human protein expressed from recombinant
cDNA clones have established that it has the same substrate specificities as the bovine enzyme, and its amino acid sequence contains motifs
associated with mitochondrial localization (Johannson and Karlsson 1996; Wang et al. 1996; Zhu et al. 1998). Crystallographic studies of the
human enzyme have confirmed its dimeric structure and allowed identification of key amino acid residues responsible for its substrate specificity
(Johansson et al. 2001).
References
C Zhu, M Johansson, J Permert, A Karlsson, "Phosphorylation of anticancer nucleoside analogs by human mitochondrial deoxyguanosine
kinase", Biochem Pharmacol, 56, 1998, 1035-1040.
I Park, DH Ives, "Properties of a highly purified mitochondrial deoxyguanosine kinase", Arch Biochem Biophys, 266, 1988, 51-60.
K Johansson, S Ramaswamy, C Ljungcrantz, W Knecht, J Piskur, B Munch-Petersen, S Eriksson, H Eklund, "Structural basis for substrate
specificities of cellular deoxyribonucleoside kinases", Nat Struct Biol, 8, 2001, 616-620.
L Wang, A Karlsson, ES Arner, S Eriksson, "Substrate specificity of mitochondrial 2'-deoxyguanosine kinase", J Biol Chem, 268, 1993,
22847-22852.
22.6.3.1 2'-deoxyguanosine + ATP => 2'-deoxyguanosine 5'-monophosphate + ADP
Description
References
Reaction
22.6.3.2 2'-deoxyinosine + ATP => 2'-deoxyinosine 5'-monophosphate + ADP
Description
At the beginning of this reaction, 1 molecule of 'ATP', and 1 molecule of '2'-deoxyinosine' are present. At the end of this reaction, 1 molecule of
'ADP', and 1 molecule of '2'-deoxyinosine 5'-monophosphate' are present.
References
Reaction
Description
References
Reaction
22.6.4 Phosphorylation of mitochondrial nucleosides by thymidine kinase 2, mitochondrial
Authors
Editors
Description
Thymidine kinase 2, mitochondrial, has been purified from human spleen. The enzyme is active as a monomer, and phosphorylates
deoxypyrimidine nucleosides (thymidine, deoxyuridine, and deoxycytidine), using ATP or, less efficiently, CTP as a phosphate donor in vitro
(Munch-Petersen et al. 1991). The enzyme requires divalent cations for activity (Mg++ is preferred) but the nature of the association between the
metal ion and the enzyme polypeptide is unclear (Lee and Cheng 1976). The mitochondrial localization of the enzyme has been established
experimentally for rats and cattle (Jansson et al. 1992); its mitochondrial localization in humans is inferred from these results and the presence of
a mitochondrial localization motif at the amino terminus of the open reading frame of a cloned human cDNA (Wang et al. 1999).
References
O Jansson, C Bohman, B Munch-Petersen, S Eriksson, "Mammalian thymidine kinase 2. Direct photoaffinity labeling with [32P]dTTP of the
enzyme from spleen, liver, heart and brain", Eur J Biochem, 206, 1992, 485-490.
LS Lee, YC Cheng, "Human deoxythymidine kinase. I. Purification and general properties of the cytoplasmic and mitochondrial isozymes derived
from blast cells of acute myelocytic leukemia", J Biol Chem, 251, 1976, 2600-2604.
L Wang, B Munch-Petersen, A Herrstrom-Sjoberg, U Hellman, T Bergman, H Jornvall, S Eriksson, "Human thymidine kinase 2: molecular
cloning and characterisation of the enzyme activity with antiviral and cytostatic nucleoside substrates", FEBS Lett, 443, 1999, 170-174.
22.6.4.1 thymidine + ATP => thymidine 5'-monophosphate + ADP
Description
This reaction takes place in the 'mitochondrial matrix' and is mediated by the 'nucleoside kinase activity' of 'thymidine kinase 2, mitochondrial'.
References
Reaction
22.6.4.2 2'-deoxyuridine + ATP => 2'-deoxyuridine 5'-monophosphate + ADP
Description
At the beginning of this reaction, 1 molecule of '2'-deoxyuridine', and 1 molecule of 'ATP' are present. At the end of this reaction, 1 molecule of
'ADP', and 1 molecule of '2'-deoxyuridine 5'-monophosphate' are present.
References
Reaction
22.6.4.3 2'-deoxycytidine + ATP => 2'-deoxycytidine 5'-monophosphate + ADP
Description
References
Reaction
22.6.5 Phosphorylation of cytosolic nucleosides by thymidine kinase 1, soluble
Authors
Editors
Description
Thymidine kinase 1, soluble, has been purified from human spleen and from cultured cells. The enzyme is active as a homotetramer, and
phosphorylates thymidine and deoxyuridine using ATP as a phosphate donor in vitro (Sherley and Kelly 1988a; Munch-Petersen et al. 1991).
Divalent cations are required for enzyme activity (Mg++ is preferred) (Lee and Cheng 1986), and ATP stabilizes the tetrameric form of the
enzyme (Munch-Petersen et al. 1993). In cells, enzyme activity is high during S phase of the cell cycle and low otherwise, correlated with
intracellular levels of thymidine kinase 1 protein (Sherley and Kelly 1988b).
References
LS Lee, YC Cheng, "Human deoxythymidine kinase. I. Purification and general properties of the cytoplasmic and mitochondrial isozymes derived
from blast cells of acute myelocytic leukemia", J Biol Chem, 251, 1976, 2600-2604.
JL Sherley, TJ Kelly, "Human cytosolic thymidine kinase. Purification and physical characterization of the enzyme from HeLa cells", J Biol Chem,
263, 1988, 375-382.
B Munch-Petersen, G Tyrsted, L Cloos, "Reversible ATP-dependent transition between two forms of human cytosolic thymidine kinase with
different enzymatic properties", J Biol Chem, 268, 1993, 15621-15625.
JL Sherley, TJ Kelly, "Regulation of human thymidine kinase during the cell cycle", J Biol Chem, 263, 1988, 8350-8358.
22.6.5.1 thymidine + ATP => thymidine 5'-monophosphate (dTMP) + ADP
Description
This reaction takes place in the 'cytosol' and is mediated by the 'nucleoside kinase activity' of 'thymidine kinase 1, soluble holoenzyme'.
References
Reaction
22.6.6 Phosphorylation of cytosolic nucleosides by uridine-cytidine kinase 1
Authors
Editors
Description
Human uridine-cytidine kinase 1 is a cytosolic, magnesium-dependent enzyme (Greenberg et al. 1977). It appears to be widely expressed in
human tissues, but its level in normal cells is typically very low (Ropp and Traut 1996). The active form of the human enzyme has not been
established experimentally, but it is inferred to be a tetramer, stabilized by ATP and destabilized by UTP and CTP, from studies of purified
mouse uridine kinase (Cheng et al. 1986; Ropp and Traut 1998). The enzyme catalyzes the phosphorylation of uridine and cytidine, and can use
ATP or, less efficiently, GTP as a phosphate donor.
References
N Greenberg, DE Schumm, TE Webb, "Uridine kinase activities and pyrimidine nucleoside phosphorylation in fluoropyrimidine-sensitive and
-resistant cell lines of the Novikoff hepatoma", Biochem J, 164, 1977, 379-387.
PA Ropp, TW Traut, "Cloning and expression of a cDNA encoding uridine kinase from mouse brain", Arch Biochem Biophys, 336, 1996,
105-112.
N Cheng, RC Payne, TW Traut, "Regulation of uridine kinase. Evidence for a regulatory subunit", J Biol Chem, 261, 1986, 13006-13012.
PA Ropp, TW Traut, "Uridine kinase: altered enzyme with decreased affinities for uridine and CTP", Arch Biochem Biophys, 359, 1998, 63-68.
Description
References
Reaction
Description
References
Reaction
22.6.7 Phosphorylation of cytosolic nucleosides by uridine-cytidine kinase 2
Authors
Editors
Description
Human uridine-cytidine kinase 2 was found in a search for human cDNA clones homologous to a mouse uridine-cytidine kinase gene. Purified
recombinant uridine-cytidine kinase 2 protein catalyzes the phosphorylation of uridine and cytidine in vitro; both ATP and GTP are efficient
phosphate donors (Van Rompay et al. 2001). The subunit structure and cation dependence of the enzyme have not been established. When it is
expressed as a fusion construct with green fluorescent protein, the protein localizes primarily to the cytosol of transfected CHO cells (Van
Rompay et al. 2003).
References
AR Van Rompay, M Johansson, A Karlsson, "Substrate specificity and phosphorylation of antiviral and anticancer nucleoside analogues by
human deoxyribonucleoside kinases and ribonucleoside kinases", Pharmacol Ther, 100, 2003, 119-139.
Description
This reaction takes place in the 'cytosol' and is mediated by the 'nucleoside kinase activity' of 'Uridine-cytidine kinase 2 '.
References
Reaction

Description
This reaction takes place in the 'cytosol' and is mediated by the 'nucleoside kinase activity' of 'Uridine-cytidine kinase 2 '.
References
Reaction
22.7 Reversible phosphorylation of nucleoside monophosphates
Authors
Editors
Description
Nucleoside monophosphate kinases catalyze the reversible phosphorylation of nucleoside and deoxynucleoside 5'-monophosphates to form the
corresponding nucleoside 5'-diphosphates. Most appear to have restricted specificities for nucleoside monophosphates, and to use ATP
preferentially (Van Rompay et al. 2000; Anderson 1973; Noda 1973). The total number of human enzymes that catalyze these reactions in vivo
is not clear. In six cases, a well-defined biochemical activity has been associated with a purified protein, and these are annotated here. However,
additional nucleoside monophosphate kinase-like human proteins have been identified in molecular cloning studies whose enzymatic activities
are unknown, and several distinctive nucleoside monophosphate kinase activities detected in cell extracts, e.g., a GTP-requiring adenylate
kinase activity (Wilson et al. 1976) and one or more guanylate kinase activities (Jamil et al. 1975) have not been unambiguously associated with
specific human proteins.
The nucleoside monophosphates against which each of the six well-characterized enzymes is active is shown in the table (Van Rompay et al.
2000). All six efficiently use ATP as a phosphate donor, but have some activity with other nucleoside triphosphates as well in vitro. The high
concentrations of ATP relative to other nucleoside triphosphates in vivo makes it the likely major phosphate donor in these reactions under most
conditions.
All of these phosphorylation reactions are freely reversible in vitro when carried out with purified enzymes and substrates, having equilibrium
constants near 1. In vivo, high ratios of ATP to ADP are likely to favor the forward direction of these reactions, i.e., the conversion of (d)NMP and
ATP to (d)NDP and ADP. At the same time, the reversibility of the reactions and the overlapping substrate specificities of the enzymes raises the
possibility that this group of reactions can buffer the intracellular nucleotide pool and regulate the relative concentrations of individual nucleotides
in the pool: if any one molecule builds up to unusually high levels, multiple routes appear to be open not only to dispose of it but to use it to
increase the supply of less abundant nucleotides.
References
L Noda, "Adenylate kinase", The Enzymes, 3rd ed (Boyer PD, editor), 8, 1973, 279-305.
T Jamil, RA Fisher, H Harris, "Studies on the properties and tissue distribution of the isozymes of guanylate kinase in man", Human Hered, 25,
1975, 402-413.
DE Wilson, Jr, S Povey, H Harris, "Adenylate kinases in man: evidence for a third locus", Ann Hum Genet, 39, 1976, 305-313.
EP Anderson, "Nucleoside and nucleotide kinases", The Enzymes, 3rd ed (Boyer PD, editor), 9, 1973, 49-96.
AR Van Rompay, M Johansson, A Karlsson, "Phosphorylation of nucleosides and nucleoside analogs by mammalian nucleoside
monophosphate kinases", Pharmacol Ther, 87, 2000, 189-198.
22.7.1 Reversible phosphorylation of cytosolic nucleoside monophosphates by deoxythymidylate kinase

(thymidylate kinase)
Authors
Editors
Description
Deoxythymidylate kinase (thymidylate kinase) phosphorylates thymidine and deoxyuridine monophosphates, with ATP or dATP as the preferred
phosphate donor. The active form of the enzyme is a dimer, found in the cytosol (although its presence in other subcellular compartments cannot
be rigorously excluded) (Anderson 1973; Lee and Cheng 1977).
References
EP Anderson, "Nucleoside and nucleotide kinases", The Enzymes, 3rd ed (Boyer PD, editor), 9, 1973, 49-96.
22.7.1.1 thymidine 5'-monophosphate (TMP) + ATP <=> thymidine 5'-diphosphate (TDP) + ADP
Description
At the beginning of this reaction, 1 molecule of 'thymidine 5'-monophosphate', and 1 molecule of 'ATP' are present. At the end of this reaction, 1
molecule of 'thymidine 5'-diphosphate', and 1 molecule of 'ADP' are present.
holoenzyme'.
References
Reaction
22.7.1.2 2'-deoxyuridine 5'-monophosphate (dUMP) + ATP <=> 2'-deoxyuridine 5'-diphosphate (dUDP) + ADP
Description
At the beginning of this reaction, 1 molecule of 'ATP', and 1 molecule of '2'-deoxyuridine 5'-monophosphate' are present. At the end of this
reaction, 1 molecule of 'ADP', and 1 molecule of '2'-deoxyuridine 5'-diphosphate' are present.
holoenzyme'.
References
Reaction
22.7.1.3 2'-deoxyuridine 5'-diphosphate (dUDP) + ADP <=> 2'deoxyuridine 5'-monophosphate (dUMP) + ATP
Description
At the beginning of this reaction, 1 molecule of 'ADP', and 1 molecule of '2'-deoxyuridine 5'-diphosphate' are present. At the end of this reaction,
1 molecule of 'ATP', and 1 molecule of '2'-deoxyuridine 5'-monophosphate' are present.
holoenzyme'.
References
Reaction
22.7.1.4 thymidine 5'-diphosphate (TDP) + ADP <=> thymidine 5'-monophosphate (TMP) + ATP
Description
At the beginning of this reaction, 1 molecule of 'thymidine 5'-diphosphate', and 1 molecule of 'ADP' are present. At the end of this reaction, 1
molecule of 'thymidine 5'-monophosphate', and 1 molecule of 'ATP' are present.
holoenzyme'.
References
Reaction
22.7.2 Reversible phosphorylation of cytosolic nucleoside monophosphates by UMP-CMP kinase
Authors
Editors
Description
UMP-CMP kinase reversibly phosphorylates uridine, cytidine, and deoxycytidine monophosphates, with ATP or dATP as the preferred
phosphate donor (Liou et al. 2002; Scott and Wright 1979). The enzyme is cytosolic, and its active form is inferred to be a monomer, from the
close correspondence between the measured mass of purified native rat protein (Arima et al. 1977) and the mass of the cloned human protein
calculated from its predicted amino acid sequence (Liou et al. 2002).
References
571, 1979, 45-54.
T Arima, H Akiyoshi, S Fujii, "Characterization of pyrimidine nucleoside monophosphokinase in normal and malignant tissues", Cancer Res, 37,
1977, 1593-1597.
Description
References
571, 1979, 45-54.
Reaction
22.7.2.2 uridine 5'-monophosphate (UMP) + ATP <=> uridine 5'-diphosphate (UDP) + ADP
Description
At the beginning of this reaction, 1 molecule of 'ATP', and 1 molecule of 'uridine 5'-monophosphate' are present. At the end of this reaction, 1
molecule of 'uridine 5'-diphosphate', and 1 molecule of 'ADP' are present.
References
571, 1979, 45-54.
Reaction
22.7.2.3 2'-deoxycytidine 5'-monophosphate (dCMP) + ATP <=> deoxycytidine 5'-diphosphate (dCDP) + ADP
Description
References
571, 1979, 45-54.
Reaction
22.7.2.4 uridine 5'-diphosphate (UDP) + ADP <=> uridine 5'-monophosphate (UMP) + ATP
Description
At the beginning of this reaction, 1 molecule of 'uridine 5'-diphosphate', and 1 molecule of 'ADP' are present. At the end of this reaction, 1
molecule of 'ATP', and 1 molecule of 'uridine 5'-monophosphate' are present.
References
571, 1979, 45-54.
Reaction
22.7.2.5 2'-deoxycytidine 5'-diphosphate (dCDP) + ADP <=> deoxycytidine 5'-monophosphate (dCMP) + ATP
Description
References
571, 1979, 45-54.
Reaction
Description
References
571, 1979, 45-54.
Reaction
22.7.3 Reversible phosphorylation of cytosolic nucleoside monophosphates by guanylate kinase 1
Authors
Editors
Description
Studies of human guanylate kinase isozymes suggest the existence of at least three genes encoding enzymes with this activity (Jamil et al.
1975), but only one human protein has been well characterized. This protein, guanylate kinase 1, is a cytosolic monomer that catalyzes the
phosphorylation of guanosine and deoxyguanosine monophosphates with ATP as the preferred phosphate donor (Agarwal et al. 1978; Miller and
Miller 1980).
References
T Jamil, RA Fisher, H Harris, "Studies on the properties and tissue distribution of the isozymes of guanylate kinase in man", Human Hered, 25,
1975, 402-413.
WH Miller, RL Miller, "Phosphorylation of acyclovir (acycloguanosine) monophosphate by GMP kinase", J Biol Chem, 255, 1980, 7204-7207.
483-491.
22.7.3.1 guanosine 5'-monophosphate (GMP) + ATP <=> guanosine 5'-diphosphate (GDP) + ADP
Description
At the beginning of this reaction, 1 molecule of 'ATP', and 1 molecule of 'GMP' are present. At the end of this reaction, 1 molecule of 'GDP', and
1 molecule of 'ADP' are present.
References
483-491.
Reaction
22.7.3.2 2'-deoxyguanosine 5'-monophosphate (dGMP) + ATP <=> 2'-deoxyguanosine 5'-diphosphate (dGDP) + ADP
Description
At the beginning of this reaction, 1 molecule of '2'-deoxyguanosine 5'-monophosphate', and 1 molecule of 'ATP' are present. At the end of this
reaction, 1 molecule of 'ADP', and 1 molecule of '2'-deoxyguanosine 5'-diphosphate' are present.
References
483-491.
Reaction
22.7.3.3 guanosine 5'-diphosphate (GDP) + ADP <=> guanosine 5'-monophosphate (GMP) + ATP
Description
At the beginning of this reaction, 1 molecule of 'ADP', and 1 molecule of 'GDP' are present. At the end of this reaction, 1 molecule of 'ATP', and 1
molecule of 'GMP' are present.
References
483-491.
Reaction
22.7.3.4 2'-deoxyguanosine 5'-diphosphate (dGDP) + ADP <=> 2'-deoxyguanosine 5'-monophosphate (dGMP) + ATP
Description
At the beginning of this reaction, 1 molecule of 'ADP', and 1 molecule of '2'-deoxyguanosine 5'-diphosphate' are present. At the end of this
reaction, 1 molecule of '2'-deoxyguanosine 5'-monophosphate', and 1 molecule of 'ATP' are present.
References
483-491.
Reaction
22.7.4 Reversible phosphorylation of cytosolic nucleoside monophosphates by adenylate kinase 1
Authors
Editors
Description
Adenylate kinase 1 is a cytosolic, monomeric enzyme that catalyzes the phosphorylation of adenosine and deoxyadenosine monophosphates,
with ATP or dATP as the phosphate donor (Criss 1970; Tsuboi 1978).
References
WE Criss, "Rat liver adenosine triphosphate:adenosine monophosphate phosphotransferase activity. II. Subcellular localization of adenylate
kinase isozymes", J Biol Chem, 245, 1970, 6352-6356.
KK Tsuboi, "AMP (dAMP) kinase from human erythrocytes", Methods Enzymol, 51, 1978, 467-473.
22.7.4.1 adenosine 5'-monophosphate (AMP) + ATP <=> adenosine 5'-diphosphate (ADP) + ADP
Description
Cytosolic adenylate kinase 1 catalyzes the reaction of ATP and AMP to form two molecules of ADP.
References
S Matsuura, M Igarashi, Y Tanizawa, M Yamada, F Kishi, T Kajii, H Fujii, S Miwa, M Sakurai, A Nakazawa, "Human adenylate kinase deficiency
associated with hemolytic anemia. A single base substitution affecting solubility and catalytic activity of the cytosolic adenylate kinase.", J Biol
Chem, 264, 1989, 10148-55.
Reaction
22.7.4.2 2'-deoxyadenosine 5'-diphosphate (dADP) + ADP <=> 2'-deoxyadenosine 5'-monophosphate (dAMP) + ATP
Description
At the beginning of this reaction, 1 molecule of 'ADP', and 1 molecule of '2'-deoxyadenosine 5'-diphosphate' are present. At the end of this
reaction, 1 molecule of '2'-deoxyadenosine 5'-monophosphate', and 1 molecule of 'ATP' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'nucleoside kinase activity' of 'adenylate kinase 1'.
References
Reaction
22.7.4.3 2'-deoxyadenosine 5'-monophosphate (dAMP) + ATP <=> 2'-deoxyadenosine 5'-diphosphate (dADP) + ADP
Description
References
Reaction
22.7.4.4 adenosine 5'-diphosphate (ADP) + ADP <=> adenosine 5'-monophosphate (AMP) + ATP
Description
At the beginning of this reaction, 2 molecules of 'ADP' is present. At the end of this reaction, 1 molecule of 'AMP', and 1 molecule of 'ATP' are
present.
References
Reaction
22.7.5 Reversible phosphorylation of cytosolic nucleoside monophosphates by adenylate kinase 5
Authors
Editors
Description
Adenylate kinase 5 is a cytosolic enzyme with an unusually broad specificity, and unusually narrow tissue distribution (only brain, of seven adult
tissues examined by Northern blotting) (Van Rompay et al. 1999). Its subunit structure is unknown, so it is annotated here as a monomer by
default.
References
AR Van Rompay, M Johansson, A Karlsson, "Identification of a novel human adenylate kinase. cDNA cloning, expression analysis, chromosome
localization and characterization of the recombinant protein", Eur J Biochem, 1999, 1999, 509-516.
22.7.5.1 2'-deoxyadenosine 5'-diphosphate (dADP) + ADP <=> 2'-deoxyadenosine 5'-monophosphate (dAMP) + ATP
Description
At the beginning of this reaction, 1 molecule of 'ADP', and 1 molecule of '2'-deoxyadenosine 5'-diphosphate' are present. At the end of this
reaction, 1 molecule of '2'-deoxyadenosine 5'-monophosphate', and 1 molecule of 'ATP' are present.
References
Reaction
22.7.5.2 2'-deoxyadenosine 5'-monophosphate (dAMP) + ATP <=> 2'-deoxyadenosine 5'-diphosphate (dADP) + ADP
Description
References
Reaction
22.7.5.3 2'-deoxycytidine 5'-diphosphate (dCDP) + ADP <=> 2'-deoxycytidine 5'-monophosphate (dCMP) + ATP
Description
References
Reaction
22.7.5.4 2'-deoxycytidine 5'-monophosphate (dCMP) + ATP <=> 2'-deoxycytidine 5'-diphosphate (dCDP) + ADP
Description
References
Reaction
Description
present.
References
Reaction
Description
At the beginning of this reaction, 1 molecule of 'AMP', and 1 molecule of 'ATP' are present. At the end of this reaction, 2 molecules of 'ADP' is
present.
References
Reaction
Description
References
Reaction
Description
References
Reaction
22.7.6 Reversible phosphorylation of mitochondrial nucleoside monophosphates by adenylate kinase 2
Authors
Editors
Description
Adenylate kinase 2, localized to the mitochondrial intermembrane space, catalyzes the phosphorylation of adenosine 5'-monophosphate by ATP.
The purified enzyme is active as a monomer (Criss 1970; Hamada et al. 1982).
References
M Hamada, M Sumida, H Okuda, T Watanabe, M Nojima, SA Kuby, "Adenosine triphosphate-adenosine-5'-monophosphate phosphotransferase

from normal human liver mitochondria. Isolation, chemical properties, and immunochemical comparison with Duchenne muscular dystrophic
serum aberrant adenylate kinase", J Biol Chem, 257, 1982, 13120-13128.
WE Criss, "Rat liver adenosine triphosphate:adenosine monophosphate phosphotransferase activity. II. Subcellular localization of adenylate
kinase isozymes", J Biol Chem, 245, 1970, 6352-6356.
Description
present.
This reaction takes place in the 'mitochondrial intermembrane space' and is mediated by the 'nucleoside kinase activity' of 'adenylate kinase 2'.
References

Reaction
Description
At the beginning of this reaction, 1 molecule of 'AMP', and 1 molecule of 'ATP' are present. At the end of this reaction, 2 molecules of 'ADP' is
present.
This reaction takes place in the 'mitochondrial intermembrane space' and is mediated by the 'nucleoside kinase activity' of 'adenylate kinase 2'.
References

Reaction
22.8 Reversible phosphorylation of nucleoside diphosphates
Authors
Editors
Description
Nucleoside diphosphate kinases catalyze the reversible phosphorylation of nucleoside and deoxynucleoside 5'-diphosphates to form the
corresponding nucleoside 5'-triphosphates. Mammalian nucleoside diphosphate kinases, first identified by Berg and Joklik, are typically
ubiquitously expressed enzymes with broad substrate specificities (Berg and Joklik 1954; Parks and Agarwal 1973). More recent work has
suggested that some of these proteins may be transcription factors, and that mutations in some of them may be associated with tumor
progression (Lacombe et al. 2000). This possible tumor-associated function is responsible for the remarkable names that have been assigned to
these proteins: "non-metastatic cells 1, protein (NM23A) expressed in", "non-metastatic cells 2, protein (NM23B) expressed in", and so forth.
Analyses of human DNA sequences and predicted amino acid sequences suggest the existence of a nucleoside diphosphate kinase protein
family containing at least eight members, many with well-conserved homologues in other eukaryotes (Lacombe et al. 2000). Only four of these
proteins have been purified, characterized biochemically, and unambiguously localized to subcellular compartments; these four proteins and
their activities are annotated here.
Nucleoside diphosphate kinases efficiently use ATP as a phosphate donor, but are typically also active with other nucleoside triphosphates in
vitro. The high concentrations of ATP relative to other nucleoside triphosphates in vivo makes it the likely major phosphate donor in these
reactions under most conditions, and only reactions with ATP as the phosphate donor are annotated. All of these phosphorylation reactions are
freely reversible in vitro when carried out with purified enzymes and substrates (Parks and Agarwal 1973; Schaertl et al. 1998). In vivo, high
ratios of ATP to ADP are likely to favor the forward direction of these reactions, i.e., the conversion of (d)NDP and ATP to (d)NTP and ADP. At
the same time, the reversibility of the reactions and the overlapping substrate specificities of the enzymes raises the possibility that this group of
reactions can buffer the intracellular nucleotide pool and regulate the relative concentrations of individual nucleoside di- and tri-phosphates in the
pool: if any one molecule builds up to unusually high levels, multiple routes appear to be open not only to dispose of it but to use it to increase
the supply of less abundant nucleotides, so both the forward and the reverse direction of each reaction is annotated.
References
Chem, 273, 1998, 5662-5669.
ML Lacombe, L Milon, A Munier, JG Mehus, DO Lambeth, "The human Nm23/nucleoside diphosphate kinases", J Bioenerg Biomembr, 32,
2000, 247-258.
P Berg, WK Joklik, "Enzymatic phosphorylation of nucleoside diphosphates", J Biol Chem, 210, 1954, 657-672.
RE Parks, Jr, RP Agarwal, "Nucleoside diphosphokinases", The Enzymes, 3rd ed (Boyer PD, editor), 8, 1973, 307-333.
22.8.1 Reversible phosphorylation of cytosolic nucleoside diphosphates by nucleoside diphosphate kinase

A hexamer
Authors
Editors
Description
The first two members of the nucleoside diphosphate kinase enzyme family, non-metastatic cells 1, protein (NM23A) expressed in and
non-metastatic cells 2, protein (NM23B) expressed in, abundant in erythrocytes, have been most extensively studied. The active form of the
enzyme is a hexamer, and the two isoenzymes can associate in all proportions to form active hexamers (Gilles et al. 1991; Webb et al. 1995).
No functional differences have been found for hexamers of different compositions. Here, three hexameric forms, A6, A3B3, and B6, have been
annotated, to reflect the fact that either isoenzyme alone is capable of carrying out the full range of reactions associated with the complex. These
reactions are the reversible phosphorylation of ADP, GDP, UDP, CDP, dADP, dGDP, dUDP, dCDP, or TDP, using any of ATP, GTP, UTP or
CTP as a phosphate donor (Schaertl et al. 1998).
While immunofluorescence studies suggest that non-metastatic cells 2, protein (NM23B) expressed in can be localized to the nucleus, consistent
with its possible role as a transcription factor (Pinon et al. 1999), non-metastatic cells 1, protein (NM23A) expressed in is cytosolic, consistent
with biochemical studies that localize the nucleoside diphosphate kinase activity of these proteins to the cytosol (Parks and Agarwal 1973).
References
Chem, 273, 1998, 5662-5669.
VP Pinon, G Millot, A Munier, J Vassy, G Linares-Cruz, J Capeau, F Calvo, ML Lacombe, "Cytoskeletal association of the A and B nucleoside
diphosphate kinases of interphasic but not mitotic human carcinoma cell lines: specific nuclear localization of the B subunit", Exp Cell Res, 246,
1999, 355-367.
PA Webb, O Perisic, CE Mendola, JM Backer, RL Williams, "The crystal structure of a human nucleoside diphosphate kinase, NM23-H2", J Mol
Biol, 251, 1995, 574-587.
22.8.1.1 cytidine 5'-diphosphate (CDP) + ATP <=> CTP + adenosine 5'-diphosphate (ADP)
Description
This reaction takes place in the 'cytosol' and is mediated by the 'nucleoside diphosphate kinase activity' of 'nucleoside diphosphate kinase A
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
22.8.1.4 adenosine 5'-diphosphate (ADP) + dATP <=> ATP + 2'-deoxyadenosine 5'-diphosphate (dADP)
Description
At the beginning of this reaction, 1 molecule of 'ADP', and 1 molecule of 'dATP' are present. At the end of this reaction, 1 molecule of
'2'-deoxyadenosine 5'-diphosphate', and 1 molecule of 'ATP' are present.
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
22.8.1.8 adenosine 5'-diphosphate (ADP) + dGTP <=> ATP + 2'-deoxyguanosine 5'-diphosphate (dGDP)
Description
At the beginning of this reaction, 1 molecule of 'ADP', and 1 molecule of 'dGTP' are present. At the end of this reaction, 1 molecule of
'2'-deoxyguanosine 5'-diphosphate', and 1 molecule of 'ATP' are present.
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
22.8.1.9 2'-deoxyuridine 5'-diphosphate (dUDP) + ATP <=> dUTP + adenosine 5'-diphosphate (ADP)
Description
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
22.8.1.10 adenosine 5'-diphosphate (ADP) + dUTP <=> ATP + 2'-deoxyuridine 5'-diphosphate (dUDP)
Description
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
22.8.1.11 adenosine 5'-diphosphate (ADP) + GTP <=> ATP + guanosine 5'-diphosphate (GDP)
Description
At the beginning of this reaction, 1 molecule of 'GTP', and 1 molecule of 'ADP' are present. At the end of this reaction, 1 molecule of 'GDP', and
1 molecule of 'ATP' are present.
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
22.8.1.12 guanosine 5'-diphosphate (GDP) + ATP <=> GTP + adenosine 5'-diphosphate (ADP)
Description
At the beginning of this reaction, 1 molecule of 'ATP', and 1 molecule of 'GDP' are present. At the end of this reaction, 1 molecule of 'GTP', and 1
molecule of 'ADP' are present.
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
22.8.1.14 thymidine 5'-diphosphate (TDP) + ATP <=> TTP + adenosine 5'-diphosphate (ADP)
Description
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
22.8.1.16 uridine 5'-diphosphate (UDP) + ATP <=> UTP + adenosine 5'-diphosphate (ADP)
Description
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction

B hexamer
Authors
Editors
Description
References
Chem, 273, 1998, 5662-5669.
1999, 355-367.
Biol, 251, 1995, 574-587.
Description
This reaction takes place in the 'cytosol' and is mediated by the 'nucleoside diphosphate kinase activity' of 'nucleoside diphosphate kinase B
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
hexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction

C hexamer
Authors
Editors
Description
This nucleoside diphosphate kinase is very similar structurally to non-metastatic cells 1, protein (NM23A) expressed in and non-metastatic cells
2, protein (NM23B) expressed in. The enzyme is cytosolic and active as a hexamer. Its activities with combinations of nucleoside diphosphates
and triphosphates have not been exhaustively examined, but its very close similarity to two very well-studied members of the nucleoside
diphosphate kinase isozyme family allows these activities to be inferred with confidence. A distinct function in vivo for this protein, either as a
kinase or as a regulator of cell growth and proliferation, remains to be established (Erent et al. 2001).
References
M Erent, P Gonin, J Cherfils, P Tissier, G Raschella, A Giartosio, F Agou, C Sarger, ML Lacombe, M Konrad, I Lascu, "Structural and catalytic
properties and homology modelling of the human nucleoside diphosphate kinase C, product of the DRnm23 gene", Eur J Biochem, 268, 2001,
1972-1981.
Description
This reaction takes place in the 'cytosol' and is mediated by the 'nucleoside diphosphate kinase activity' of 'nucleoside diphosphate kinase C
hexamer'.
References
1972-1981.
Reaction
Description
hexamer'.
References
1972-1981.
Reaction
Description
hexamer'.
References
1972-1981.
Reaction
Description
hexamer'.
References
1972-1981.
Reaction
Description
hexamer'.
References
1972-1981.
Reaction
Description
hexamer'.
References
1972-1981.
Reaction
Description
hexamer'.
References
1972-1981.
Reaction
Description
hexamer'.
References
1972-1981.
Reaction
Description
hexamer'.
References
1972-1981.
Reaction
Description
hexamer'.
References
1972-1981.
Reaction
Description
hexamer'.
References
1972-1981.
Reaction
Description
hexamer'.
References
1972-1981.
Reaction
Description
hexamer'.
References
1972-1981.
Reaction
Description
hexamer'.
References
1972-1981.
Reaction
Description
hexamer'.
References
1972-1981.
Reaction
Description
hexamer'.
References
1972-1981.
Reaction
22.8.4 Reversible phosphorylation of mitochondrial nucleoside diphosphates by nucleoside diphosphate

kinase D, mitochondrial hexamer
Authors
Editors
Description
This nucleoside diphosphate kinase is very similar structurally to non-metastatic cells 1, protein (NM23A) expressed in and non-metastatic cells
2, protein (NM23B) expressed in. The enzyme is associated with the inner mitochondrial membrane and active as a hexamer. Its activities with
combinations of nucleoside diphosphates and triphosphates have not been exhaustively examined, but its very close similarity to two very
well-studied members of the nucleoside diphosphate kinase isozyme family allows these activities to be inferred with confidence (Milon et al.
2000).
The enzyme's orientation in the inner mitochondrial membrane has not been established experimentally. It is the only well-characterized member
of the isozyme family associated with mitochondria, however, so it is inferred to be the catalyst responsible for phosphorylation of nucleoside
diphosphates other than ADP in the mitochondrial matrix (Parks and Agarwal 1973). (Another possible mitochondrial nucleoside diphosphate
kinase has been described, but its sequence similarity to other members of the nucleoside diphosphate kinase isozyme family is low, and its
catalytic activity appears to be weak (Lacombe et al. 2000).)
References
ML Lacombe, L Milon, A Munier, JG Mehus, DO Lambeth, "The human Nm23/nucleoside diphosphate kinases", J Bioenerg Biomembr, 32,
2000, 247-258.
L Milon, P Meyer, M Chiadmi, A Munier, M Johansson, A Karlsson, I Lascu, J Capeau, J Janin, ML Lacombe, "The human nm23-H4 gene
product is a mitochondrial nucleoside diphosphate kinase", J Biol Chem, 275, 2000, 14264-14272.
Description
This reaction takes place in the 'mitochondrial inner membrane' and is mediated by the 'nucleoside diphosphate kinase activity' of 'nucleoside
diphosphate kinase D, mitochondrial hexamer '.
References
Reaction
Description
References
Reaction
Description
References
Reaction
Description
References
Reaction
Description
References
Reaction
Description
References
Reaction
Description
References
Reaction
Description
References
Reaction
Description
References
Reaction
Description
References
Reaction
Description
References
Reaction
Description
References
Reaction
Description
References
Reaction
Description
References
Reaction
Description
References
Reaction
Description
References
Reaction

A,B heterohexamer
Authors
Editors
Description
References
Chem, 273, 1998, 5662-5669.
1999, 355-367.
Biol, 251, 1995, 574-587.
22.8.5.1 cytidine 5'-diphosphate + ATP <=> cytidine 5'-triphosphate + ADP
Description
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
22.8.5.2 uridine 5'-diphosphate (UDP) + ATP <=> uridine 5'-triphosphate (UTP) + ADP
Description
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
22.8.5.4 thymidine 5'-diphosphate (TDP) + ATP <=> thymidine 5'-triphosphate (TTP) + ADP
Description
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
22.8.5.5 2'-deoxyuridine 5'-diphosphate (dUDP) + ATP <=> deoxyuridine 5'-triphosphate (dUTP) + ADP
Description
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
22.8.5.11 2'-deoxyuridine 5'-triphosphate (dUTP) + ADP <=> 2'-deoxyuridine 5'-diphosphate (dUDP) + ATP
Description
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
Description
heterohexamer'.
References
Chem, 273, 1998, 5662-5669.
Reaction
23 Porphyrin metabolism
Reviewers
Sassa, S, 2007-01-24.
Description
Porphyrins are heterocyclic macrocycles, consisting of four pyrrole subunits (tetrapyrrole) linked by four methine (=CH-) bridges. The extensive
conjugated porphyrin macrocycle is chromatic and the name itself, porphyrin, is derived from the Greek word for purple. The aromatic character
of porphyrins can be seen by NMR spectroscopy.
Porphyrins readily combine with metals by coordinating them in the central cavity. Iron (heme) and magnesium (chlorophyll) are two well known
examples although zinc, copper, nickel and cobalt form other known metal-containing phorphyrins. A porphyrin which has no metal in the cavity
is called a free base.
Some iron-containing porphyrins are called hemes (heme-containing proteins or hemoproteins) and these are found extensively in nature ie.
hemoglobin. Hemoglobin is quantitatively the most important hemoprotein. The hemoglobin iron is the transfer site of oxygen and carries it in the
blood all round the body for cell respiration. Other examples are cytochromes present in mitochondria and endoplasmic reticulum which takes
part in electron transfer events, catalase and peroxidase whic protect the body against the oxidant hydrogen peroxide and tryptophan oxygenase
which is present in intermediary metabolism. Hemoproteins are synthesized in all mammalian cells and the major sites are erythropoietic tissue
and the liver.
A chlorin is the ring structure found in chlorophyll. Chlorins are produced when one of the four pyrrole subunits is reduced to pyrroline. If two of
the four pyrrole subunits are reduced then a bacteriochlorin is produced (found in some photosynthestic bacteria).
23.1 Heme biosynthesis
Authors
Editors
Reviewers
Sassa, S, 2007-01-24.
Description
Eight enzymes are involved in heme biosynthesis, four each in the mitochondria and the cytosol. The process starts in the mitochondria with the
condensation of succinyl CoA (from the TCA cycle) and glycine to form 5-aminolevulinate (ALA). The next four steps take place in the cytosol.
Two molecules of ALA are condensed to form the monopyrrole porphobilinogen (PBG). The next two steps convert four molecules of PBG into
the cyclic tetrapyrrole uroporphyringen III, which is then decarboxylated into coproporphyrinogen III. The last three steps occur in the
mitochondria and involve modifications to the tetrapyrrole side chains and finally, insertion of iron. In addition to these synthetic steps, a
spontaneous cytosolic reaction allows the formation of uroporphyringen I which is then enzymatically decarboxylated to coproporphyrinogen I,
which cannot be metabolized further in humans. Also, lead can inactivate ALAD, the enzyme that catalyzes PBG synthesis, and ferrochelatase,
the enzyme that catalyzes heme synthesis.
The porphyrias are disorders that arise from defects in the enzymes of heme biosynthesis. Defective pathway enzymes after ALA synthase
result in accumulated substrates which can cause either skin problems, neurological complications, or both due to their toxicity in higher
concentrations. They are broadly classified as hepatic porphyrias or erythropoietic porphyrias, based on the site of the overproduction of the
substrate. Each defect is described together with the reaction it affects.
References
P Ponka, "Cell biology of heme", Am J Med Sci, 318, 1999, 241-56.
RS Ajioka, JD Phillips, JP Kushner, "Biosynthesis of heme in mammals", Biochim Biophys Acta, 1763, 2006, 723-36.
23.1.1 Succinyl CoA and glycine condense to form 5-aminolevulinate (ALA)
Authors
Editors
Reviewers
Sassa, S, 2007-01-24.
Description
The committed step for porphyrin synthesis is the formation of 5-aminolevulinate (ALA) by condensation of glycine (from the general amino acid
pool) and succinyl-CoA (from the TCA cycle), in the mitochondrial matrix. The reaction is catalyzed by two different ALA synthases, one
expressed ubiquitously (ALAS1) and the other only expressed in erythroid precursors (ALAS2). Both enzymes are expressed as homodimers
and require pyridoxal 5-phosphate as a cofactor.
No disease-causing mutations of ALAS1 have been recognised in humans. Mutations in ALAS2 cause X-linked sideroblastic anaemia (XLSA),
characterised by a microcytic hypochromic anaemia.
References
I Astner, JO Schulze, J van den Heuvel, D Jahn, WD Schubert, DW Heinz, "Crystal structure of 5-aminolevulinate synthase, the first enzyme of
heme biosynthesis, and its link to XLSA in humans", EMBO J, 24, 2005, 3166-77.
LC Gardner, SJ Smith, TM Cox, "Biosynthesis of delta-aminolevulinic acid and the regulation of heme formation by immature erythroid cells in
man", J Biol Chem, 266, 1991, 22010-8.
Reaction
23.1.2 ALA is transported from the mitochondrial matrix to the cytosol
Authors
Editors
Reviewers
Sassa, S, 2007-01-24.
Description
5-aminolevulinate is transported from the mitochondrial matrix to the cytosol. The transporter that enables it to cross the inner mitochondrial
membrane is unknown.
Reaction
23.1.3 ALAD octamer associates with Pb++, forming a catalytically inactive complex
Authors
Editors

Reviewers
Sassa, S, 2007-01-24.
Description
Lead binds to ALAD enzyme displacing zinc ions essential for its catalytic activity and inactivating it. Lead is a major environmental toxin and this
enzyme is one of its principal molecular targets (Jaffe et al. 2001).
References
EK Jaffe, J Martins, J Li, J Kervinen, Jr Dunbrack RL, "The molecular mechanism of lead inhibition of human porphobilinogen synthase", J Biol
Chem, 276, 2001, 1531-7.
Reaction
23.1.4 Two molecules of ALA condense to form porphobilinogen (PBG)
Authors
Editors
Reviewers
Sassa, S, 2007-01-24.
Description
5-Aminolevulinic acid dehydratase (ALAD), also known as porphobilinogen synthase (PBGS), catalyzes the asymmetric condensation of two
molecules of ALA to form porphobilinogen. The substrate that becomes the acetyl side chain-containing half of PBG is called A-side ALA; the
half that becomes the propionyl side chains and the pyrrole nitrogen is called P-ALA (Jaffe 2004). Porphobilinogen (PBG) is the first pyrrole
formed, the precursor to all tetrapyrrole pigments such as heme and chlorophyll. There are at least eight bonds that are made or broken during
this reaction. The active form of the ALAD enzyme is an octamer complexed with eight Zn++ ions, four that are strongly bound and four that are
weakly bound. The four weakly bound ones are dispensible for enzyme activity in vitro (Bevan et al. 1980; Mitchell et al. 2001).
Deficiencies of ALAD enzyme in vivo are associated with 5-aminolevulinate dehydratase-deficient porphyria (e.g., Akagi et al. 2000).
References
EK Jaffe, J Martins, J Li, J Kervinen, Jr Dunbrack RL, "The molecular mechanism of lead inhibition of human porphobilinogen synthase", J Biol
Chem, 276, 2001, 1531-7.
R Akagi, R Shimizu, K Furuyama, MO Doss, S Sassa, "Novel molecular defects of the delta-aminolevulinate dehydratase gene in a patient with
inherited acute hepatic porphyria", Hepatology, 31, 2000, 704-8.
DR Bevan, P Bodlaender, D Shemin, "Mechanism of porphobilinogen synthase. Requirement of Zn2+ for enzyme activity.", J Biol Chem, 255,
1980, 2030-5.
EK Jaffe, "The porphobilinogen synthase catalyzed reaction mechanism", Bioorg Chem, 32, 2004, 316-25.
LW Mitchell, M Volin, J Martins, EK Jaffe, "Mechanistic implications of mutations to the active site lysine of porphobilinogen synthase", J Biol
Chem, 276, 2001, 1538-44.
Reaction
23.1.5 Four PBGs combine through deamination to form hydroxymethylbilane (HMB)
Authors

Editors
Reviewers
Sassa, S, 2007-01-24.
Description
Cytosolic porphobilinogen deaminase catalyzes the polymerization of four molecules of porphobilinogen (PBG) to generate hydroxymethylbilane
(HMB), an unstable tetrapyrrole. This reaction is the first step in the formation of the tetrapyrrole macrocycle. Two isoforms of porphobilinogen
deaminase are generated by alternative splicing, one expresssed in erythroid tissues and one ubiquitously expressed in the body. Deficiencies
of both forms of PBG deaminase are associated with acute intermittent porphyria.
References
PM Shoolingin-Jordan, A Al-Dbass, LA McNeill, M Sarwar, D Butler, "Human porphobilinogen deaminase mutations in the investigation of the
mechanism of dipyrromethane cofactor assembly and tetrapyrrole formation", Biochem Soc Trans, 31, 2003, 731-5.
PM Anderson, RJ Desnick, "Purification and properties of uroporphyrinogen I synthase from human erythrocytes. Identification of stable
enzyme-substrate intermediates.", J Biol Chem, 255, 1980, 1993-9.
Reaction
23.1.6 Conversion of HMB to uroporphyrinogen III
Authors

Editors
Reviewers
Sassa, S, 2007-01-24.
Description
Cytosolic uroporphyrinogen III synthase (URO3S) catalyzes the conversion of HMB (hydroxymethylbilane) to uroporphyrinogen III, a reaction
involving ring closure and intramolecular rearrangement. Uroporphyrinogen III represents a branch point for the pathways leading to formation of
heme, chlorophyll and corrins. HMB is rapidly converted from a linear tetrapyrrole to the cyclic form. Deficiencies of URO3S in vivo are
associated with congenital erythropoietic porphyria.
References
PM Shoolingin-Jordan, "Porphobilinogen deaminase and uroporphyrinogen III synthase: structure, molecular biology, and mechanism", J
Bioenerg Biomembr, 27, 1995, 181-95.
SF Tsai, DF Bishop, RJ Desnick, "Purification and properties of uroporphyrinogen III synthase from human erythrocytes", J Biol Chem, 262,
1987, 1268-73.
Reaction
23.1.7 Spontaneous conversion of HMB to uroporphyrinogen I
Authors

Editors
Reviewers
Sassa, S, 2007-01-24.
Description
Hydroxymethybilane (HMB) can spontaneously cyclize and rearrange to form uroporphyrinogen I.
References
SF Tsai, DF Bishop, RJ Desnick, "Purification and properties of uroporphyrinogen III synthase from human erythrocytes", J Biol Chem, 262,
1987, 1268-73.
Reaction
23.1.8 Uroporphyrinogen III is decarboxylated to form coproporphyrinogen III
Authors
Editors
Reviewers
Sassa, S, 2007-01-24.
Description
Cytosolic uroporphyrinogen decarboxylase (UROD) catalyzes the sequntial removal of four carboxylic groups from the acetic acid side chains of
uroporphyrinogen III (uro'gen III) to form coproporphyrinogen III (copro'gen III) (de Verneuil et al. 1983). Human UROD is a dimer (Whitby et al.
1998). Heterogenous and homogenous deficiencies of UROD are associated with porphyria cutanea tarda and hepatoerythropoietic porphyria
respectively in vivo (Moran-Jiminez et al. 1996).
References
H de Verneuil, S Sassa, A Kappas, "Purification and properties of uroporphyrinogen decarboxylase from human erythrocytes. A single enzyme
catalyzing the four sequential decarboxylations of uroporphyrinogens I and III.", J Biol Chem, 258, 1983, 2454-60.
MJ Moran-Jimenez, C Ged, M Romana, R Enriquez De Salamanca, A Taieb, G Topi, L D'Alessandro, H de Verneuil, "Uroporphyrinogen
decarboxylase: complete human gene sequence and molecular study of three families with hepatoerythropoietic porphyria", Am J Hum Genet,
58, 1996, 712-21.
FG Whitby, JD Phillips, JP Kushner, CP Hill, "Crystal structure of human uroporphyrinogen decarboxylase", EMBO J, 17, 1998, 2463-71.
Reaction
23.1.9 Uroporphyrinogen I is decarboxylated to form coproporphyrinogen I
Authors
Editors
Reviewers
Sassa, S, 2007-01-24.
Description
Cytosolic uroporphyrinogen decarboxylase (UROD) catalyzes the sequential removal of four carboxylic groups from the acetic acid side chains
of uroporphyrinogen I (uro'gen I) to form coproporphyrinogen I (copro'gen I). UROD catalyzes this reaction less efficiently than the
decarboxylation of uroporphyrinogen III (de Verneuil et al. 1983).
References
H de Verneuil, S Sassa, A Kappas, "Purification and properties of uroporphyrinogen decarboxylase from human erythrocytes. A single enzyme
catalyzing the four sequential decarboxylations of uroporphyrinogens I and III.", J Biol Chem, 258, 1983, 2454-60.
MJ Moran-Jimenez, C Ged, M Romana, R Enriquez De Salamanca, A Taieb, G Topi, L D'Alessandro, H de Verneuil, "Uroporphyrinogen
decarboxylase: complete human gene sequence and molecular study of three families with hepatoerythropoietic porphyria", Am J Hum Genet,
58, 1996, 712-21.
FG Whitby, JD Phillips, JP Kushner, CP Hill, "Crystal structure of human uroporphyrinogen decarboxylase", EMBO J, 17, 1998, 2463-71.
Reaction
23.1.10 Translocation of coproporphyrinogen III from the cytosol to the mitochondrial intermembrane space
Authors
Editors
Reviewers
Sassa, S, 2007-01-24.
Description
Coproporpyrinogen III enters the mitochondrial intermembrane space from the cytosol. It is not known whether this process is facilitated by a
transporter.
Reaction
23.1.11 Conversion of coproporphyrinogen III to protoporphyrinogen IX
Authors
Editors
Reviewers
Sassa, S, 2007-01-24.
Description
O2-dependent coproporpyrinogen oxidase (CPO) catalyzes the conversion of coproporphyrinogen III to protoporphyrinogen IX. The localization
of the human enzyme to the mitochondrial intermembrane space is inferred from studies of the homologous rat enzyme (Elder and Evans 1978).
The human enzyme functions as a homodimer (Lee et al. 2005). Enzyme deficiency is associated with hereditary coproporphyria in vivo.
References
CL Cooper, TD Lash, MA Jones, "Kinetic evaluation of human cloned coproporphyrinogen oxidase using a ring isomer of the natural substrate",
Med Sci Monit, 11, 2005, BR420-5.
DS Lee, E Flachsova, M Bodnarova, B Demeler, P Martasek, CS Raman, "Structural basis of hereditary coproporphyria", Proc Natl Acad Sci U
S A, 102, 2005, 14232-7.
GH Elder, JO Evans, "Evidence that the coproporphyrinogen oxidase activity of rat liver is situated in the intermembrane space of mitochondria",
Biochem J, 172, 1978, 345-7.
Reaction
23.1.12 Oxidation of protoporphyrinogen IX to protoporphyrin IX
Authors
Editors
Reviewers
Sassa, S, 2007-01-24.
Description
Six electrons are oxidized in proto'gen IX to form the planar macrocycle protoporphyrin IX (proto IX). This reaction is performed by the enzyme
protoporphyrinogen oxidase (PPO). PPO functions as a homodimer containing one non-covalently-bound FAD. The protein resides on the outer
surface of the inner mitochondrial membrane. PPO deficiency is associated with variegate porphyria in vivo.
References
TA Dailey, HA Dailey, "Human protoporphyrinogen oxidase: expression, purification, and characterization of the cloned enzyme", Protein Sci, 5,
1996, 98-105.
Reaction
23.1.13 Protoporphyrin IX is transported from the mitochondrial intermembrane space into the
mitochondrial matrix
Authors
Editors
Reviewers
Sassa, S, 2007-01-24.
Description
Protoporphyrin IX is transported into the mitochondrial matrix where it becomes available for the last step in the heme biosynthetic pathway. The
transporter that mediates this event is unknown.
Reaction
23.1.14 Ferrous iron is inserted into protoporphyrin IX to form heme
Authors
Editors

Reviewers
Sassa, S, 2007-01-24.
Description
Ferrochelatase (FECH) catalyzes the insertion of ferrous iron into protoporphyrin IX to form heme. FECH is localized on the matrix surface of the
inner mitochondrial membrane and this reaction takes place within the mitochondrial matrix. The enzyme functions as a homodimer with each
monomer containing a nitric oxide-sensitive 2Fe-2S cluster. Enzyme deficiency is associated with erythropoietic protoporphyria in vivo, and
inhibition of ferrochelatase activity is a clinically important consequence of lead poisoning (Piomelli et al. 1987).
References
CK Wu, HA Dailey, JP Rose, A Burden, VM Sellers, BC Wang, "The 2.0 A structure of human ferrochelatase, the terminal enzyme of heme
biosynthesis.", Nat Struct Biol, 8, 2001, 156-60.
S Piomelli, C Seaman, S Kapoor, "Lead-induced abnormalities of porphyrin metabolism. The relationship with iron deficiency.", Ann N Y Acad
Sci, 514, 1987, 278-88.
Reaction
24 Pyruvate metabolism and TCA cycle
Authors
Birney, E, D'Eustachio, P, Schmidt, EE, 2003-11-03.
Editors
Description
These two pathways convert pyruvate (derived from glucose by glycolysis or from alanine by transamination) to acetyl-CoA, which in turn can be
oxidized via the citric acid cycle.
24.1 Oxidative decarboxylation of pyruvate to acetyl CoA by pyruvate

dehydrogenase
Authors
Birney, E, Schmidt, EE, 2003-01-28.
Editors
Description
This process, carried out by the mitochondrial pyruvate dehydrogenase complex, consists of five distinct reactions. First, pyruvate is oxidatively
decarboxylated, catalyzed by the E1 component of the complex (pyruvate dehydrogenase). Lipoamide associated with E1 is reduced at the
same time. Next, the acetyl group derived from pyruvate is transferred to coenzyme A in two steps catalyzed by the E2 component of the
complex (dihydrolipolyl transacetylase). Finally, the oxidized form of lipoamide is regenerated and electrons are transferred to NAD+ in two steps
catalyzed by the E3 component of the complex (dihydrolipoyl dehydrogenase).
References
24.1.1 pyruvate + TPP => 2-(alpha-hydroxyethyl)-TPP + CO2
Description
Reaction 1: E1 accepts a two-carbon aldehyde group from pyruvate and binds it to TPP, forming hydroxyethyl-TPP.
References
ZH Zhou, DB McCarthy, CM O'Connor, LJ Reed, JK Stoops, "The remarkable structural and functional organization of the eukaryotic pyruvate
dehydrogenase complexes.", Proc Natl Acad Sci U S A, 98, 2001, 14802-7.
Reaction
24.1.2 2-(alpha-hydroxyethyl)-TPP + lipoamide => S-acetyldihydrolipoamide + TPP
Description
Reaction 2: The aldehyde group is transferred by E1 to the first lipoamide swinging arm on E2 and simultaneously oxidised to an acetyl group.
Reaction 3: The acetyl group is transferred to the second swinging arm, which positions it for transfer to CoA-SH.
References
Reaction
24.1.3 S-acetyldihydrolipoamide + CoA => acetyl-CoA + dihydrolipoamide
Description
Reaction 4: The acetyl group is transferred to CoA-SH, producing acetyl-CoA.
References
Reaction
24.1.4 dihydrolipoamide + FAD => lipoamide + FADH2 [pyruvate dehydrogenase]
Description
Reaction 5: E3 oxidises the reduced lipoamide swinging arm by transferring two hydrogens to FAD.
References
Reaction
24.1.5 FADH2 + NAD+ => FAD + NADH + H+ [pyruvate dehydrogenase]
Description
Reaction 6: The reduced flavin (FADH2) is oxidised by NAD+.
References
Reaction
24.2 Citric acid cycle (TCA cycle)
Authors
Birney, E, 2003-01-28.
Editors
Description
In a series of reactions called the citric acid, or tricarboxylic acid (TCA) cycle, a two-carbon unit, the acetyl group of acetyl CoA (which is derived
primarily from oxidative decarboxylation of pyruvate, the beta-oxidation of long-chain fatty acids, and the catabolism of several amino acids) is
completely oxidized to CO2 in reactions that also yield one high-energy phosphate bond (as GTP or ATP) and four reducing equivalents (three
NADH + H+, and one FADH2). The NADH and FADH2 are then oxidized by the electron transport chain to yield nine more high-energy
phosphate bonds (as ATP). All reactions of the citric acid cycle take place in the mitochondrion.
The cyclical nature of the reactions responsible for the exidation of pyruvate was first suggested by Hans Krebs, from biochemical studies of
pigeon breast muscle (Krebs et al. 1938; Krebs and Eggleston 1940). Many of the molecular details of individual reactions were worked out by
Ochoa and colleagues, largely through studies of enzymes purified from pig heart (Ochoa 1980). While the human homologues of these
enzymes have all been identified, their biochemcial characterization has in general been limited and molecular details of the human reactions
are inferred from those worked out in studies of the model systems.
References
HA Krebs, E Salvin, WA Johnson, "The formation of citric and alpha-ketoglutaric acids in the mammalian body", Biochem J, 32, 1938, 113-117.
S Ochoa, "The pursuit of a hobby", Annu Rev Biochem, 49, 1980, 1-30.
HA Krebs, LV Eggleston, "The oxidation of pyruvate in pigeon breast muscle", Biochem J, 34, 1940, 442-459.
24.2.1 Acetyl-CoA + H2O + Oxaloacetate => Citrate + CoA
Description
Mitochondrial citrate synthesis catalyzes the irreversible reaction of acetyl-CoA, water, and oxaloacetate to form citrate and coenzyme A. This
reaction is the entry point of two-carbon units into the citric acid cycle. The reaction is subject to allosteric regulation. The gene encoding the
human enzyme has been cloned (Goldenthal et al. 1998), but the enzyme has not been characterized in detail - its properties are inferred from
those of the well-studied homologous pig enzyme (e.g., Morgunov and Srere 1998).
References
MJ Goldenthal, J Marin-Garcia, R Ananthakrishnan, "Cloning and molecular analysis of the human citrate synthase gene", Genome, 41, 1998,
733-738.
273, 1998, 29540-29544.
Reaction
24.2.2 Citrate <=> cis-Aconitate + H2O
Description
Mitochondrial aconitase reversibly converts citrate to isocitrate via cis-aconitate intermediate.
References
GS Baldwin, KL Seet, J Callaghan, G Toncich, BH Toh, RL Moritz, MR Rubira, R Simpson, "Purification and partial amino acid sequence of
human aconitase", Protein Seq Data Anal, 4, 1991, 63-67.
Reaction
24.2.3 cis-Aconitate + H2O <=> Isocitrate
Description
Mitochondrial aconitase reversibly converts citrate to isocitrate via cis-aconitate intermediate.
References
GS Baldwin, KL Seet, J Callaghan, G Toncich, BH Toh, RL Moritz, MR Rubira, R Simpson, "Purification and partial amino acid sequence of
human aconitase", Protein Seq Data Anal, 4, 1991, 63-67.
Reaction
24.2.4 Isocitrate + NAD+ => alpha-ketoglutarate + CO2 + NADH + H+
Description
Mitochondrial isocitrate dehydrogenase catalyzes the irreversible reaction of isocitrate and NAD+ to form alpha-ketoglutarate, CO2, and NADH +
H+. This is the first of four oxidation reactions in the citric acid cycle, and the first decarboxylation.
References
S Soundar, JH Park, RF Colman, "Evaluation by mutagenesis of the importance of 3 arginines in alpha, beta, and gamma subunits of human
NAD-dependent isocitrate dehydrogenase", J Biol Chem, 278, 2003, 52146-52153.
Reaction
24.2.5 Oxidative decarboxylation of alpha-ketoglutarate to succinyl CoA by alpha-ketoglutarate

dehydrogenase
Description
This process, carried out by the mitochondrial alpha-ketoglutarate dehydrogenase complex, consists of five distinct reactions. First,
alpha-ketoglutarate is oxidatively decarboxylated, catalyzed by the E1 component of the complex. Lipoamide associated with E1 is reduced at
the same time. Next, the succinyl group derived from alpha-ketoglutarate is transferred to coenzyme A in two steps catalyzed by the E2
component of the complex (dihydrolipolyl transacetylase). Finally, the oxidized form of lipoamide is regenerated and electrons are transferred to
References
24.2.5.1 alpha-ketoglutarate + TPP => 3-carboxy-1-hydroxypropyl-TPP + CO2
Description
The conversion of alpha-ketoglutarate (2-oxoglutarate) to succinyl-CoA is the second of four oxidation reactions in the citric acid cycle, and the
second decarboxylation. Its mechanism exactly parallels that for the oxidative decarboxylation of pyruvate to acetyl CoA. The pyruvate
dehydrogenase and alpha-ketoglutarate dehydrogenase complexes are structurally homologous, containing homologous E1 and E2 proteins,
and identical E3 proteins and cofactors.
References
Reaction
24.2.5.2 3-carboxy-1-hydroxypropyl-TPP + lipoamide => S-succinyldihydrolipoamide + TPP
Description
References
Reaction
24.2.5.3 S-succinyldihydrolipoamide + CoA => succinyl-CoA + dihydrolipoamide
Description
References
Reaction
24.2.5.4 dihydrolipoamide + FAD => lipoamide + FADH2 [alpha-ketoglutarate dehydrogenase]
Description
'Lipoamide', and 1 molecule of 'FADH2' are present.
dehydrogenase complex, dihydrolipoamide linked'.
References
Reaction
24.2.5.5 FADH2 + NAD+ => FAD + NADH + H+ [alpha-ketoglutarate dehydrogenase]
Description
dehydrogenase complex, FADH2 linked'.
References
Reaction
24.2.6 GDP + Orthophosphate + Succinyl-CoA <=> GTP + Succinate + CoA
Editors
Description
Enzymes from yeast, bovine heart, and pig kidney capable of catalyzing the conversion of succinyl-CoA to succinate plus Coenzyme A, coupled
to the conversion of GDP and orthophosphate to GTP, were first described by Sanadi et al. (1954).
More recent work has established that the enzyme catalyzing the reaction in vertebrates is a heterodimer that occurs in two isoforms. The
enzymes have been purified from pigeon and rat tissue and characterized in detail. More limited studies indicate that the human enzymes
function in the same way. Both isoforms, an alpha:betaA heterodimer and an alpha:betaG heterodimer, catalyze the reversible conversion of
succinyl-CoA to succinate plus Coenzyme A. The alpha:betaA heterodimer couples this conversion to the synthesis of ATP from ADP and
orthophosphate, while the alpha:betaG heterodimer couples it to the synthesis of GTP from GDP and orthophosphate (Johnson et al. 1998a,b;
Lambeth et al. 2004).
Both isoforms are found in vivo, and appear to be expressed at different levels in various tissues. Their relative contributions to the flux of carbon
atoms through the TCA cycle are unknown. Genetic and biochemical data suggest that the alpha:betaA isoform may be required to catalyze the
reverse reaction, conversion of succinate, Coenzyme A, and ATP to succinyl-CoA, ADP, and orthophosphate for heme biosynthesis (Furuyama
and Sassa 2000).
References
K Furuyama, S Sassa, "Interaction between succinyl CoA synthetase and the heme-biosynthetic enzyme ALAS-E is disrupted in sideroblastic
DR Sanadi, DM Gibson, P Ayengar, "Guanosine triphosphate, the primary product of phosphorylation coupled to the breakdown of succinyl
coenzyme A", Biochim Biophys Acta, 14, 1954, 434-436.
JD Johnson, JG Mehus, KN Tews, BI Milavetz, DO Lambeth, "Genetic evidence for the expression of ATP- and GTP-specific succinyl-CoA
synthetases in multicellular eucaryotes", J Biol Chem, 273, 1998, 27580-6.
DO Lambeth, KN Tews, S Adkins, D Frohlich, BI Milavetz, "Expression of two succinyl-CoA synthetases with different nucleotide specificities in
mammalian tissues", J Biol Chem, 279, 2004, 36621-4.
JD Johnson, WW Muhonen, DO Lambeth, "Characterization of the ATP- and GTP-specific succinyl-CoA synthetases in pigeon. The enzymes
incorporate the same alpha-subunit.", J Biol Chem, 273, 1998, 27573-9.
Reaction
24.2.7 ADP + Orthophosphate + Succinyl-CoA <=> ATP + Succinate + CoA
Editors
Description
Enzymes from yeast, bovine heart, and pig kidney capable of catalyzing the conversion of succinyl-CoA to succinate plus Coenzyme A, coupled
to the conversion of GDP and orthophosphate to GTP, were first described by Sanadi et al. (1954).
More recent work has established that the enzyme catalyzing the reaction in vertebrates is a heterodimer that occurs in two isoforms. The
enzymes have been purified from pigeon and rat tissue and characterized in detail. More limited studies indicate that the human enzymes
function in the same way. Both isoforms, an alpha:betaA heterodimer and an alpha:betaG heterodimer, catalyze the reversible conversion of
succinyl-CoA to succinate plus Coenzyme A. The alpha:betaA heterodimer couples this conversion to the synthesis of ATP from ADP and
orthophosphate, while the alpha:betaG heterodimer couples it to the synthesis of GTP from GDP and orthophosphate (Johnson et al. 1998a,b;
Lambeth et al. 2004).
Both isoforms are found in vivo, and appear to be expressed at different levels in various tissues. Their relative contributions to the flux of carbon
atoms through the TCA cycle are unknown. Genetic and biochemical data suggest that the alpha:betaA isoform may be required to catalyze the
reverse reaction, conversion of succinate, Coenzyme A, and ATP to succinyl-CoA, ADP, and orthophosphate for heme biosynthesis (Furuyama
and Sassa 2000).
References
K Furuyama, S Sassa, "Interaction between succinyl CoA synthetase and the heme-biosynthetic enzyme ALAS-E is disrupted in sideroblastic
DR Sanadi, DM Gibson, P Ayengar, "Guanosine triphosphate, the primary product of phosphorylation coupled to the breakdown of succinyl
coenzyme A", Biochim Biophys Acta, 14, 1954, 434-436.
JD Johnson, JG Mehus, KN Tews, BI Milavetz, DO Lambeth, "Genetic evidence for the expression of ATP- and GTP-specific succinyl-CoA
synthetases in multicellular eucaryotes", J Biol Chem, 273, 1998, 27580-6.
DO Lambeth, KN Tews, S Adkins, D Frohlich, BI Milavetz, "Expression of two succinyl-CoA synthetases with different nucleotide specificities in
mammalian tissues", J Biol Chem, 279, 2004, 36621-4.
JD Johnson, WW Muhonen, DO Lambeth, "Characterization of the ATP- and GTP-specific succinyl-CoA synthetases in pigeon. The enzymes
incorporate the same alpha-subunit.", J Biol Chem, 273, 1998, 27573-9.
Reaction
24.2.8 Succinate <=> Fumarate (with FAD redox reaction on enzyme)
Description
The succinate dehydrogenase complex, associated with the inner mitochondrial membrane, catalyzes the dehydrogenation of succinate to
fumarate, reducing the FAD cofactor bound to the enzyme. This redox potential is then used in the electron transfer chain to drive a proton
motive force to generate ATP.
References
JJ Briere, J Favier, V El Ghouzzi, F Djouadi, P Benit, AP Gimenez, P Rustin, "Succinate dehydrogenase deficiency in human", Cell Mol Life Sci,
62, 2005, 2317â€“2324.
Reaction
24.2.9 Fumarate + H2O <=> (S)-Malate
Description
Mitochondrial fumarate hydratase catalyzes the reaction of fumarate and water to form malate, the seventh step of the TCA cycle.
References
T Bourgeron, D Chretien, J Poggi-Bach, S Doonan, D Rabier, P LetouzÃ©, A Munnich, A RÃ¶tig, P Landrieu, P Rustin, "Mutation of the
fumarase gene in two siblings with progressive encephalopathy and fumarase deficiency", J Clin Invest, 93, 1994, 2514-2518.
Reaction
24.2.10 (S)-Malate + NAD+ <=> Oxaloacetate + NADH + H+
Description
Mitochondrial malate dehydrogenase catalyzes the reaction of malate and NAD+ to form oxaloacetate and NADH + H+, the eighth reaction in
the TCA cycle. This reaction is highly endergonic but does proceed due to the low concentration of oxaloacetate, due to the effect of the first
reaction in the TCA cycle. The dimeric structure of the human dehydrogenase is inferred from that established for its well-studied pig homolog
(Sanchez et al. 1998).
References
2184-2189.
273, 1998, 29540-29544.
Reaction
25 Post-translational protein modification
Authors
Editors
Reviewers
Orlean, P, Stafford, DW, 0000-00-00.
Description
After translation, many newly formed proteins undergo further covalent modifications that alter their functional properties and that are essentially
irreversible under physiological conditions in the body. These modifications include the internal peptide bond cleavages that activate
proenzymes, the attachment of oligosaccharide moieties to membrane-bound and secreted proteins, the attachment of lipid or glycolipid
moieties that serve to anchor proteins to cellular membranes, and the vitamin K-dependent attachment of carboxyl groups to glutamate residues.
25.1 Gamma-carboxylation, transport, and amino-terminal cleavage of

proteins
Authors
Editors
Reviewers
Stafford, DW, 0000-00-00.

Description
A number of proteins, including eight required for normal blood clot formation and its regulation (Prothrombin (factor II), factor VII, factor IX,
factor X, protein C, protein S, protein Z, and Gas6) share a sequence motif rich in glutamate residues near their amino termini. Carboxylation of
the glutamate residues within this motif followed by removal of an aminoterminal propeptide is required for each of these proteins to function.
These modifications occur as the proteins move through the endoplasmic reticulum and Golgi apparatus.
25.1.1 Gamma-carboxylation of protein precursors
Authors
Editors
Description
Gamma-carboxylation of a cluster of glutamate residues near the amino termini of thrombin, factor VII, factor IX, factor X, protein C, protein S,
protein Z, and Gas 6 is required for these proteins to bind Ca++ and function efficiently in blood clotting. A single enzyme, vitamin K-dependent
gamma-carboxylase, catalyzes the gamma-carboxylation of all eight proteins involved in clotting (Morris et al. 1995; Brenner et al. 1998; Spronk
et al. 2000). In the carboxylation reaction, the enzyme binds its substrate protein via a sequence motif on the amino terminal side of the
glutamate residues to be carboxylated (Furie et al. 1999), then processively carboxylates all glutamates in the cluster before releasing the
substrate (Morris et al. 1995; Berkner 2000; Stenina et al. 2001). The reaction occurs in the endoplasmic reticulum (Bristol et al. 1996).
References
DP Morris, RD Stevens, DJ Wright, DW Stafford, "Processive post-translational modification. Vitamin K-dependent carboxylation of a peptide
substrate.", J Biol Chem, 270, 1995, 30491-8.
HM Spronk, RA Farah, GR Buchanan, C Vermeer, BA Soute, "Novel mutation in the gamma-glutamyl carboxylase gene resulting in congenital
combined deficiency of all vitamin K-dependent blood coagulation factors", Blood, 96, 2000, 3650-2.
JA Bristol, JV Ratcliffe, DA Roth, MA Jacobs, BC Furie, B Furie, "Biosynthesis of prothrombin: intracellular localization of the vitamin
K-dependent carboxylase and the sites of gamma-carboxylation", Blood, 88, 1996, 2585-93.
O Stenina, BN Pudota, BA McNally, EL Hommema, KL Berkner, "Tethered processivity of the vitamin K-dependent carboxylase: factor IX is
efficiently modified in a mechanism which distinguishes Gla's from Glu's and which accounts for comprehensive carboxylation in vivo",
Biochemistry, 40, 2001, 10301-9.
B Furie, BA Bouchard, BC Furie, "Vitamin K-dependent biosynthesis of gamma-carboxyglutamic acid", Blood, 93, 1999, 1798-808.
KL Berkner, "The vitamin K-dependent carboxylase", J Nutr, 130, 2000, 1877-80.
B Brenner, B Sanchez-Vega, SM Wu, N Lanir, DW Stafford, J Solera, "A missense mutation in gamma-glutamyl carboxylase gene causes
combined deficiency of all vitamin K-dependent blood coagulation factors", Blood, 92, 1998, 4554-9.
25.1.1.1 pro-factor IX, uncarboxylated + 12 CO2 + 12 O2 + 12 vitamin K hydroquinone -> pro-factor IX + 12 H2O + 12 vitamin K epoxide
Description
At the beginning of this reaction, 12 molecules of 'Oxygen', 12 molecules of 'vitamin K hydroquinone', 12 molecules of 'CO2', and 1 molecule of
'pro-factor IX, uncarboxylated' are present. At the end of this reaction, 12 molecules of 'H2O', 12 molecules of 'vitamin K epoxide', and 1
molecule of 'pro-factor IX' are present.
This reaction takes place in the 'endoplasmic reticulum membrane' and is mediated by the 'gamma-glutamyl carboxylase activity' of 'vitamin
K-dependent gamma-carboxylase'.
References
Biochemistry, 40, 2001, 10301-9.
J Ware, DL Diuguid, HA Liebman, MJ Rabiet, CK Kasper, BC Furie, B Furie, DW Stafford, "Factor IX San Dimas. Substitution of glutamine for
Arg-4 in the propeptide leads to incomplete gamma-carboxylation and altered phospholipid binding properties.", J Biol Chem, 264, 1989,
11401-6.
Reaction
25.1.1.2 pro-factor VII, uncarboxylated + 10 CO2 + 10 O2 + 10 vitamin K hydroquinone -> pro-factor VII + 10 H2O + 10 vitamin K epoxide
Description
'pro-factor VII, uncarboxylated' are present. At the end of this reaction, 1 molecule of 'pro-factor VII', 10 molecules of 'H2O', and 10 molecules of
'vitamin K epoxide' are present.
References
Biochemistry, 40, 2001, 10301-9.
Reaction
25.1.1.3 pro-factor X, uncarboxylated + 11 CO2 + 11 O2 + 11 vitamin K hydroquinone -> pro-factor X + 11 H2O + 11 vitamin K epoxide
Description
At the beginning of this reaction, 11 molecules of 'Oxygen', 1 molecule of 'pro-factor X, uncarboxylated', 11 molecules of 'vitamin K
hydroquinone', and 11 molecules of 'CO2' are present. At the end of this reaction, 1 molecule of 'pro-factor X', 11 molecules of 'H2O', and 11
molecules of 'vitamin K epoxide' are present.
References
Biochemistry, 40, 2001, 10301-9.

Reaction
25.1.1.4 pro-prothrombin, uncarboxylated + 10 CO2 + 10 O2 + 10 vitamin K hydroquinone -> pro-prothrombin + 10 H2O + 10 vitamin K
epoxide
Description
'pro-prothrombin (factor II), uncarboxylated' are present. At the end of this reaction, 1 molecule of 'pro-prothrombin (factor II)', 10 molecules of
'H2O', and 10 molecules of 'vitamin K epoxide' are present.
References
Biochemistry, 40, 2001, 10301-9.
Reaction
25.1.1.5 pro-protein C, uncarboxylated + 8 CO2 + 8 O2 + 8 vitamin K hydroquinone -> pro-protein C + 8 H2O + 8 vitamin K epoxide
Description
'pro-protein C, uncarboxylated' are present. At the end of this reaction, 8 molecules of 'H2O', 8 molecules of 'vitamin K epoxide', and 1 molecule
of 'pro-protein C' are present.
References
Biochemistry, 40, 2001, 10301-9.
Reaction
25.1.1.6 pro-protein S, uncarboxylated + 11 CO2 + 11 O2 + 11 vitamin K hydroquinone -> pro-protein S + 11 H2O + 11 vitamin K epoxide
Description
'pro-protein S, uncarboxylated' are present. At the end of this reaction, 1 molecule of 'pro-protein S', 11 molecules of 'H2O', and 11 molecules of
'vitamin K epoxide' are present.
References
Biochemistry, 40, 2001, 10301-9.
Reaction
25.1.1.7 pro-protein Z, uncarboxylated + 13 CO2 + 13 O2 + 13 vitamin K hydroquinone -> pro-protein Z + 13 H2O + 13 vitamin K epoxide
Description
At the beginning of this reaction, 1 molecule of 'pro-protein Z, uncarboxylated', 13 molecules of 'Oxygen', 13 molecules of 'vitamin K
hydroquinone', and 13 molecules of 'CO2' are present. At the end of this reaction, 1 molecule of 'pro-protein Z', 13 molecules of 'H2O', and 13
molecules of 'vitamin K epoxide' are present.
References
Biochemistry, 40, 2001, 10301-9.

Reaction
25.1.1.8 pro-GAS6, uncarboxylated + 11 CO2 + 11 O2 + 11 vitamin K hydroquinone -> pro-GAS6 + 11 H2O + 11 vitamin K epoxide
Authors
Editors
Description
The details of the gamma-carboxylation of GAS6 have not been determined directly, but are inferred from those worked out for protein S
(Manfioletti et al. 1993).
References
G Manfioletti, C Brancolini, G Avanzi, C Schneider, "The protein encoded by a growth arrest-specific gene (gas6) is a new member of the vitamin
K-dependent proteins related to protein S, a negative coregulator in the blood coagulation cascade", Mol Cell Biol, 13, 1993, 4976-85.
Source reaction
This reaction was inferred from the corresponding reaction "pro-protein S, uncarboxylated + 11 CO2 + 11 O2 + 11 vitamin K hydroquinone ->
pro-protein S + 11 H2O + 11 vitamin K epoxide" in species Homo sapiens.
Biochemistry, 40, 2001, 10301-9.

Reaction
25.1.2 Transport of gamma-carboxylated protein precursors from the endoplasmic reticulum to the Golgi
apparatus
Authors
Editors
Description
The details of the vesicle-mediated transport of proteins from the endoplasmic reticulum to the Golgi apparatus will be annotated in a future
release of Reactome.
25.1.2.1 Pro-factor IX is transported from the endoplasmic reticulum to the Golgi apparatus
Description
In this reaction, 1 molecule of 'pro-factor IX' is translocated from endoplasmic reticulum lumen to Golgi lumen.
This reaction takes place in the 'ER to Golgi transport vesicle'.
Reaction
25.1.2.2 Pro-factor VII is transported from the endoplasmic reticulum to the Golgi apparatus
Description
In this reaction, 1 molecule of 'pro-factor VII' is translocated from endoplasmic reticulum lumen to Golgi lumen.
Reaction
25.1.2.3 Pro-factor X is transported from the endoplasmic reticulum to the Golgi apparatus
Description
In this reaction, 1 molecule of 'pro-factor X' is translocated from endoplasmic reticulum lumen to Golgi lumen.
Reaction
25.1.2.4 Pro-prothrombin is transported from the endoplasmic reticulum to the Golgi apparatus
Description
In this reaction, 1 molecule of 'pro-prothrombin (factor II)' is translocated from endoplasmic reticulum lumen to Golgi lumen.

Reaction
25.1.2.5 Pro-protein C is transported from the endoplasmic reticulum to the Golgi apparatus
Description
In this reaction, 1 molecule of 'pro-protein C' is translocated from endoplasmic reticulum lumen to Golgi lumen.
Reaction
25.1.2.6 Pro-protein S is transported from the endoplasmic reticulum to the Golgi apparatus
Description
In this reaction, 1 molecule of 'pro-protein S' is translocated from endoplasmic reticulum membrane to Golgi membrane.
Reaction
25.1.2.7 Pro-protein Z is transported from the endoplasmic reticulum to the Golgi apparatus
Description
In this reaction, 1 molecule of 'pro-protein Z' is translocated from endoplasmic reticulum lumen to Golgi lumen.
Reaction
25.1.2.8 Pro-GAS6 is transported from the endoplasmic reticulum to the Golgi apparatus
Description
In this reaction, 1 molecule of 'pro-GAS6' is translocated from endoplasmic reticulum lumen to Golgi lumen.
Reaction
25.1.3 Removal of aminoterminal propeptides from gamma-carboxylated proteins
Authors
Editors
Description
Furin is an endopeptidase localized to the Golgi membrane that cleaves many proteins on the carboxyterminal side of the sequence motif
Arg-[any residue]-(Lys or Arg)-Arg (Jones et al. 1995; Leduc et al. 1992). In the case of gamma-carboxylated proteins, if this cleavage does not
occur, the proteins are still secreted but do not function properly (Bristol et al. 1993; Lind et al. 1997). The aminoterminal fragments,
"propeptides", generated in this reaction have no known function; the carboxylated, cleaved proteins are delivered to the cell membrane or
secreted from the cell via pathways to be annotated in a future release of Reactome.
References
R Leduc, SS Molloy, BA Thorne, G Thomas, "Activation of human furin precursor processing endoprotease occurs by an intramolecular
autoproteolytic cleavage", J Biol Chem, 267, 1992, 14304-8.
JA Bristol, BC Furie, B Furie, "Propeptide processing during factor IX biosynthesis. Effect of point mutations adjacent to the propeptide cleavage
site.", J Biol Chem, 268, 1993, 7577-84.
BG Jones, L Thomas, SS Molloy, CD Thulin, MD Fry, KA Walsh, G Thomas, "Intracellular trafficking of furin is modulated by the phosphorylation
state of a casein kinase II site in its cytoplasmic tail", EMBO J, 14, 1995, 5869-83.
B Lind, AH Johnsen, S Thorsen, "Naturally occurring Arg(-1) to His mutation in human protein C leads to aberrant propeptide processing and
secretion of dysfunctional protein C", Blood, 89, 1997, 2807-16.
25.1.3.1 pro-factor IX -> factor IX + factor IX propeptide
Description
At the beginning of this reaction, 1 molecule of 'pro-factor IX' is present. At the end of this reaction, 1 molecule of 'factor IX', and 1 molecule of
'factor IX propeptide' are present.
This reaction takes place in the 'Golgi membrane' and is mediated by the 'furin activity' of 'furin'.
Reaction
25.1.3.2 pro-factor VII -> factor VII + factor VII propeptide
Description
At the beginning of this reaction, 1 molecule of 'pro-factor VII' is present. At the end of this reaction, 1 molecule of 'factor VII', and 1 molecule of
'factor VII propeptide' are present.
Reaction
25.1.3.3 pro-factor X -> factor X + factor X propeptide
Description
At the beginning of this reaction, 1 molecule of 'pro-factor X' is present. At the end of this reaction, 1 molecule of 'factor X light chain propeptide',
and 1 molecule of 'factor X' are present.
Reaction
25.1.3.4 pro-prothrombin -> prothrombin + prothrombin propeptide
Description
At the beginning of this reaction, 1 molecule of 'pro-prothrombin (factor II)' is present. At the end of this reaction, 1 molecule of 'prothrombin
(factor II) propeptide', and 1 molecule of 'prothrombin (factor II)' are present.
Reaction
25.1.3.5 pro-protein C -> protein C + protein C propeptide
Description
At the beginning of this reaction, 1 molecule of 'pro-protein C' is present. At the end of this reaction, 1 molecule of 'protein C', and 1 molecule of
'protein C light chain propeptide' are present.
This reaction takes place in the 'Golgi lumen' and is mediated by the 'furin activity' of 'furin'.
Reaction
25.1.3.6 pro-protein S -> protein S + protein S propeptide
Description
At the beginning of this reaction, 1 molecule of 'pro-protein S' is present. At the end of this reaction, 1 molecule of 'protein S propeptide', and 1
molecule of 'protein S' are present.
Reaction
25.1.3.7 pro-protein Z -> protein Z + protein Z propeptide
Description
At the beginning of this reaction, 1 molecule of 'pro-protein Z' is present. At the end of this reaction, 1 molecule of 'protein Z propeptide', and 1
molecule of 'protein Z' are present.
Reaction
25.1.3.8 pro-GAS6 -> GAS6 + GAS6 propeptide
Authors
Editors
Description
The details of the gamma-carboxylation of GAS6 have not been determined directly, but are inferred from those worked out for protein S
(Manfioletti et al. 1993).
References
G Manfioletti, C Brancolini, G Avanzi, C Schneider, "The protein encoded by a growth arrest-specific gene (gas6) is a new member of the vitamin
K-dependent proteins related to protein S, a negative coregulator in the blood coagulation cascade", Mol Cell Biol, 13, 1993, 4976-85.
Reaction
25.2 Synthesis of GPI-anchored proteins
Description
Glycosylphosphatidyl inositol (GPI) acts as a membrane anchor for many cell surface proteins. GPI is synthesized in the endoplasmic reticulum.
In humans, a single pathway consisting of eleven reactions appears to be responsible for the synthesis of the major GPI species involved in
membrane protein anchoring.
As a nascent protein fated to become GPI-anchored moves into the lumen of the endoplasmic reticulum, it is attacked by a transamidase
complex that cleaves it near its carboxy terminus and attaches an acylated GPI moiety. The GPI moiety is deacylated, yielding a protein-GPI
conjugate that can be efficiently transported to the Golgi apparatus.
25.2.1 Synthesis of dolichol-phosphate mannose
Authors
Editors
Reviewers
Orlean, P, 0000-00-00.
Description
Dolichol-phosphate-mannose (DPM) is the donor of mannose groups in the synthesis of the dolichol pyrophosphate-linked precursor
oligosaccharide in asparagine-linked glycosylation, in the synthesis of the glycosyl phosphatidylinositol (GPI) anchor precursor, in protein
O-mannosylation and in protein C-mannosylation. Its synthesis proceeds in two steps. First, cytosolic GDP-mannose reacts with dolichol
phosphate exposed on the cytosolic face of the endoplasmic reticulum membrane to form DPM with its mannose moiety oriented toward the
cytosol. The DPM molecule then flips in the endoplasmic reticulum membrane, so that its mannose moiety is in the endoplasmic reticulum
lumen, accessible to the enzymes that catalyze its transfer to growing glycolipids and glycoproteins (Kinoshita and Inoue 2000; Maeda et al.
2000).
References
T Kinoshita, N Inoue, "Dissecting and manipulating the pathway for glycosylphosphatidylinositol-anchor biosynthesis", Curr Opin Chem Biol, 4,
2000, 632-8.
Y Maeda, S Tanaka, J Hino, K Kangawa, T Kinoshita, "Human dolichol-phosphate-mannose synthase consists of three subunits, DPM1, DPM2
and DPM3", EMBO J, 19, 2000, 2475-82.
25.2.1.1 dolichol phosphate + GDP-alpha-D-mannose -> dolichyl phosphate D-mannose
Authors
Editors
Description
Cytosolic GDP-mannose reacts with dolichol phosphate in the endoplasmic reticulum membrane to form dolichyl phosphate D-mannose. The
reaction is catalyzed by dolichol-phosphate mannosyltransferase, a heterotrimeric protein embedded in the endoplasmic reticulum membrane.
The first subunit of the heterotrimer appears to be the actual catalyst, and the other two subunits appear to stabilize it (Maeda et al. 2000).
References
Y Maeda, S Tanaka, J Hino, K Kangawa, T Kinoshita, "Human dolichol-phosphate-mannose synthase consists of three subunits, DPM1, DPM2
and DPM3", EMBO J, 19, 2000, 2475-82.
Reaction
25.2.1.2 Reorientation of dolichyl phosphate D-mannose in the endoplasmic reticulum membrane
Authors
Editors
Description
Dolichyl phosphate D-mannose is flipped in the endoplasmic reticulum membrane so that its mannose moiety is oriented inwards, towards the
endoplasmic reticulum lumen, where it is accessible to transferases catalyzing the synthesis of glycolipids and glycoproteins (Kinoshita and
Inoue 2000).
References
2000, 632-8.
Reaction
25.2.2 Synthesis of glycosylphosphatidylinositol (GPI)
Authors
Editors
Reviewers
Orlean, P, 0000-00-00.
Description
Glycosylphosphatidyl inositol (GPI) acts as a membrane anchor for many cell surface proteins. GPI is synthesized in the endoplasmic reticulum.
In humans, a single pathway consisting of nine reactions appears to be responsible for the synthesis of the major GPI species involved in
membrane protein anchoring. This pathway is shown in the figure. Two additional reactions, not shown, allow the synthesis of variant forms of
GPI, one with an additional mannose residue and one with an additional ethanolamine (Tauron et al. 2004; Shishioh et al. 2005). These variant
GPI molecules may be used for tissue-specific protein modifications, or may function independently.
The steps of GPI synthesis were first identified by isolating large numbers of mutant cell lines that had lost the ability to express GPI anchored
proteins on their surfaces. Somatic cell hybrid analyses of these lines allowed the definition of complementation groups corresponding to distinct
mutated genes, and cDNAs corresponding to normal forms of these genes were identified on the basis of their abilities to restore normal cell
surface protein expression in mutant cells. Co-precipitation experiments with tagged cloned proteins have allowed the identification of additional
proteins involved in GPI anchor biosynthesis.
References
N Shishioh, Y Hong, K Ohishi, H Ashida, Y Maeda, T Kinoshita, "GPI7 is the second partner of PIG-F and involved in modification of
glycosylphosphatidylinositol", J Biol Chem, 280, 2005, 9728-34.
2000, 632-8.
BW Taron, PA Colussi, JM Wiedman, P Orlean, CH Taron, "Human Smp3p adds a fourth mannose to yeast and human
glycosylphosphatidylinositol precursors in vivo", J Biol Chem, 279, 2004, 36083-92.
25.2.2.1 phosphatidylinositol + UDP-N-acetyl-D-glucosamine -> N-acetylglucosaminyl-PI + UDP

Authors
Editors
Description
The first step of GPI synthesis is the transfer of N-acetylglucosamine from cytosolic UDP-N-acetylglucosamine to phosphatidyl inositol (PI) in the
endoplasmic reticulum membrane. The reaction is catalyzed by a multimeric enzyme, also localized to the endoplasmic reticulum membrane, six
components of which have been identified to date by mutagenesis studies in cultured cells and by co-recipitation studies in vitro (Watanabe et al.
1996, 1998, 2000).
References
R Watanabe, T Kinoshita, R Masaki, A Yamamoto, J Takeda, N Inoue, "PIG-A and PIG-H, which participate in glycosylphosphatidylinositol
anchor biosynthesis, form a protein complex in the endoplasmic reticulum", J Biol Chem, 271, 1996, 26868-75.
R Watanabe, Y Murakami, MD Marmor, N Inoue, Y Maeda, J Hino, K Kangawa, M Julius, T Kinoshita, "Initial enzyme for
glycosylphosphatidylinositol biosynthesis requires PIG-P and is regulated by DPM2", EMBO J, 19, 2000, 4402-11.
R Watanabe, N Inoue, B Westfall, CH Taron, P Orlean, J Takeda, T Kinoshita, "The first step of glycosylphosphatidylinositol biosynthesis is
mediated by a complex of PIG-A, PIG-H, PIG-C and GPI1", EMBO J, 17, 1998, 877-85.
Reaction
25.2.2.2 N-acetylglucosaminyl-PI + H2O -> glucosaminyl-PI + acetate
Authors
Editors
Description
In the second step of GPI synthesis, N-acetylglucosaminyl-PI is hydrolyzed to yield glucosaminyl-PI and acetate. The phosphatidylinositol (PI)
derivatives involved in this reaction are located in the endoplasmic reticulum membrane, as is the PIG-L enzyme that catalyzes it (Sharma et al.
1999; Pottekat and Menon 2004).
References
A Pottekat, AK Menon, "Subcellular localization and targeting of N-acetylglucosaminyl phosphatidylinositol de-N-acetylase, the second enzyme
in the glycosylphosphatidylinositol biosynthetic pathway", J Biol Chem, 279, 2004, 15743-51.
DK Sharma, TK Smith, CT Weller, A Crossman, JS Brimacombe, MA Ferguson, "Differences between the trypanosomal and human GlcNAc-PI
de-N-acetylases of glycosylphosphatidylinositol membrane anchor biosynthesis", Glycobiology, 9, 1999, 415-22.
Reaction
25.2.2.3 Reorientation of glucosaminyl-acyl-PI in the endoplasmic reticulum membrane
Authors
Editors
Description
GPI moieties are synthesized anchored to dolichol phosphate in the membrane of the endoplasmic reticulum. The first two steps of the synthetic
pathway, leading to the production of glucosaminyl-PI, occur on the cytosolic face of the membrane, while addition of an acyl group (step 4) and
all subsequent steps occur on the lumenal face (Murakami et al. 2003). No mutant cell lines defective in the reorientation step have been
identified, and the mechanism by which it occurs is unknown.
References
2000, 632-8.
Y Murakami, U Siripanyapinyo, Y Hong, JY Kang, S Ishihara, H Nakakuma, Y Maeda, T Kinoshita, "PIG-W is critical for inositol acylation but not
for flipping of glycosylphosphatidylinositol-anchor", Mol Biol Cell, 14, 2003, 4285-95.
Reaction
25.2.2.4 glucosaminyl-PI + fatty acyl-CoA -> glucosaminyl-acyl-PI + CoA-SH
Authors
Editors
Description
In the fourth step of GPI synthesis, an acyl group (typically palmitate) is transferred from acyl CoA to glucosaminyl-PI. Mutagenesis and cloning
studies suggest that a single protein, PIG-W, catalyzes this reaction (Murakami et al. 2003).
References
Y Murakami, U Siripanyapinyo, Y Hong, JY Kang, S Ishihara, H Nakakuma, Y Maeda, T Kinoshita, "PIG-W is critical for inositol acylation but not
for flipping of glycosylphosphatidylinositol-anchor", Mol Biol Cell, 14, 2003, 4285-95.
Reaction
25.2.2.5 glucosaminyl-acyl-PI + dolichol phosphate D-mannose -> mannose(al1-4)glucosaminyl-acyl-PI + dolichol phosphate
Authors
Editors
Description
In the fifth step of GPI synthesis, a mannose residue is added to glucosaminyl-acyl-PI. The reaction takes place at the lumenal surface of the
endoplasmic reticulum membrane. It is catalyzed by a complex of at least two components, PIG-M and PIG-X (Maeda et al. 2001; Ashida et al.
2005).
References
H Ashida, Y Hong, Y Murakami, N Shishioh, N Sugimoto, YU Kim, Y Maeda, T Kinoshita, "Mammalian PIG-X and yeast Pbn1p are the essential
components of glycosylphosphatidylinositol-mannosyltransferase I", Mol Biol Cell, 16, 2005, 1439-48.
Y Maeda, R Watanabe, CL Harris, Y Hong, K Ohishi, K Kinoshita, T Kinoshita, "PIG-M transfers the first mannose to glycosylphosphatidylinositol
on the lumenal side of the ER", EMBO J, 20, 2001, 250-61.
Reaction
25.2.2.6 mannose(a1-4)glucosaminyl-acyl-PI + phosphatidylethanolamine -> (ethanolamineP) mannose(al1-4)glucosaminyl-acyl-PI +

diacylglycerol
Authors
Editors
Description
In the sixth step of GPI synthesis, a phosphoethanolamine group is transferred from phosphatidylethanolamine onto the first mannose of the GPI
precursor. The human protein that catalyzes this reaction was first identified because it could complement a yeast mutant strain defective for GPI
synthesis (Gaynor et al. 1999); its specific function in phosphoethanolamine transfer is inferred from functional studies of the homologous mouse
protein (Hong et al. 1999). The reaction is annotated here with phosphatidylethanolamine as the donor of the phosphoethanolamine group on
the basis of studies in yeast (Imhof et al. 2000).
References
Y Hong, Y Maeda, R Watanabe, K Ohishi, M Mishkind, H Riezman, T Kinoshita, "Pig-n, a mammalian homologue of yeast Mcd4p, is involved in
transferring phosphoethanolamine to the first mannose of the glycosylphosphatidylinositol", J Biol Chem, 274, 1999, 35099-106.
I Imhof, E Canivenc-Gansel, U Meyer, A Conzelmann, "Phosphatidylethanolamine is the donor of the phosphorylethanolamine linked to the
alpha1,4-linked mannose of yeast GPI structures", Glycobiology, 10, 2000, 1271-5.
EC Gaynor, G Mondesert, SJ Grimme, SI Reed, P Orlean, SD Emr, "MCD4 encodes a conserved endoplasmic reticulum membrane protein
essential for glycosylphosphatidylinositol anchor synthesis in yeast", Mol Biol Cell, 10, 1999, 627-48.
Reaction
25.2.2.7 (ethanolamineP) mannose (a1-4) glucosaminyl-acyl-PI + dolichol phosphate D-mannose -> mannose (a1-6) (ethanolamineP)
mannose (a1-4) glucosaminyl-acyl-PI + dolichol phosphate
Authors
Editors
Description
In the seventh reaction of GPI synthesis, a second mannose residue is added to the glycolipid on the lumenal face of the endoplasmic reticulum
membrane. PIG-V was identified as the catylyst, or a component of the catalyst, of this reaction, on the basis of its ability to correct the metabolic
defects of yeast and mammalian mutant cells arrested at this stage of the GPI synthetic process (Fabre et al. 2005; Kang et al. 2005).
References
JY Kang, Y Hong, H Ashida, N Shishioh, Y Murakami, YS Morita, Y Maeda, T Kinoshita, "PIG-V involved in transferring the second mannose in
AL Fabre, P Orlean, CH Taron, "Saccharomyces cerevisiae Ybr004c and its human homologue are required for addition of the second mannose
during glycosylphosphatidylinositol precursor assembly", FEBS J, 272, 2005, 1160-8.
Reaction
25.2.2.8 mannose (a1-6) (ethanolamineP) mannose (a1-4) glucosaminyl-acyl-PI + dolichol phosphate D-mannose -> mannose (a1-2)
mannose (a1-6) (ethanolamineP) mannose (a1-4) glucosaminyl-acyl-PI + dolichol phosphate
Authors
Editors
Description
In the eighth reaction of GPI synthesis, a third mannose residue is added, catalyzed by PIG-B (Takahashi et al. 1996).
References
M Takahashi, N Inoue, K Ohishi, Y Maeda, N Nakamura, Y Endo, T Fujita, J Takeda, T Kinoshita, "PIG-B, a membrane protein of the
endoplasmic reticulum with a large lumenal domain, is involved in transferring the third mannose of the GPI anchor", EMBO J, 15, 1996,
4254-61.
Reaction
25.2.2.9 mannose (a1-2) mannose (a1-6) (ethanolamineP) mannose (a1-4) glucosaminyl-acyl-PI + phosphatidylethanolamine ->
(ethanolamineP) mannose (a1-2) mannose (a1-6) (ethanolamineP) mannose (a1-4) glucosaminyl-acyl-PI (acyl-GPI) + diacylglycerol
Authors
Editors
Description
The final step in the main pathway for the synthesis of GPI moieties in human cells is the addition of an ethanolamine phosphate to the third
mannose residue of the glycolipid, donated by phosphatidylethanolamine. This reaction has been experimentally characterized in the mouse,
where studies with mutated cell lines defective in GPI biosynthesis have established the role of two proteins, PIG-F and PIG-O, in this reaction
(Hong et al. 2000). While a human PIG-F protein has been identified and shown to be involved in this event (Inoue et al. 1993), the human event
has not been fully characterized and is therefore annotated here as inferred from studies of the mouse event.
References
N Inoue, T Kinoshita, T Orii, J Takeda, "Cloning of a human gene, PIG-F, a component of glycosylphosphatidylinositol anchor biosynthesis, by a
novel expression cloning strategy", J Biol Chem, 268, 1993, 6882-5.
Y Hong, Y Maeda, R Watanabe, N Inoue, K Ohishi, T Kinoshita, "Requirement of PIG-F and PIG-O for transferring phosphoethanolamine to the
third mannose in glycosylphosphatidylinositol", J Biol Chem, 275, 2000, 20911-9.
Source reaction
This reaction was inferred from the corresponding reaction "mannose (a1-2) mannose (a1-6) (ethanolamineP) mannose (a1-4)
glucosaminyl-acyl-PI + phosphatidylethanolamine -> (ethanolamineP) mannose (a1-2) mannose (a1-6) (ethanolamineP) mannose (a1-4)
glucosaminyl-acyl-PI (acyl-GPI) + diacylglycerol" in species Mus musculus.
N Inoue, T Kinoshita, T Orii, J Takeda, "Cloning of a human gene, PIG-F, a component of glycosylphosphatidylinositol anchor biosynthesis, by a
novel expression cloning strategy", J Biol Chem, 268, 1993, 6882-5.
Y Hong, Y Maeda, R Watanabe, N Inoue, K Ohishi, T Kinoshita, "Requirement of PIG-F and PIG-O for transferring phosphoethanolamine to the
third mannose in glycosylphosphatidylinositol", J Biol Chem, 275, 2000, 20911-9.
Reaction
25.2.2.10 mannose (a1-2) mannose (a1-6) (ethanolamineP) mannose (a1-4) glucosaminyl-acyl-PI -> mannose (a1) mannose (a1-2)
mannose (a1-6) (ethanolamineP) mannose (a1-4) glucosaminyl-acyl-PI
Authors
Editors
Description
Most human GPI anchors are thought to contain three mannose residues, while most yeast GPI anchors contain four. Recently, a human
homologue of the yeast enzyme responsible for addition of the fourth mannose residue to GPI molecules was identified and shown to mediate
synthesis of human GPI molecules with four mannose residues. While the mannose donor and the nature of the bond linking the third and fourth
mannose residues have not been established directly in studies with the human enzyme, these features are known for yeast and the normal
human gene restores GPI synthesis in mutant yeast. This observation, tegether with the sequence similarities among PIG-B and SMP3, it is
reasonable to infer that the human enzyme uses dolichol-P-mannose as a donor. The functional distinction between GPI anchors with three and
four mannose residues is unknown, although the latter appear to be abundant in many human tissues (Tauron et al. 2004).
References
BW Taron, PA Colussi, JM Wiedman, P Orlean, CH Taron, "Human Smp3p adds a fourth mannose to yeast and human
glycosylphosphatidylinositol precursors in vivo", J Biol Chem, 279, 2004, 36083-92.
Reaction
25.2.2.11 (ethanolamineP) mannose (a1-2) mannose (a1-6) (ethanolamineP) mannose (a1-4) glucosaminyl-acyl-PI -> (ethanolamineP)
mannose (a1-2) (ethanolamineP) mannose (a1-6) (ethanolamineP) mannose (a1-4) glucosaminyl-acyl-PI
Authors
Editors
Description
Most human GPI anchors have ethanolamine phosphate groups attached to their first and third mannose residues, but GPI anchors with
ethanolamine phosphates attached to all three mannose residues have also been identified. Addition of the third ethanolamine phosphate can be
catalyzed by a complex of PIG-F and a newly described human protein, GPI7. The donor of the ethanolamine phosphate for this reaction is
unknown (Shishioh et al. 2005).
References
N Shishioh, Y Hong, K Ohishi, H Ashida, Y Maeda, T Kinoshita, "GPI7 is the second partner of PIG-F and involved in modification of
Reaction
25.2.3 Attachment of GPI anchor to uPAR
Authors
Editors
Description
The mature form of urokinase plasminogen activator receptor (uPAR) is attached to the plasma membrane by a glycosylphosphatidylinositol
(GPI) anchor (Ploug et al. 1991). As nascent uPAR polypeptide moves into the lumen of the endoplasmic reticulum, it is attacked by a
transamidase complex that cleaves the uPAR polypeptide after residue 305, releasing the carboxyterminal peptide of uPAR and replacing it with
an acylated GPI moiety. In a second step, the GPI moiety is deacylated, yielding a uPAR-GPI conjugate that can be efficiently transported to the
Golgi apparatus. This transport process and the other events needed to generate fully glycosylated uPAR-GPI associated with the plasma
membrane will be annotated in a future release of Reactome.
References
M Ploug, E Ronne, N Behrendt, AL Jensen, F Blasi, K Dano, "Cellular receptor for urokinase plasminogen activator. Carboxyl-terminal
processing and membrane anchoring by glycosyl-phosphatidylinositol.", J Biol Chem, 266, 1991, 1926-33.
25.2.3.1 uPAR precursor + acyl-GPI -> uPAR-acyl-GPI + uPAR propeptide
Authors
Editors
Description
In the endoplasmic reticulum, the precursor form of urokinase plasminogen activator receptor is transamidated at residue 305, replacing the 30
carboxyterminal residues of the protein with an acylated glucosylphosphatidylinositol (acyl-GPI) moiety. The released carboxyterminal
propeptide has no known function. The reaction is catalyzed by GPI transamidase, a complex of at least five proteins associated with the
lumenal surface of the endoplasmic reticulum membrane (Yu et al. 1997; Ohishi et al. 2001; Hong et al. 2003).
References
K Ohishi, N Inoue, T Kinoshita, "PIG-S and PIG-T, essential for GPI anchor attachment to proteins, form a complex with GAA1 and GPI8",
EMBO J, 20, 2001, 4088-98.
J Yu, S Nagarajan, JJ Knez, S Udenfriend, R Chen, ME Medof, "The affected gene underlying the class K glycosylphosphatidylinositol (GPI)
surface protein defect codes for the GPI transamidase", Proc Natl Acad Sci U S A, 94, 1997, 12580-5.
Y Hong, K Ohishi, JY Kang, S Tanaka, N Inoue, J Nishimura, Y Maeda, T Kinoshita, "Human PIG-U and yeast Cdc91p are the fifth subunit of
GPI transamidase that attaches GPI-anchors to proteins", Mol Biol Cell, 14, 2003, 1780-9.
Reaction
25.2.3.2 uPAR-acyl-GPI + H2O -> uPAR + long-chain fatty acid
Authors
Editors
Description
The fatty acid group added to inositol in the fourth step of GPI biosynthsis is removed from GPI-conjugated uPAR. This hydrolysis event occurs
in the endoplasmic reticulum and appears to be associated with efficient transport of the conjugated protein from the endoplasmic reticulum to
the Golgi apparatus (Tanaka et al. 2004).
References
S Tanaka, Y Maeda, Y Tashima, T Kinoshita, "Inositol deacylation of glycosylphosphatidylinositol-anchored proteins is mediated by mammalian
PGAP1 and yeast Bst1p", J Biol Chem, 279, 2004, 14256-63.
Reaction
25.3 Hypusine synthesis from eIF5A-lysine
Authors
Johansson, HE, 2007-11-28.
Editors
Reviewers
Jassal, B, 2008-01-28.
Description
Cytosolic eIF5A undergoes a unique two-step post-translational modification at Lys 50 via deoxyhypusine (Dhp) to hypusine (Hyp). In the first
step deoxyhypusine synthase transfers the aminobutyl group of spermidine to the epsilon-amino group of lysine 50, using NAD+ as a cofactor.
Hydroxylation of the C2 of the newly added moiety in the second step is catalyzed by deoxyhypusine hydroxylase/monooxygenase with
molecular oxygen as the source. The molecular function of eIF5A is unknown, but the protein is required for viability in eukaryotic cells and its
normal function requires hypusinylation. eIF5A is the only protein known to undergo hypusinylation (Park 2006).
References
MH Park, "The post-translational synthesis of a polyamine-derived amino acid, hypusine, in the eukaryotic translation initiation factor 5A
(eIF5A)", J Biochem (Tokyo), 139, 2006, 161-9.
25.3.1 EIF5A + spermidine <=> EIF5A(Dhp) + 1,3-diaminopropane
Authors
Editors
Reviewers
Jassal, B, 2008-01-28.
Description
Cytosolic deoxyhypusine synthase catalyzes the reaction of EIF5A protein, spermidine, and NAD+ to convert lysine-50 of EIF5A to
deoxyhypusine, generating 1,3-diaminopropane and NADH + H+ in the process (Park 2006). Although the reaction is reversible, the reverse
reaction is probably minimized under physiological conditions by the rapid, irreversible conversion of EIF5A(Dhp) to EIF5A(Hyp).
References
MH Park, "The post-translational synthesis of a polyamine-derived amino acid, hypusine, in the eukaryotic translation initiation factor 5A
(eIF5A)", J Biochem (Tokyo), 139, 2006, 161-9.
YA Joe, EC Wolff, MH Park, "Cloning and expression of human deoxyhypusine synthase cDNA. Structure-function studies with the recombinant
enzyme and mutant proteins.", J Biol Chem, 270, 1995, 22386-92.
EC Wolff, JE Folk, MH Park, "Enzyme-substrate intermediate formation at lysine 329 of human deoxyhypusine synthase", J Biol Chem, 272,
1997, 15865-71.
PM Clement, CA Henderson, ZA Jenkins, Z Smit-McBride, EC Wolff, JW Hershey, MH Park, HE Johansson, "Identification and characterization
of eukaryotic initiation factor 5A-2", Eur J Biochem, 270, 2003, 4254-63.
Reaction
25.3.2 EIF5A(Dhp) + O2 => EIF5A(Hyp)
Authors

Editors
Reviewers
Jassal, B, 2008-01-28.
Description
Cytosolic deoxyhypusine hydroxylase catalyzes the irreversible conversion of peptidyl-deoxyhypusine to peptidyl-hypusine. The only known
substrate for this enzyme is the modified lysine at residue 50 of eIF5A (Kang et al. 2007; Kim et al. 2006).
References
KR Kang, YS Kim, EC Wolff, MH Park, "Specificity of the deoxyhypusine hydroxylase-eukaryotic translation initiation factor (eIF5A) interaction:
identification of amino acid residues of the enzyme required for binding of its substrate, deoxyhypusine-containing eIF5A", J Biol Chem, 282,
2007, 8300-8.
YS Kim, KR Kang, EC Wolff, JK Bell, P McPhie, MH Park, "Deoxyhypusine hydroxylase is a Fe(II)-dependent, HEAT-repeat enzyme.
Identification of amino acid residues critical for Fe(II) binding and catalysis [corrected].", J Biol Chem, 281, 2006, 13217-25.
PM Clement, CA Henderson, ZA Jenkins, Z Smit-McBride, EC Wolff, JW Hershey, MH Park, HE Johansson, "Identification and characterization
of eukaryotic initiation factor 5A-2", Eur J Biochem, 270, 2003, 4254-63.
Reaction
26 Regulation of beta-cell development
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The normal development of the pancreas during gestation has been intensively investigated over the past decade especially in the mouse
(Servitja and Ferrer 2004; Chakrabarti and Mirmira 2003). Studies of genetic defects associated with maturity onset diabetes of the young
(MODY) has provided direct insight into these processes as they take place in humans (Fajans et al. 2001). During embryogenesis, committed
epithelial cells from the early pancreatic buds differentiate into mature endocrine and exocrine cells. It is helpful to schematize this process into
four consecutive cellular stages, to begin to describe the complex interplay of signal transduction pathways and transcriptional networks. The
annotations here are by no means complete - factors in addition to the ones described here must be active, and even for the ones that are
described, only key examples of their regulatory effects and interactions have been annotated.
It is also important to realize that in the human, unlike the mouse, cells of the different stages can be present simultaneously in the developing
pancreas and the linear representation of these developmental events shown here is an over-simplification of the actual developmental process
(e.g., Sarkar et al. 2008).
The first stage of this process involves the predifferentiated epithelial cells of the two pancreatic anlagen that arise from the definitive endoderm
at approximately somite stages 11-15 and undergo budding from somite stages 20-22. This period corresponds to gestational days 8.75-9.5 in
the mouse, and 26 in the human.
Pancreatic buds subsequently coalesce to form a single primitive gland, while concomitantly a ductal tree lined by highly proliferative epithelial
cells is formed. A subset of such epithelial cells is thought to differentiate into either endocrine or acinar exocrine cells. A third cellular stage is
defined by the endocrine-committed progenitors that selectively express the basic helix-loop-helix transcription factor NEUROG3. NEUROG3 is
known to activate a complex transcriptional network that is essential for the specification of endocrine cells. Many transcription factors that are
activated by NEUROG3 are also involved in islet-subtype cellular specification and in subsequent stages of differentiation of endocrine cells.
This transient cellular stage thus leads to the generation of all known pancreatic endocrine cells, including insulin-producing beta-cells, and
glucagon-producing alpha cells, the final stage of this schematic developmental process.
The diagram below summarizes interactions that take place between transcription factors and transcription factor target genes during these
cellular stages, and shows cases where there is both functional evidence that a transcription factor is required for the target gene to be
expressed, and biochemical evidence that this interaction is direct. We also describe instances where a signaling pathway is known to regulate a
transcription factor gene in this process, even if the intervening signaling pathway is not fully understood.
References
JM Servitja, J Ferrer, "Transcriptional networks controlling pancreatic development and beta cell function", Diabetologia, 47, 2004, 597-613.
SA Sarkar, S Kobberup, R Wong, AD Lopez, N Quayum, T Still, A Kutchma, JN Jensen, R Gianani, GM Beattie, J Jensen, A Hayek, JC Hutton,
"Global gene expression profiling and histochemical analysis of the developing human fetal pancreas", Diabetologia, 51, 2008, 285-97.
SS Fajans, GI Bell, KS Polonsky, "Molecular mechanisms and clinical pathophysiology of maturity-onset diabetes of the young", N Engl J Med,
345, 2001, 971-80.
SK Chakrabarti, RG Mirmira, "Transcription factors direct the development and function of pancreatic beta cells", Trends Endocrinol Metab, 14,
2003, 78-84.
26.1 Regulation of gene expression in early pancreatic precursor cells
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The properties of transcriptional networks early in the differentiation of human pancreatic cells are inferred from the properties of well-studied
networks in mouse models. In mice, the first visible sign of pancreatic development is the appearance of pancreatic buds at about embryonic day
9. The cells in these buds are already committed to differentiate into specialized cells of the exocrine and endocrine pancreas. Expression of
transcription factors including Hnf1b, Hnf6, and Pdx1, as well as responsiveness to Fgf10 (fibroblast growth factor 10), up-regulates the
expression of factors including Ptf1, Onecut3, Lrh1, and Nkx6.1 (Servitja and Ferrer 2004; Chakrabarti and Mirmira 2003).
References
2003, 78-84.
26.1.1 HNF1B-dependent synthesis of HNF6 protein
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The HNF6 gene is transcribed, its mRNA is translated, and the protein product is transported to the nucleus. HNF6 transcription requires the
activity of the HNF1B transcription factor. These events and interactions have not been studied directly in humans, but are inferred from
corresponding ones worked out in the mouse.
Source reaction
This reaction was inferred from the corresponding reaction "HNF1b transactivates Hnf6" in species Mus musculus.
AV Poll, CE Pierreux, L Lokmane, C Haumaitre, Y Achouri, P Jacquemin, GG Rousseau, S Cereghini, FP Lemaigre, "A vHNF1/TCF2-HNF6
cascade regulates the transcription factor network that controls generation of pancreatic precursor cells", Diabetes, 55, 2006, 61-9.
Reaction
26.1.2 HNF1B- and FGF10-dependent synthesis of PTF1A protein
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The PTF1A gene is transcribed, its mRNA is translated, and the protein product is transported to the nucleus. PTF1A transcription requires the
activity of the HNF1B transcription factor and FGF10. These events and interactions have not been studied directly in humans, but are inferred
from corresponding ones worked out in the mouse.
Source reaction
This reaction was inferred from the corresponding reaction "FGF10 activates Ptf1 expression in early pancreatic buds" in species Mus musculus.
P Jacquemin, H Yoshitomi, Y Kashima, GG Rousseau, FP Lemaigre, KS Zaret, "An endothelial-mesenchymal relay pathway regulates early
phases of pancreas development", Dev Biol, 290, 2006, 189-99.
This reaction was inferred from the corresponding reaction "HNF1b transactivates Ptf1a" in species Mus musculus.
C Haumaitre, E Barbacci, M Jenny, MO Ott, G Gradwohl, S Cereghini, "Lack of TCF2/vHNF1 in mice leads to pancreas agenesis", Proc Natl
Acad Sci U S A, 102, 2005, 1490-5.
Reaction
26.1.3 HNF6-dependent synthesis of ONECUT3 protein during early pancreas specification
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The ONECUT3 gene is transcribed, its mRNA is translated, and the protein product is transported to the nucleus. ONECUT3 transcription
requires the activity of the HNF6 transcription factor. These events and interactions have not been studied directly in humans, but are inferred
Source reaction
This reaction was inferred from the corresponding reaction "HNF6 transactivates Onecut3 during early pancreas specification" in species Mus
musculus.
CE Pierreux, V Vanhorenbeeck, P Jacquemin, FP Lemaigre, GG Rousseau, "The transcription factor hepatocyte nuclear factor-6/Onecut-1
controls the expression of its paralog Onecut-3 in developing mouse endoderm", J Biol Chem, 279, 2004, 51298-304.
Reaction
26.1.4 HNF6- and FGF10-dependent synthesis of PDX1 protein
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The PDX1 gene is transcribed, its mRNA is translated, and the protein product is transported to the nucleus. PDX1 transcription requires the
activities of the HNF6 transcription factor and FGF10. These events and interactions have not been studied directly in humans, but are inferred
Source reaction
This reaction was inferred from the corresponding reaction "HNF6 transactivates Pdx1" in species Mus musculus.
P Jacquemin, FP Lemaigre, GG Rousseau, "The Onecut transcription factor HNF-6 (OC-1) is required for timely specification of the pancreas
and acts upstream of Pdx-1 in the specification cascade", Dev Biol, 258, 2003, 105-16.
This reaction was inferred from the corresponding reaction "FGF10 activates Pdx1 expression in the developing early pancreatic bud" in species
Mus musculus.
A Bhushan, N Itoh, S Kato, JP Thiery, P Czernichow, S Bellusci, R Scharfmann, "Fgf10 is essential for maintaining the proliferative capacity of
epithelial progenitor cells during early pancreatic organogenesis", Development, 128, 2001, 5109-17.
P Jacquemin, H Yoshitomi, Y Kashima, GG Rousseau, FP Lemaigre, KS Zaret, "An endothelial-mesenchymal relay pathway regulates early
phases of pancreas development", Dev Biol, 290, 2006, 189-99.
Reaction
26.1.5 PDX1-dependent synthesis of NR5A2 protein
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The NR5A2 gene is transcribed, its mRNA is translated, and the protein product is transported to the nucleus. NR5A2 transcription requires the
activity of the PDX1 transcription factor. These events and interactions have not been studied in vivo in humans, but are inferred from
corresponding ones worked out in the mouse and from in vitro studies of PDX1 protein binding to the Nr5A2 gene (Annicotte et al. 2003).
References
JS Annicotte, E Fayard, GH Swift, L Selander, H Edlund, T Tanaka, T Kodama, K Schoonjans, J Auwerx, "Pancreatic-duodenal homeobox 1
regulates expression of liver receptor homolog 1 during pancreas development", Mol Cell Biol, 23, 2003, 6713-24.
Source reaction
This reaction was inferred from the corresponding reaction "PDX1 transactivates Lrh1" in species Mus musculus.
JS Annicotte, E Fayard, GH Swift, L Selander, H Edlund, T Tanaka, T Kodama, K Schoonjans, J Auwerx, "Pancreatic-duodenal homeobox 1
regulates expression of liver receptor homolog 1 during pancreas development", Mol Cell Biol, 23, 2003, 6713-24.
Reaction
26.1.6 PDX1-dependent synthesis of NKX6-1 protein

Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The NKX6-1 gene is transcribed, its mRNA is translated, and the protein product is transported to the nucleus. NKX6-1 transcription requires the
activity of the PDX1 transcription factor. These events and interactions have not been studied directly in humans, but are inferred from
Source reaction
This reaction was inferred from the corresponding reaction "PDX1 transactivates Nkx6.1" in species Mus musculus.
JK Pedersen, SB Nelson, MC Jorgensen, KD Henseleit, Y Fujitani, CV Wright, M Sander, P Serup, "Endodermal expression of Nkx6 genes
depends differentially on Pdx1", Dev Biol, 288, 2005, 487-501.
U Ahlgren, J Jonsson, L Jonsson, K Simu, H Edlund, "beta-cell-specific inactivation of the mouse Ipf1/Pdx1 gene results in loss of the beta-cell
phenotype and maturity onset diabetes", Genes Dev, 12, 1998, 1763-8.
H Wang, P Maechler, B Ritz-Laser, KA Hagenfeldt, H Ishihara, J Philippe, CB Wollheim, "Pdx1 level defines pancreatic gene expression pattern
and cell lineage differentiation", J Biol Chem, 276, 2001, 25279-86.
H Watada, RG Mirmira, J Leung, MS German, "Transcriptional and translational regulation of beta-cell differentiation factor Nkx6.1.", J Biol
Chem, 275, 2000, 34224-30.
SB Nelson, AE Schaffer, M Sander, "The transcription factors Nkx6.1 and Nkx6.2 possess equivalent activities in promoting beta-cell fate
specification in Pdx1+ pancreatic progenitor cells", Development, 134, 2007, 2491-500.
Reaction
26.2 Regulation of gene expression in late stage (branching morphogenesis)

pancreatic bud precursor cells
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The properties of transcriptional networks in late stage (branching morphogenesis) pancreatic bud precursor cells are inferred from the
properties of well-studied networks in mouse models. In mice, committed but undifferentiated epithelial cells are organized into branching ductal
structures. At a molecular level, expression of Pdx1, Nkx2.2, and Nkx6.1 is reduced while Hnf6 expression remains high. Hnf6 mediates the
continued expression of Onecut3 and Hnf1 beta and epithelial cell proliferation. As expression of Ngn3 (corresponds to human NEUROG3)
rises, endocrine differentiation of the epithelial cells begins (Servitja and Ferrer 2004; Chakrabarti and Mirmira 2003).
References
2003, 78-84.
26.2.1 HNF6-dependent synthesis of HNF1B protein
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The HNF1B gene is transcribed, its mRNA is translated, and the protein products are transported to the nucleus. HNF1B transcription requires
the activity of the HNF6 transcription factor. These events and interactions have not been studied directly in humans, but are inferred from
Source reaction
This reaction was inferred from the corresponding reaction "HNF6 positively regulates Hnf1b expression" in species Mus musculus.
MA Maestro, SF Boj, RF Luco, CE Pierreux, J Cabedo, JM Servitja, MS German, GG Rousseau, FP Lemaigre, J Ferrer, "Hnf6 and Tcf2
(MODY5) are linked in a gene network operating in a precursor cell domain of the embryonic pancreas", Hum Mol Genet, 12, 2003, 3307-14.
Reaction
26.2.2 HNF6-dependent synthesis of ONECUT3 protein during morphogenesis
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The ONECUT3 gene is transcribed, its mRNA is translated, and the protein product is transported to the nucleus during morphogenesis of the
pancreas. ONECUT3 transcription requires the activity of the HNF6 transcription factor. These events and interactions have not been studied
directly in humans, but are inferred from corresponding ones worked out in the mouse.
Source reaction
This reaction was inferred from the corresponding reaction "HNF6 transactivates Onecut3 during morphogenesis" in species Mus musculus.
Reaction
26.2.3 HNF6-dependent synthesis of NEUROG3 protein during morphogenesis
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The NEUROG3 gene is transcribed, its mRNA is translated, and the protein product is transported to the nucleus during morphogenesis of the
pancreas. NEUROG3 transcription requires the activity of the HNF6 transcription factor. HES1 represses NEUROG3 transcription. In vivo, the
interplay between HNF6 and HES6 deteremines the timing and level of NEUROG3 expression, which is critical for normal development of the
pancreas. These events and interactions have not been studied directly in humans, but are inferred from corresponding ones worked out in the
mouse.
Source reaction
This reaction was inferred from the corresponding reaction "Hes1 represses Ngn3 expression" in species Mus musculus.
JC Lee, SB Smith, H Watada, J Lin, DW Scheel, J Wang, RG Mirmira, MS German, "Regulation of the pancreatic pro-endocrine gene
neurogenin3", Diabetes, 50, 2001, 928-36.
J Jensen, EE Pedersen, P Galante, J Hald, RS Heller, M Ishibashi, R Kageyama, F Guillemot, P Serup, OD Madsen, "Control of endodermal
endocrine development by Hes-1", Nat Genet, 24, 2000, 36-44.
A Apelqvist, H Li, L Sommer, P Beatus, DJ Anderson, T Honjo, M Hrabe de Angelis, U Lendahl, H Edlund, "Notch signalling controls pancreatic
cell differentiation", Nature, 400, 1999, 877-81.
This reaction was inferred from the corresponding reaction "HNF6 transactivates Ngn3 during morphogenesis" in species Mus musculus.
P Jacquemin, SM Durviaux, J Jensen, C Godfraind, G Gradwohl, F Guillemot, OD Madsen, P Carmeliet, M Dewerchin, D Collen, GG Rousseau,
FP Lemaigre, "Transcription factor hepatocyte nuclear factor 6 regulates pancreatic endocrine cell differentiation and controls expression of the
proendocrine gene ngn3", Mol Cell Biol, 20, 2000, 4445-54.
Reaction
26.2.4 RBPJ- and NOTCH1-dependent synthesis of HES1 protein during morphogenesis
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The HES1 gene is transcribed, its mRNA is translated, and the protein product is transported to the nucleus during morphogenesis of the
pancreas. HES1 transcription requires the activity of the RBPJ transcription factor and the NOTCH1 intracellular domain, probably as a complex
(Aster et al. 1997). These events and interactions have not been studied directly in humans, but are inferred from corresponding ones worked
out in the mouse.
References
JC Aster, ES Robertson, RP Hasserjian, JR Turner, E Kieff, J Sklar, "Oncogenic forms of NOTCH1 lacking either the primary binding site for
RBP-Jkappa or nuclear localization sequences retain the ability to associate with RBP-Jkappa and activate transcription", J Biol Chem, 272,
1997, 11336-43.
Source reaction
This reaction was inferred from the corresponding reaction "RBP-Jkappa transactivates Hes1 in the presence of NICD" in species Mus
musculus.
S Jarriault, Bail O Le, E Hirsinger, O Pourquie, F Logeat, CF Strong, C Brou, NG Seidah, l A Isra, "Delta-1 activation of notch-1 signaling results
in HES-1 transactivation", Mol Cell Biol, 18, 1998, 7423-31.
J Jensen, EE Pedersen, P Galante, J Hald, RS Heller, M Ishibashi, R Kageyama, F Guillemot, P Serup, OD Madsen, "Control of endodermal
endocrine development by Hes-1", Nat Genet, 24, 2000, 36-44.
A Apelqvist, H Li, L Sommer, P Beatus, DJ Anderson, T Honjo, M Hrabe de Angelis, U Lendahl, H Edlund, "Notch signalling controls pancreatic
cell differentiation", Nature, 400, 1999, 877-81.
M Baron, "An overview of the Notch signalling pathway", Semin Cell Dev Biol, 14, 2003, 113-9.
Reaction
26.3 Regulation of gene expression in endocrine-committed (NEUROG3+)

progenitor cells
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
Studies in mouse model systems indicate that the transcription factor neurogenin 3 plays a central role in the induction of endocrine
differentiation in the developing pancreas (Servitja and Ferrer 2004; Chakrabarti and Mirmira 2003). In both mice and humans critical events in
this induction process include the neurogenin 3 (NEUROG3)-dependent transcription of PAX4, NEUROD1, NKX2-2, and INSM1.
References
2003, 78-84.
26.3.1 NEUROG3-dependent synthesis of PAX4 protein
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The PAX4 gene is transcribed, its mRNA is translated, and the protein product is transported to the nucleus. PAX4 transcription requires the
activity of the NEUROG3 transcription factor (Heremans et al. 2002; Mellitzer et al. 2006).
References
G Mellitzer, S Bonne, RF Luco, M Van De Casteele, N Lenne-Samuel, P Collombat, A Mansouri, J Lee, M Lan, D Pipeleers, FC Nielsen, J
Ferrer, G Gradwohl, H Heimberg, "IA1 is NGN3-dependent and essential for differentiation of the endocrine pancreas", EMBO J, 25, 2006,
1344-52.
Y Heremans, M Van De Casteele, P in't Veld, G Gradwohl, P Serup, OD Madsen, D Pipeleers, H Heimberg, "Recapitulation of embryonic
neuroendocrine differentiation in adult human pancreatic duct cells expressing neurogenin 3", J Cell Biol, 159, 2002, 303-12.
Reaction
26.3.2 NEUROG3-dependent synthesis of NEUROD1
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The NEUROD1 gene is transcribed, its mRNA is translated, and the protein product is transported to the nucleus. NEUROD1 transcription
requires the activity of the NEUROG3 transcription factor (Heremans et al. 2002; Mellitzer et al. 2006).
References
1344-52.
Reaction
26.3.3 NEUROG3-dependent synthesis of NKX2-2
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The NKX2-2 gene is transcribed, its mRNA is translated, and the protein product is transported to the nucleus. NKX2-2 transcription requires the
activity of the NEUROG3 transcription factor (Heremans et al. 2002; Mellitzer et al. 2006).
References
1344-52.
Reaction
26.3.4 NEUROG3-dependent synthesis of INSM1
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The INSM1 gene is transcribed, its mRNA is translated, and the protein product is transported to the nucleus. INSM1 transcription requires the
activity of the NEUROG3 transcription factor (Mellitzer et al. 2006).
References
1344-52.
Reaction
26.4 Regulation of gene expression in beta cells
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
Two transcription factors, PDX1 and HNF1A, play key roles in maintaining the gene expression pattern characteristic of mature beta cells in the
endocrine pancreas. Targets of these regulatory molecules include genes encoding insulin, the GLUT2 glucose transporter, the liver- (and
pancreas) specific form of pyruvate kinase and other transcription factors including HNF4A, HNF4G, and FOXA3. PDX1 expression in turn is
controlled by the activities of MAFA, FOXA2, and PAX6, and negatively regulated via AKT (Chakrabarti and Mirmira 2003; Servitja and Ferrer
2004).
References
2003, 78-84.
26.4.1 AKT-mediated inactivation of FOXO1A
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The unphosphorylated form of FOXO1A shuttles between the nucleus and cytoplasm, maintaining a substantial concentration of this protein in
the nucleoplasm, where it functions as a transcription factor. Phosphorylation of the protein, catalyzed by activated AKT, causes its exclusion
from the nucleus (Zhang et al. 2002).
References
X Zhang, L Gan, H Pan, S Guo, X He, ST Olson, A Mesecar, S Adam, TG Unterman, "Phosphorylation of serine 256 suppresses transactivation
by FKHR (FOXO1) by multiple mechanisms. Direct and indirect effects on nuclear/cytoplasmic shuttling and DNA binding", J Biol Chem, 277,
2002, 45276-84.
26.4.1.1 AKT phosphorylates FOXO1A
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
One or more of the isoforms of AKT catalyzes the phosphorylation of FOXO1A protein at three sites, threonine-24, serine-256, and serine-319
(Zhang et al. 2002, 2006). This reaction occurs in the nucleoplasm, and thus is dependent on the phosphorylation and nuclear import of AKT in
response to upstream regulatory factors (Burgering and Kops 2002).
References
X Zhang, S Zhang, H Yamane, R Wahl, A Ali, JA Lofgren, RL Kendall, "Kinetic mechanism of AKT/PKB enzyme family", J Biol Chem, 281, 2006,
13949-56.
2002, 45276-84.
BM Burgering, GJ Kops, "Cell cycle and death control: long live Forkheads", Trends Biochem Sci, 27, 2002, 352-60.
Reaction
26.4.1.2 Phosphorylated FOXO1A is excluded from the nucleus
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
Phosphorylated FOXO1A is transported from the nucleoplasm to the cytosol (Zhang et al. 2002).
References
2002, 45276-84.
Reaction
26.4.2 FOXOA2-, MAFA-, and PAX6-dependent synthesis of PDX1 protein
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The PDX1 (IPF1) gene is transcribed, its mRNA is translated, and the protein product is transported to the nucleus. PDX1 transcription is
positively regulated by the activities of the FOXA2, MAFA, and PAX6 transcription factors. It is negatively regulated by FOXO1A, so events that
deplete the nucleoplasmic pool of FOXO1A increase expression of PDX1. These events and interactions have not been studied directly in
humans, but are inferred from corresponding ones worked out in the mouse.
Source reaction
This reaction was inferred from the corresponding reaction "FOXa2/HNF3b transactivates Pdx1" in species Mus musculus.
CS Lee, NJ Sund, MZ Vatamaniuk, FM Matschinsky, DA Stoffers, KH Kaestner, "Foxa2 controls Pdx1 gene expression in pancreatic beta-cells
in vivo", Diabetes, 51, 2002, 2546-51.
SE Samaras, MA Cissell, K Gerrish, CV Wright, M Gannon, R Stein, "Conserved sequences in a tissue-specific regulatory region of the pdx-1
gene mediate transcription in Pancreatic beta cells: role for hepatocyte nuclear factor 3 beta and Pax6", Mol Cell Biol, 22, 2002, 4702-13.
This reaction was inferred from the corresponding reaction "MafA transactivates Pdx1" in species Mus musculus.
SE Samaras, L Zhao, A Means, E Henderson, TA Matsuoka, R Stein, "The islet beta cell-enriched RIPE3b1/Maf transcription factor regulates
pdx-1 expression", J Biol Chem, 278, 2003, 12263-70.
C Zhang, T Moriguchi, M Kajihara, R Esaki, A Harada, H Shimohata, H Oishi, M Hamada, N Morito, K Hasegawa, T Kudo, JD Engel, M
Yamamoto, S Takahashi, "MafA is a key regulator of glucose-stimulated insulin secretion", Mol Cell Biol, 25, 2005, 4969-76.
This reaction was inferred from the corresponding reaction "PAX6 transactivates Pdx1" in species Mus musculus.
SE Samaras, MA Cissell, K Gerrish, CV Wright, M Gannon, R Stein, "Conserved sequences in a tissue-specific regulatory region of the pdx-1
gene mediate transcription in Pancreatic beta cells: role for hepatocyte nuclear factor 3 beta and Pax6", Mol Cell Biol, 22, 2002, 4702-13.
Reaction
26.4.3 MAFA-, NKX2-2-, PAX6-, and PDX1-dependent synthesis of insulin precursor protein
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The INS1 gene, encoding insulin precursor protein, is transcribed and its mRNA is translated on membrane-associated ribosomes. INS1
transcription is positively regulated by the activities of the MAFA, NKX2-2, PAX6, and PDX1 transcription factors. These events and interactions
are inferred from corresponding ones studied in molecular detail in the mouse.
Source reaction
This reaction was inferred from the corresponding reaction "NKX2.2 transactivates the Insulin gene" in species Mus musculus.
L Sussel, J Kalamaras, DJ Hartigan-O'Connor, JJ Meneses, RA Pedersen, JL Rubenstein, MS German, "Mice lacking the homeodomain
transcription factor Nkx2.2 have diabetes due to arrested differentiation of pancreatic beta cells.", Development, 125, 1998, 2213-21.
MA Cissell, L Zhao, L Sussel, E Henderson, R Stein, "Transcription factor occupancy of the insulin gene in vivo. Evidence for direct regulation by
Nkx2.2.", J Biol Chem, 278, 2003, 751-6.
This reaction was inferred from the corresponding reaction "PAX6 transactivates the Insulin gene" in species Mus musculus.
Nkx2.2.", J Biol Chem, 278, 2003, 751-6.
M Sander, A Neubuser, J Kalamaras, HC Ee, GR Martin, MS German, "Genetic analysis reveals that PAX6 is required for normal transcription of
pancreatic hormone genes and islet development", Genes Dev, 11, 1997, 1662-73.
L St-Onge, B Sosa-Pineda, K Chowdhury, A Mansouri, P Gruss, "Pax6 is required for differentiation of glucagon-producing alpha-cells in mouse
pancreas", Nature, 387, 1997, 406-9.
This reaction was inferred from the corresponding reaction "PDX1 transactivates the Insulin gene" in species Mus musculus.
SK Chakrabarti, JC James, RG Mirmira, "Quantitative assessment of gene targeting in vitro and in vivo by the pancreatic transcription factor,
Pdx1. Importance of chromatin structure in directing promoter binding.", J Biol Chem, 277, 2002, 13286-93.
H Ohlsson, K Karlsson, T Edlund, "IPF1, a homeodomain-containing transactivator of the insulin gene", EMBO J, 12, 1993, 4251-9.
Reaction
26.4.4 PDX1-dependent synthesis of IAPP protein
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The IAPP gene, encoding islet amyloid precursor protein, is transcribed and its mRNA is translated. IAPP transcription is positively regulated by
PDX1 transcription factor. These events and interactions are inferred from corresponding ones studied in molecular detail in the mouse.
Source reaction
This reaction was inferred from the corresponding reaction "PDX1 transactivates Iapp" in species Mus musculus.
Reaction
26.4.5 PDX1-dependent synthesis of NKX6-1 protein
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
In mature beta-cells of the pancreas, the NKX6-1 gene is transcribed, its mRNA is translated, and the protein product is transported to the
nucleus. NKX6-1 transcription is positively regulated by the activity of the PDX1 transcription factor. These events and interactions have not
been studied directly in humans, but are inferred from corresponding ones worked out in the mouse.
Source reaction
This reaction was inferred from the corresponding reaction "PDX1 transactivates Nkx6.1" in species Mus musculus.
JK Pedersen, SB Nelson, MC Jorgensen, KD Henseleit, Y Fujitani, CV Wright, M Sander, P Serup, "Endodermal expression of Nkx6 genes
depends differentially on Pdx1", Dev Biol, 288, 2005, 487-501.
H Wang, P Maechler, B Ritz-Laser, KA Hagenfeldt, H Ishihara, J Philippe, CB Wollheim, "Pdx1 level defines pancreatic gene expression pattern
and cell lineage differentiation", J Biol Chem, 276, 2001, 25279-86.
H Watada, RG Mirmira, J Leung, MS German, "Transcriptional and translational regulation of beta-cell differentiation factor Nkx6.1.", J Biol
Chem, 275, 2000, 34224-30.
SB Nelson, AE Schaffer, M Sander, "The transcription factors Nkx6.1 and Nkx6.2 possess equivalent activities in promoting beta-cell fate
specification in Pdx1+ pancreatic progenitor cells", Development, 134, 2007, 2491-500.
Reaction
26.4.6 NEUROD1- and PDX1-dependent synthesis of glucokinase (GCK) protein

Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The glucokinase (GCK) gene is transcribed and its mRNA is translated. GCK transcription is positively regulated by the activity of the NEUROD1
and PDX1 transcription factors. These events and interactions are inferred from corresponding ones studied in molecular detail in the mouse.
Source reaction
This reaction was inferred from the corresponding reaction "NeuroD1 transactivates the glucokinase gene" in species Mus musculus.
JM Moates, S Nanda, MA Cissell, MJ Tsai, R Stein, "BETA2 activates transcription from the upstream glucokinase gene promoter in islet
beta-cells and gut endocrine cells", Diabetes, 52, 2003, 403-8.
Nkx2.2.", J Biol Chem, 278, 2003, 751-6.
This reaction was inferred from the corresponding reaction "PDX1 transactivates the glucokinase gene" in species Mus musculus.
H Watada, Y Kajimoto, Y Umayahara, T Matsuoka, H Kaneto, Y Fujitani, T Kamada, R Kawamori, Y Yamasaki, "The human glucokinase gene
beta-cell-type promoter: an essential role of insulin promoter factor 1/PDX-1 in its activation in HIT-T15 cells", Diabetes, 45, 1996, 1478-88.
Nkx2.2.", J Biol Chem, 278, 2003, 751-6.
Reaction
26.4.7 HNF1A-dependent synthesis of GLUT2 protein
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The GLUT2 gene is transcribed, its mRNA is translated, and the protein product is localized to the plasma membrane. GLUT2 expression is
positively regulated by HNF1A. In vivo, pancreatic GLUT2 expression is positively regulated by HNF1A. Mutations in HNF1A are associated with
a form of MODY (maturity onset diabetes of the young) (Fanjans et al. 2001) and interactions between the HNF1A protein product and the
GLUT2 promoter have been demomstrated in vitro (Ban et al. 2002). However, the molecular details of GLUT2 expression in intact pancreatic
beta cells have not been studied in humans, but are inferred from corresponding ones worked out in the mouse.
References
N Ban, Y Yamada, Y Someya, K Miyawaki, Y Ihara, M Hosokawa, S Toyokuni, K Tsuda, Y Seino, "Hepatocyte nuclear factor-1alpha recruits the
transcriptional co-activator p300 on the GLUT2 gene promoter", Diabetes, 51, 2002, 1409-18.
345, 2001, 971-80.
Source reaction
This reaction was inferred from the corresponding reaction "HNF1a transactivates Glut2" in species Mus musculus.
SF Boj, M Parrizas, MA Maestro, J Ferrer, "A transcription factor regulatory circuit in differentiated pancreatic cells", Proc Natl Acad Sci U S A,
98, 2001, 14481-6.
H Wang, P Maechler, KA Hagenfeldt, CB Wollheim, "Dominant-negative suppression of HNF-1alpha function results in defective insulin gene
transcription and impaired metabolism-secretion coupling in a pancreatic beta-cell line", EMBO J, 17, 1998, 6701-13.
M Parrizas, MA Maestro, SF Boj, A Paniagua, R Casamitjana, R Gomis, F Rivera, J Ferrer, "Hepatic nuclear factor 1-alpha directs nucleosomal
hyperacetylation to its tissue-specific transcriptional targets", Mol Cell Biol, 21, 2001, 3234-43.
Reaction
26.4.8 HNF1A-dependent synthesis of the L isoform of PKLR protein
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The PKLR gene is transcribed, its mRNA is translated, spliced, and translated to yield the L isoform of PKLR protein. PKLR expression is
positively regulated by HNF1A. Mutations in HNF1A are associated with a form of MODY (maturity onset diabetes of the young) (Fanjans et al.
2001) but the molecular details of PKLR expression in intact pancreatic beta cells have not been studied in humans, and are inferred from
References
345, 2001, 971-80.
Source reaction
This reaction was inferred from the corresponding reaction "HNF1a transactivates Pklr" in species Mus musculus.
98, 2001, 14481-6.
H Wang, P Maechler, KA Hagenfeldt, CB Wollheim, "Dominant-negative suppression of HNF-1alpha function results in defective insulin gene
transcription and impaired metabolism-secretion coupling in a pancreatic beta-cell line", EMBO J, 17, 1998, 6701-13.
M Parrizas, MA Maestro, SF Boj, A Paniagua, R Casamitjana, R Gomis, F Rivera, J Ferrer, "Hepatic nuclear factor 1-alpha directs nucleosomal
hyperacetylation to its tissue-specific transcriptional targets", Mol Cell Biol, 21, 2001, 3234-43.
Reaction
26.4.9 HNF1A-dependent synthesis of HNF4A
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The HNF4A gene is transcribed from either of two promoters, P1 and P2, the resulting mRNA is translated, and the protein products localize in
the nucleoplasm. Transcription is positively regulated by HNF1A. Many of the molecular details of these events have not been studied
experimentally in humans, but are inferred from mouse model systems (Boj et al. 2001). Transcription in mouse and human pancreatic beta cells
is P2-dependent and in humans yields three isoforms of mature HNF4A protein. A point mutation in the human P2 genomic DNA sequence is
associated with MODY (maturity onset diabetes of the young), consistent with the hypothesis that P2-mediated transcription is essential for
HNF4A expression and normal beta cell function (Hansen et al. 2002).
References
98, 2001, 14481-6.
SK Hansen, M Parrizas, ML Jensen, S Pruhova, J Ek, SF Boj, A Johansen, MA Maestro, F Rivera, H Eiberg, M Andel, J Lebl, O Pedersen, J
Ferrer, T Hansen, "Genetic evidence that HNF-1alpha-dependent transcriptional control of HNF-4alpha is essential for human pancreatic beta
cell function", J Clin Invest, 110, 2002, 827-33.
Source reaction
This reaction was inferred from the corresponding reaction "HNF1a regulates Hnf4a" in species Mus musculus.
98, 2001, 14481-6.
SK Hansen, M Parrizas, ML Jensen, S Pruhova, J Ek, SF Boj, A Johansen, MA Maestro, F Rivera, H Eiberg, M Andel, J Lebl, O Pedersen, J
Ferrer, T Hansen, "Genetic evidence that HNF-1alpha-dependent transcriptional control of HNF-4alpha is essential for human pancreatic beta
cell function", J Clin Invest, 110, 2002, 827-33.
DQ Shih, S Screenan, KN Munoz, L Philipson, M Pontoglio, M Yaniv, KS Polonsky, M Stoffel, "Loss of HNF-1alpha function in mice leads to
abnormal expression of genes involved in pancreatic islet development and metabolism", Diabetes, 50, 2001, 2472-80.
Reaction
26.4.10 HNF1A-dependent synthesis of HNF4G protein
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The HNF4G gene is transcribed, its mRNA is translated, and the protein product is localized to the nucleoplasm. HNF4G expression is positively
regulated by HNF1A. The molecular details of HNF4G expression in intact pancreatic beta cells have not been studied in humans, but are
inferred from corresponding ones worked out in the mouse (Boj et al. 2001).
References
98, 2001, 14481-6.
Source reaction
This reaction was inferred from the corresponding reaction "HNF1a regulates Hnf4g" in species Mus musculus.
98, 2001, 14481-6.
Reaction
26.4.11 HNF1A-dependent synthesis of FOXA3
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
The FOXA3 gene is transcribed, its mRNA is translated, and the protein product is localized to the nucleoplasm. FOXA3 expression is positively
regulated by HNF1A. The molecular details of FOXA3 expression in intact pancreatic beta cells have not been studied in humans, but are
inferred from corresponding ones worked out in the mouse (Boj et al. 2001).
References
98, 2001, 14481-6.
Source reaction
This reaction was inferred from the corresponding reaction "HNF1a regulates Foxa3" in species Mus musculus.
98, 2001, 14481-6.
H Hiemisch, G Schutz, KH Kaestner, "Transcriptional regulation in endoderm development: characterization of an enhancer controlling Hnf3g
expression by transgenesis and targeted mutagenesis", EMBO J, 16, 1997, 3995-4006.
Reaction
27 Regulatory RNA pathways
Reviewers
Karginov, F, Hannon, GJ, 2008-02-08.
Description
In this module, the biology of various types of non-coding RNA will be annotated starting from microRNAs.
27.1 MicroRNA biogenesis
Authors
Gopinathrao, G, May, B, 2007-11-18.
Reviewers
Description
1. Transcription.
MicroRNA (miRNA) transcripts may come from autonomously transcribed genes or they may be contained in cotranscripts with other genes.
Most miRNAs are transcribed by RNA polymerase II, however a few miRNAs originate as RNA polymerase III cotranscripts with neighboring
repetitive elements. The initial transcript, termed a primary microRNA (pri-miRNA), contains an imperfectly double-stranded region within a
hairpin loop. Longer sequences extend from the 5' and 3' ends of the hairpin and may also contain double-stranded regions.
2. Cleavage by Drosha.
The 5' and 3' ends of the pri-miRNA are removed during endoribonucleolytic cleavage by the Drosha nuclease in a complex with the
RNA-binding protein DGCR8 (the Microprocessor complex). The cleavage product is a short hairpin of about 60 to 70 nt called the
pre-microRNA (pre-miRNA).
3. Nuclear export by Exportin-5.
The resulting pre-miRNA is bound by Exportin-5 in a complex with Ran and GTP. The complex translocates the pre-miRNA through the nuclear
pore into the cytoplasm.
4. Cleavage by Dicer.
Once in the cytoplasm the pre-miRNA is bound and cleaved by the Dicer ribonuclease in complex with the RNA-binding protein TRBP (TAR
binding protein). The product is an imperfectly double-stranded miRNA of about 21 to 23 nucleotides. At this stage the double-stranded miRNA
has protruding single-stranded 3' ends of 2-3 nt.
5. Strand selection and incorporation into RNA-Induced Silencing Complex (RISC).
At this stage the miRNA has two strands: the passenger strand, which will be removed and degraded, and the guide strand, which will be
retained and guides the RISC to target mRNAs. An Argonaute protein is already bound to the Dicer:miRNA complex or subsequently binds the
complex (the order is unknown). The human genome encodes 4 Argonaute proteins, however only Argonaute2 can cleave target mRNAs with
perfect or nearly perfect complementarity to the guide miRNA. In mammalian cells, such endogenous interactions are very rare; most miRNAs
only cause translational repression. For complexes that contain Argonaute2, cleavage of the passenger strand of the double-stranded miRNA
accompanies removal of the passenger strand. Complexes containing other Argonautes may use a helicase to remove the passenger strand but
this is not fully known. The resulting single-stranded miRNA (the guide strand) remains in a complex containing Argonaute, Dicer, TRBP, and
other proteins termed the RNA-induced Silencing Complex (RISC). The full set of proteins composing RISC is not yet known.
References
RC Lee, RL Feinbaum, V Ambros, "The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14",
Cell, 75, 1993, 843-54.
A Rodriguez, S Griffiths-Jones, JL Ashurst, A Bradley, "Identification of mammalian microRNA host genes and transcription units", Genome Res,
14, 2004, 1902-10.
DP Bartel, "MicroRNAs: genomics, biogenesis, mechanism, and function", Cell, 116, 2004, 281-97.
X Cai, CH Hagedorn, BR Cullen, "Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs",
RNA, 10, 2004, 1957-66.
L He, JM Thomson, MT Hemann, E Hernando-Monge, D Mu, S Goodson, S Powers, C Cordon-Cardo, SW Lowe, GJ Hannon, SM Hammond, "A
microRNA polycistron as a potential human oncogene", Nature, 435, 2005, 828-33.
GM Borchert, W Lanier, BL Davidson, "RNA polymerase III transcribes human microRNAs", Nat Struct Mol Biol, 13, 2006, 1097-101.
Y Lee, M Kim, J Han, KH Yeom, S Lee, SH Baek, VN Kim, "MicroRNA genes are transcribed by RNA polymerase II", EMBO J, 23, 2004,
4051-60.
EP Murchison, GJ Hannon, "miRNAs on the move: miRNA biogenesis and the RNAi machinery", Curr Opin Cell Biol, 16, 2004, 223-9.
L He, GJ Hannon, "MicroRNAs: small RNAs with a big role in gene regulation", Nat Rev Genet, 5, 2004, 522-31.
27.1.1 Pol II mediated transcription of microRNA genes
Authors
Editors
Reviewers
Description
1. Transcription of miRNA genes.
Most miRNAs are transcribed by RNA polymerase II. The miRNAs may be autonomous transcription units or they may be located in other
transcripts, including locations within introns and other untranslated regions. Of the polymerase II transcribed miRNAs, about 60% are located in
introns of protein coding genes, 12 % are in introns of non-coding RNAs, 18% are in exons of non-coding RNAs, and 10% uncertain.
A second class of miRNA genes are associated with Alu and other repetitive elements and are cotranscribed with these elements by RNA
polymerase III. There are currently only a few proven examples of polymerase III transcribed miRNAs.
References
A Rodriguez, S Griffiths-Jones, JL Ashurst, A Bradley, "Identification of mammalian microRNA host genes and transcription units", Genome Res,
14, 2004, 1902-10.
X Cai, CH Hagedorn, BR Cullen, "Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs",
RNA, 10, 2004, 1957-66.
GM Borchert, W Lanier, BL Davidson, "RNA polymerase III transcribes human microRNAs", Nat Struct Mol Biol, 13, 2006, 1097-101.
Y Lee, M Kim, J Han, KH Yeom, S Lee, SH Baek, VN Kim, "MicroRNA genes are transcribed by RNA polymerase II", EMBO J, 23, 2004,
4051-60.
Reaction
27.1.2 Microprocessor complex cleaves pri-miRNA to pre-miRNA
Authors
Reviewers
Description
2. Nuclear processing by Drosha Microprocessor complex.
The primary-microRNA (pri-miRNA) is bound by the Microprocessor complex (Drosha:DGCR8) and both strands are cleaved by Drosha near the
free 5' and 3' ends of the pri-miRNA, that is, at the ends distal from the internal loop. The product is a double-stranded RNA having 2 nucleotides
protruding at each 3' end and having an internal loop.
References
Y Lee, K Jeon, JT Lee, S Kim, VN Kim, "MicroRNA maturation: stepwise processing and subcellular localization", EMBO J, 21, 2002, 4663-70.
J Han, Y Lee, KH Yeom, YK Kim, H Jin, VN Kim, "The Drosha-DGCR8 complex in primary microRNA processing", Genes Dev, 18, 2004,
3016-27.
AM Denli, BB Tops, RH Plasterk, RF Ketting, GJ Hannon, "Processing of primary microRNAs by the Microprocessor complex", Nature, 432,
2004, 231-5.
Y Lee, C Ahn, J Han, H Choi, J Kim, J Yim, J Lee, P Provost, O Radmark, S Kim, VN Kim, "The nuclear RNase III Drosha initiates microRNA
processing", Nature, 425, 2003, 415-9.
RI Gregory, KP Yan, G Amuthan, B Doratotaj, N Cooch, R Shiekhattar, TP Chendrimada, "The Microprocessor complex mediates the genesis of
microRNAs", Nature, 432, 2004, 235-40.
Reaction
27.1.3 Exportin-5 recognizes 3' overhang of pre-miRNA
Authors
Reviewers
Reaction
27.1.4 Exportin complex translocates pre-miRNA to cytosol
Authors

Reviewers
Description
3. Nuclear Export by Exportin-5.
The pre-microRNA is bound by the Exportin-5:RanGTP complex in the nucleus and the complex is translocated through the nuclear pore into the
cytoplasm. In the process GTP is hydrolyzed to GDP.
References
R Yi, Y Qin, IG Macara, BR Cullen, "Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs", Genes Dev, 17, 2003,
3011-6.
MT Bohnsack, K Czaplinski, D Gorlich, "Exportin 5 is a RanGTP-dependent dsRNA-binding protein that mediates nuclear export of
pre-miRNAs", RNA, 10, 2004, 185-91.
E Lund, S GÃ¼ttinger, A Calado, JE Dahlberg, U Kutay, "Nuclear export of microRNA precursors", Science, 303, 2004, 95-8.
Reaction
27.1.5 Dicer cleaves pre-miRNA to mature miRNA
Authors
Reviewers
Description
4. Cytoplasmic processing by Dicer.
The pre-miRNA has a protruding 3' end created by cleavage by Drosha:DGCR8 and an internal loop. The Dicer:TRBP complex binds the
pre-miRNA and Dicer cleaves near the loop. The product is a double-stranded RNA of 21-24 bp having 2-nucleotide protrusions at each 3' end.
References
TP Chendrimada, RI Gregory, E Kumaraswamy, J Norman, N Cooch, K Nishikura, R Shiekhattar, "TRBP recruits the Dicer complex to Ago2 for
microRNA processing and gene silencing", Nature, 436, 2005, 740-4.
AD Haase, L Jaskiewicz, H Zhang, S Laine, R Sack, A Gatignol, W Filipowicz, "TRBP, a regulator of cellular PKR and HIV-1 virus expression,
interacts with Dicer and functions in RNA silencing", EMBO Rep, 6, 2005, 961-7.
RF Ketting, SE Fischer, E Bernstein, T Sijen, GJ Hannon, RH Plasterk, "Dicer functions in RNA interference and in synthesis of small RNA
involved in developmental timing in C. elegans", Genes Dev, 15, 2001, 2654-9.
G HutvÃ¡gner, J McLachlan, AE Pasquinelli, E BÃ¡lint, T Tuschl, PD Zamore, "A cellular function for the RNA-interference enzyme Dicer in the
maturation of the let-7 small temporal RNA", Science, 293, 2001, 834-8.
E Bernstein, SY Kim, MA Carmell, EP Murchison, H Alcorn, MZ Li, AA Mills, SJ Elledge, KV Anderson, GJ Hannon, "Dicer is essential for mouse
development", Nat Genet, 35, 2003, 215-7.
Reaction
28 Signaling by BMP
Authors
Huminiecki, L, Moustakas, A, 2007-11-07.
Reviewers
Heldin, CH, 2007-11-12.
Description
The TGF-beta/BMP (bone morphogenetic protein) pathway incorporates several signalling pathways that share most, but not all, components of
a central signal transduction engine. The general signalling scheme is rather simple: upon binding of a ligand, an activated plasma membrane
receptor complex is formed, which passes on the signal towards the nucleus through a phosphorylated receptor-activated SMAD (r-SMAD). In
the nucleus, the activated r-SMAD promotes transcription in a complex with a closely-related helper molecule termed the Co-SMAD. However,
this simple linear pathway expands into a network when various regulatory components and mechanisms are taken into account. The signalling
pathway includes a great variety of different TGF-beta/BMP superfamily ligands and receptors, several types of the r-SMAD, and functionally
critical negative feedback loops. The r-SMAD/Co-SMAD can interact with a great number of transcriptional co-activators/co-repressors to
regulate positively or negatively effector genes, so that the interpretation of a signal depends on the cell-type and cross talk with other signalling
pathways such as Notch, MAPK and Wnt. The pathway plays a number of different biological roles in the control of embryonic and adult cell
proliferation and differentiation, and it is implicated in a great number of human diseases.
References
D Chen, M Zhao, GR Mundy, "Bone morphogenetic proteins", Growth Factors, 22, 2004, 233-41.
28.1 The ligand trap binds the ligand BMP2, blocking BMP signalling
Authors
Reviewers
Heldin, CH, 2007-11-12.
Description
BMP ligand traps are cystine-knot containing proteins which bind BMPs and antagonise their actions. They are active during organ development
and morphogenesis. Different BMP ligand traps show specific spatio-temporal expression during development, and selective activity against
specific BMP ligands.
References
E Gazzerro, E Canalis, "Bone morphogenetic proteins and their antagonists", Rev Endocr Metab Disord, 7, 2006, 51-65.
Reaction
28.2 Formation of a heteromeric BMP receptor complex
Authors
Reviewers
Heldin, CH, 2007-11-12.
Description
BMP receptors, unlike TGF-beta receptors are known to form hetero-oligomeric complexes in the endoplasmic reticulum and are transported as
oligomers to the plasma membrane where they bind ligand. However, evidence for ligand-induced heteromeric BMP receptor complexes on the
cell surface has also been published, leading to a model where both pre-formed and ligand-induced receptor oligomers are encountered on the
plasma membrane. Based on the latter, a theory has been formulated that suggests that the signaling outcome from pre-formed and
ligand-induced BMP receptor complexes is different. The mechanism that might explain this theory must involve different ways of internalization
and trafficking of the BMP receptor complexes.
References
A Nohe, S Hassel, M Ehrlich, F Neubauer, W Sebald, YI Henis, P Knaus, "The mode of bone morphogenetic protein (BMP) receptor
oligomerization determines different BMP-2 signaling pathways", J Biol Chem, 277, 2002, 5330-8.
GP Allendorph, WW Vale, S Choe, "Structure of the ternary signaling complex of a TGF-beta superfamily member", Proc Natl Acad Sci U S A,
103, 2006, 7643-8.
L Gilboa, A Nohe, T Geissendorfer, W Sebald, YI Henis, P Knaus, "Bone morphogenetic protein receptor complexes on the surface of live cells:
a new oligomerization mode for serine/threonine kinase receptors", Mol Biol Cell, 11, 2000, 1023-35.
Reaction
28.3 BMP2 binds to the receptor complex
Authors
Reviewers
Heldin, CH, 2007-11-12.
Description
The mature dimeric BMP2 binds with high affinity to its signalling receptor, the type II receptor serine/threonine kinase. The type II receptor is
known to form dimeric complexes even in the absence of BMP2.
Reaction
28.4 Type II receptor phosphorylates type I receptor
Authors
Reviewers
Heldin, CH, 2007-11-12.
Description
Formation of the hetero-tetrameric BMP2:receptor complex induces receptor rotation, so that their cytoplasmic kinase domains face each other
in a catalytically favourable configuration. The constitutively active type II receptor kinase (which auto-phosphorylates in the absence of ligand),
trans-phosphorylates specific serine residues at the conserved Gly-Ser-rich juxtapositioned domain of the type I receptor. It is not known if
exactly 8 ATPs are required for the phosphorylation of type I receptor, there could be more or less than this number.
References
BL Rosenzweig, T Imamura, T Okadome, GN Cox, H Yamashita, P ten Dijke, CH Heldin, K Miyazono, "Cloning and characterization of a human
type II receptor for bone morphogenetic proteins", Proc Natl Acad Sci U S A, 92, 1995, 7632-6.
F Liu, F Ventura, J Doody, J Massague, "Human type II receptor for bone morphogenic proteins (BMPs): extension of the two-kinase receptor
model to the BMPs", Mol Cell Biol, 15, 1995, 3479-86.
Reaction
28.5 An anchoring protein, Endofin, recruits R-Smad1/5/8
Authors
Reviewers
Heldin, CH, 2007-11-12.
Description
Endofin is a FYVE domain-containing protein that strongly resembles SARA, the Smad anchor for receptor activation that facilitates TGF-beta
signalling. Endofin acts in a similar manner as SARA, it binds to BMP-specific R-Smads, it localizes in early endosomes and it facilitates their
phosphorylation, thus promoting signal transduction by the BMP receptors. However, it should be noted that endofin has also been reported to
bind to the Co-Smad, Smad4, and to the TGF-beta type receptor, thus enhancing TGF-beta signalling. Since Smad4 is a common Smad that
operates in the BMP-specific pathways, the latter observation might imply that endofin could regulate both TGF-beta and BMP signalling, a
hypothesis still open for investigation.
References
W Shi, C Chang, S Nie, S Xie, M Wan, X Cao, "Endofin acts as a Smad anchor for receptor activation in BMP signaling", J Cell Sci, 120, 2007,
1216-24.
Reaction
28.6 Ubiquitin-dependent degradation controls basal levels of R-Smad1/5/8
Authors
Reviewers
Heldin, CH, 2007-11-12.
Description
r-SMAD levels are kept low within cells. This is achieved by constant ubiquitination of r-SMADs by Smurf ubiquitin ligases and ensuing
proteasomal degradation.
References
H Zhu, P Kavsak, S Abdollah, JL Wrana, GH Thomsen, "A SMAD ubiquitin ligase targets the BMP pathway and affects embryonic pattern
formation", Nature, 400, 1999, 687-93.
Y Zhang, C Chang, DJ Gehling, A Hemmati-Brivanlou, R Derynck, "Regulation of Smad degradation and activity by Smurf2, an E3 ubiquitin
ligase", Proc Natl Acad Sci U S A, 98, 2001, 974-9.
X Lin, M Liang, XH Feng, "Smurf2 is a ubiquitin E3 ligase mediating proteasome-dependent degradation of Smad2 in transforming growth
factor-beta signaling", J Biol Chem, 275, 2000, 36818-22.
Reaction
28.7 I-Smad binds to type I receptor, preventing Smad1/5/8 from being

activated
Authors
Reviewers
Heldin, CH, 2007-11-12.
Description
Smad6 and Smad7, the two I-Smads, bind directly to the BMP type I receptors and recruit the ubiquitin ligase Smurf1. This reaction leads to
competitive inhibition of R-Smad binding to the type I receptor and activating phosphorylation by the receptor, and also leads to BMP receptor
ubiquitination and degradation.
References
T Ebisawa, M Fukuchi, G Murakami, T Chiba, K Tanaka, T Imamura, K Miyazono, "Smurf1 interacts with transforming growth factor-beta type I
receptor through Smad7 and induces receptor degradation", J Biol Chem, 276, 2001, 12477-80.
C Suzuki, G Murakami, M Fukuchi, T Shimanuki, Y Shikauchi, T Imamura, K Miyazono, "Smurf1 regulates the inhibitory activity of Smad7 by
targeting Smad7 to the plasma membrane", J Biol Chem, 277, 2002, 39919-25.
G Murakami, T Watabe, K Takaoka, K Miyazono, T Imamura, "Cooperative inhibition of bone morphogenetic protein signaling by Smurf1 and
inhibitory Smads", Mol Biol Cell, 14, 2003, 2809-17.
Reaction
28.8 Activated type I receptor phosphorylates R-Smad1/5/8 directly
Authors
Reviewers
Heldin, CH, 2007-11-12.
Description
Activated type I receptor kinase directly phosphorylates two of the C-terminal serine residues of the R-SMAD. Binding of the R-SMAD to the L45
loop of the type I receptor is critical for this event.
References
M Kretzschmar, F Liu, A Hata, J Doody, J Massague, "The TGF-beta family mediator Smad1 is phosphorylated directly and activated
functionally by the BMP receptor kinase", Genes Dev, 11, 1997, 984-95.
PA Hoodless, T Haerry, S Abdollah, M Stapleton, MB O'Connor, L Attisano, JL Wrana, "MADR1, a MAD-related protein that functions in BMP2
signaling pathways", Cell, 85, 1996, 489-500.
Reaction
28.9 Phospho-R-Smad1/5/8 dissociates from the receptor complex
Authors
Reviewers
Heldin, CH, 2007-11-12.
Description
Upon phosphorylation of the R-SMAD, the conformation of the C-terminal (MH2) domain of the R-SMAD changes, lowering its affinity to the type
I receptor and endofin. As a result, the phosphorylated-R-SMAD dissociates from the activated receptor complex.
References
M Kretzschmar, F Liu, A Hata, J Doody, J Massague, "The TGF-beta family mediator Smad1 is phosphorylated directly and activated
functionally by the BMP receptor kinase", Genes Dev, 11, 1997, 984-95.
G Lagna, A Hata, A Hemmati-Brivanlou, J Massague, "Partnership between DPC4 and SMAD proteins in TGF-beta signalling pathways",
Nature, 383, 1996, 832-6.
W Shi, C Chang, S Nie, S Xie, M Wan, X Cao, "Endofin acts as a Smad anchor for receptor activation in BMP signaling", J Cell Sci, 120, 2007,
1216-24.
Reaction
28.10 I-Smad competes with Co-Smad for R-Smad1/5/8
Authors
Reviewers
Heldin, CH, 2007-11-12.
Description
I-SMAD selectively antagonizes BMP-activated Smad1/5/9 by acting as a CO-SMAD decoy.
References
A Hata, G Lagna, J Massague, A Hemmati-Brivanlou, "Smad6 inhibits BMP/Smad1 signaling by specifically competing with the Smad4 tumor
suppressor", Genes Dev, 12, 1998, 186-97.
Reaction
28.11 Phospho-R-Smad1/5/8 forms a complex with Co-Smad
Authors
Reviewers
Heldin, CH, 2007-11-12.

Description
The phosphorylated C-terminal tail of r-SMAD induces a conformation change of the MH2 domain, which now acquires high affinity towards
Co-SMAD (common mediator of signal transduction in TGF-beta/BMP signalling). The r-SMAD:Co-SMAD complex most likely is a trimer of two
r-SMADs with one Co-SMAD. It is important to notice that the Co-SMAD itself cannot be phosphorylated as it lacks the C-terminal serine motif.
References
BY Qin, BM Chacko, SS Lam, MP de Caestecker, JJ Correia, K Lin, "Structural basis of Smad1 activation by receptor kinase phosphorylation",
Mol Cell, 8, 2001, 1303-12.
G Lagna, A Hata, A Hemmati-Brivanlou, J Massague, "Partnership between DPC4 and SMAD proteins in TGF-beta signalling pathways",
Nature, 383, 1996, 832-6.
Reaction
28.12 The phospho-R-Smad1/5/8:Co-Smad transfers to the nucleus
Authors
Reviewers
Heldin, CH, 2007-11-12.
Description
The phosphorylated-r-SMAD1/5/8:Co-SMAD complex rapidly translocates to the nucleus where it binds directly to DNA and interacts with a
plethora of transcription co-factors. Regulation of target gene expression can be either positive or negative. A classic example of a target gene of
the pathway are the genes encoding for i-SMADs. Thus, BMP2/SMAD signalling induces the expression of the negative regulators of the
pathway (a negative feedback loop).
References
Z Xiao, R Latek, HF Lodish, "An extended bipartite nuclear localization signal in Smad4 is required for its nuclear import and transcriptional
activity", Oncogene, 22, 2003, 1057-69.
Z Xiao, AM Brownawell, IG Macara, HF Lodish, "A novel nuclear export signal in Smad1 is essential for its signaling activity", J Biol Chem, 278,
2003, 34245-52.
Z Xiao, N Watson, C Rodriguez, HF Lodish, "Nucleocytoplasmic shuttling of Smad1 conferred by its nuclear localization and nuclear export
signals", J Biol Chem, 276, 2001, 39404-10.
Reaction
28.13 Ubiquitin-dependent degradation of the Smad complex terminates

BMP2 signalling
Authors
Reviewers
Heldin, CH, 2007-11-12.
Description
The nuclear r-SMAD:Co-SMAD complex recruits ubiquitin conjugating enzymes that ubiquitinate the complex and eventually lead to its
proteasomal degradation. This provides an end point to the signaling pathway.
References
CH Heldin, P ten Dijke, "SMAD destruction turns off signalling", Nat Cell Biol, 1, 1999, E195-7.
C Gruendler, Y Lin, J Farley, T Wang, "Proteasomal degradation of Smad1 induced by bone morphogenetic proteins", J Biol Chem, 276, 2001,
46533-43.
Reaction
28.14 SKI complexes with the Smad complex, suppressing BMP2 signalling
Authors
Reviewers
Heldin, CH, 2007-11-12.
Description
SKI resides in both the nucleus and the cytoplasm. Cytoplasmic SKI binds to the R-SMAD:C-SMAD complex and disrupts it (the mechanism is
not clear). Nuclear SKI binds to the active R-SMAD:CO-SMAD complex while bound to chromatin and recruits co-repressors that inhibit
transcription mediated by the active SMAD complex.
References
W Wang, FV Mariani, RM Harland, K Luo, "Ski represses bone morphogenic protein signaling in Xenopus and mammalian cells", Proc Natl Acad
Sci U S A, 97, 2000, 14394-9.
Reaction
29 Signaling by EGFR
Authors
Jassal, B, Castagnoli, L, 2008-02-28.
Reviewers
Muthuswamy, S, Heldin, CH, 2008-02-28.
Description
The epidermal growth factor receptor (EGFR) is one member of the ErbB family of transmembrane glycoprotein tyrosine receptor kinases (RTK).
Binding of EGFR to its ligands leads to autophosphorylation of tyrosine residues on the receptor and subsequent activation of signal transduction
pathways that are involved in regulating cellular proliferation, differentiation, and survival. Ligand binding with EGFR results in receptor homo- or
heterodimerization at the cell surface. Trans-autophosphorylation of the EGFR tyrosine kinase domains occurs and the phosphorylated tyrosine
kinase residues serve as binding sites for the recruitment of signal transducers and activators of intracellular substrates, such as Ras, which
then stimulate an intracellular signal transduction cascade.
References
RS Herbst, "Review of epidermal growth factor receptor biology", Int J Radiat Oncol Biol Phys, 59, 2004, 21-6.
G Carpenter, "Employment of the epidermal growth factor receptor in growth factor-independent signaling pathways", J Cell Biol, 146, 1999,
697-702.
A Wells, "EGF receptor", Int J Biochem Cell Biol, 31, 1999, 637-43.
J Schlessinger, "Ligand-induced, receptor-mediated dimerization and activation of EGF receptor", Cell, 110, 2002, 669-72.
29.1 Pro-EGF is cleaved to form mature EGF
Authors
Castagnoli, L, 2006-10-10.
Reviewers
Muthuswamy, S, 2007-02-16.
Description
Ligands of the epidermal growth factor receptor (EGFR) are shed from the plasma membrane by metalloproteases. Identification of the
sheddases for EGFR ligands using mouse embryonic cells lacking candidate sheddases (a disintegrin and metalloprotease; ADAM) has
revealed that ADAM10, -12 and -17 are the sheddases of the EGFR ligands in response to various shedding stimulants such as GPCR agonists,
growth factors, cytokines, osmotic stress, wounding and phorbol ester. Among the EGFR ligands, heparin-binding EGF-like growth factor
(HB-EGF), EGF and TGF-alpha are the best characterized.
Source reaction
This reaction was inferred from the corresponding reaction "Mouse pro-EGF is cleaved by ADAM sheddases" in species Mus musculus.
S Higashiyama, D Nanba, "ADAM-mediated ectodomain shedding of HB-EGF in receptor cross-talk", Biochim Biophys Acta, 1751, 2005, 110-7.
Reaction
29.2 EGFR binds EGF ligand
Authors
Reviewers
Description
The prototypic receptor tyrosine kinase (RTK) EGFR is composed of 3 major domains; an extracellular domain linked via a single
membrane-spanning domain to a cytoplasmic domain. EGF binds to the extracellular domain from where the signal is transmitted to the
cytoplasmic domain.
References
JM Sherrill, J Kyte, "Activation of epidermal growth factor receptor by epidermal growth factor", Biochemistry, 35, 1996, 5705-18.
Reaction
29.3 EGFR dimerization
Authors
Reviewers
Description
EGF and other growth factors induce oligomerization of their specific receptors. Inactive EGFR monomers are in equilibrium with active EGFR
dimers and binding of the EGF ligand stabilizes the active dimeric form.
References
JM Sherrill, J Kyte, "Activation of epidermal growth factor receptor by epidermal growth factor", Biochemistry, 35, 1996, 5705-18.
Reaction
29.4 EGFR autophosphorylation
Authors
Reviewers
Description
The cytoplasmic domain of EGFR contains tyrosine, serine and threonine phosphorylation sites. Dimerization of EGFR activates its intrinsic
protein kinase activity and results in autophosphorylation of 5 of these sites (Y992, Y1068, Y1086, Y1148 and Y1173). Tyrosine
autophosphorylation is crucial for normal receptor signalling as it provides specific binding sites for cytosolic target proteins involved in signal
transmission.
References
BL Margolis, I Lax, R Kris, M Dombalagian, AM Honegger, R Howk, D Givol, A Ullrich, J Schlessinger, "All autophosphorylation sites of
epidermal growth factor (EGF) receptor and HER2/neu are located in their carboxyl-terminal tails. Identification of a novel site in EGF receptor.",
J Biol Chem, 264, 1989, 10667-71.
K Helin, L Beguinot, "Internalization and down-regulation of the human epidermal growth factor receptor are regulated by the carboxyl-terminal
tyrosines", J Biol Chem, 266, 1991, 8363-8.
GM Walton, WS Chen, MG Rosenfeld, GN Gill, "Analysis of deletions of the carboxyl terminus of the epidermal growth factor receptor reveals
self-phosphorylation at tyrosine 992 and enhanced in vivo tyrosine phosphorylation of cell substrates", J Biol Chem, 265, 1990, 1750-4.
Reaction
29.5 Phosphorylation of EGFR by SRC kinase
Authors
Reviewers
Description
As well as autophosphorylation, EGFR can become tyrosine-phosphorylated by the action of the proto-oncogene tyrosine-protein kinase, c-src.
This Src homology 2 (SH2) domain-containing protein is one of many such proteins which bind to phosphorylated sites on EGFR to effect signal
transmission into the cell.
References
CR Lombardo, TG Consler, DB Kassel, "In vitro phosphorylation of the epidermal growth factor receptor autophosphorylation domain by c-src:
identification of phosphorylation sites and c-src SH2 domain binding sites", Biochemistry, 34, 1995, 16456-66.
Reaction
29.6 EGFR interacts with phospholipase C-gamma
Authors
Jassal, B, 2008-02-13.
Reviewers
Heldin, CH, 2008-02-12.
Description
Activated epidermal growth factor receptors (EGFR) can stimulate phosphatidylinositol (PI) turnover. Activated EGFR can activate
phospholipase C-gamma1 (PLC-gamma1) which hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-triphosphate (IP3) and
diacylglycerol (DAG). IP3 is instrumental in the release of calcium from intracellular stores and DAG is involved in protein kinase C activation.
References
SM HernÃ¡ndez-Sotomayor, G Carpenter, "Epidermal growth factor receptor: elements of intracellular communication", J Membr Biol, 128, 1992,
81-9.
29.6.1 Phospholipase C-gamma1 binds to the activated EGF receptor
Authors
Jassal, B, 2008-02-13.
Reviewers
Heldin, CH, 2008-02-12.
Description
Inactive phospholipase C-gamma1 (PLC-gamma1) binds to activated epidermal growth factor receptor (EGFR).
References
J Meisenhelder, PG Suh, SG Rhee, T Hunter, "Phospholipase C-gamma is a substrate for the PDGF and EGF receptor protein-tyrosine kinases
in vivo and in vitro", Cell, 57, 1989, 1109-22.
B Margolis, F Bellot, AM Honegger, A Ullrich, J Schlessinger, A Zilberstein, "Tyrosine kinase activity is essential for the association of
phospholipase C-gamma with the epidermal growth factor receptor", Mol Cell Biol, 10, 1990, 435-41.
Reaction
29.6.2 EGFR activates PLC-gamma1 by phosphorylation
Authors
Jassal, B, 2008-02-13.
Reviewers
Heldin, CH, 2008-02-12.
Description
EGFR phosphorylates PLC-gamma1, thus activating it.
References
MI Wahl, S Nishibe, JW Kim, H Kim, SG Rhee, G Carpenter, "Identification of two epidermal growth factor-sensitive tyrosine phosphorylation
sites of phospholipase C-gamma in intact HSC-1 cells", J Biol Chem, 265, 1990, 3944-8.
J Meisenhelder, PG Suh, SG Rhee, T Hunter, "Phospholipase C-gamma is a substrate for the PDGF and EGF receptor protein-tyrosine kinases
in vivo and in vitro", Cell, 57, 1989, 1109-22.
Reaction
29.6.3 Active PLC-gamma1 dissociates from EGFR
Authors
Jassal, B, 2008-02-13.
Reviewers
Heldin, CH, 2008-02-12.
Description
Once activated PLC-gamma1 dissociates from EGFR, it can hydrolyze PIP2.
Reaction
29.7 Grb2 events in EGFR signaling
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
Autophosphorylation of tyrosine residues are docking sites for many downstream effectors in EGFR signaling. SH2-containing
phosphotyrosine-binding domains of adaptor proteins like GRB2 is one such example. GRB2 is constitutively associated with SOS, a guanine
nucleotide exchange factor of Ras. GRB2 binding to phosphorylated EGFR results in the recruitment of SOS to the plasma membrane where it
comes in proximity to Ras. This mechanism has been seen to be the model for Ras activation.
References
A Sorkin, "Internalization of the epidermal growth factor receptor: role in signalling", Biochem Soc Trans, 29, 2001, 480-4.
AM Tari, G Lopez-Berestein, "GRB2: a pivotal protein in signal transduction", Semin Oncol, 28, 2001, 142-7.
29.7.1 Grb2 binds Sos
Authors
Charalambous, M, 2005-01-07.
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Heldin, CH, 2008-02-12.
Description
In the cytoplasm of unstimulated cells, SOS is found in a complex with GRB2. The interaction occurs between the carboxy terminal domain of
SOS and the Src homology 3 (SH3) domains of GRB2.
References
Reaction
29.7.2 Sos:Grb2 complex binds to EGF:EGFR complex
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
Cytoplasmic target proteins containing the SH2 domain can bind to activated EGFR. One such protein, growth factor receptor-bound protein 2
(GRB2), can bind activated EGFR with its SH2 domain whilst in complex with SOS through its SH3 domain. GRB2 can bind at either Y1068
and/or Y1086 tyrosine autophosphorylation sites on the receptor.
References
T Okutani, Y Okabayashi, Y Kido, Y Sugimoto, K Sakaguchi, K Matuoka, T Takenawa, M Kasuga, "Grb2/Ash binds directly to tyrosines 1068
and 1086 and indirectly to tyrosine 1148 of activated human epidermal growth factor receptors in intact cells", J Biol Chem, 269, 1994, 31310-4.
AG Batzer, D Rotin, JM Urena, EY Skolnik, J Schlessinger, "Hierarchy of binding sites for Grb2 and Shc on the epidermal growth factor
receptor", Mol Cell Biol, 14, 1994, 5192-201.
Reaction
29.7.3 Sos-mediated nucleotide exchange of Ras (EGF:EGFR-Sos:Grb2)
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
The guanine nucleotide exchange factor SOS interacts with EGFR through the adaptor protein, GRB2. Upon formation of this complex, SOS
activates Ras by promoting GDP release and GTP binding.
References
P Chardin, JH Camonis, NW Gale, L Van Aelst, J Schlessinger, MH Wigler, D Bar-Sagi, "Human Sos1: a guanine nucleotide exchange factor for
Ras that binds to GRB2", Science, 260, 1993, 1338-43.
Reaction
29.7.4 Transient dissociation of 14-3-3 upon Ras binding
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Heldin, CH, 2008-02-12.
Description
Activated p21ras (the GTP-bound form) is associated with the plasma membrane. Inactive Raf-1 is associated in the cytoplasm with 14-3-3.
14-3-3 binds to Raf-1 via the Ser259 phosphorylation site (S1). This interaction stabilises the inactive conformation of Raf-1 in which the
Ras-binding Cysteine-rich domain (CRD) is obscured. The Raf-1 molecule contains an additional p21ras-binding domain (RBD), a second serine
phosphorylation site at S621 (S2) and two tyrosine phosphorylation sites (at 340, Y1 and 341, Y2).
Raf-1 binds activated p21ras via the RBD. This displaces 14-3-3 from Ser259 and unmasks the CRD.
References
DK Morrison, RE Cutler, "The complexity of Raf-1 regulation.", Curr Opin Cell Biol, 9, 1997, 174-9.
Reaction
29.8 Shc events in EGFR signaling
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
Grb2 can bind EGFR directly or through another SH2-containing protein, Shc. This association is involved in Ras activation.
References
L Bonfini, E Migliaccio, G Pelicci, L Lanfrancone, PG Pelicci, "Not all Shc's roads lead to Ras", Trends Biochem Sci, 21, 1996, 257-61.
A Sorkin, "Internalization of the epidermal growth factor receptor: role in signalling", Biochem Soc Trans, 29, 2001, 480-4.
29.8.1 Shc binds to the phospho-receptor:ligand complex
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
SHC (Src homology 2 domain-containing) transforming protein can bind to either tyrosine 1148 and/or tyrosine 1173 sites on the EGF receptor.
References
K Sakaguchi, Y Okabayashi, Y Kido, S Kimura, Y Matsumura, K Inushima, M Kasuga, "Shc phosphotyrosine-binding domain dominantly
interacts with epidermal growth factor receptors and mediates Ras activation in intact cells", Mol Endocrinol, 12, 1998, 536-43.
Reaction
29.8.2 Shc phosphorylation by phospho-EGFR:EGF
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
Once bound to EGFR, SHC is phosphorylated on two tyrosines (Y349, Y350).

References
C Soler, CV Alvarez, L Beguinot, G Carpenter, "Potent SHC tyrosine phosphorylation by epidermal growth factor at low receptor density or in the
absence of receptor autophosphorylation sites", Oncogene, 9, 1994, 2207-15.
J VanderKuur, G Allevato, N Billestrup, G Norstedt, C Carter-Su, "Growth hormone-promoted tyrosyl phosphorylation of SHC proteins and SHC
association with Grb2", J Biol Chem, 270, 1995, 7587-93.
Reaction
29.8.3 Sos:Grb2 binds to Phospho-Shc:EGF:Phospho-EGFR
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
The tyrosine sites on SHC become possible binding sites for the GRB2:SOS complex.
References
Reaction
29.8.4 Sos-mediated nucleotide exchange of Ras (EGF:EGFR-Sos:Grb2:Shc)

Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
SOS is the guanine nucleotide exchange factor (GEF) for Ras. SOS activates Ras nucleotide exchange from the inactive form (bound to GDP)
to an active form (bound to GTP).
References
Reaction
29.8.5 Transient dissociation of 14-3-3 upon Ras binding
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Heldin, CH, 2008-02-12.
Description
References
Reaction
29.9 Gab1 signalosome
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
SHP2 down-regulates PI3K activation by dephosphorylating GAB1.
References
DR Mattoon, B Lamothe, I Lax, J Schlessinger, "The docking protein Gab1 is the primary mediator of EGF-stimulated activation of the PI-3K/Akt
cell survival pathway", BMC Biol, 2, 2004, 24.
29.9.1 Binding of Grb2 to Gab1
Authors
Reviewers
Heldin, CH, 2008-02-12.

Description
GRB2 (Growth factor receptor-bound protein 2) binds to GAB1 (GRB2-associated binding protein 1).
References
LS Lock, MM Frigault, C Saucier, M Park, "Grb2-independent recruitment of Gab1 requires the C-terminal lobe and structural integrity of the Met
receptor kinase domain", J Biol Chem, 278, 2003, 30083-90.
LS Lock, I Royal, MA Naujokas, M Park, "Identification of an atypical Grb2 carboxyl-terminal SH3 domain binding site in Gab docking proteins
reveals Grb2-dependent and -independent recruitment of Gab1 to receptor tyrosine kinases", J Biol Chem, 275, 2000, 31536-45.
Reaction
29.9.2 Binding of PI3K regulatory alpha subunit to Gab1:Grb2
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
The Src homology 2 (SH2) domain of the phosphatidylinositol 3-kinase (PI3K) regulatory subunit (PI3Kp85) binds to GAB1 in a
phosphorylation-independent manner. GAB1 serves as a docking protein which recruits a number of downstream signalling proteins. PI3Kp85
can bind to either GAB1 or phosphorylated GAB1.
References
Y Onishi-Haraikawa, M Funaki, N Gotoh, M Shibuya, K Inukai, H Katagiri, Y Fukushima, M Anai, T Ogihara, H Sakoda, H Ono, M Kikuchi, Y
Oka, T Asano, "Unique phosphorylation mechanism of Gab1 using PI 3-kinase as an adaptor protein", Biochem Biophys Res Commun, 288,
2001, 476-82.
Reaction
29.9.3 Gab1:Grb2:PI3K binds to EGF:Phospho-EGFR
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
The regulatory subunit of PI3K mediates the association of GAB1 and receptor protein-tyrosine kinases such as the EGF receptor, which can
phosphorylate GAB1. It appears that the PI3K regulatory subunit acts as an adaptor protein allowing GAB1 to serve as a substrate for several
tyrosine kinases.
References
M Holgado-Madruga, DK Moscatello, DR Emlet, R Dieterich, AJ Wong, "Grb2-associated binder-1 mediates phosphatidylinositol 3-kinase
activation and the promotion of cell survival by nerve growth factor", Proc Natl Acad Sci U S A, 94, 1997, 12419-24.
Reaction
29.9.4 PI3K catalytic subunit binds to Gab1:Grb2:PI3K:EGF:EGFR
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
The 110 kDa catalytic subunit binds to the 85 kDa regulatory subunit to create the active PI3K.
References
GA Rodrigues, M Falasca, Z Zhang, SH Ong, J Schlessinger, "A novel positive feedback loop mediated by the docking protein Gab1 and
phosphatidylinositol 3-kinase in epidermal growth factor receptor signaling", Mol Cell Biol, 20, 2000, 1448-59.
Reaction
29.9.5 PI3K converts Phosphatidylinositol-4,5-bisphosphate to Phosphatidylinositol-3,4,5-trisphosphate
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
The kinase activity of PI3K mediates the phosphorylation of PIP2 to form PIP3
References
Reaction
29.9.6 Gab1 binds phosphatidylinositol-3,4,5-trisphosphate
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
The pleckstrin homology (PH) domain of GAB1 binds to PIP3 and can target GAB1 to the plasma membrane in response to EGF stimulation.
This mechanism provides a positive feedback loop with respect to PI3K activation, to enhance EGFR signalling.
References
Reaction
29.9.7 Gab1:Grb2 binds to EGF:Phospho-EGFR
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
GAB1 binds to EGF receptors via tyrosine autophosphorylation sites on the receptor.
References
LS Lock, I Royal, MA Naujokas, M Park, "Identification of an atypical Grb2 carboxyl-terminal SH3 domain binding site in Gab docking proteins
reveals Grb2-dependent and -independent recruitment of Gab1 to receptor tyrosine kinases", J Biol Chem, 275, 2000, 31536-45.
Reaction
29.9.8 Gab1 phosphorylation by EGFR kinase
Authors
Reviewers
Heldin, CH, 2008-02-12.

Description
EGFR kinase phosphorylates the phosphorylation sites tyrosine 627 and 659 on GAB1
References
YX Fan, L Wong, TB Deb, GR Johnson, "Ligand regulates epidermal growth factor receptor kinase specificity: activation increases preference for
GAB1 and SHC versus autophosphorylation sites", J Biol Chem, 279, 2004, 38143-50.
S Lehr, J Kotzka, A Herkner, E Klein, C Siethoff, B Knebel, V Noelle, JC Bruning, HW Klein, HE Meyer, W Krone, D Muller-Wieland,
"Identification of tyrosine phosphorylation sites in human Gab-1 protein by EGF receptor kinase in vitro", Biochemistry, 38, 1999, 151-9.
Reaction
29.9.9 Activation of SHP2 through the binding to phospho-Gab1
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
The SH2 domains repress phosphatase activity of SHP2. Binding of these domains to phosphotyrosine-containing proteins relieves this
autoinhibition, possibly by inducing a conformational change in the enzyme.
References
GS Kapoor, Y Zhan, GR Johnson, DM O'Rourke, "Distinct domains in the SHP-2 phosphatase differentially regulate epidermal growth factor
receptor/NF-kappaB activation through Gab1 in glioblastoma cells", Mol Cell Biol, 24, 2004, 823-36.
JM Cunnick, L Mei, CA Doupnik, J Wu, "Phosphotyrosines 627 and 659 of Gab1 constitute a bisphosphoryl tyrosine-based activation motif
(BTAM) conferring binding and activation of SHP2", J Biol Chem, 276, 2001, 24380-7.
Reaction
29.9.10 Dephosphorylation of Gab1 by SHP2
Authors
Reviewers
Heldin, CH, 2008-02-12.

Description
Phosphorylated GAB1 can bind PI3 kinase by its regulatory alpha subunit. SHP2 dephosphorylation of the tyrosine residues 447, 472 and 589
on GAB1 means PI3 kinase can no longer bind to the complex in the plasma membrane and cannot be activated.
References
P Gual, S Giordano, TA Williams, S Rocchi, E Van Obberghen, PM Comoglio, "Sustained recruitment of phospholipase C-gamma to Gab1 is
required for HGF-induced branching tubulogenesis", Oncogene, 19, 2000, 1509-18.
Reaction
29.9.11 SHP2 dephosphorylates Tyr 992 on EGFR
Authors
Reviewers
Heldin, CH, 2008-02-12.

Description
The tyrosine-protein phosphatase SHP2 is a positive effector of EGFR signalling. SHP2 inhibits the tyrosine-dependent translocation of RasGAP
(catalyses Ras inactivation) to the plasma membrane, thereby keeping it away from Ras-GTP (its substrate). This inhibition is achieved by the
dephosphorylation of a RasGAP binding site on the EGF receptor.
References
YM Agazie, MJ Hayman, "Molecular mechanism for a role of SHP2 in epidermal growth factor receptor signaling", Mol Cell Biol, 23, 2003,
7875-86.
Reaction
29.9.12 Dephosphorylation of PAG by SHP2
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
Dephosphorylation of CBP/PAG negatively regulates the recruitment of the Src inhibiting kinase, Csk. Src is not negatively regulated by
phosphorylation by Csk.
References
Y Ren, S Meng, L Mei, ZJ Zhao, R Jove, J Wu, "Roles of Gab1 and SHP2 in paxillin tyrosine dephosphorylation and Src activation in response
to epidermal growth factor", J Biol Chem, 279, 2004, 8497-505.
Reaction
29.9.13 Sustained activation of SRC kinase by SHP2
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
SHP2 can dephosphorylate paxillin, which leads to Csk dissociation from the paxillin-Src complex and Src activation. Src is an SHP2 effector in
EGF-stimulated Erk activation and cell migration.
References
A Montagner, A Yart, M Dance, B Perret, JP Salles, P Raynal, "A novel role for Gab1 and SHP2 in epidermal growth factor-induced Ras
activation", J Biol Chem, 280, 2005, 5350-60.
Reaction
29.10 EGFR downregulation
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
Regulation of receptor tyrosine kinase (RTK) activity is implicated in the control of almost all cellular functions. One of the best understood RTKs
is epidermal growth factor receptor (EGFR). Growth factors can bind to EGFR and activate it to initiate signalling cascades within the cell.
EGFRs can also be recruited to clathrin-coated pits which can be internalised into endocytic vesicles. From here, EGFRs can either be recycled
back to the plasma membrane or directed to lysosomes for destruction.This provides a mechanism by which EGFR signalling is negatively
regulated and controls the strength and duration of EGFR-induced signals. It also prevents EGFR hyperactivation as commonly seen in
tumorigenesis.
The proto-oncogene Cbl can negatively regulate EGFR signalling. The Cbl family of RING-type ubiquitin ligases are able to poly-ubiquitinate
EGFR, an essential step in EGFR degradation. All Cbl proteins have a unique domain that recognises phosphorylated tyrosine residues on
activated EGFRs. They also direct the ubiquitination and degradation of activated EGFRs by recruiting ubiquitin-conjugation enzymes. Cbl
proteins function by specifically targeting activated EGFRs and mediating their down-regulation, thus providing a means by which signaling
processes can be negatively regulated.
Cbl also promotes receptor internalization via it's interaction with an adaptor protein, CIN85 (Cbl-interacting protein of 85kDa). CIN85 binds to
Cbl via it's SH3 domain and is enhanced by the EGFR-induced tyrosine phosphorylation of Cbl. The proline-rich region of CIN85 interacts with
endophilins which are regulatory components of clathrin-coated vesicles (CCVs). Endophilins bind to membranes and induce membrane
curvature, in conjunction with other proteins involved in CCV formation. The rapid recruitment of endophilin to the activated receptor complex by
CIN85 is the mechanism which controls receptor internalization.
References
CB Thien, WY Langdon, "Cbl: many adaptations to regulate protein tyrosine kinases", Nat Rev Mol Cell Biol, 2, 2001, 294-307.
MD Marmor, Y Yarden, "Role of protein ubiquitylation in regulating endocytosis of receptor tyrosine kinases", Oncogene, 23, 2004, 2057-70.
I Dikic, "Mechanisms controlling EGF receptor endocytosis and degradation", Biochem Soc Trans, 31, 2003, 1178-81.
29.10.1 binding of Cbl to EGFR
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
Phosphorylation at Tyrosine 1045 of EGFR creates a major docking site for E3 ubiquitin-protein ligase, CBL (Casitas B-lineage lymphoma proto-
oncogene) and is required to sort the EGFR to lysosomes for degradation. The E3 ligase Cbl plays a crucial role in these events as it dually
participates in early events of internalization via a CIN85-endophilin dependent mechanism and endocytic sorting by mediating multiple
monoubiquitylation of the receptor.
References
LM Grovdal, E Stang, A Sorkin, IH Madshus, "Direct interaction of Cbl with pTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR
to lysosomes for degradation", Exp Cell Res, 300, 2004, 388-95.
Reaction
29.10.2 Phosphorylation of Cbl (EGFR:Cbl)
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
EGF (and indeed FGF, PDGF and NGF) stimulation results in CBL phosphorylation on Tyr-371. Phosphorylation is necessary for CBL to exhibit
ubiquitin ligase activity.
References
ML Galisteo, I Dikic, AG Batzer, WY Langdon, J Schlessinger, "Tyrosine phosphorylation of the c-cbl proto-oncogene protein product and
association with epidermal growth factor (EGF) receptor upon EGF stimulation", J Biol Chem, 270, 1995, 20242-5.
CK Kassenbrock, SM Anderson, "Regulation of ubiquitin protein ligase activity in c-Cbl by phosphorylation-induced conformational change and
constitutive activation by tyrosine to glutamate point mutations", J Biol Chem, 279, 2004, 28017-27.
Reaction
29.10.3 Cbl binds and ubiquitinates Phospho-Sprouty
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
Sprouty is ubiquitinated by CBL in an EGF-dependent manner. EGF stimulation induces the tyrosine phosphorylation of Sprouty, which in turn
enhances the interaction of Sprouty with CBL.The CBL-mediated ubiquitination of Sprouty targets the protein for degradation by the 26S
proteosome.
References
AB Hall, N Jura, J DaSilva, YJ Jang, D Gong, D Bar-Sagi, "hSpry2 is targeted to the ubiquitin-dependent proteasome pathway by c-Cbl", Curr
Biol, 13, 2003, 308-14.
Reaction
29.10.4 Ubiquitination of stimulated EGFR (Cbl)
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
CBL down-regulates receptor tyrosine kinases by conjugating ubiquitin to them. This leads to receptor internalization and degradation. The
ubiquitin protein ligase activity of CBL (abbreviated as E3 activity) is mediated by its RING finger domain.
References
CA Joazeiro, SS Wing, H Huang, JD Leverson, T Hunter, YC Liu, "The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent
ubiquitin-protein ligase", Science, 286, 1999, 309-12.
Reaction
29.10.5 Cbl binds to Grb2
Authors
Reviewers
Heldin, CH, 2008-02-12.

Description
CBL binds multiple signalling proteins including GRB2. The CBL:GRB2 complex translocates to the plasma membrane where it can bind to
GRB2-specific docking sites on the EGF receptor.
References
G Panchamoorthy, T Fukazawa, S Miyake, S Soltoff, K Reedquist, B Druker, S Shoelson, L Cantley, H Band, "p120cbl is a major substrate of
tyrosine phosphorylation upon B cell antigen receptor stimulation and interacts in vivo with Fyn and Syk tyrosine kinases, Grb2 and Shc
adaptors, and the p85 subunit of phosphatidylinositol 3-kinase", J Biol Chem, 271, 1996, 3187-94.
Reaction
29.10.6 Localization of Cbl:Grb2 to the membrane
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
Upon EGF stimulation and consequent EGFR phosphorylation, GRB2 binds phosphorylated tyrosines
References
H Waterman, M Katz, C Rubin, K Shtiegman, S Lavi, A Elson, T Jovin, Y Yarden, "A mutant EGF-receptor defective in ubiquitylation and
endocytosis unveils a role for Grb2 in negative signaling", EMBO J, 21, 2002, 303-13.
Reaction
29.10.7 Phosphorylation of Cbl (EGFR:Cbl:Grb2)
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
EGF (and indeed FGF, PDGF and NGF) stimulation results in CBL phosphorylation on Tyr-371. Phosphorylation is necessary for CBL to exhibit
ubiquitin ligase activity.
References
ML Galisteo, I Dikic, AG Batzer, WY Langdon, J Schlessinger, "Tyrosine phosphorylation of the c-cbl proto-oncogene protein product and
association with epidermal growth factor (EGF) receptor upon EGF stimulation", J Biol Chem, 270, 1995, 20242-5.
CK Kassenbrock, SM Anderson, "Regulation of ubiquitin protein ligase activity in c-Cbl by phosphorylation-induced conformational change and
constitutive activation by tyrosine to glutamate point mutations", J Biol Chem, 279, 2004, 28017-27.
Reaction
29.10.8 Ubiquitination of stimulated EGFR (Cbl:Grb2)
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
CBL down-regulates receptor tyrosine kinases by conjugating ubiquitin to them. This leads to receptor internalization and degradation. The
ubiquitin protein ligase activity of CBL (abbreviated as E3 activity) is mediated by its RING finger domain.
References
CA Joazeiro, SS Wing, H Huang, JD Leverson, T Hunter, YC Liu, "The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent
ubiquitin-protein ligase", Science, 286, 1999, 309-12.
Reaction
29.10.9 Sprouty lures cytosolic Cbl away from EGFR
Authors
Reviewers
Heldin, CH, 2008-02-12.

Description
The NEYTEG motif is very similar to the CBL binding motif around Tyr-1045 in EGFR. Tyrosine-phosphorylated Sprouty (hSpry) binds to CBL,
which then can't ubiquitinate EGFR. Sprouty acts as a decoy to lure CBL away from EGFR and targets it for degradation.
References
C Rubin, V Litvak, H Medvedovsky, Y Zwang, S Lev, Y Yarden, "Sprouty fine-tunes EGF signaling through interlinked positive and negative
feedback loops", Curr Biol, 13, 2003, 297-307.
ES Wong, CW Fong, J Lim, P Yusoff, BC Low, WY Langdon, GR Guy, "Sprouty2 attenuates epidermal growth factor receptor ubiquitylation and
endocytosis, and consequently enhances Ras/ERK signalling", EMBO J, 21, 2002, 4796-808.
Reaction
29.10.10 Sprouty lures membrane-bound Cbl away from EGFR
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
The NEYTEG motif is very similar to the CBL binding motif around Tyr-1045 in EGFR. Tyrosine-phosphorylated Sprouty (hSpry) binds to CBL,
which then can't ubiquitinate EGFR. Sprouty acts as a decoy to lure CBL away from EGFR and targets it for degradation.
References
C Rubin, V Litvak, H Medvedovsky, Y Zwang, S Lev, Y Yarden, "Sprouty fine-tunes EGF signaling through interlinked positive and negative
feedback loops", Curr Biol, 13, 2003, 297-307.
ES Wong, CW Fong, J Lim, P Yusoff, BC Low, WY Langdon, GR Guy, "Sprouty2 attenuates epidermal growth factor receptor ubiquitylation and
endocytosis, and consequently enhances Ras/ERK signalling", EMBO J, 21, 2002, 4796-808.
Reaction
29.10.11 Cbl ubiquitinates Sprouty
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
Sprouty is ubiquitinated by CBL in an EGF-dependent manner. EGF stimulation induces the tyrosine phosphorylation of Sprouty, which in turn
enhances the interaction of Sprouty with CBL. The CBL-mediated ubiquitination of Sprouty targets the protein for degradation by the 26S
proteasome.
References
AB Hall, N Jura, J DaSilva, YJ Jang, D Gong, D Bar-Sagi, "hSpry2 is targeted to the ubiquitin-dependent proteasome pathway by c-Cbl", Curr
Biol, 13, 2003, 308-14.
Reaction
29.10.12 Cdc42 lures Cbl away from the receptor
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
Activated CDC42 binds to p85Cool-1/beta-Pix, a protein that directly associates with CBL. This inhibits the binding of CBL by the EGF receptor
and thus prevents CBL from catalyzing receptor ubiquitination.
References
WJ Wu, S Tu, RA Cerione, "Activated Cdc42 sequesters c-Cbl and prevents EGF receptor degradation", Cell, 114, 2003, 715-25.
Reaction
29.10.13 betaPIX pushes CIN85 away from Cbl
Authors
Reviewers
Heldin, CH, 2008-02-12.

Description
High concentrations of active CDC42 and bPix may promote the binding of bPix to CBL, pushing out the usually preferred binding partner CIN85
from the CBL complex. This competitive mechanism could block the CIN85-imposed clustering phenomenon on CBL that is required for tighter
binding.
References
MH Schmidt, K Husnjak, I Szymkiewicz, K Haglund, I Dikic, "Cbl escapes Cdc42-mediated inhibition by downregulation of the adaptor molecule
betaPix", Oncogene, 25, 2006, 3071-8.
Reaction
29.10.14 Cbl escapes Cdc42-mediated inhibition by down-regulating the adaptor molecule betaPix
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
BetaPix/Cool-1 associates with CBL, which appears to be a critical step in Cdc42-mediated inhibition of EGFR ubiquitylation and
downregulation. The SH3 domain of betaPix specifically interacts with a proline-arginine motif (PxxxPR) present within CBL, which mediates
ubiquitylation and subsequent degradation of betaPix.
References
MH Schmidt, K Husnjak, I Szymkiewicz, K Haglund, I Dikic, "Cbl escapes Cdc42-mediated inhibition by downregulation of the adaptor molecule
betaPix", Oncogene, 25, 2006, 3071-8.
Reaction
29.10.15 Assembly of EGFR complex in clathrin-coated vesicles
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
CBl-CIN85-Endophilin complex mediates ligand-induced down-regulation of the EGF receptor. The BAR domain of endophilin induces
membrane curvature. The three SH3 domains of CIN85 bind to atypical proline-arginine motifs (PxxxPR) present in the carboxyl termini of CBL
and CBL-b. In this way, CIN85 clusters CBL molecules, which is crucial for efficient EGFR endocytosis and degradation.
References
P Soubeyran, K Kowanetz, I Szymkiewicz, WY Langdon, I Dikic, "Cbl-CIN85-endophilin complex mediates ligand-induced downregulation of
EGF receptors", Nature, 416, 2002, 183-7.
Reaction
29.10.16 Assembly in clathrin-coated vesicles (CCVs)
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
Epsin directly modifies membrane curvature on binding to PIP2 in conjunction with clathrin polymerisation.
References
JA Rosenthal, H Chen, VI Slepnev, L Pellegrini, AE Salcini, PP Di Fiore, P De Camilli, "The epsins define a family of proteins that interact with
components of the clathrin coat and contain a new protein module", J Biol Chem, 274, 1999, 33959-65.
Reaction
29.10.17 EGFR non-clathrin mediated endocytosis
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
At higher concentrations of ligand, a substantial fraction of the receptor (>50%) is endocytosed through a clathrin-dependent,
lipid-raft-dependent route as the receptor becomes ubiquitnated and Y1045 phosphorylated. Eps15 and Epsin are found in caveolae. Eps15 and
Epsin are immunoprecipated with the EGF receptor.
Non-clathrin internalisation of ubiquitinated EGFR depends on its interaction with proteins harbouring the UIM Ub-interacting motif, as shown
through the ablation of three Ub-interactingmotif-containing proteins, Eps15, Eps15R and Epsin.
References
S Sigismund, T Woelk, C Puri, E Maspero, C Tacchetti, P Transidico, PP Di Fiore, S Polo, "Clathrin-independent endocytosis of ubiquitinated
cargos", Proc Natl Acad Sci U S A, 102, 2005, 2760-5.
E Klapisz, I Sorokina, S Lemeer, M Pijnenburg, AJ Verkleij, PM van Bergen en Henegouwen, "A ubiquitin-interacting motif (UIM) is essential for
Eps15 and Eps15R ubiquitination", J Biol Chem, 277, 2002, 30746-53.
Reaction
29.10.18 Cbl-mediated ubiquitination of CIN85
Authors
Reviewers
Heldin, CH, 2008-02-12.

Description
The adaptor protein CIN85 is monoubiquitinated by CBL after EGF stimulation. Monoubiquitination is thought to regulate receptor internalization
and endosomal sorting.
References
K Haglund, N Shimokawa, I Szymkiewicz, I Dikic, "Cbl-directed monoubiquitination of CIN85 is involved in regulation of ligand-induced
degradation of EGF receptors", Proc Natl Acad Sci U S A, 99, 2002, 12191-6.
Reaction
29.10.19 Sprouty sequesters Cbl away from active EGFR
Authors
Reviewers
Heldin, CH, 2008-02-12.
Description
Sprouty can constitutively interact with two SH3 domains of CIN85 whereas the third SH3 domain of CIN85 can still associate with CBL on cell
activation with EGF. This allows Sprouty to block CIN85-mediated clustering of CBL molecules, stablisation of CBL-EGFR interactions and
efficient ubiquitination and down-regulation of EGFR.
References
K Haglund, MH Schmidt, ES Wong, GR Guy, I Dikic, "Sprouty2 acts at the Cbl/CIN85 interface to inhibit epidermal growth factor receptor
downregulation", EMBO Rep, 6, 2005, 635-41.
Reaction
30 Signaling by FGFR
Authors
de Bono, B, 2007-01-10.
Editors
de Bono, B, D'Eustachio, P, 2007-02-10.
Reviewers
Mohammadi, M, 2007-02-06.
Description
The 22 members of the fibroblast growth factor (FGF) family of growth factors mediate their cellular responses by binding to and activating the
different isoforms encoded by the four receptor tyrosine kinases (RTKs) designated FGFR1, FGFR2, FGFR3 and FGFR4. These receptors are
key regulators of several developmental processes in which cell fate and differentiation to various tissue lineages are determined. Unlike other
growth factors, FGFs act in concert with heparin or heparan sulfate proteoglycan (HSPG) to activate FGFRs and to induce the pleiotropic
responses that lead to the variety of cellular responses induced by this large family of growth factors. An alternative, FGF-independent, source of
FGFR activation originates from the interaction with cell adhesion molecules, typically in the context of interactions on neural cell membranes
and is crucial for neuronal survival and development. Upon ligand binding, receptor dimers are formed and their intrinsic tyrosine kinase is
activated causing phosphorylation of multiple tyrosine residues on the receptors. These then serve as docking sites for the recruitment of SH2
(src homology-2) or PTB (phosphotyrosine binding) domains of adaptors, docking proteins or signaling enzymes. Signaling complexes are
assembled and recruited to the active receptors resulting in a cascade of phosphorylation events. This leads to stimulation of intracellular
signaling pathways that control cell proliferation, cell differentiation, cell migration, cell survival and cell shape, depending on the cell type or
stage of maturation.
References
DM Ornitz, PJ Marie, "FGF signaling pathways in endochondral and intramembranous bone development and human genetic disease", Genes
Dev, 16, 2002, 1446-65.
VP Eswarakumar, I Lax, J Schlessinger, "Cellular signaling by fibroblast growth factor receptors", Cytokine Growth Factor Rev, 16, 2005,
139-49.
J Schlessinger, "Common and distinct elements in cellular signaling via EGF and FGF receptors", Science, 306, 2004, 1506-7.
EJ Williams, J Furness, FS Walsh, P Doherty, "Activation of the FGF receptor underlies neurite outgrowth stimulated by L1, N-CAM, and
N-cadherin", Neuron, 13, 1994, 583-94.
X Zhang, OA Ibrahimi, SK Olsen, H Umemori, M Mohammadi, DM Ornitz, "Receptor specificity of the fibroblast growth factor family. The
complete mammalian FGF family.", J Biol Chem, 281, 2006, 15694-700.
L Dailey, D Ambrosetti, A Mansukhani, C Basilico, "Mechanisms underlying differential responses to FGF signaling", Cytokine Growth Factor
Rev, 16, 2005, 233-47.
J Schlessinger, "Cell signaling by receptor tyrosine kinases", Cell, 103, 2000, 211-25.
30.1 FGFR ligand binding and activation
Authors
de Bono, B, 2007-01-10.
Editors
Reviewers
Description
FGFs bind and activate alternatively spliced forms of four tyrosine kinase FGF receptors in the region between the second and third
immunoglobulin domain (known as D2 and D3). The first immunoglobulin domain (D1) and a stretch of acidic residues known as an acid box in
the linker between D1 and D2 impart a lower affinity to FGFs compared to receptors in which such regions are removed. FGFRs also contain a
short amino acid motif within the second immunoglobulin domain that shares sequence homology with functional motifs present in neural
adhesion molecules such as NCAM and N-cadherin. This so called CAM homology domain (CHD) forms a contiguous sequence with the acid
box region and is crucial for this mode of activation. Interactions between the neural cell adhesion molecules are important for a number of
developmental events and have also been implicated in tumor progression. Although the interaction can be seen over most of the cell surface, it
is not seen at points of cell-cell contact where the adhesion molecules accumulate at stable junctions. The FGFR interaction with N-cadherin and
NCAM (but not FGF) is absolutely dependant on the presence of the acid box motif. As this motif can be spliced out of all four FGFRs, this
suggests a mechanism that can regulate the interaction of the receptor with different ligand classes. From a hormonal point of view, the spatial
and temporal expression patterns of FGFs and FGFRs and the ability of specific ligand-receptor pairs to actively signal are important factors
regulating FGF activity in a variety of biological processes. FGF signaling activity is regulated by the binding specificity of ligands and receptors
and is modulated by extrinsic cofactors such as heparan sulfate proteoglycans.
References
F Wang, M Kan, J Xu, G Yan, WL McKeehan, "Ligand-specific structural domains in the fibroblast growth factor receptor", J Biol Chem, 270,
1995, 10222-30.
P Doherty, FS Walsh, "CAM-FGF Receptor Interactions: A Model for Axonal Growth", Mol Cell Neurosci, 8, 1996, 99-111.
F Wang, M Kan, G Yan, J Xu, WL McKeehan, "Alternately spliced NH2-terminal immunoglobulin-like Loop I in the ectodomain of the fibroblast
growth factor (FGF) receptor 1 lowers affinity for both heparin and FGF-1", J Biol Chem, 270, 1995, 10231-5.
30.1.1 FGFR1 ligand binding and activation
Authors
de Bono, B, 2007-01-10.
Editors
Reviewers
Description
The vertebrate fibroblast growth factor receptor 1 (FGFR1) is alternatively spliced generating multiple variants that are differentially expressed
during embryo development and in the adult body. The restricted expression patterns of FGFR1 isoforms, together with differential expression
and binding of specific ligands, leads to activation of common FGFR1 signal transduction pathways, but may result in distinctively different
biological responses as a result of differences in cellular context. FGFR1 isoforms are also present in the nucleus in complex with various
fibroblast growth factors where they function to regulate transcription of target genes.
FGFR is probably activated by NCAM very differently from the way by which it is activated by FGFs, reflecting the different conditions for
NCAMÃ¢â‚¬"FGFR and FGFÃ¢â‚¬"FGFR interactions. The affinity of FGF for FGFR is approximately 10e6 times higher than that of NCAM for
FGFR. Moreover, in the brain NCAM is constantly present on the cell surface at a much higher (micromolar) concentration than FGFs, which
only appear transiently in the extracellular environment in the nanomolar range.
References
VV Kiselyov, G Skladchikova, AM Hinsby, PH Jensen, N Kulahin, V Soroka, N Pedersen, V Tsetlin, FM Poulsen, V Berezin, E Bock, "Structural
basis for a direct interaction between FGFR1 and NCAM and evidence for a regulatory role of ATP", Structure, 11, 2003, 691-701.
VV Kiselyov, V Soroka, V Berezin, E Bock, "Structural biology of NCAM homophilic binding and activation of FGFR", J Neurochem, 94, 2005,
1169-79.
C Groth, M Lardelli, "The structure and function of vertebrate fibroblast growth factor receptor 1", Int J Dev Biol, 46, 2002, 393-400.
30.1.1.1 FGFR1b ligand binding and activation
Authors
de Bono, B, 2007-01-10.
Reviewers
Description
This pathway depicts the binding of an experimentally-verified range of ligands to FGFR1b. While binding affinities may vary considerably within
this set, the ligands listed have been established to bring about receptor activation at their reported physiological concentrations.
References
30.1.1.1.1 FGFR1b binds to FGF
Authors
de Bono, B, 2007-01-10.
Reviewers
Description
In this reaction, FGF receptor in the plasma membrane binds an associating extracellular ligand, a requisite step for subsequent activation. The
resulting complex consists of dimerized receptor, two ligand molecules, and heparan sulfate (Mohammadi et al. 2005). NCAM and other
members of the CAM protein family directly or indirectly modulate this interaction in a variety of neural tissues. The details of this interaction in
vivo have not been definitively established at the molecular level, but are thought to play a central role in the regulation of the development of
these tissues (Williams et al. 1994; Kiselyov et al. 2003, 2005; Christensen et al. 2006).
References
1169-79.
EJ Williams, J Furness, FS Walsh, P Doherty, "Activation of the FGF receptor underlies neurite outgrowth stimulated by L1, N-CAM, and
N-cadherin", Neuron, 13, 1994, 583-94.
C Christensen, JB Lauridsen, V Berezin, E Bock, VV Kiselyov, "The neural cell adhesion molecule binds to fibroblast growth factor receptor 2",
FEBS Lett, 580, 2006, 3386-90.
M Mohammadi, SK Olsen, OA Ibrahimi, "Structural basis for fibroblast growth factor receptor activation", Cytokine Growth Factor Rev, 16, 2005,
107-37.
Reaction
30.1.1.1.2 Autocatalytic phosphorylation of FGFR1b
Authors
de Bono, B, 2007-01-10.
Reviewers
Description
The intrinsic protein tyrosine kinase activity of activated FGF receptor 1 catalyzes multiple phosphorylation events (Mohammadi et al. 1997),
creating a number of binding sites on its cytoplasmic tail for membrane bound docking proteins to gather intracellular signaling mediators.
References
M Mohammadi, I Dikic, A Sorokin, WH Burgess, M Jaye, J Schlessinger, "Identification of six novel autophosphorylation sites on fibroblast
growth factor receptor 1 and elucidation of their importance in receptor activation and signal transduction", Mol Cell Biol, 16, 1996, 977-89.
Reaction
30.1.1.2 FGFR1c ligand binding and activation
Authors
de Bono, B, 2007-01-10.
Reviewers
Description
This pathway depicts the binding of an experimentally-verified range of ligands to FGFR1c. While binding affinities may vary considerably within
References
30.1.1.2.1 FGFR1c binds to FGF
Authors
de Bono, B, 2007-01-10.
Reviewers
Description
resulting complex consists of dimerized receptor, two ligand molecules, and heparan sulfate.
References
107-37.
Reaction
30.1.1.2.2 Autocatalytic phosphorylation of FGFR1c
Authors
de Bono, B, 2007-01-10.
Reviewers
Description
References
M Mohammadi, G McMahon, L Sun, C Tang, P Hirth, BK Yeh, SR Hubbard, J Schlessinger, "Structures of the tyrosine kinase domain of
fibroblast growth factor receptor in complex with inhibitors", Science, 276, 1997, 955-60.
Reaction
30.1.1.3 FGFR1c and Klotho ligand binding and activation
Authors
de Bono, B, 2007-01-10.
Reviewers
Description
FGF23 is a unique member of the fibroblast growth factor (FGF) family because it acts as a hormone that derives from bone and regulates
kidney functions, whereas most other family members are thought to regulate various cell functions at a local level. Klotho is essential for
endogenous FGF23 function as it converts FGFR1(IIIc) into a specific FGF23 receptor.
References
T Fujita, S Fukumoto, H Hasegawa, K Iijima, K Okawa, T Shimada, I Urakawa, Y Yamazaki, T Yameshita, "Klotho converts canonical FGF
receptor into a specific receptor for FGF23", Nature, 2006.
30.1.1.3.1 FGFR1c binds to Klotho-bound FGF23
Authors
de Bono, B, 2007-01-10.
Reviewers
Description
References
107-37.
Reaction
30.1.1.3.2 Autocatalytic phosphorylation of Klotho-bound FGFR1c
Authors
de Bono, B, 2007-01-10.
Reviewers
Description
References
Reaction
Authors
de Bono, B, 2007-01-10.
Editors
Reviewers
Description
Dominant mutations in the fibroblast growth factor receptor 2 (FGFR2) gene have been identified as causes of four phenotypically distinct
craniosynostosis syndromes, including Crouzon, Jackson- Weiss, Pfeiffer, and Apert syndromes. FGFR2 binds a number of different FGFs
preferentially, as illustrated in this pathway. FGFR is probably activated by NCAM very differently from the way by which it is activated by FGFs,
reflecting the different conditions for NCAMÃ¢â‚¬"FGFR and FGFÃ¢â‚¬"FGFR interactions. The affinity of FGF for FGFR is approximately 10e6
times higher than that of NCAM for FGFR. Moreover, in the brain NCAM is constantly present on the cell surface at a much higher (micromolar)
concentration than FGFs, which only appear transiently in the extracellular environment in the nanomolar range.
References
NE Hatch, M Hudson, ML Seto, ML Cunningham, M Bothwell, "Intracellular retention, degradation, and signaling of glycosylation-deficient
FGFR2 and craniosynostosis syndrome-associated FGFR2C278F", J Biol Chem, 281, 2006, 27292-305.
1169-79.
AB Moffa, SP Ethier, "Differential signal transduction of alternatively spliced FGFR2 variants expressed in human mammary epithelial cells", J
Cell Physiol, 210, 2007, 720-31.
Authors
de Bono, B, 2007-01-10.
Reviewers
Description
References
Authors
de Bono, B, 2007-01-10.
Reviewers
Description
In this reaction, FGF receptor 2b in the plasma membrane binds an associating extracellular ligand, a requisite step for subsequent activation.
The resulting complex consists of dimerized receptor, two ligand molecules, and heparan sulfate. Two isoforms of FGFR2b generated by
alternative splicing and differing only by the presence ("long") or absence ("short") of two amino acid residues at positions 428-429 are equally
active in ligand binding and dimerization but differ in their abilities to interact with downstream targets.
References
107-37.
Reaction
Authors
de Bono, B, 2007-01-10.
Reviewers
Description
The intrinsic protein tyrosine kinase activity of the activated FGFR2b receptor leads to multiple phosphorylation events, creating a number of
binding sites on its cytoplasmic tail for membrane bound docking proteins to gather intracellular signaling mediators. Two isoforms of FGFR2c
generated by alternative splicing and differing only by the presence ("long") or absence ("short") of two amino acid residues at positions 428-429
are equally active in autophosphorylation, but differ in their abilities to interact with downstream targets.
References
KC Hart, SC Robertson, DJ Donoghue, "Identification of tyrosine residues in constitutively activated fibroblast growth factor receptor 3 involved
in mitogenesis, Stat activation, and phosphatidylinositol 3-kinase activation", Mol Biol Cell, 12, 2001, 931-42.
Reaction
Authors
de Bono, B, 2007-01-10.
Reviewers
Description
References
Authors
de Bono, B, 2007-01-10.
Reviewers
Description
In this reaction, FGF receptor 2c in the plasma membrane binds an associating extracellular ligand, a requisite step for subsequent activation.
The resulting complex consists of dimerized receptor, two ligand molecules, and heparan sulfate. Two isoforms of FGFR2c generated by
alternative splicing and differing only by the presence ("long") or absence ("short") of two amino acid residues at positions 428-429 are equally
active in ligand binding and dimerization but differ in their abilities to interact with downstream targets.
References
107-37.
Reaction
Authors
de Bono, B, 2007-01-10.
Reviewers
Description
The intrinsic protein tyrosine kinase activity of the activated FGFR2c receptor leads to multiple phosphorylation events, creating a number of
binding sites on its cytoplasmic tail for membrane bound docking proteins to gather intracellular signaling mediators. Two isoforms of FGFR2c
generated by alternative splicing and differing only by the presence ("long") or absence ("short") of two amino acid residues at positions 428-429
are equally active in autophosphorylation, but differ in their abilities to interact with downstream targets.
References
CA Dionne, G Crumley, F Bellot, JM Kaplow, G Searfoss, M Ruta, WH Burgess, M Jaye, J Schlessinger, "Cloning and expression of two distinct
high-affinity receptors cross-reacting with acidic and basic fibroblast growth factors", EMBO J, 9, 1990, 2685-92.
Reaction
Authors
de Bono, B, 2007-01-10.
Editors
Reviewers
Description
FGFR3 is a receptor tyrosine kinase of the FGF receptor family, known to have a negative regulatory effect on long bone growth. Somatically,
some of the same activating mutations are associated with hypochondroplasia, multiple myeloma, and cervical and vesical carcinoma.
References
CG L'Hote, MA Knowles, "Cell responses to FGFR3 signalling: growth, differentiation and apoptosis", Exp Cell Res, 304, 2005, 417-31.
Authors
de Bono, B, 2007-01-10.
Reviewers
Description
References
Authors
de Bono, B, 2007-01-10.
Reviewers
Description
References
107-37.
Reaction
Authors
de Bono, B, 2007-01-10.
Reviewers
Description
References
Reaction
Authors
de Bono, B, 2007-01-10.
Reviewers
Description
References
Authors
de Bono, B, 2007-01-10.
Reviewers
Description
References
107-37.
Reaction
Authors
de Bono, B, 2007-01-10.
Reviewers
Description
The intrinsic protein tyrosine kinase activity of activated FGF receptor 3 catalyzes multiple phosphorylation events, creating a number of binding
sites on its cytoplasmic tail for membrane bound docking proteins to gather intracellular signaling mediators.
Reaction
Authors
de Bono, B, 2007-01-10.
Editors
Reviewers
Description
FGFR4 is expressed mainly in mature skeletal muscle, and disruption of FGFR4 signaling interrupts limb muscle formation in vertebrates.
This pathway depicts the binding of an experimentally-verified range of ligands to FGFR4. While binding affinities may vary considerably within
References
S Yu, L Zheng, DK Trinh, SL Asa, S Ezzat, "Distinct transcriptional control and action of fibroblast growth factor receptor 4 in differentiating
skeletal muscle cells", Lab Invest, 84, 2004, 1571-80.
30.1.4.1 FGFR4 binds to FGF
Authors
de Bono, B, 2007-01-10.
Editors
Reviewers
Description
References
107-37.
Reaction
30.1.4.2 Autocatalytic phosphorylation of FGFR4
Authors
de Bono, B, 2007-01-10.
Editors
Reviewers
Description
References
Reaction
31 Signaling in Immune system
Authors
de Bono, B, Gillespie, ME, Luo, F, Ouwehand, W.H., 2006--0-3-.
Reviewers
D'Eustachio, P, Gay, NJ, Gale M, Jr, Zwaginga, JJ, 2006--0-4-.
Description
Humans are exposed to millions of potential pathogens daily, through contact, ingestion, and inhalation. Our ability to avoid infection depends on
the adaptive immune system and during the first critical hours and days of exposure to a new pathogen, our innate immune system.
31.1 Innate Immunity Signaling
Description
Innate immunity encompases the nonspecific part of immunity tha are part of an individual's natural biologic makeup
31.1.1 Toll Receptor Cascades
Authors
de Bono, B, Gillespie, ME, Luo, F, Gay, NJ, 0000-00-00.
Reviewers
D'Eustachio, P, Gale M, Jr, Gay, NJ, 2006-10-31.
Description
In human, ten members of the Toll-like receptor (TLR) family (TLR1-TLR10) have been identified (TLR11 has been found in mouse, but not in
human). All TLRs have a similar Toll/IL-1 receptor (TIR) domain in their cytoplasmic region and an Ig-like domain in the extracellular region,
where each is enriched with a varying number of leucine-rich repeats (LRRs). Each TLR can recognize specific microbial pathogen components.
The binding pathogens component of the TLRs initializes signaling pathways that lead to induction of Interferon alpha/beta. There are three main
signaling pathways: the first is a MyD88-dependent pathway that is common to all TLRs, except TLR3; the second is a TRAM-dependent
pathway that is peculiar to TLR3 and TLR4 and is mediated by TRIF and RIP1; and the third is a TRAF6-mediated pathway peculiar to TLR3.
References
S Akira, "Toll-like receptor signaling", J Biol Chem, 278, 2003, 38105-8.
K Takeda, S Akira, "TIRAP/Mal, TRIF and TRAM. Differential utilization of these TIR", Int Immunol, 17, 2005, 1-14.
H Heine, AJ Ulmer, "Recognition of bacterial products by toll-like receptors", Chem Immunol Allergy, 86, 2005, 99-119.
T Kawai, "Pathogen recognition with Toll-like receptors", Curr Opin Immunol, 17, 2005, 338-44.
K Takeda, S Akira, "TIR domains, which are conserved among all TLRs. Recent accumulating", Semin Immunol, 16, 2004, 3-9.
31.1.1.1 Toll Like Receptor 10 (TLR10) Cascade

Authors
Luo, F, 2005-11-10.
Reviewers
Gale M, Jr, 2006-10-31.
Description
Little is known about TLR10 ligands. It has been established that the receptor homodimerizes upon binding and signals in an MyD88-dependent
manner. It may also heterodimerize with TLRs 1 and 2. It is expressed in a restricted fashion as a highly N-glycosylated protein detectable in B
cells and dendritic cells.
References
U Hasan, C Chaffois, C Gaillard, V Saulnier, E Merck, S Tancredi, C Guiet, F Briere, J Vlach, S Lebecque, G Trinchieri, EE Bates, "Human
TLR10 is a functional receptor, expressed by B cells and plasmacytoid dendritic cells, which activates gene transcription through MyD88", J
Immunol, 174, 2005, 2942-50.
31.1.1.1.1 Ligand binds to TLR10
Authors
Luo, F, 2005-11-10.
Reviewers
Gale M, Jr, 2006-10-31.
Description
The ligand to the TLR10 dimer is as yet unknown.
References
U Hasan, C Chaffois, C Gaillard, V Saulnier, E Merck, S Tancredi, C Guiet, F Briere, J Vlach, S Lebecque, G Trinchieri, EE Bates, "Human
TLR10 is a functional receptor, expressed by B cells and plasmacytoid dendritic cells, which activates gene transcription through MyD88", J
Immunol, 174, 2005, 2942-50.
Reaction
31.1.1.1.2 MyD88 cascade
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
TLRÃ¢â‚¬â„¢s TIR intracellular domain binds to a homologous domain in an adaptor protein, MyD88, which also contains a death domain. This
death domain undergoes homophilic interaction with the death domain of a serine/threonine protein kinase known as IRAK; this leads to the
autophosphorylation of IRAK. Autophosphorylated IRAK then forms a complex with TRAF6 and this, in turn, results in the oligomerization of
TRAF6. The oligomerization of TRAF6 activates TAK-1, a member of the MAP 3-kinase family, and this leads to the activation of the IkB
kinases. These kinases, in turn, phosphorylate IkB, leading to its proteolytic degradation and the translocation of NF-kB to the nucleus.
Concomitantly, members of the activator protein-1 (AP-1) transcription factor family, Jun and Fos, are activated, and both AP-1 transcription
factors and NF-kB are required for cytokine production, which in turn produces downstream inflammatory effects.
References
M Gangloff, NJ Gay, "MD-2: the Toll 'gatekeeper' in endotoxin signalling", Trends Biochem Sci, 29, 2004, 294-300.
31.1.1.1.2.1 MyD88 and Mal combine with the activated TLR receptor in human
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
MyD88 binds to both IRAK (IL-1 receptor-associated kinase) and the receptor heterocomplex (the signaling complex) and thereby mediates the
association of IRAK with the receptor. MyD88 therefore couples a serine/threonine protein kinase to the receptor complex.
References
H Wesche, WJ Henzel, W Shillinglaw, S Li, Z Cao, "MyD88: an adapter that recruits IRAK to the IL-1 receptor complex", Immunity, 7, 1997,
837-47.
T Horng, GM Barton, RA Flavell, R Medzhitov, "The adaptor molecule TIRAP provides signalling specificity for Toll-like receptors", Nature, 420,
2002, 329-33.
M Yamamoto, S Sato, H Hemmi, H Sanjo, S Uematsu, T Kaisho, K Hoshino, O Takeuchi, M Kobayashi, T Fujita, K Takeda, S Akira, "Essential
role for TIRAP in activation of the signalling cascade shared by TLR2 and TLR4", Nature, 420, 2002, 324-9.
Reaction
31.1.1.1.2.2 IRAK4 combines with activated TLR receptor complexed with Mal:MyD88
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.

Description
IRAK-4 is the mammalian homolog of Drosophila melanogaster Pelle. Like Pelle, and unlike the other mammalian IRAKs, IRAK-4 plays a critical
role in Toll receptor signalling - any interference with IRAK-4's kinase activity virtually abolishes downstream events. This is not the case with
other members of the IRAK family.
References
S Li, A Strelow, EJ Fontana, H Wesche, "IRAK-4: a novel member of the IRAK family with the properties of an", Proc Natl Acad Sci U S A, 99,
2002, 5567-72.
DN Edwards, P Towb, SA Wasserman, "An activity-dependent network of interactions links the Rel protein Dorsal with its cytoplasmic
regulators", Development, 124, 1997, 3855-64.
P Towb, A Bergmann, SA Wasserman, "The protein kinase Pelle mediates feedback regulation in the Drosophila Toll signaling pathway",
Development, 128, 2001, 4729-36.
N Suzuki, S Suzuki, WC Yeh, "IRAK-4 as the central TIR signaling mediator in innate immunity", Trends Immunol, 23, 2002, 503-6.
Reaction
31.1.1.1.2.3 IRAK1 combines with activated TLR complexed with IRAK4 and MyD88
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
At the receptor, IRAK1 associates with MyD88, and phosphorylation by IRAK4 triggers IRAK1 autophosphorylation. This results in disassociation
and release from the receptor-MyD88 complex, leading to the formation of a new protein complex consisting of hyperphosphorylated IRAK1 and
TRAF6, a prerequisite for TRAF6-mediated NF-B activation and induction of an inflammatory response.
References
N Rao, S Nguyen, K Ngo, WP Fung-Leung, "A novel splice variant of interleukin-1 receptor (IL-1R)-associated kinase 1 plays a negative
regulatory role in toll/IL-1R-induced inflammatory", Mol Cell Biol, 25, 2005, 6521-32.
Reaction
31.1.1.1.2.4 First phosphorylation of IRAK1 by IRAK4 bound to activated TLR
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
First, IRAK-1 is phosphorylated at Thr209. This results in a conformational change of the kinase domain, permitting further phosphorylations to
take place. Substitution of Thr209 by alanine results in a kinase-inactive IRAK-1.
References
2002, 5567-72.
C Kollewe, AC Mackensen, D Neumann, J Knop, P Cao, S Li, H Wesche, MU Martin, "Sequential autophosphorylation steps in the
interleukin-1", J Biol Chem, 279, 2004, 5227-36.
Reaction
31.1.1.1.2.5 Second phosphorylation of IRAK1 by IRAK4 bound to activated TLR
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
Second, Thr387 in the activation loop is phosphorylated, leading to full enzymatic activity.
References
Reaction
31.1.1.1.2.6 Multiple IRAK1 autophosphorylation steps
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.

Description
Third, IRAK-1 autophosphorylates several times in the proline-, serine-, and threonine-rich ProST region between the N-terminal death domain
and kinase domain. The five IRAK1 phosphorylation sites annotated in this reaction are predicted to occur in the molecule's ProST region which
was identified by Kollewe et al. (2004). While there is no direct confirmation as to the precise amino acid position of each phosphorylation site,
the five sample positions selected here represent positions where phosphorylation is extremely likely.
References
Reaction
31.1.1.1.2.7 Dissociation of IRAK1-P(n) from TLR
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
Hyperphosphorylation of this region leads to dissociation of IRAK-1 from the upstream adapters like MyD88 but leaves its interaction with the
downstream adapter TRAF6 unaffected.
References
Reaction
31.1.1.1.2.8 IRAK1-P(n) combines with TRAF6
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
Hyperphosphorylated IRAK-1 leaves the receptor complex and interacts with TRAF6.
References
K Ross, L Yang, S Dower, F Volpe, F Guesdon, "Identification of threonine 66 as a functionally critical residue of the", J Biol Chem, 277, 2002,
37414-21.
Reaction
31.1.1.1.2.9 IRAK1-P(n):TRAF6 binds MEKK1
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
TRAF6 binds to MAPK kinase kinases (MEKK1), and the adapter protein ECSIT is thought mediate this interaction.
References
E Kopp, R Medzhitov, J Carothers, C Xiao, I Douglas, CA Janeway, S Ghosh, "ECSIT is an evolutionarily conserved intermediate in the Toll/IL-1
signal transduction pathway", Genes Dev, 13, 1999, 2059-71.
Reaction
Authors
Luo, F, 2005-11-10.
Reviewers
Gay, NJ, 2006-04-24.

Description
Type I interferon (IFN-alpha/beta) production in mammalian cells is a primary anti-viral immune response. The toll like receptor-3 (TLR3)
cascade is activated by the presence of dsRNA. The downstream targets of the TLR3 pathway is the stimulation the interferon response.
References
M Matsumoto, K Funami, H Oshiumi, T Seya, "Toll-like receptor 3: a link between toll-like receptor, interferon and viruses", Microbiol Immunol,
48, 2004, 147-54.
31.1.1.2.1 Viral dsRNA binds the Toll-Like Receptor 3 (TLR3)
Authors
Luo, F, 2005-11-10.
Reviewers
Gay, NJ, 2006-04-24.
Description
Viral double-stranded RNA binds to toll-like receptor 3.
References
L Alexopoulou, AC Holt, R Medzhitov, RA Flavell, "Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3",
Nature, 413, 2001, 732-8.
Reaction
31.1.1.2.2 Viral dsRNA bound TRL3 Recruits TRIF
Authors
Luo, F, 2005-11-10.
Reviewers
Gay, NJ, 2006-04-24.
Description
TIR domain-containing adaptor inducing IFN-beta (TRIF) associates with activated toll-like receptor 3 (TLR3).
References
M Yamamoto, S Sato, H Hemmi, K Hoshino, T Kaisho, H Sanjo, O Takeuchi, M Sugiyama, M Okabe, K Takeda, "Role of adaptor TRIF in the
MyD88-independent toll-like receptor signaling pathway", Science, 301, 2003, 640-3.
Reaction
31.1.1.2.3 Viral dsRNA:TLR3:TRIF Complex Activates RIP1

Authors
Luo, F, 2005-11-10.
Reviewers
Gay, NJ, 2006-04-24.
Description
Receptor-interacting protein 1 (RIP1) mediates the activation of interferon-alpha/beta via intermediate activation of IKK/TBK1 or NFkB pathways.
References
E Meylan, K Burns, K Hofmann, V Blancheteau, F Martinon, M Kelliher, J Tschopp, "RIP1 is an essential mediator of Toll-like receptor 3-induced
NF-kappa B", Nat Immunol, 5, 2004, 503-7.
31.1.1.2.3.1 Viral dsRNA:TLR3:TRIF Complex Recruits RIP1
Authors
Luo, F, 2005-11-10.
Reviewers
Gay, NJ, 2006-04-24.
Description
The Viral dsRNA:TLR3:TRIF complex recruits RIP1
References
Reaction
31.1.1.2.3.2 Viral dsRNA:TLR3:TRIF Complex Releases Activated RIP1
Authors
Luo, F, 2005-11-10.
Reviewers
Gay, NJ, 2006-04-24.
Description
The Viral dsRNA:TLR3:TRIF complex releases activated RIP1
References
Reaction
31.1.1.2.3.3 RIP1 phosphorylates IKKs
Authors
Luo, F, 2005-11-10.
Reviewers
Gay, NJ, 2006-04-24.

Description
At the beginning of this reaction, 2 molecules of 'ATP', and 1 molecule of 'Inhibitor of KappaB kinase (IKK) Complex' are present. At the end of
this reaction, 1 molecule of 'Activated IKK Complex', and 2 molecules of 'ADP' are present.
This reaction is mediated by the 'protein kinase activity' of 'RIP1 (Actived)'.
References
S Doyle, S Vaidya, R O'Connell, H Dadgostar, P Dempsey, T Wu, G Rao, R Sun, M Haberland, R Modlin, G Cheng, "IRF3 mediates a
TLR3/TLR4-specific antiviral gene program", Immunity, 17, 2002, 251-63.
A Civas, P Genin, P Morin, R Lin, J Hiscott, "Promoter organization of the interferon-A genes differentially affects virus-induced expression and
responsiveness to TBK1 and IKKepsilon", J Biol Chem, 281, 2006, 4856-66.
Reaction
31.1.1.2.3.4 Phospho-IKK Complex phosphorylates IkB within the IkB:NFkB Complex
Authors
Luo, F, 2005-11-10.
Reviewers
Gay, NJ, 2006-04-24.
Description
At the beginning of this reaction, 2 molecules of 'ATP', and 1 molecule of 'IkBs:NFkB Complex' are present. At the end of this reaction, 1
molecule of 'NFkB Complex', 1 molecule of 'Phospho-NF-kappaB Inhibitor', and 2 molecules of 'ADP' are present.
This reaction is mediated by the 'protein kinase activity' of 'Activated IKK Complex'.
References
J Gil, J Alcami, M Esteban, "Activation of NF-kappa B by the dsRNA-dependent protein kinase, PKR involves the I kappa B kinase complex",
Oncogene, 19, 2000, 1369-78.
Reaction
31.1.1.2.3.5 Released NFkB complex is transported to the Nucleus
Authors
Luo, F, 2005-11-10.
Reviewers
Gay, NJ, 2006-04-24.
Description
In this reaction, 1 molecule of 'NFkB Complex' is translocated from cytosol to nucleoplasm.
References
Oncogene, 19, 2000, 1369-78.
Reaction
31.1.1.2.4 TRAF6 Mediated Induction of the antiviral cytokine IFN-alpha/beta cascade
Reviewers
Gay, NJ, 2006-04-24.
Description
TRIF recruits TRAF6 via a specific TRAF6-binding sequence and initiates the NF-kB signaling pathway. Recruitment of TRAF6 by TRIF leads to
recruitment of TAB2, TAK1 and PKR followed by release of the whole complex to the cytoplasm and activation of the IKK complex and MAP
kinases. TRAF6 is recruited to the activated TLR receptor complex and is itself activated by IRAK1 that binds to its TRAF domain. The
IRAK1/TRAF6 complex then dissociates from the receptor and associates with TGF-activated kinase 1 (TAK1) and TAK1 binding proteins, TAB1
and TAB2.
References
Z Jiang, M Zamanian-Daryoush, H Nie, AM Silva, BR Williams, X Li, "Poly(I-C)-induced Toll-like receptor 3 (TLR3)-mediated activation of
NFkappa B and MAP kinase is through an interleukin-1 receptor-associated kinase (IRAK)-independent pathway employing the signaling
components TLR3-TRAF6-TAK1-TAB2-PKR ", J Biol Chem, 278, 2003, 16713-9.
RH Arch, RW Gedrich, CB Thompson, "Tumor necrosis factor receptor-associated factors (TRAFs)--a family of adapter proteins that regulates
life and death", Genes Dev, 12, 1998, 2821-30.
MA Lomaga, WC Yeh, I Sarosi, GS Duncan, C Furlonger, A Ho, S Morony, C Capparelli, G Van, S Kaufman, A van der Heiden, A Itie, A
Wakeham, W Khoo, T Sasaki, Z Cao, JM Penninger, CJ Paige, DL Lacey, CR Dunstan, WJ Boyle, DV Goeddel, TW Mak, "TRAF6 deficiency
results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling", Genes Dev, 13, 1999, 1015-24.
A Naito, S Azuma, S Tanaka, T Miyazaki, S Takaki, K Takatsu, K Nakao, K Nakamura, M Katsuki, T Yamamoto, J Inoue, "Severe osteopetrosis,
defective interleukin-1 signalling and lymph node organogenesis in TRAF6-deficient mice", Genes Cells, 4, 1999, 353-62.
31.1.1.2.4.1 TRAF6 is Recruited to the Viral dsRNA:TLR3:TRIF Complex
Reviewers
Gay, NJ, 2006-04-24.
Description
TRAF6 is recruited to the Viral dsRNA:TLR3:TRIF complex
References
Reaction
31.1.1.2.4.2 Formation of the Viral dsRNA:TLR3:TRIF:TRAF6:TAB1:TAB2 Complex
Reviewers
Gay, NJ, 2006-04-24.
Description
At the beginning of this reaction, 1 molecule of 'TAK1', 1 molecule of 'TAB2', 1 molecule of 'TAB1', and 1 molecule of 'Viral
dsRNA:TLR3:TRIF:TRAF6 Complex' are present. At the end of this reaction, 1 molecule of 'Viral dsRNA:TLR3:TRIF:TRAF6:TAK1:TAB1:TAB2
Complex' is present.
References
Reaction
31.1.1.2.4.3 Activation and Release of the TRAF6:Phospho-Tak1:Tab1:Phospho-Tab2 Complex
Reviewers
Gay, NJ, 2006-04-24.

Description
At the beginning of this reaction, 1 molecule of 'Viral dsRNA:TLR3:TRIF:TRAF6:TAK1:TAB1:TAB2 Complex', and 2 molecules of 'ATP' are
present. At the end of this reaction, 1 molecule of 'Activated TRAF6:Phospho-TAK1:TAB1:Phospho-Tab2 Complex', 1 molecule of 'Viral
dsRNA:TLR3:TRIF complex', and 2 molecules of 'ADP' are present.
References
Reaction
31.1.1.2.4.4 Phosphorylation of JNK by the Activated TRAF6:Phospho-TAK1:TAB1:Phospho-Tab2 Complex
Authors
Luo, F, 2005-11-10.
Reviewers
Gay, NJ, 2006-04-24.
Description
MKK4 phosphorylates and activates JNK
References
K Deacon, JL Blank, "Characterization of the mitogen-activated protein kinase kinase 4 (MKK4)/c-Jun NH2-terminal kinase 1 and MKK3/p38
pathways regulated by MEK kinases 2 and 3. MEK kinase 3 activates MKK3 but does not cause activation of p38 kinase in vivo.", J Biol Chem,
272, 1997, 14489-96.
Reaction
31.1.1.2.4.5 TRAF6:Phosho-TAK1:Tab1:PhosphoTAB2 mediated phosphorylation of MAPK1/p38
Authors
Luo, F, 2005-11-10.
Reviewers
Gay, NJ, 2006-04-24.
Description
At the beginning of this reaction, 1 molecule of 'ATP', and 1 molecule of 'MAPK1' are present. At the end of this reaction, 1 molecule of
'MAPK1-P', and 1 molecule of 'ADP' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'protein kinase activity' of 'Activated TRAF6:Phospho-TAK1:TAB1:Phospho-Tab2
Complex'.
References
RJ Lee, C Albanese, RJ Stenger, G Watanabe, G Inghirami, 3rd Haines GK, M Webster, WJ Muller, JS Brugge, RJ Davis, RG Pestell,
"pp60(v-src) induction of cyclin D1 requires collaborative interactions between the extracellular signal-regulated kinase, p38, and Jun kinase
pathways. A role for cAMP response element-binding protein and activating transcription factor-2 in pp60(v-src) signaling in breast cancer cells.",
J Biol Chem, 274, 1999, 7341-50.
Reaction
31.1.1.2.4.6 Phosphorylated MAPK1 phosphorylates ATF-2
Authors
Luo, F, 2005-11-10.
Reviewers
Gay, NJ, 2006-04-24.
Description
At the beginning of this reaction, 1 molecule of 'ATP', and 1 molecule of 'ATF-2' are present. At the end of this reaction, 1 molecule of 'ADP', and
1 molecule of 'ATF-2-P' are present.
This reaction is mediated by the 'protein kinase activity' of 'MAPK1-P'.
References
E Martin-Blanco, "p38 MAPK signalling cascades: ancient roles and new functions", Bioessays, 22, 2000, 637-45.
Reaction
31.1.1.2.4.7 Dimerisation of Activated Protein 1 (AP-1) heterodimer
Authors
Luo, F, 2005-11-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
At the beginning of this reaction, 1 molecule of 'c-Jun-P', and 1 molecule of 'ATF-2-P' are present. At the end of this reaction, 1 molecule of
'AP-1' is present.
References
E Ainbinder, S Bergelson, R Pinkus, V Daniel, "Regulatory mechanisms involved in activator-protein-1 (AP-1)-mediated activation of
glutathione-S-transferase gene expression by chemical agents", Eur J Biochem, 243, 1997, 49-57.
Reaction
31.1.1.2.4.8 TRAF6:Phosho-TAK1:Tab1:PhosphoTAB2 mediated phosphorylation of the IKK Complex
Authors
Luo, F, 2005-11-10.
Reviewers
Gay, NJ, 2006-04-24.

Description
At the beginning of this reaction, 1 molecule of 'ATP', and 1 molecule of 'Inhibitor of KappaB kinase (IKK) Complex' are present. At the end of
this reaction, 1 molecule of 'Activated IKK Complex', and 1 molecule of 'ADP' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'protein kinase activity' of 'Activated TRAF6:Phospho-TAK1:TAB1:Phospho-Tab2
Complex'.
References
RH Arch, RW Gedrich, CB Thompson, "Tumor necrosis factor receptor-associated factors (TRAFs)--a family of adapter proteins that regulates
life and death", Genes Dev, 12, 1998, 2821-30.
Reaction
31.1.1.2.4.9 Phospho-IKK Complex phosphorylates IkB within the IkB:NFkB Complex
Authors
Luo, F, 2005-11-10.
Reviewers
Gay, NJ, 2006-04-24.
Description
At the beginning of this reaction, 2 molecules of 'ATP', and 1 molecule of 'IkBs:NFkB Complex' are present. At the end of this reaction, 1
molecule of 'NFkB Complex', 1 molecule of 'Phospho-NF-kappaB Inhibitor', and 2 molecules of 'ADP' are present.
This reaction is mediated by the 'protein kinase activity' of 'Activated IKK Complex'.
References
Oncogene, 19, 2000, 1369-78.
Reaction
31.1.1.2.4.10 Released NFkB complex is transported to the Nucleus
Authors
Luo, F, 2005-11-10.
Reviewers
Gay, NJ, 2006-04-24.
Description
In this reaction, 1 molecule of 'NFkB Complex' is translocated from cytosol to nucleoplasm.
References
Oncogene, 19, 2000, 1369-78.
Reaction
31.1.1.2.5 Viral dsRNA:TLR3:TRIF Complex Activates TBK1
Reviewers
Gay, NJ, 2006-04-24.

References
GC Sen, SN Sarkar, "Transcriptional signaling by double-stranded RNA: role of TLR3", Cytokine Growth Factor Rev, 16, 2005, 1-14.
31.1.1.2.5.1 TBK1 is Recruited to the Viral dsRNA:TLR3:TRIF Complex
Authors
Luo, F, 2005-11-10.
Reviewers
Gay, NJ, 2006-04-24.
Description
At the beginning of this reaction, 1 molecule of 'ATP', 1 molecule of 'TBK1', and 1 molecule of 'Viral dsRNA:TLR3:TRIF complex' are present. At
the end of this reaction, 1 molecule of 'Viral dsRNA:TLR3:TRIF:TBK1 Complex', and 1 molecule of 'ADP' are present.
This reaction is mediated by the 'protein kinase activity' of 'RIP1 (Actived)'.
References
KA Fitzgerald, SM McWhirter, KL Faia, DC Rowe, E Latz, DT Golenbock, AJ Coyle, SM Liao, T Maniatis, "IKKepsilon and TBK1 are essential
components of the IRF3 signaling pathway", Nat Immunol, 4, 2003, 491-6.
Reaction
31.1.1.2.5.2 Viral dsRNA:TLR3:TRIF:TBK1 complex recruits IRF3

Authors
Luo, F, 2005-11-10.
Reviewers
Gay, NJ, 2006-04-24.
Description
At the beginning of this reaction, 1 molecule of 'IRF3', and 1 molecule of 'Viral dsRNA:TLR3:TRIF:TBK1 Complex' are present. At the end of this
reaction, 1 molecule of 'Viral dsRNA:TLR3:TRIF:TBK1:IRF3 Complex' is present.
References
Reaction
31.1.1.2.5.3 Phosphorylation and Release of IRF3
Authors
Luo, F, 2005-11-10.
Reviewers
Gay, NJ, 2006-04-24.
Description
At the beginning of this reaction, 4 molecules of 'ATP', and 1 molecule of 'Viral dsRNA:TLR3:TRIF:TBK1:IRF3 Complex' are present. At the end
of this reaction, 1 molecule of 'IRF3-P', 1 molecule of 'Viral dsRNA:TLR3:TRIF:TBK1 Complex', and 4 molecules of 'ADP' are present.
This reaction is mediated by the 'protein kinase activity' of 'Viral dsRNA:TLR3:TRIF:TBK1:IRF3 Complex'.
References
Reaction
31.1.1.2.5.4 Dimerization of Phospho-IRF3
Reviewers
Gay, NJ, 2006-04-24.
Description
At the beginning of this reaction, 2 molecules of 'IRF3-P' is present. At the end of this reaction, 1 molecule of 'IRF3-P:IRF3-P' is present.
References
Reaction
31.1.1.2.5.5 Dimerized Phospho-IRF3 is Transported To The Nucleus

Reviewers
Gay, NJ, 2006-04-24.
Description
In this reaction, 1 molecule of 'IRF3-P:IRF3-P' is translocated from cytosol to nucleoplasm.
References
Reaction
Authors
Luo, F, 2005-11-10.
Reviewers
Gale M, Jr, 2006-10-31.
Description
TLR5 is the receptor for flagellin, the protein that forms bacterial flagella. Unlike most other Pathogen-Associated Molecular Patterns (PAMPs),
flagellin does not undergo any posttranslational modifications that would distinguish it from cellular proteins. However, flagellin is extremely
conserved at its amino- and carboxyl-termini, which presumably explains why it was selected as a ligand for innate immune recognition. TLR5 is
expressed on epithelial cells as well as on macrophages and dendritic cells. Expression of TLR5 on intestinal epithelium is polarized such that
TLR5 is expressed only on the basolateral side of the cell, as pathogenic but not commensal microbes cross the epithelial barrier. This ensures
that innate immune responses are confined to pathogenic but not commensal microbes.
References
F Hayashi, KD Smith, A Ozinsky, TR Hawn, EC Yi, DR Goodlett, JK Eng, S Akira, DM Underhill, A Aderem, "The innate immune response to
bacterial flagellin is mediated by Toll-like receptor 5", Nature, 410, 2001, 1099-103.
AT Gewirtz, TA Navas, S Lyons, PJ Godowski, JL Madara, "Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce
epithelial proinflammatory gene expression", J Immunol, 167, 2001, 1882-5.
W Paul, "Fundamental Immunology", Fundamental Immunology, 5, 2004.
31.1.1.3.1 Flagellin binds to TLR5
Reviewers
Gale M, Jr, 2006-10-31.
Description
Fragments of extracelllar flagellin bind the TLR5 homodimer.
References
F Hayashi, KD Smith, A Ozinsky, TR Hawn, EC Yi, DR Goodlett, JK Eng, S Akira, DM Underhill, A Aderem, "The innate immune response to
bacterial flagellin is mediated by Toll-like receptor 5", Nature, 410, 2001, 1099-103.
N McNamara, M Gallup, A Sucher, I Maltseva, D McKemy, C Basbaum, "AsialoGM1 and TLR5 cooperate in flagellin-induced nucleotide
signaling to activate Erk1/2", Am J Respir Cell Mol Biol, 34, 2006, 653-60.
B Beutler, "Inferences, questions and possibilities in Toll-like receptor signalling", Nature, 430, 2004, 257-63.
Jr Smith MF, A Mitchell, G Li, S Ding, AM Fitzmaurice, K Ryan, S Crowe, JB Goldberg, "Toll-like receptor (TLR) 2 and TLR5, but not TLR4, are
required for Helicobacter pylori-induced NF-kappa B activation and chemokine expression by epithelial cells", J Biol Chem, 278, 2003, 32552-60.
Reaction

Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
837-47.
2002, 329-33.
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
2002, 5567-72.
Development, 128, 2001, 4729-36.
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
2002, 5567-72.
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.

Description
References
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
37414-21.
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
31.1.1.4 Toll Like Receptor 7/8 (TLR7/8) Cascade
Authors
Luo, F, 2005-11-10.
Reviewers
Gale M, Jr, 2006-10-31.
Description
RNA can serve as a danger signal, both in its double-stranded form (that is associated with viral infection), as well as single-stranded RNA
(ssRNA). Specifically, guanosine (G)- and uridine (U)-rich ssRNA oligonucleotides derived from human immunodeficiency virus-1 (HIV-1), for
example, stimulate dendritic cells (DC) and macrophages to secrete interferon-alpha and proinflammatory, as well as regulatory, cytokines. This
has been found to be mediated by TLR7, as well as TLR8. Separate studies showed that imidazoquinoline compounds (e.g. imiquimod and
R-848, low-molecular-weight immune response modifiers that can induce the synthesis of interferon-alpha) also exert their effects in a
MyD88-dependent fashion independently through TLR7 and 8.
References
F Heil, H Hemmi, H Hochrein, F Ampenberger, C Kirschning, S Akira, G Lipford, H Wagner, S Bauer, "Species-specific recognition of
single-stranded RNA via toll-like receptor 7 and 8", Science, 303, 2004, 1526-9.
31.1.1.4.1 Ligand binds to TLR7 or TLR8
Authors
Luo, F, 2005-11-10.
Reviewers
Gay, NJ, 2006-04-24.
Description
Endosomal recognition of influenza genomic RNA and imidazoquinoline compounds occurs by means of TLR7 and TLR8.
References
SS Diebold, T Kaisho, H Hemmi, S Akira, C Reis e Sousa, "Innate antiviral responses by means of TLR7-mediated recognition of
single-stranded RNA", Science, 303, 2004, 1529-31.
F Heil, H Hemmi, H Hochrein, F Ampenberger, C Kirschning, S Akira, G Lipford, H Wagner, S Bauer, "Species-specific recognition of
single-stranded RNA via toll-like receptor 7 and 8", Science, 303, 2004, 1526-9.
M Jurk, F Heil, J Vollmer, C Schetter, AM Krieg, H Wagner, G Lipford, S Bauer, "Human TLR7 or TLR8 independently confer responsiveness to
the antiviral compound R-848", Nat Immunol, 3, 2002, 499.
H Hemmi, T Kaisho, O Takeuchi, S Sato, H Sanjo, K Hoshino, T Horiuchi, H Tomizawa, K Takeda, S Akira, "Small anti-viral compounds activate
immune cells via the TLR7 MyD88-dependent signaling pathway", Nat Immunol, 3, 2002, 196-200.
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.

Description
References
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
837-47.
2002, 329-33.
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
2002, 5567-72.
Development, 128, 2001, 4729-36.
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.

Description
References
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
2002, 5567-72.
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
37414-21.
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
Authors
Luo, F, 2005-11-10.
Reviewers
Gale M, Jr, 2006-10-31.
Description
CpG DNA is an unusual Pathogen-Associated Molecular Pattern (PAMP). Cytosine methylation exists in mammalian but not bacterial cells, and
most (but not all) CpG in the mammalian genome is methylated. Therefore, unmethylated CpG DNA may signal the presence of microbial
infection. Evidence of CpG recognition by TLR9 was demonstrated both in human and mouse, and this type of signaling requires its
internalization into late endosomal/lysosomal compartments. TLR9 has been reported to be able to discern different types of CpG motifs, and
therefore that it presumably recognizes CpG DNA directly. It appears that over evolutionary periods, TLR9 molecules expressed by different
species have diverged. This has led to differences in the precise sequence motif (CpG dinucleotide plus flanking regions) that optimally stimulate
the innate immune system of different animals.
References
AM Krieg, AK Yi, S Matson, TJ Waldschmidt, GA Bishop, R Teasdale, GA Koretzky, DM Klinman, "CpG motifs in bacterial DNA trigger direct
B-cell activation", Nature, 374, 1995, 546-9.
H Hemmi, O Takeuchi, T Kawai, T Kaisho, S Sato, H Sanjo, M Matsumoto, K Hoshino, H Wagner, K Takeda, S Akira, "A Toll-like receptor
recognizes bacterial DNA", Nature, 408, 2000, 740-5.
H Hacker, H Mischak, T Miethke, S Liptay, R Schmid, T Sparwasser, K Heeg, GB Lipford, H Wagner, "CpG-DNA-specific activation of
antigen-presenting cells requires stress kinase activity and is preceded by non-specific endocytosis and endosomal maturation", EMBO J, 17,
1998, 6230-40.
F Takeshita, CA Leifer, I Gursel, KJ Ishii, S Takeshita, M Gursel, DM Klinman, "Cutting edge: Role of Toll-like receptor 9 in CpG DNA-induced
activation of human cells", J Immunol, 167, 2001, 3555-8.
S Bauer, CJ Kirschning, H Hacker, V Redecke, S Hausmann, S Akira, H Wagner, GB Lipford, "Human TLR9 confers responsiveness to bacterial
DNA via species-specific CpG motif recognition", Proc Natl Acad Sci U S A, 98, 2001, 9237-42.
F Takeshita, I Gursel, KJ Ishii, K Suzuki, M Gursel, DM Klinman, "Signal transduction pathways mediated by the interaction of CpG DNA with
Toll-like receptor 9", Semin Immunol, 16, 2004, 17-22.
31.1.1.5.1 Engulfed CpG DNA binds to endosomal TLR9
Description
Synthetic oligodeoxynucleotides (ODN) expressing non-methylated CpG motifs patterned after those present in bacterial DNA have
characteristic immunomodulatory effects. CpG DNA is recognized as a pathogen-associated molecular pattern by TLR9, and triggers a rapid
innate immune response.
References
H Wagner, "The immunobiology of the TLR9 subfamily", Trends Immunol, 25, 2004, 381-6.
Reaction
31.1.1.5.2 Rab5-mediated recruitment of class III PI3K to TLR9
Description
TLR9 signaling has the uncommon property of triggering PI3K-mediated cascades via Rab5.
References
F Takeshita, I Gursel, KJ Ishii, K Suzuki, M Gursel, DM Klinman, "Signal transduction pathways mediated by the interaction of CpG DNA with
Toll-like receptor 9", Semin Immunol, 16, 2004, 17-22.
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
837-47.
2002, 329-33.
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
2002, 5567-72.
Development, 128, 2001, 4729-36.
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.

Description
References
2002, 5567-72.
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
37414-21.
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
Toll-like Receptor 4 is a Microbe Associated Molecular Pattern receptor well known for it's sensitivity to Bacterial Lipopolysaccharides (LPS).
LPS is assembled within diverse Gram-negative bacteria, many of which are human or plant pathogens. It is a component of the bacterial cell
wall, and a potent activator of the innate immune response in humans, causing reactions including fever, headache, nausea, diarrhoea, changes
in leukocyte and platelet counts, disseminated intravascular coagulation, multiorgan failure, shock and death. All these reactions are induced by
cytokines and other endogenous mediators which are produced after interaction of the LPS with the humoral and cellular targets of the host. In
macrophages, lipid A (a form of LPS) activation of TLR4 triggers the biosynthesis of diverse mediators of inflammation, such as TNF-alpha and
IL1-beta, and activates the production of costimulatory molecules required for the adaptive immune response. In mononuclear and endothelial
cells, lipid A also stimulates tissue factor production. These events are desirable for clearing local infections, but when these various mediators
and clotting factors are overproduced, they can damage small blood vessels and precipitate shock accompanied by disseminated intravascular
coagulation and multiple organ failure.
31.1.1.6.1 Association of LBP with LPS
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
Lipopolysaccharide-binding protein (LBP) is a 65-kDa plasma protein which acts as a lipid transfer protein for LPS. LBP is an acute-phase
opsonin that binds gram-negative bacteria and bacterial fragments and promote the interaction of coated bacteria with phagocytes.
References
SD Wright, PS Tobias, RJ Ulevitch, RA Ramos, "Lipopolysaccharide (LPS) binding protein opsonizes LPS-bearing particles", J Exp Med, 170,
1989, 1231-41.
Reaction
31.1.1.6.2 LPS transferred from LBP carrier to CD14
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
LBP delivers lipid A from bacteria (or bacterial membrane fragments) to CD14 on the surfaces of animal cells, where it is recognised by Toll-like
receptor 4 (TLR4). Thus, LBP is an opsonin and CD14 is an opsonic receptor for complexes of LPS (or LPS-containing particles such as
bacteria) and LBP. CD14 may assume two isoforms - it may either be secreted into the extracellular compartment, or it may be anchored to the
plasma membrane via at GPI module.
References
MJ Fenton, DT Golenbock, "LPS-binding proteins and receptors", J Leukoc Biol, 64, 1998, 25-32.
31.1.1.6.2.1 GPI-bound CD14 binds LPS
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
At the beginning of this reaction, 1 molecule of 'GPI-anchored form of CD14', and 1 molecule of 'LBP complexed with bacterial LPS' are present.
At the end of this reaction, 1 molecule of 'LPS complexed with GPI-anchored CD14', and 1 molecule of 'LBP' are present.
References
Reaction
31.1.1.6.2.2 Secreted CD14 binds LPS
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
At the beginning of this reaction, 1 molecule of 'Secreted form of CD14', and 1 molecule of 'LBP complexed with bacterial LPS' are present. At
the end of this reaction, 1 molecule of 'LBP', and 1 molecule of 'LPS complexed with secreted CD14' are present.
References
Reaction
31.1.1.6.3 Transfer of LPS onto TLR4 from CD14
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
The Toll-like receptor 4 (TLR4) is a membrane-spanning protein distantly related to the IL1 receptor. Both CD14 and members of the Toll family
contain multiple leucine-rich repeats. In addition, the latter possess a Toll-homology domain in the cytoplasmic tail, which is important in the
generation of a transmembrane signal linked to LPS-induced cell activation. Of all Toll family members, TLR4 is probably the exclusive receptor
for LPS from most Gram negative organisms. It is thought that a signal-competent receptor complex consists of two TLR4 proteins and possibly
two MD2 units as well, binding at least one LPS molecule. The TLR4 pathway triggers the inflammatory responses induced by LPS in a process
that requires the interaction of LPS-bound myeloid differentiation-2 (MD-2) with TLR4.
References
Reaction
31.1.1.6.4 Activated TLR4 signalling
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
The first known downstream component of TLR4 signalling is the adaptor MyD88. An MyD88-independent pathway involving the adaptor
MyD88-adaptor-like (Mal; also known as TIR-domain-containing adaptor protein or TIRAP) has also been established for TLR4 signalling.
MyD88 comprises an N-terminal Death Domain (DD) and a C-terminal TIR, whereas Mal lacks the DD. The TIR homotypic interactions bring
adaptors into contact with the activated TLRs, whereas the DD modules recruit serine/threonine kinases such as
interleukin-1-receptor-associated kinase (IRAK). Recruitment of these protein kinases is accompanied by autophosphorylation, which in turn
results in the interaction of IRAK with TNF-receptor-associated factor 6 (TRAF6). This leads ultimately to activation of the IÃŽÂºB kinase (IKK)
complex, with degradation of the inhibitor IÃŽÂºB and activation of NF-ÃŽÂºB, resulting in the production of pro-inflammatory cytokines.
References
KA Fitzgerald, DC Rowe, DT Golenbock, "Endotoxin recognition and signal transduction by the TLR4/MD2-complex", Microbes Infect, 6, 2004,
1361-7.
SM Sacre, E Andreakos, M Feldmann, BM Foxwell, "Endotoxin signaling in human macrophages: signaling via an alternate mechanism", J
Endotoxin Res, 10, 2004, 445-52.
31.1.1.6.4.1 MyD88 cascade

Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
31.1.1.6.4.1.1 MyD88 and Mal combine with the activated TLR receptor in human
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
837-47.
2002, 329-33.
Reaction
31.1.1.6.4.1.2 IRAK4 combines with activated TLR receptor complexed with Mal:MyD88
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
2002, 5567-72.
Development, 128, 2001, 4729-36.
Reaction
31.1.1.6.4.1.3 IRAK1 combines with activated TLR complexed with IRAK4 and MyD88
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
31.1.1.6.4.1.4 First phosphorylation of IRAK1 by IRAK4 bound to activated TLR
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
2002, 5567-72.
Reaction
31.1.1.6.4.1.5 Second phosphorylation of IRAK1 by IRAK4 bound to activated TLR
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
31.1.1.6.4.1.6 Multiple IRAK1 autophosphorylation steps
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.

Description
References
Reaction
31.1.1.6.4.1.7 Dissociation of IRAK1-P(n) from TLR
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
31.1.1.6.4.1.8 IRAK1-P(n) combines with TRAF6
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
37414-21.
Reaction
31.1.1.6.4.1.9 IRAK1-P(n):TRAF6 binds MEKK1
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
31.1.1.6.4.2 TRAM Cascade
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
The MyD88-independent TRAM-TRIF signalling route activates NF-kB and induces expression of inflammatory cytokines in primary human
macrophages. IRF3 is also activated through the participation of cytosolic TBK1.
References
31.1.1.6.4.2.1 Association of TRAM to activated TLR4
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.

Description
TRIF-related adapter molecule (TRAM, also called TIRP or TICAM-2) is 235 amino acids in length, and its TIR domain is most closely related to
TRIF (and hence its name). Notably, both human and mouse TRAM contain a cysteine (C117 in humans) at the position where other adapters
and TLRs have a conserved proline, although an adjacent proline (P116) is present. TRAM associates with TLR4 and TRIF, as well as the
non-canonical IÃŽÂºB kinases, IKK epsilon, and TBK1, which phosphorylate IRF3. TRAM provides specificity for the MyD88-independent
component of TLR4 signaling.
References
KA Fitzgerald, DC Rowe, DT Golenbock, "Endotoxin recognition and signal transduction by the TLR4/MD2-complex", Microbes Infect, 6, 2004,
1361-7.
M Yamamoto, S Sato, K Mori, K Hoshino, O Takeuchi, K Takeda, "Cutting edge: a novel Toll/IL-1 receptor domain-containing adapter that", J
Immunol, 169, 2002, 6668-72.
M Yamamoto, S Sato, H Hemmi, S Uematsu, K Hoshino, T Kaisho, O Takeuchi, K Takeda, "TRAM is specifically involved in the Toll-like
receptor 4-mediated", Nat Immunol, 4, 2003, 1144-50.
Reaction
31.1.1.6.4.2.2 Association of TRIF to TRAM complexed to activated TLR4
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
TRIF mediates the MyD88-independent pathway from TLR4.

References
M Yamamoto, S Sato, H Hemmi, S Uematsu, K Hoshino, T Kaisho, O Takeuchi, K Takeda, "TRAM is specifically involved in the Toll-like
receptor 4-mediated", Nat Immunol, 4, 2003, 1144-50.
Reaction
31.1.1.6.4.2.3 TBK1-mediated phosphorylation of IRF-3
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
TANK-binding kinase 1 (TBK1) plays a crucial role in the induction of IFN-beta and IFN-inducible genes. TBK1 interacts with TRIF and
phosphorylates IRF-3.
References
KA Fitzgerald, DC Rowe, BJ Barnes, DR Caffrey, A Visintin, E Latz, B Monks, PM Pitha, DT Golenbock, "LPS-TLR4 signaling to IRF-3/7 and
NF-kappaB involves the toll adapters", J Exp Med, 198, 2003, 1043-55.
H Hemmi, O Takeuchi, S Sato, M Yamamoto, T Kaisho, H Sanjo, T Kawai, K Hoshino, K Takeda, "The roles of two IkappaB kinase-related
kinases in lipopolysaccharide and", J Exp Med, 199, 2004, 1641-50.
R Lin, C Heylbroeck, PM Pitha, J Hiscott, "Virus-dependent phosphorylation of the IRF-3 transcription factor regulates nuclear translocation,
transactivation potential, and proteasome-mediated degradation", Mol Cell Biol, 18, 1998, 2986-96.
Reaction
31.1.1.6.4.2.4 Dimerisation of phosphorylated IRF3
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
The interferon regulatory factors (IRF) consist of a growing family of related transcription proteins first identified as regulators of the alpha beta
interferon (IFN-/) gene promoters, as well as the interferon-stimulated response element (ISRE) of some IFN-stimulated genes. IRF-3 is
posttranslationally modified by protein phosphorylation at multiple serine and threonine residues, which are located in the carboxy terminus of
IRF-3. Phosphorylation results in the cytoplasm-to-nucleus translocation of IRF-3, DNA binding, and increased transcriptional activation. This
phosphorylation induces dimerization and association with the coactivators CREB-binding protein/p300, and the resultant complex activates the
target genes in the nucleus.
References
KA Fitzgerald, DC Rowe, BJ Barnes, DR Caffrey, A Visintin, E Latz, B Monks, PM Pitha, DT Golenbock, "LPS-TLR4 signaling to IRF-3/7 and
NF-kappaB involves the toll adapters", J Exp Med, 198, 2003, 1043-55.
H Hemmi, O Takeuchi, S Sato, M Yamamoto, T Kaisho, H Sanjo, T Kawai, K Hoshino, K Takeda, "The roles of two IkappaB kinase-related
kinases in lipopolysaccharide and", J Exp Med, 199, 2004, 1641-50.
R Lin, C Heylbroeck, PM Pitha, J Hiscott, "Virus-dependent phosphorylation of the IRF-3 transcription factor regulates nuclear translocation,
transactivation potential, and proteasome-mediated degradation", Mol Cell Biol, 18, 1998, 2986-96.
M Mori, M Yoneyama, T Ito, K Takahashi, F Inagaki, T Fujita, "Identification of Ser-386 of interferon regulatory factor 3 as critical", J Biol Chem,
279, 2004, 9698-702.
Reaction
31.1.1.6.4.2.5 TRIF:TRAM:TLR4 complex recruits RIP1
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
At the beginning of this reaction, 1 molecule of 'TRIF:TRAM:activated TLR4', and 1 molecule of 'RIP1' are present. At the end of this reaction, 1
molecule of 'RIP1:TRIF:TRAM:activated TLR4' is present.
Reaction
31.1.1.6.4.2.6 TRIF:TRAM:TLR4 complex activates RIP1
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
At the beginning of this reaction, 1 molecule of 'RIP1:TRIF:TRAM:activated TLR4' is present. At the end of this reaction, 1 molecule of
'TRIF:TRAM:activated TLR4', and 1 molecule of 'RIP1 (Actived)' are present.
Reaction
31.1.1.6.5 Chlamydial HSP60 binding to TLR4
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
References
Y Bulut, E Faure, L Thomas, H Karahashi, KS Michelsen, O Equils, SG Morrison, RP Morrison, M Arditi, "Chlamydial heat shock protein 60
activates macrophages and endothelial cells through Toll-like receptor 4 and MD2 in a MyD88-dependent pathway", J Immunol, 168, 2002,
1435-40.
K Ohashi, V Burkart, S Flohe, H Kolb, "Cutting edge: heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex", J
Immunol, 164, 2000, 558-61.
Reaction
31.1.1.7 Toll Like Receptor 2 Cascade
Authors
Reviewers
Description
TLR2 is involved in recognition of peptidoglycan from gram-positive bacteria, bacterial lipoproteins, mycoplasma lipoprotein and mycobacterial
products. It is quite possible that recognition of at least some other TLR2 ligands may be assisted by additional accessory proteins, particularly in
association with TLR1 or TLR6. TLR2 is expressed constitutively on macrophages, dendritic cells, and B cells, and can be induced in some other
cell types, including epithelial cells. TLR1 and TLR6, on the other hand, are expressed almost ubiquitously (Muzio et al. 2000). TLR2 may be a
sensor and inductor of specific defense processes, including oxidative stress and cellular necrosis initially spurred by microbial compounds.
References
M Muzio, D Bosisio, N Polentarutti, G D'amico, A Stoppacciaro, R Mancinelli, C van't Veer, G Penton-Rol, LP Ruco, P Allavena, A Mantovani,
"Differential expression and regulation of toll-like receptors (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells", J
Immunol, 164, 2000, 5998-6004.
31.1.1.7.1 Toll Like Receptor TLR1:TLR2 Cascade
Authors
Reviewers
Description
TLR1 is expressed by monocytes. TLR1 and TLR2 cotranslationally form heterodimeric complexes on the cell surface and in the cytosol. The
TLR2:TLR1 complex recognizes Neisserial PorB and Mycobacterial triacylated lipoproteins and peptides, amongst others, triggering
up-regulation of nuclear factor-kappaB production and apoptotic cascades. Such cooperation between TLR1 and TLR2 on the cell surface of
normal human peripheral blood mononuclear cells, for instance, leads to the activation of pro-inflammatory cytokine secretion (Sandor et al.
2003).
References
F Sandor, E Latz, F Re, L Mandell, G Repik, DT Golenbock, T Espevik, EA Kurt-Jones, RW Finberg, "Importance of extra- and intracellular
domains of TLR1 and TLR2 in NFkappa B signaling", J Cell Biol, 162, 2003, 1099-110.
31.1.1.7.1.1 Ligand bound to TLR1:TLR2
Authors
Reviewers
Description
The TLR2:TLR1 complex recognizes Neisserial PorB and Mycobacterial triacylated lipoproteins and peptides, amongst others.
References
F Sandor, E Latz, F Re, L Mandell, G Repik, DT Golenbock, T Espevik, EA Kurt-Jones, RW Finberg, "Importance of extra- and intracellular
domains of TLR1 and TLR2 in NFkappa B signaling", J Cell Biol, 162, 2003, 1099-110.
AO Aliprantis, RB Yang, MR Mark, S Suggett, B Devaux, JD Radolf, GR Klimpel, P Godowski, A Zychlinsky, "Cell activation and apoptosis by
bacterial lipoproteins through toll-like receptor-2", Science, 285, 1999, 736-9.
P Massari, A Visintin, J Gunawardana, KA Halmen, CA King, DT Golenbock, LM Wetzler, "Meningococcal porin PorB binds to TLR2 and
requires TLR1 for signaling", J Immunol, 176, 2006, 2373-80.
B Beutler, "Inferences, questions and possibilities in Toll-like receptor signalling", Nature, 430, 2004, 257-63.
Reaction
31.1.1.7.1.2 MyD88 cascade
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
837-47.
2002, 329-33.
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
2002, 5567-72.
Development, 128, 2001, 4729-36.
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.

Description
References
2002, 5567-72.
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
37414-21.
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
31.1.1.7.2 Toll Like Receptor TLR6:TLR2 Cascade
Authors
Reviewers
Description
TLR2 and TLR4 recognize different bacterial cell wall components. While TLR4 is trained onto Gram-negative lipopolysaccharide components,
TLR2 - in combination with TLR6 - plays a major role in recognizing peptidoglycan wall products from Gram-positive bacteria, as well as
Mycobacterial diacylated lipopeptides. In particular, TLR6 appears to participate in discriminating the subtle differences between dipalmitoyl and
tripalmitoyl cysteinyl residues (Okusawa et al. 2004).
References
T Okusawa, M Fujita, J Nakamura, T Into, M Yasuda, A Yoshimura, Y Hara, A Hasebe, DT Golenbock, M Morita, Y Kuroki, T Ogawa, K Shibata,
"Relationship between structures and biological activities of mycoplasmal diacylated lipopeptides and their recognition by toll-like receptors 2 and
6", Infect Immun, 72, 2004, 1657-65.
31.1.1.7.2.1 Ligand bound to TLR6:TLR2
Authors
Reviewers
Description
TLR2 - in combination with TLR6 - plays a major role in recognizing peptidoglycan wall products from Gram-positive bacteria, as well as
Mycobacterial diacylated lipopeptides.
References
T Okusawa, M Fujita, J Nakamura, T Into, M Yasuda, A Yoshimura, Y Hara, A Hasebe, DT Golenbock, M Morita, Y Kuroki, T Ogawa, K Shibata,
"Relationship between structures and biological activities of mycoplasmal diacylated lipopeptides and their recognition by toll-like receptors 2 and
6", Infect Immun, 72, 2004, 1657-65.
TK Means, S Wang, E Lien, A Yoshimura, DT Golenbock, MJ Fenton, "Human toll-like receptors mediate cellular activation by Mycobacterium
tuberculosis", J Immunol, 163, 1999, 3920-7.
AO Aliprantis, RB Yang, MR Mark, S Suggett, B Devaux, JD Radolf, GR Klimpel, P Godowski, A Zychlinsky, "Cell activation and apoptosis by
bacterial lipoproteins through toll-like receptor-2", Science, 285, 1999, 736-9.
O Takeuchi, K Hoshino, T Kawai, H Sanjo, H Takada, T Ogawa, K Takeda, S Akira, "Differential roles of TLR2 and TLR4 in recognition of
gram-negative and gram-positive bacterial cell wall components", Immunity, 11, 1999, 443-51.
AM Hajjar, DS O'Mahony, A Ozinsky, DM Underhill, A Aderem, SJ Klebanoff, CB Wilson, "Cutting edge: functional interactions between toll-like
receptor (TLR) 2 and TLR1 or TLR6 in response to phenol-soluble modulin", J Immunol, 166, 2001, 15-9.
R Schwandner, R Dziarski, H Wesche, M Rothe, CJ Kirschning, "Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by
toll-like receptor 2", J Biol Chem, 274, 1999, 17406-9.
Reaction
31.1.1.7.2.2 MyD88 cascade
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
837-47.
2002, 329-33.
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
2002, 5567-72.
Development, 128, 2001, 4729-36.
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.

Description
References
2002, 5567-72.
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
37414-21.
Reaction
Authors
de Bono, B, 2005-08-16.
Reviewers
Gay, NJ, 2006-04-24.
Description
References
Reaction
31.1.1.8 Toll Like Receptor 11 (TLR11) Cascade (Murine)
Authors
de Bono, B, 2006-09-30.
Reviewers
Gale M, Jr, 2006-10-31.
Description
TLR11 is present in mice, but not humans. It is known to recognize uropathogenic Escherichia coli, as well as a profilin-like protein from
Toxoplasma gondii. The TLR11 gene in human contains several stop codons and does not code for a full-length protein. It has been shown that
T. gondii profilin shares significant similarity only with profilin genes from other apicomplexan protozoa, such as the malaria parasite. It is
therefore likely that TLR11 might be involved in the recognition of these parasites. Phylogenetic analysis of all vertebrate TLRs shows clustering
of a set of TLRs that includes the TLR11Ã¢â‚¬"13 and TLR21Ã¢â‚¬"23 subfamilies, which have representatives from mice, fish and frogs.
References
JC Roach, G Glusman, L Rowen, A Kaur, MK Purcell, KD Smith, LE Hood, A Aderem, "The evolution of vertebrate Toll-like receptors", Proc Natl
Acad Sci U S A, 102, 2005, 9577-82.
31.1.1.8.1 Toxomplasma gondii Profilin binds to murine TLR11
Authors
de Bono, B, 2006-09-30.
Reviewers
Gale M, Jr, 2006-10-31.
Description
The protozoan parasite Toxoplasma gondii generates a potent interleukin-12 (IL-12) response in murine DCs that is dependent on MyD88,
through TLR11 binding.
References
FN Lauw, DR Caffrey, DT Golenbock, "Of mice and man: TLR11 (finally) finds profilin", Trends Immunol, 26, 2005, 509-11.
F Yarovinsky, D Zhang, JF Andersen, GL Bannenberg, CN Serhan, MS Hayden, S Hieny, FS Sutterwala, RA Flavell, S Ghosh, A Sher, "TLR11
activation of dendritic cells by a protozoan profilin-like protein", Science, 308, 2005, 1626-9.
Reaction
31.1.2 Complement cascade
Authors
de Bono, B, 2004-08-04.
Reviewers
References
P Gasque, "Complement: a unique innate immune sensor for danger signals", Mol Immunol, 41, 2004, 1089-98.
HJ Muller-Eberhard, "Molecular organization and function of the complement system", Annu Rev Biochem, 57, 1988, 321-47.
M Nonaka, F Yoshizaki, "Evolution of the complement system", Mol Immunol, 40, 2004, 897-902.
BZ Schmidt, HR Colten, "Complement: a critical test of its biological importance", Immunol Rev, 178, 2000, 166-76.
RB Sim, A Laich, "Serine proteases of the complement system", Biochem Soc Trans, 28, 2000, 545-50.
31.1.2.1 Initial triggering of complement

Authors
de Bono, B, 2004-08-04.
Reviewers
Description
Complement activation is due to a cascade of proteolytic steps, performed by serine protease domains in some of the components. Three
different pathways of activation are distinguished triggered by target-bound antibody (the classical pathway); microbial polysaccharide structures
(the lectin pathway); or recognition of other "foreign" surface structures (the alternative pathway) by C3b. All three merge in the pivotal activation
of C3 and, subsequently, of C5 by highly specific enzymatic complexes, the so-called C3/C5 convertases. A complement system with three C3
activation pathways and a common lytic pathway is found only in jawed vertebrates.
References
31.1.2.1.1 Creation of C4 and C2 activators
Authors
de Bono, B, 2004-08-04.
Reviewers
31.1.2.1.1.1 Lectin pathway of complement activation
Authors
de Bono, B, 2004-08-04.
Reviewers
Description
Mannose-binding lectin (MBL), a Ca-dependent (C-type) lectin, initiates the complement cascade after binding to specific carbohydrate patterns
on pathogenic cell surfaces. The MBL polypeptide chain consists of a short N-terminal cysteine-rich region, a collagen-like region comprising 19
Gly-X-Y triplets, a 34-residue hydrophobic stretch, and a C-terminal C-type lectin domain. MBL monomers associate via their cysteine-rich and
collagen-like regions to form homotrimers, and these in turn associate into oligomers. The predominant oligomers found in human serum contain
three (MBL-I) or four (MBL-II) homotrimers (Fujita et al. 2004; Teillet et al. 2005). These oligomers are associated with homodimers of the
MASP2 serine protease (Fujita et al. 2004; Hajela et al. 2002). MBL-II is associated with one or two MASP homodimers (Chen and Wallis 2001,
2004). The carbohydrate recognition domain (CRD) of MBL binds carbohydrates with 3- and 4- OH groups in the pyranose ring, such as
mannose and N-acetyl-D-glucosamine, in the presence of Ca2+. This binding results in a change in conformation of the MBL and activation of
MASP by cleavage (Fujita et al. 2004). MASP2a cleaves C4 to generate C4a and C4b. C4b binds to the bacterial or foreign cell surface via its
thioester bond (Law and Dodds 1997) and binds circulating C2. Bound C2 is then cleaved by MASP2 to yield the C3 convertase C4b-C2a.
MASP1, a serine protease encoded by an alternatively spliced transcript of the same gene that encodes MASP2, can also be activated by
binding of the MBL complex to carbohydrate patterns. MASP-1a cleaves fibrinogen to yield fibrinopeptide B, and cleaves and activates factor
XIII. While MASP1 can also cleave C2, it is not thought to mediate the initial cleavage and activation of C2 in vivo (Chen and Wallis 2004).
MASP-1a may have a role in cleavage of 'dead C3', i.e. C3(H2O) (Hajela et al. 2002).
References
F Teillet, B Dublet, JP Andrieu, C Gaboriaud, GJ Arlaud, NM Thielens, "The two major oligomeric forms of human mannan-binding lectin:
chemical characterization, carbohydrate-binding properties, and interaction with MBL-associated serine proteases", J Immunol, 174, 2005,
2870-7.
CB Chen, R Wallis, "Stoichiometry of complexes between mannose-binding protein and its associated serine proteases. Defining functional units
for complement activation.", J Biol Chem, 276, 2001, 25894-902.
SV Petersen, S Thiel, JC Jensenius, "The mannan-binding lectin pathway of complement activation: biology and disease association", Mol
Immunol, 38, 2001, 133-49.
SK Law, AW Dodds, "The internal thioester and the covalent binding properties of the complement proteins C3 and C4", Protein Sci, 6, 1997,
263-74.
CB Chen, R Wallis, "Two mechanisms for mannose-binding protein modulation of the activity of its associated serine proteases", J Biol Chem,
279, 2004, 26058-65.
K Hajela, M Kojima, G Ambrus, KH Wong, BE Moffatt, J Ferluga, S Hajela, P Gal, RB Sim, "The biological functions of MBL-associated serine
proteases (MASPs)", Immunobiology, 205, 2002, 467-75.
T Fujita, M Matsushita, Y Endo, "The lectin-complement pathway--its role in innate immunity and evolution", Immunol Rev, 198, 2004, 185-202.
31.1.2.1.1.1.1 MBL binds to repetitive carbohydrate structures on the surfaces of viruses, bacteria, fungi, and protozoa
Authors
de Bono, B, 2004-08-04.
Reviewers
Description
Extracellular MBL oligomer complexed with MASP2 binds to a repetitive carbohydrate motif on a target surface to form a MASP2:MBL
oligomer:carbohydrate complex on the surface. Such motifs occur on the surfaces of viruses, bacteria, fungi and protozoa. The affinity of any
one MBL binding site for a carbohydrate ligand is low, but interaction between multiple binding sites on an MBL oligomer and a repetitive
carbohydrate motif on a target surface allow high-avidity binding. The specificity of the MBL binding site (it does not bind glucose or sialic acid)
and the requirement for a repeated target motif may account for the failure of MBL to bind human glycoproteins under normal conditions
(Petersen et al. 2001). This reaction in particular represents the interaction of MBL with bacterial mannose repeats.
References
SV Petersen, S Thiel, JC Jensenius, "The mannan-binding lectin pathway of complement activation: biology and disease association", Mol
Immunol, 38, 2001, 133-49.
O Neth, DL Jack, AW Dodds, H Holzel, NJ Klein, MW Turner, "Mannose-binding lectin binds to a range of clinically relevant microorganisms and
promotes complement deposition", Infect Immun, 68, 2000, 688-93.
Reaction
31.1.2.1.1.1.2 Activation of MBL
Authors
de Bono, B, 2004-08-04.
Reviewers
Description
In this reaction, the MASP2 serine protease moiety of an MBL oligomer:MASP2 complex bound to a target surface becomes catalytically active.
References
T Vorup-Jensen, SV Petersen, AG Hansen, K Poulsen, W Schwaeble, RB Sim, KB Reid, SJ Davis, S Thiel, JC Jensenius, "Distinct pathways of
mannan-binding lectin (MBL)- and C1-complex autoactivation revealed by reconstitution of MBL with recombinant MBL-associated serine
protease-2", J Immunol, 165, 2000, 2093-100.
Reaction
31.1.2.1.1.2 Classical antibody-mediated complement activation
Authors
de Bono, B, 2004-08-04.
Reviewers
Description
C1, the first component of complement is a complex containing three protein species, C1q, C1r, and C1s. C1q is assembled from six identical
subunits each of which consists of three homologous chains (A, B, and C). These chains form a globular domain at the C-terminus, followed by
the "neck" and a coil in the "stalk." The six subunits are held together by the collagenous stalk parts (giving rise to the comparison of C1q with a
"bunch of six tulips"). The stalks also interact with the [C1r:C1s]x2 tetramer assembled in a linear chain. Binding of an antigen to an antibody of
the IgM or IgG class induces a conformational change in the Fc domain of the antibody that allows it to bind to the C1q component of C1. C1
activation requires interaction with two separate Fc domains, so pentavalent IgM antibody is far more efficient at complement activation than IgG
antibody. Antibody binding results in a conformational change in the C1r component of the C1 complex and a proteolytic cleavage of C1r,
activating it. Active C1r then cleaves and activates the C1s component of the C1 complex (Muller-Eberhard 1988).
References
LA Gregory, NM Thielens, GJ Arlaud, JC Fontecilla-Camps, C Gaboriaud, "X-ray structure of the Ca2+-binding interaction domain of C1s.
Insights into the assembly of the C1 complex of complement.", J Biol Chem, 278, 2003, 32157-64.
GJ Arlaud, V Rossi, NM Thielens, C Gaboriaud, B Bersch, JF Hernandez, "Structural and functional studies on C1r and C1s: new insights into
the mechanisms involved in C1 activity and assembly", Immunobiology, 199, 1998, 303-16.
31.1.2.1.1.2.1 Activation of C1R
Authors
de Bono, B, 2004-08-04.
Reviewers
Description
C1 activation requires interaction with two separate Fc domains, so pentavalent IgM antibody is far more efficient at complement activation than
IgG antibody (Muller-Eberhard and Kunkel 1961). Antibody binding results in a conformational change in the C1r component of the C1 complex
and a proteolytic cleavage of C1r, activating it (Ziccardi and Cooper 1976). This reaction is irreversible under physiological conditions.
References
RJ Ziccardi, NR Cooper, "Activation of C1r by proteolytic cleavage", J Immunol, 116, 1976, 504-9.
HJ Muller-Eberhard, HG Kunkel, "Isolation of a thermolabile serum protein which precipitates gamma-globulin aggregates and participates in
immune hemolysis", Proc Soc Exp Biol Med, 106, 1961, 291-5.
Reaction
31.1.2.1.1.2.2 Activation of C1S
Authors
de Bono, B, 2004-08-04.
Reviewers
Description
In this irreversible reaction, the activated C1r subunit of the C1:antibody:antigen complex cleaves the C1s subunit of the complex, activating it in
turn (Ziccardi and Cooper 1976).
References
RJ Ziccardi, NR Cooper, "Physicochemical and functional characterization of the C1r subunit of the first complement component", J Immunol,
116, 1976, 496-503.
Reaction
31.1.2.1.2 Conversion of C4 into C4a and C4b
Authors
de Bono, B, 2004-08-04.
Reviewers
Description
C4 is cleaved into the small C4a and the large C4b fragment (termed nascent C4b), which undergoes a gross conformational change. The
internal thioester in C4b becomes exposed and able to form covalent bonds with surrounding molecules (Law and Dodds 1997). This
irreversible, extracellular reaction can be catalyzed by activated MBL, generated through the lectin pathway of complement activation (Fujita et
al. 2004; Hajela et al. 2002), and by activated C1, generated through the classical pathway (Muller-Eberhard and Lepow 1965).
References
SK Law, AW Dodds, "The internal thioester and the covalent binding properties of the complement proteins C3 and C4", Protein Sci, 6, 1997,
263-74.
HJ Muller-Eberhard, IH Lepow, "C'1 esterase effect on activity and physicochemical properties of the fourth component of complement", J Exp
Med, 121, 1965, 819-33.
Reaction
31.1.2.1.3 Conversion of C2 into C2a and C2b
Authors
de Bono, B, 2004-08-04.
Reviewers
Description
C2 is cleaved into the large C2a and the small C2b fragment. This irreversible, extracellular reaction can be catalyzed by activated MBL,
generated through the lectin pathway of complement activation (Vorup-Jensen et al. 2000), and by activated C1, generated through the classical
pathway (Nasagawa and Stroud 1977).
References
T Vorup-Jensen, SV Petersen, AG Hansen, K Poulsen, W Schwaeble, RB Sim, KB Reid, SJ Davis, S Thiel, JC Jensenius, "Distinct pathways of
mannan-binding lectin (MBL)- and C1-complex autoactivation revealed by reconstitution of MBL with recombinant MBL-associated serine
protease-2", J Immunol, 165, 2000, 2093-100.
HJ Muller-Eberhard, MJ Polley, MA Calcott, "Formation and functional significance of a molecular complex derived from the second and the
fourth component of human complement", J Exp Med, 125, 1967, 359-80.
S Nagasawa, RM Stroud, "Cleavage of C2 by C1s into the antigenically distinct fragments C2a and C2b: demonstration of binding of C2b to
C4b", Proc Natl Acad Sci U S A, 74, 1977, 2998-3001.
Reaction
31.1.2.1.4 Formation of C3 convertase (C4b:C2a complex)
Authors
de Bono, B, 2004-08-04.
Reviewers
Description
C4b and C2a form a complex termed the classical pathway C3 convertase (Muller-Eberhard et al. 1967). C2a that fails to bind C4b is rapidly
inactivated.
References
HJ Muller-Eberhard, MJ Polley, MA Calcott, "Formation and functional significance of a molecular complex derived from the second and the
fourth component of human complement", J Exp Med, 125, 1967, 359-80.
Reaction
31.1.2.1.5 Alternative complement activation
Authors
de Bono, B, 2004-08-04.
Reviewers
Description
The proteins participating in alternative pathway activation are C3 (and C3b), the factors B, D, and properdin. In the first place, alternative
pathway activation is a positive feedback mechanism to increase C3b. When C3b binds covalently to sugars on a cell surface, it can become
protected. Then Factor B binds to C3b. In the presence of Factor D, bound Factor B is cleaved to Ba and Bb. Bb contains the active site for a C3
convertase. Properdin then binds to C3bBb to stabilize the C3bBb convertase on cell surface leading to cleavage of C3. Finally, a C3bBb3b
complex forms and this is a C5 convertase.
References
DT Fearon, "Activation of the alternative complement pathway", CRC Crit Rev Immunol, 1, 1979, 1-32.
31.1.2.1.5.1 Spontaneous hydrolysis of C3 thioester
Authors
de Bono, B, 2004-08-04.
Reviewers
Description
The thioester linkage between cysteine residue 1010 and glutamate residue 1013 in the alpha chain of complement factor 3 (C3) can
spontaneously hydrolyze, yielding so-called C3(H2O) (Tack et al. 1980; Pangburn and Muller-Eberhard 1980; Pangburn et al. 1981).
References
MK Pangburn, HJ Muller-Eberhard, "Relation of putative thioester bond in C3 to activation of the alternative pathway and the binding of C3b to
biological targets of complement", J Exp Med, 152, 1980, 1102-14.
BF Tack, RA Harrison, J Janatova, ML Thomas, JW Prahl, "Evidence for presence of an internal thiolester bond in third component of human
complement", Proc Natl Acad Sci U S A, 77, 1980, 5764-8.
MK Pangburn, RD Schreiber, HJ Muller-Eberhard, "Formation of the initial C3 convertase of the alternative complement pathway. Acquisition of
C3b-like activities by spontaneous hydrolysis of the putative thioester in native C3.", J Exp Med, 154, 1981, 856-67.
Reaction
31.1.2.1.5.2 Factor B binds to Complement factor 3(H2O) (C3(H2O))
Authors
de Bono, B, 2004-08-04.
Reviewers
Description
Complement Factor 3(H2O) (C3(H2O)), the product of the spontaneous hydrolysis of the thioester form of C3, can bind to Factor B to form a
soluble complex (Schreiber et al. 1978).
References
RD Schreiber, MK Pangburn, PH Lesavre, HJ Muller-Eberhard, "Initiation of the alternative pathway of complement: recognition of activators by
bound C3b and assembly of the entire pathway from six isolated proteins", Proc Natl Acad Sci U S A, 75, 1978, 3948-52.
Reaction
31.1.2.1.5.3 Factor D cleaves C3(H2O)-bound Factor B
Authors
de Bono, B, 2004-08-04.
Reviewers
Description
Factor D, a constitutively active serine protease found in trace amounts in the blood, cleaves a specific Arg-Lys bond in the Factor B component
of the soluble C3(H2O):Factor B complex, yielding C3(H2O):Factor Bb and an inactive polypeptide, Factor Ba (Fearon and Austin 1975; Lesavre
and Muller-Eberhard 1978; Lesavre et al. 1979; Schreiber et al. 1978). The Factor Bb component of the C3(H2O):Factor Bb catalyzes the
hydrolysis of soluble C3 complexes to produce C3b complex and C3a polypeptide. This reaction is not known to be directly coupled to the
association of the newly generated C3b complexes with a cell surface. Rather, surface association appears to stabilize C3b, which otherwise is
rapidly, spontaneously inactivated (Muller-Eberhard 1988).
References
PH Lesavre, TE Hugli, AF Esser, HJ Muller-Eberhard, "The alternative pathway C3/C5 convertase: chemical basis of factor B activation", J
Immunol, 123, 1979, 529-34.
DT Fearon, KF Austen, "Initiation of C3 cleavage in the alternative complement pathway", J Immunol, 115, 1975, 1357-61.
PH Lesavre, HJ Muller-Eberhard, "Mechanism of action of factor D of the alternative complement pathway", J Exp Med, 148, 1978, 1498-509.
Reaction
31.1.2.1.5.4 Properdin stabilizes C3b:Bb bound to cell surfaces
Authors
de Bono, B, 2004-08-04.
Reviewers
Description
Properdin binds and stabilizes C3b:Bb complexes. It is found in the blood as a mixture of multivalent oligomers: 30% dimers, 45% trimers, 10%
tetramers, and 15% higher oligomers. Monomers associate with one another in a head-to-tail arrangement, producing closed circular structures
(Smith et al. 1984). These features suggest that the properdin oligomer associated with a C3b:Bb complex on a surface such as a cell
membrane can facilitate recruitment of additional C3b:Bb complexes to the site (Farries et al. 1988; Hourcade 2006).
References
RG Medicus, O Gotze, HJ Muller-Eberhard, "Alternative pathway of complement: recruitment of precursor properdin by the labile C3/C5
convertase and the potentiation of the pathway", J Exp Med, 144, 1976, 1076-1093.
DE Hourcade, "The role of properdin in the assembly of the alternative pathway C3 convertases of complement", J Biol Chem, 281, 2006,
2128-32.
CA Smith, MK Pangburn, CW Vogel, HJ Muller-Eberhard, "Molecular architecture of human properdin, a positive regulator of the alternative
pathway of complement", J Biol Chem, 259, 1984, 4582-8.
TC Farries, PJ Lachmann, RA Harrison, "Analysis of the interactions between properdin, the third component of complement (C3), and its
physiological activation products", Biochem J, 252, 1988, 47-54.
Reaction
31.1.2.1.5.5 Formation of alternate C5 convertase

Authors
de Bono, B, 2004-08-04.
Reviewers
Description
The complex of C3b:Factor Bb, stabilized on a cell surface by properdin, catalyzes the cleavage of C3 to yield C3b and C3a. The C3b is
recruited to the C3b:Factor B complex through its interaction with properdin (Daha et al. 1976; Medicus et al. 1976; Hourcade 2006).
References
RG Medicus, O Gotze, HJ Muller-Eberhard, "Alternative pathway of complement: recruitment of precursor properdin by the labile C3/C5
convertase and the potentiation of the pathway", J Exp Med, 144, 1976, 1076-1093.
DE Hourcade, "The role of properdin in the assembly of the alternative pathway C3 convertases of complement", J Biol Chem, 281, 2006,
2128-32.
MR Daha, DT Fearon, KF Austen, "C3 requirements for formation of alternative pathway C5 convertase", J Immunol, 117, 1976, 630-4.
Reaction
31.1.2.1.5.6 Cleavage of C3 by C3 convertase
Authors
de Bono, B, 2004-08-04.
Reviewers
References
Reaction
31.1.2.1.5.7 C3(H2O):Factor Bb-mediated C3 cleavage leads to C3b deposition on a target cell surface
Description
The Factor Bb component of the C3(H2O):Factor Bb catalyzes the hydrolysis of soluble C3 complexes to produce C3b complex and C3a
polypeptide. This reaction is not known to be directly coupled to the association of the newly generated C3b complexes with a cell surface.
Rather, surface association appears to stabilize C3b, which otherwise is rapidly, spontaneously inactivated (Muller-Eberhard 1988).
References
Reaction
31.1.2.1.5.8 Factor B binds to surface-associated C3b
Description
C3b on a surface binds Factor B from solution to form a complex (Schreiber et al. 1978; Muller-Eberhard 1988).
References
Reaction
31.1.2.1.5.9 Factor D cleaves C3b-bound Factor B
Description
Factor D, a constitutively active serine protease found in trace amounts in the blood, cleaves a specific Arg-Lys bond in the Factor B component
of the cell surface-associated C3b:Factor B complex, yielding C3b:Factor Bb on the surface and releasing an inactive polypeptide, Factor Ba
(Lesavre and Muller-Eberhard 1978; Lesavre et al. 1979; Schreiber et al. 1978).
References
PH Lesavre, TE Hugli, AF Esser, HJ Muller-Eberhard, "The alternative pathway C3/C5 convertase: chemical basis of factor B activation", J
Immunol, 123, 1979, 529-34.
Reaction
31.1.2.2 Terminal pathway of complement
Authors
de Bono, B, 2004-08-04.
Reviewers
Description
After cleavage of C5, C5b undergoes conformational changes and exposes a binding site for C6. C5b6 binds C7 resulting in the exposure of
membrane binding sites and incorporation into target membranes. The membrane-bound C5b-7 complex can then bind C8. C5b-8 acts as a
polymerizing agent for C9. The first C9 bound to C5b-8 undergoes major structural changes enabling formation of an elongated molecule and
allows binding of additional C9 molecules and insertion of C9 cylinders into the target membrane. The number of C9 molecules varies from 1-12
in the membrane, although polymers containing up to fifteen C9 molecules are also possible.
References
31.1.2.2.1 Formation of C5b:C6 complex
Authors
de Bono, B, 2004-08-04.
Reviewers
References
Reaction
31.1.2.2.2 Formation of C5b:C6:C7 complex
Authors
de Bono, B, 2004-08-04.
Reviewers
References
Reaction
31.1.2.2.3 C7 allows complex to insert into membrane under attack

Authors
de Bono, B, 2004-08-04.
Reviewers
References
Reaction
31.1.2.2.4 Formation of C5b:C6:C7:C8 complex
Authors
de Bono, B, 2004-08-04.
Reviewers
References
Reaction
31.1.2.2.5 Creation of the Membrane Attack Complex (MAC)
Authors
de Bono, B, 2004-08-04.
Reviewers
Reaction
31.1.2.3 Activation of C3 and C5
Authors
de Bono, B, 2004-08-04.
Reviewers
Description
The 3 pathways of complement activation converge on the cleavage of C3 by C3 convertases. C3 convertase cleaves C3 into C3a and C3b - a
central step of complement activation. C3a remains in the fluid phase and acts as an anaphylatoxin, whereas C3b can bind to C3 convertases to
form C5 convertase, can act as an opsonin, or is degraded into fragments which cannot form an active convertase.
31.1.2.3.1 Cleavage of C3 by C3 convertase

Authors
de Bono, B, 2004-08-04.
Reviewers
References
Reaction
31.1.2.3.2 Formation of classic C5 convertase
Authors
de Bono, B, 2004-08-04.
Reviewers
References
N Rawal, MK Pangburn, "Formation of high affinity C5 convertase of the classical pathway of complement", J Biol Chem, 278, 2003, 38476-83.
Reaction
31.1.2.3.3 Activation of C5
Authors
de Bono, B, 2004-08-04.
Reviewers
Description
The same complexes as for C3 activation are employed for the cleavage of C5. C3 convertases with an additional C3b molecule covalently
deposited in the immediate vicinity form the C5 convertases C3b,Bb,C3b and C4b,2a,3b, respectively. The second C3b acts like an anvil for C5:
it interacts with C5 and presents C5 in the correct conformation for cleavage by the C2a or Bb enzyme.
References
Reaction
31.2 Immunoregulatory interactions between a Lymphoid and a

non-Lymphoid cell
Authors
de Bono, B, 2007-07-08.
Reviewers
Description
A number of receptors and cell adhesion molecules play a key role in modifying the response of cells of lymphoid origin (such as B-, T- and NK
cells) to self and tumor antigens, as well as to pathogenic organisms.
Molecules such as KIRs and LILRs form part of a crucial surveillance system that looks out for any derangement, usually caused by cancer or
viral infection, in MHC Class I presentation. Somatic cells are also able to report internal functional impairment by displaying surface stress
markers such as MICA. The presence of these molecules on somatic cells is picked up by C-lectin NK immune receptors.
Lymphoid cells are able to regulate their location and movement in accordance to their state of activation, and home in on tissues expressing the
appropriate complementary ligands. For example, lymphoid cells may fine tune the presence and concentration of adhesion molecules belonging
to the IgSF, Selectin and Integrin class that interact with a number of vascular markers of inflammation.
Furthermore, there are a number of avenues through which lymphoid cells may interact with antigen. This may be presented directly to a specific
T-cell receptor in the context of an MHC molecule. Antigen-antibody complexes may anchor to the cell via a small number of lymphoid-specific
Fc receptors that may, in turn, influence cell function further. Activated complement factor C3d binds to both antigen and to cell surface receptor
CD21. In such cases, the far-reaching influence of CD19 on B-lymphocyte function is tempered by its interaction with CD21.
References
YR Carrasco, FD Batista, "B cell recognition of membrane-bound antigen: an exquisite way of sensing ligands", Curr Opin Immunol, 18, 2006,
286-91.
S Cemerski, A Shaw, "Immune synapses in T-cell activation", Curr Opin Immunol, 18, 2006, 298-304.
SK Bromley, WR Burack, KG Johnson, K Somersalo, TN Sims, C Sumen, MM Davis, AS Shaw, PM Allen, ML Dustin, "The immunological
synapse", Annu Rev Immunol, 19, 2001, 375-96.
S Nedvetzki, S Sowinski, RA Eagle, J Harris, F Vely, D Pende, J Trowsdale, E Vivier, S Gordon, DM Davis, "Reciprocal regulation of human
natural killer cells and macrophages associated with distinct immune synapses", Blood, 109, 2007, 3776-85.
31.2.1 VCAM-1interacts with VLA-4
Authors
de Bono, B, 2007-07-08.
Reviewers
Description
Integrins play a central role in mediating lymphocyte adhesion to a number of surfaces. LFA-1 interacts with ICAMs 1-3 that are typically
expressed on other immune system cells. ICAM-4 also interacts with LFA-1, and is known to be expressed on telencepahlic neurons.
VCAM-1 regulates lymphocyte adhesion to activated endothelial cells via Very Late Antigen-4 (VLA-4).
To function in a circulating mode, leukocytes express LFA-1 and VLA-4 in a low ligand binding capacity. When leukocytes reach sites of
imflammation, these integrins are switched to a higher binding state to guide the complex process of transmigration, tethering, rolling, arrest,
adhesion and shape change.
Signal cascades between LFA-1 and VLA-4 may cross-talk affecting binding affinities in a reciprocal fashion.
References
H Yusuf-Makagiansar, ME Anderson, TV Yakovleva, JS Murray, TJ Siahaan, "Inhibition of LFA-1/ICAM-1 and VLA-4/VCAM-1 as a therapeutic
approach to inflammation and autoimmune diseases", Med Res Rev, 22, 2002, 146-67.
EY Jones, K Harlos, MJ Bottomley, RC Robinson, PC Driscoll, RM Edwards, JM Clements, TJ Dudgeon, DI Stuart, "Crystal structure of an
integrin-binding fragment of vascular cell adhesion molecule-1 at 1.8 A resolution.", Nature, 373, 1995, 539-44.
Reaction
31.2.2 TCR complex interacts with peptide antigen-presenting MHC Class I
Authors
de Bono, B, 2007-07-08.
Reviewers
Description
T cells distinguish foreign material from self through presentation of fragments of the antigen by the MHC cell surface receptors. Only if an MHC
molecule presents an appropriate antigenic peptide will a cellular immune response be triggered. The orchestration of recognition and signaling
events, from the initial recognition of antigenic peptides to the lysis of the target cell, is performed in a localized environment on the T cell, called
the immunological synapse, and requires the coordinated activities of several T-Cell Receptor (TCR)-associated molecules. This particular
reaction depicts the interaction of the TCR with MHC Class I molecules on somatic cell, requiring the support of CD3 and CD8 proteins.
References
KL Arnett, SC Harrison, DC Wiley, "Crystal structure of a human CD3-epsilon/delta dimer in complex with a UCHT1 single-chain antibody
fragment", Proc Natl Acad Sci U S A, 101, 2004, 16268-73.
L Kjer-Nielsen, MA Dunstone, L Kostenko, LK Ely, T Beddoe, NA Mifsud, AW Purcell, AG Brooks, J McCluskey, J Rossjohn, "Crystal structure of
the human T cell receptor CD3 epsilon gamma heterodimer complexed to the therapeutic mAb OKT3", Proc Natl Acad Sci U S A, 101, 2004,
7675-80.
MG Rudolph, RL Stanfield, IA Wilson, "How TCRs bind MHCs, peptides, and coreceptors", Annu Rev Immunol, 24, 2006, 419-66.
Reaction
31.2.3 KIR2DL2/3 interacting with HLA-C group 1 (Cw4)
Authors
de Bono, B, 2007-07-08.
Reviewers
Description
A hallmark of human NK cells is the expression of HLA class I-specific killer-cell immunoglobulin-like receptors (KIR). KIRs are not only variably
expressed on the level of single NK cells but they are also highly polymorphic and polygenic (i.e. the gene content of the KIR cluster varies from
individual to individual).
There are 15 functional KIR genes known to date, 11 encoding receptors with two immunoglobulin domains (KIR2D genes) and 4 with three
domains (KIR3D genes). Inhibitory KIR genes are characterized by long cytoplasmic tails featuring immunoreceptor tyrosine-based inhibitory
motifs (ITIM), which upon engagement transmit inhibitory signals leading to the general shutdown of NK cell effector functions. There are six
inhibitory KIRs with clearly defined specificities, all of the inhibitory kind and all for HLA class I allotypes: KIR2DL2 and KIR2DL3 for HLA-C
group 1, KIR2DL1 for HLA-C group 2, KIR3DL1 for HLA-B (Bw4 epitope), KIR3DL2 with HLA-A3 and KIR2DL4 with HLA-G.
In contrast, stimulatory KIR have short cytoplasmic tails lacking ITIM, but have a charged amino acid in the transmembrane region that provides
a docking site for the activating adapter molecule DAP12. KIR2DS1 is known to bind HLA-C group 2 and KIR2DS2 binds HLA-C group 1.
References
C Vilches, P Parham, "KIR: diverse, rapidly evolving receptors of innate and adaptive immunity", Annu Rev Immunol, 20, 2002, 217-51.
M Uhrberg, "The KIR gene family: life in the fast lane of evolution", Eur J Immunol, 35, 2005, 10-5.
JC Boyington, PD Sun, "A structural perspective on MHC class I recognition by killer cell immunoglobulin-like receptors", Mol Immunol, 38, 2002,
1007-21.
Reaction
31.2.4 NKG2D homodimer interacting with ligands
Authors
de Bono, B, 2007-07-08.
Reviewers
Description
NKG2D is an activating immunoreceptor. By engaging NKG2D, HlA Class I-like molecules such as MICA, MICB, ULBP1-4 and RAE-1 provide
powerful costimulation for NK cells and T-cells and can determine the magnitude and outcome of certain effector functions. NKG2D ligands are
upregulated on the surfaces of cells under conditions of stress, for example infection or tumorigenesis, and therefore act as molecular flags to
the immune system that something is wrong.
References
L Deng, RA Mariuzza, "Structural basis for recognition of MHC and MHC-like ligands by natural killer cell receptors", Semin Immunol, 18, 2006,
159-66.
RA Eagle, JA Traherne, O Ashiru, MR Wills, J Trowsdale, "Regulation of NKG2D ligand gene expression", Hum Immunol, 67, 2006, 159-69.
RK Strong, "Asymmetric ligand recognition by the activating natural killer cell receptor NKG2D, a symmetric homodimer", Mol Immunol, 38,
2002, 1029-37.
F Borrego, J Kabat, DK Kim, L Lieto, K Maasho, J Pena, R Solana, JE Coligan, "Structure and function of major histocompatibility complex
(MHC) class I specific receptors expressed on human natural killer (NK) cells", Mol Immunol, 38, 2002, 637-60.
Reaction
31.2.5 CD96 binds Nectin-like-5
Authors
de Bono, B, 2007-07-08.
Reviewers
Description
NK cells express adhesion molecules that allow interaction with their tumour targets, promoting their lysis.
For instance, the activating receptor CD226 is known to be involved in cytotoxic lymphocyte formation, as well as platelet adhesion to the
endothelium. The cytoplasmic domain of CD226 contains binding motifs for members of the band 4.1 family of proteins, and for members of the
membrane-associated guanylate kinase homolog (MAGUK) family. These proteins connect the CD226 receptor to the cytoskeleton and may
promote clustering with LFA-1 integrin (also discussed in this pathway), which is known to participate in CD226's signaling cascade. CD226
plays a role in transendothelial migration, where it facilitates adherence to endothelial cells and migration between cell junctions.
Nectin-2 binds CD226. It is ubiquitously expressed in cells of various tissues, especially in epithelial cells, neurons and fibroblasts. Like many
other nectin and Necl proteins, nectin-2 serves as a viral entry receptor for alpha-herpesviruses including herpes simplex virus (HSV-1 and
HSV-2). The other CD226 ligand, Necl-5, was initially identified as a receptor for poliovirus.
CD96, another ligand for Necl-5, is strongly upregulated in activated NK cells.
CRTAM is similarly up-regulated, and has been shown to to bind Necl-2, promoting NK cell cytotoxicity towards otherwise poorly immunogenic
targets.
References
2006, 359-66.
A Fuchs, M Cella, E Giurisato, AS Shaw, M Colonna, "Cutting edge: CD96 (tactile) promotes NK cell-target cell adhesion by interacting with the
poliovirus receptor (CD155)", J Immunol, 172, 2004, 3994-8.
Reaction
31.2.6 MadCAM interacts with Integrin alpha-4 beta-7
Authors
de Bono, B, 2007-07-08.
Reviewers
Description
Mucosal addressin cell adhesion molecule (MAdCAM-1) is present in the endothelium of mucosa, and binds alpha-4 beta-7 integrin and
L-selectin, regulating both the passage and retention of leukocytes in mucosal tissues. MAdCAM-1 has been shown to be present as a
homodimer.
References
J Dando, KW Wilkinson, S Ortlepp, DJ King, RL Brady, "A reassessment of the MAdCAM-1 structure and its role in integrin recognition", Acta
Crystallogr D Biol Crystallogr, 58, 2002, 233-41.
J Wang, TA Springer, "Structural specializations of immunoglobulin superfamily members for adhesion to integrins and viruses", Immunol Rev,
163, 1998, 197-215.
Reaction
31.2.7 LILRs interact with MHC Class I
Authors
de Bono, B, 2007-07-08.
Reviewers
Description
Leukocyte immunoglobulin (Ig)-like receptors [LILRs, also known as Ig-like transcripts (ILTs)] are a family of inhibitory and stimulatory receptors
encoded within the leukocyte receptor complex and are expressed by immune cell types of both myeloid and lymphoid lineage. Several
members of the LILR family recognize major histocompatibility complex class I. The immunomodulatory role of LILR receptors indicates that they
may exert an influence on signaling pathways of both innate and adaptive immune systems.
Signaling mechanisms are employed that are similar to the ones adopted by the closely related killer cell inhibitory receptors (KIRs). ITIMs
recruit inhibitory phosphatases that dephosphorylate ITIM and ITAM domains in order to influence intracellular signaling cascades. In contrast,
activating LILRs, which lack any signaling domains of their own, rely on association with an adaptor protein such as FceRI-gamma to transmit
their signal through its intracellular ITAMs.
References
D Brown, J Trowsdale, R Allen, "The LILR family: modulators of innate and adaptive immune pathways in health and disease", Tissue Antigens,
64, 2004, 215-25.
Reaction
31.2.8 Ligands bind L-selectin
Authors
de Bono, B, 2007-07-08.
Reviewers
Description
L-selectin plays a major role in leukocyte traffic through lymph node high endothelial venules.
Both MAdCAM and GlyCAM-1 are major L-selectin ligands produced by these venules and mediate leukocyte rolling, particularly in lymphocytes.
They are also expressed in mammary tissue and play an important role in the transfer of immune cells into milk secretions.
The adhesive properties of CD34 and its potential role in homing lymphocytes to lymphoid tissues mimics the mechanims leukocytes adopt to
travel to inflammatory sites.
References
O Dwir, F Shimron, C Chen, MS Singer, SD Rosen, R Alon, "GlyCAM-1 supports leukocyte rolling in flow: evidence for a greater dynamic
stability of L-selectin rolling of lymphocytes than of neutrophils", Cell Adhes Commun, 6, 1998, 349-70.
T Nishimura, "Expression of potential lymphocyte trafficking mediator molecules in the mammary gland", Vet Res, 34, 2003, 3-10.
GU Gangenahalli, VK Singh, YK Verma, P Gupta, RK Sharma, R Chandra, PM Luthra, "Hematopoietic stem cell antigen CD34: role in adhesion
or homing", Stem Cells Dev, 15, 2006, 305-13.
Reaction
31.2.9 ICAMs 1-4 bind to Integrin LFA-1
Authors
de Bono, B, 2007-07-08.
Reviewers
Description
Integrins play a central role in mediating lymphocyte adhesion to a number of surfaces. LFA-1 interacts with ICAMs 1-3 that are typically
expressed on other immune system cells. ICAM-4 also interacts with LFA-1, and is known to be expressed on telencepahlic neurons.
VCAM-1 regulates lymphocyte adhesion to activated endothelial cells via Very Late Antigen-4 (VLA-4).
To function in a circulating mode, leukocytes express LFA-1 and VLA-4 in a low ligand binding capacity. When leukocytes reach sites of
imflammation, these integrins are switched to a higher binding state to guide the complex process of transmigration, tethering, rolling, arrest,
adhesion and shape change.
Signal cascades between LFA-1 and VLA-4 may cross-talk affecting binding affinities in a reciprocal fashion.
References
H Yusuf-Makagiansar, ME Anderson, TV Yakovleva, JS Murray, TJ Siahaan, "Inhibition of LFA-1/ICAM-1 and VLA-4/VCAM-1 as a therapeutic
approach to inflammation and autoimmune diseases", Med Res Rev, 22, 2002, 146-67.
Reaction
31.2.10 NKG2A-CD94 heterdimer interacts with HLA-E
Authors
de Bono, B, 2007-07-08.
Reviewers
Description
After interaction with its ligand HLA-E, which is expressed on normal cells, the C-type lectin inhibitory receptor CD94/NKG2A suppresses
activation signaling processes. CD94/NKG2A receptors continuously recycle from the cell surface through endosomal compartments and back
again in a process that requires energy and the cytoskeleton. This steady state process appears to be largely unaffected by exposure to ligand.
References
F Borrego, M Masilamani, J Kabat, TB Sanni, JE Coligan, "The cell biology of the human natural killer cell CD94/NKG2A inhibitory receptor", Mol
Immunol, 42, 2005, 485-8.
MW Sawicki, N Dimasi, K Natarajan, J Wang, DH Margulies, RA Mariuzza, "Structural basis of MHC class I recognition by natural killer cell
receptors", Immunol Rev, 181, 2001, 52-65.
Reaction
31.2.11 Epithelial cadherin binds to KLRG1
Authors
de Bono, B, 2007-07-08.
Reviewers
Description
The lectin-like NK cell receptor KLRG1 binds to cadherins on epithelial cells and transmits inhibitory signals to the leukocyte.
References
2006, 359-66.
Source reaction
This reaction was inferred from the corresponding reaction "Epithelial cadherin binds to KLRG1 in mice" in species Mus musculus.
2006, 359-66.
C Grundemann, M Bauer, O Schweier, N von Oppen, U Lassing, P Saudan, KF Becker, K Karp, T Hanke, MF Bachmann, H Pircher, "Cutting
edge: identification of E-cadherin as a ligand for the murine killer cell lectin-like receptor G1", J Immunol, 176, 2006, 1311-5.
MS Tessmer, C Fugere, F Stevenaert, OV Naidenko, HJ Chong, G Leclercq, L Brossay, "KLRG1 binds cadherins and preferentially associates
with SHIP-1", Int Immunol, 19, 2007, 391-400.
Reaction
31.2.12 CRTAM binds to NECL2
Authors
de Bono, B, 2007-07-08.
Reviewers
Description
targets.
References
2006, 359-66.
L Galibert, GS Diemer, Z Liu, RS Johnson, JL Smith, T Walzer, MR Comeau, CT Rauch, MF Wolfson, RA Sorensen, AR Van der Vuurst de
Vries, DG Branstetter, RM Koelling, J Scholler, WC Fanslow, PR Baum, JM Derry, W Yan, "Nectin-like protein 2 defines a subset of T-cell zone
dendritic cells and is a ligand for class-I-restricted T-cell-associated molecule", J Biol Chem, 280, 2005, 21955-64.
N Arase, A Takeuchi, M Unno, S Hirano, T Yokosuka, H Arase, T Saito, "Heterotypic interaction of CRTAM with Necl2 induces cell adhesion on
activated NK cells and CD8+ T cells", Int Immunol, 17, 2005, 1227-37.
Reaction
31.2.13 Poliovirus precursor binds CD226
Authors
de Bono, B, 2007-07-08.
Reviewers
Description
targets.
References
2006, 359-66.
D Pende, C Bottino, R Castriconi, C Cantoni, S Marcenaro, P Rivera, GM Spaggiari, A Dondero, B Carnemolla, N Reymond, MC Mingari, M
Lopez, L Moretta, A Moretta, "PVR (CD155) and Nectin-2 (CD112) as ligands of the human DNAM-1 (CD226) activating receptor: involvement in
tumor cell lysis", Mol Immunol, 42, 2005, 463-9.
Reaction
31.2.14 Nectin 2 binds CD226
Authors
de Bono, B, 2007-07-08.
Reviewers
Description
targets.
References
2006, 359-66.
D Pende, C Bottino, R Castriconi, C Cantoni, S Marcenaro, P Rivera, GM Spaggiari, A Dondero, B Carnemolla, N Reymond, MC Mingari, M
Lopez, L Moretta, A Moretta, "PVR (CD155) and Nectin-2 (CD112) as ligands of the human DNAM-1 (CD226) activating receptor: involvement in
tumor cell lysis", Mol Immunol, 42, 2005, 463-9.
Reaction
31.2.15 CD200 binds to CD200R
Authors
de Bono, B, 2007-07-08.
Reviewers
Description
While not ubiquitously distributed, CD200 is expressed on a wide range of cell types including thymocytes, B-cells, activated T-cells, follicular
dendritic cells, endothelium, CNS neurons in the central nervous system, cells in reproductive organs, keratinocytes and renal glomeruli.
CD200R is a myeloid-inhibitory receptor, despite the absence of classical ITIMs in the cytoplasmic portion of the protein. Interestingly, CD200 is
also expressed on neurons within the CNS and would be predicted to modulate activation of microglia through CD200R.
References
R Gorczynski, Z Chen, Y Kai, L Lee, S Wong, PA Marsden, "CD200 is a ligand for all members of the CD200R family of immunoregulatory
molecules", J Immunol, 172, 2004, 7744-9.
K Minas, J Liversidge, "Is the CD200/CD200 receptor interaction more than just a myeloid cell inhibitory signal?", Crit Rev Immunol, 26, 2006,
213-30.
Reaction
31.2.16 MHC Class I interacts with CD160
Authors
de Bono, B, 2007-07-08.
Reviewers
Description
CD160 is a GPI-anchored lymphocyte surface receptor in which expression is mostly restricted to the highly cytotoxic NK cells. MHC class I
molecules bind to CD160 receptors on circulating NK lymphocytes and this triggers their cytotoxic activity and cytokine production. NK cells
stimulated by IL-15 secrete soluble CD160 protein that binds to MHC-I molecules, resulting in the inhibition of the cytotoxic CD8+ T lymphocyte
activity and of the CD160-mediated NK cell cytotoxicity.
References
J Giustiniani, A Marie-Cardine, A Bensussan, "A soluble form of the MHC class I-specific CD160 receptor is released from human activated NK
lymphocytes and inhibits cell-mediated cytotoxicity", J Immunol, 178, 2007, 1293-300.
M Rabot, J Tabiasco, B Polgar, M Aguerre-Girr, A Berrebi, A Bensussan, N Strbo, D Rukavina, P Le Bouteiller, "HLA class I/NK cell receptor
interaction in early human decidua basalis: possible functional consequences", Chem Immunol Allergy, 89, 2005, 72-83.
Reaction
31.2.17 Fc gamma receptors interact with antigen-bound IgG
Authors
de Bono, B, 2007-07-08.
Reviewers
Description
Most cells of the immune system express receptors for the Fc region of IgG. This heterogeneous family of molecules plays a critical role in
immunity, by linking the humoral to the cellular responses. NK cells and B cells have been shown to express exclusively Fc-gamma RIIIa and
RIIb respectively.
References
S Radaev, P Sun, "Recognition of immunoglobulins by Fcgamma receptors", Mol Immunol, 38, 2002, 1073-83.
JF Cohen-Solal, L Cassard, WH Fridman, C Sautes-Fridman, "Fc gamma receptors", Immunol Lett, 92, 2004, 199-205.
Reaction
31.2.18 CD40 interacting with CD40L
Authors
de Bono, B, 2007-07-08.
Reviewers
Description
CD40 is a member of the Tumour Necrosis Factor receptor family and its ligand CD40L is a type II transmembrane protein of the TNF
superfamily. The latter is expressed preferentially on T-cells and platelets. In the immune system, CD40-CD40L interaction affects some key
processes such as immune cell activation, differentiation, proliferation, and apoptosis. CD40-CD40L interaction also upregulates costimulatory
molecules (ICAM-1, VCAM-1, E-selectin, LFA-3, B7.1, B7.2, class II MHC, and CD40 itself).
References
K Chen, J Huang, W Gong, L Zhang, P Yu, JM Wang, "CD40/CD40L dyad in the inflammatory and immune responses in the central nervous
system", Cell Mol Immunol, 3, 2006, 163-9.
Reaction
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
References
2006, 359-66.
16, 2005, 2694-703.
Reaction
31.2.20 C3d-complexed antigen binds to complement receptor
Authors
de Bono, B, 2007-07-08.
Reviewers
Description
CD19 is a lymphocyte cell surface molecule that functions as a general response regulator or rheostat, which defnes signalling thresholds.
These responses are infuenced by signals transduced through a CD19-CD21 cell surface receptor complex, where the binding of complement
C3d to CD21 links humoral immune responses with the innate immune system. The CD19-CD21 complex is composed of at least four
non-covalently associated proteins: CD19, CD21(complement receptor 2),CD81 and CD225.
References
TF Tedder, KM Haas, JC Poe, "CD19-CD21 complex regulates an intrinsic Src family kinase amplification loop that links innate immunity with
B-lymphocyte intracellular calcium responses", Biochem Soc Trans, 30, 2002, 807-11.
RH Carter, RA Barrington, "Signaling by the CD19/CD21 complex on B cells", Curr Dir Autoimmun, 7, 2004, 4-32.
FR Toapanta, TM Ross, "Complement-mediated activation of the adaptive immune responses: role of C3d in linking the innate and adaptive
immunity", Immunol Res, 36, 2006, 197-210.
CJ Del Nagro, DC Otero, AN Anzelon, SA Omori, RV Kolla, RC Rickert, "CD19 function in central and peripheral B-cell development", Immunol
Res, 31, 2005, 119-31.
Reaction
31.2.21 KIR2DL1 interacting with HLA-C group 2 (Cw3)
Authors
de Bono, B, 2007-07-08.
Reviewers
Description
References
1007-21.
Reaction
31.2.22 KIR3DL1 interacting with HLA Bw4
Authors
de Bono, B, 2007-07-08.
Reviewers
Description
References
1007-21.
Reaction
31.2.23 KIR3DL2 interacting with HLA-A3
Authors
de Bono, B, 2007-07-08.
Reviewers
Description
References
1007-21.
Reaction
31.2.24 KIR2DL4 interacting with HLA-G
Authors
de Bono, B, 2007-07-08.
Reviewers
Description
References
1007-21.
Reaction
31.2.25 KIR2DS1 interacting with HLA-C group 2 (Cw3)
Authors
de Bono, B, 2007-07-08.
Reviewers
Description
References
1007-21.
Reaction
31.2.26 KIR2DS2 interacting with HLA-C group 1 (Cw4)
Authors
de Bono, B, 2007-07-08.
Reviewers
Description
References
1007-21.
Reaction
31.3 TCR signaling
Authors
Rudd, C.E., de Bono, B, Garapati, Phani Vijay, 2008--0-1-.
Reviewers
Description
The TCR is a multisubunit complex that consists of clonotypic alpha/beta chains noncovalently associated with the invariant CD3
delta/epsilon/gamma and TCR zeta chains. T cell activation by antigen presenting cells (APCs) results in the activation of protein tyrosine
kinases (PTKs) that associate with CD3 and TCR zeta subunits and the co-receptor CD4. Members of the Src kinases (Lck), Syk kinases
(ZAP-70), Tec (Itk) and Csk families of nonreceptor PTKs play a crucial role in T cell activation. Activation of PTKs following TCR engagement
results in the recruitment and tyrosine phosphorylation of enzymes such as phospholipase C gamma1 and Vav as well as critical adaptor
proteins such as LAT, SLP-76 and Gads. These proximal activation leads to reorganization of the cytoskeleton as well as transcription activation
of multiple genes leading to T lymphocyte proliferation, differentiation and/or effector function.
References
CE Rudd, H Schneider, "Unifying concepts in CD28, ICOS and CTLA4 co-receptor signalling", Nat Rev Immunol, 3, 2003, 544-56.
CE Rudd, "Adaptors and molecular scaffolds in immune cell signaling", Cell, 96, 1999, 5-8.
JE van Leeuwen, LE Samelson, "T cell antigen-receptor signal transduction", Curr Opin Immunol, 11, 1999, 242-8.
31.3.1 Phosphorylation of CD3 and TCR zeta chains
Authors
Reviewers
Description
Prior to T cell receptor (TCR) stimulation, CD4/CD8 associated Lck remains seperated from the TCR and is maintained in an inactive state by
the action of Csk. Csk phosphorylates the negative regulatory tyrosine of Lck and inactivates the Lck kinase domain.
Upon TCR stimulation, CD4/CD8 associated Lck co-localizes with the TCR leading to the phosphorylation of the CD3 and TCR subunit. Lck
becomes activated by way of CD45-mediated dephosphorylation of negative regulatory tyrosine residues. The presence to PAG-bound Csk is
further reduced via the dephosphorylation of PAG (step 1).
Lck is further activated by trans-autophosphorylation on the tyrosine residue on its activation loop (step 2). Active Lck further phosphorylates the
tyrosine residues on CD3 chains. The signal-transducing CD3 delta/epsilon/gamma and TCR zeta chains contain a critical signaling motif known
as the immunoreceptor tyrosine-based activation motif (ITAM). The two critical tyrosines of each ITAM motif are phosphorylated by Lck (step 3).
References
31.3.1.1 Interaction of Csk with PAG
Authors
Reviewers
Description
Csk is a tyrosine kinase that phosphorylates the negative regulatory C-terminal tyrosine residue Y505 of Lck to maintain Lck in an inactive state.
In resting T cells, Csk is targeted to lipid rafts through engagement of its SH2 domain with phosphotyrosine residue pY317 of PAG. PAG is
expressed as a tyrosine phosphorylated protein in nonstimulated T-cells. This interaction of Csk and PAG allows activation of Csk and inhibition
of Lck. Given that PAG-1 T cell knock out show a weak phenotype, some other protein may substitute in activating Csk.
References
T Brdicka, D Pavlistova, A Leo, E Bruyns, V Korinek, P Angelisova, J Scherer, A Shevchenko, I Hilgert, J Cerny, K Drbal, Y Kuramitsu, B
Kornacker, V Horejsi, B Schraven, "Phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), a novel ubiquitously
expressed transmembrane adaptor protein, binds the protein tyrosine kinase csk and is involved in regulation of T cell activation", J Exp Med,
191, 2000, 1591-604.
B Marinari, L Simeoni, B Schraven, E Piccolella, L Tuosto, "The activation of Csk by CD4 interferes with TCR-mediated activatory signaling", Eur
J Immunol, 33, 2003, 2609-18.
A Leo, J Wienands, G Baier, V Horejsi, B Schraven, "Adapters in lymphocyte signaling", J Clin Invest, 109, 2002, 301-9.
Reaction
31.3.1.2 Inactivation of Lck by Csk
Authors
Reviewers
Description
Lck is a member of the Src family tyrosine kinases and these members have the following domains in common: N-terminal Myristoylation site for
saturated fatty acid addition, a unique region, a Src-homology 3 (SH3) domain, an SH2 domain, a tyrosine kinase domain (SH1), and a
C-terminal negative regulatory domain. Myristoylation engenders Lck with the ability to attach to cellular membranes. This interaction is mediated
by both myristic acid and palmitic acid that are bound to the amino terminal glycine and Cys-3 and/or Cys-5.
The unique region of Lck is thought to be involved in the interaction with the cytoplasmic tails of coreceptors CD4 and CD8. The Lck/CD4
interaction require conserved cysteine motifs: a CxCP motif in CD4 and a CxxC motif in the Lck unique domain. The SH3 and SH2 domains of
Lck are involved in intramolecular and intermolecular regulation by mediating protein-protein interactions via poly-proline and
phosphotyrosine-specific interactions, respectively.
Lck adopts specific conformation that largely dictate its level of activity. The C-ter tail has an autoinhibitory phosphorylation site (tyr 505). When
the Y505 is phosphorylated, Lck adopts a closed conformation, where the pY505 residue creates an intramolecular binding motif for the SH2
domain, effectively inactivating the kinase domain. The inactivating phosphorylation on Y505 is carried out by the Src-specific kinase Csk.
References
EH Palacios, A Weiss, "Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation", Oncogene, 23, 2004, 7990-8000.
TJ Boggon, MJ Eck, "Structure and regulation of Src family kinases", Oncogene, 23, 2004, 7918-27.
Reaction
31.3.1.3 Dephosphorylation of Lck-pY505 by CD45
Authors
Reviewers
Description
TCR stimulation induce the transient dephosphorylation of PAG thereby release the Csk from its plasma membrane anchor. The release of Csk
from its proximity with Lck may serve to facilitate the activation of Lck. The inactive Lck is dephosphorylated by CD45 phosphatase. CD45
specifically dephosphorylates the Y505 residue of Lck and induce the active open conformation.
References
Reaction
31.3.1.4 Activation of Lck
Authors

Reviewers
Description
The binding of CD4/CD8 to non-polymorphic regions of MHC brings Lck in to proximity with TCR subunits phosphorylation. Lck is further
phosphorylated to promote the active conformation and to increase their catalytic activity. The C-term domain contain a regulatory activation
loop, which is the site of activating Tyr 394 phosphorylation. This tyrosine is auto-phosphorylated to attain an active conformation on TCR
stimulation. Now Lck through its kinase activity phosphorylates the ITAMs in TCR zeta and CD3 members.
References
EH Palacios, A Weiss, "Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation", Oncogene, 23, 2004, 7990-8000.
Reaction
31.3.1.5 Phosphorylation of ITAM motifs in CD3 complexes
Authors
Reviewers
Description
The autophosphorylated, active Lck is now proximally positioned to phosphorylate specific tyrosine residues within ITAMs (immunoreceptor
tyrosine-based activation motifs) located within the CD3 and the TCR zeta signaling chains of the TCR. ITAMs consist of evolutionarily
conserved amino-acid sequence motifs of D/ExYxxLx(6-8)YxxL. Both the tyrosine residues in the motif are phosphorylated by Lck and the TCR
complex include 10 ITAMs with one ITAM in each of the CD3 chains including the three tandem ITAMs in each zeta chains.
References
B Wilkinson, JS Downey, CE Rudd, "T-cell signalling and immune system disorders", Expert Rev Mol Med, 7, 2005, 1-29.
Y Huang, RL Wange, "T cell receptor signaling: beyond complex complexes", J Biol Chem, 279, 2004, 28827-30.
Reaction
31.3.2 Translocation of ZAP-70 to Immunological synapse
Authors
Reviewers
Description
The dual phosphorylated ITAMs recruit Syk kinase ZAP-70 via their tandem SH2 domains (step 4). ZAP-70 subsequently undergoes
phosphorylation on multiple tyrosine residues for further activation. ZAP-70 includes both positive and negative regulatory sites. Tyrosine 493 is
a conserved regulatory site found within the activation loop of the kinase domain. This site has shown to be a positive regulatory site required for
ZAP-70 kinase activity and is phosphorylated by Lck (step 5). This phosphorylation contributes to the active conformation of the catalytic domain.
Later ZAP-70 undergoes trans-autophosphorylation at Y315 and Y319 (step 6). These sites appear to be positive regulatory sites. ZAP-70
achieves its full activation after the trans-autophosphorylation. Activated ZAP-70 along with Lck phosphorylates the multiple tyrosine residues in
the adaptor protein LAT (step 7).
References
31.3.2.1 Recruitment of ZAP-70 to phosphorylated ITAMs
Authors

Reviewers
Description
Phosphorylation of the ITAMs by Lck is followed by the recruitment of the ZAP-70 a member of Syk family PTK, to the receptor complex. ZAP-70
is exclusively expressed in T cells and NK cells. The dually phosphorylated ITAMs provide a high-affinity docking site for the tandem
SH2-domains of the ZAP-70. Once recruited, ZAP-70 is activated by phosphorylation and will be responsible for the phosphorylation of further
downstream molecules. Due to the presence of 10 ITAMs in the TCR complex, up to 10 ZAP-70 molecules may cluster on the fully
phosphorylated receptors.
References
DH Chu, CT Morita, A Weiss, "The Syk family of protein tyrosine kinases in T-cell activation and development", Immunol Rev, 165, 1998,
167-80.
T Mustelin, K Tasken, "Positive and negative regulation of T-cell activation through kinases and phosphatases", Biochem J, 371, 2003, 15-27.
NS van Oers, A Weiss, "The Syk/ZAP-70 protein tyrosine kinase connection to antigen receptor signalling processes", Semin Immunol, 7, 1995,
227-36.
Reaction
31.3.2.2 Phosphorylation of ZAP-70 by Lck
Authors
Reviewers
Description
In ZAP-70 there are multiple phosphorylation sites (Y292, Y315, Y319, Y492, Y493) which have both positive and negative regulatory effect on
its catalytic activity. Tyrosine 493 is a conserved regulatory site found within the activation loop of the kinase domain. This site has shown to be a
positive regulatory site required for ZAP-70 kinase activity and is phosphorylated by Lck. This phosphorylation contribute to the active
conformation of the catalytic domain.
References
167-80.
227-36.
BL Williams, BJ Irvin, SL Sutor, CC Chini, E Yacyshyn, J Bubeck Wardenburg, M Dalton, AC Chan, RT Abraham, "Phosphorylation of Tyr319 in
ZAP-70 is required for T-cell antigen receptor-dependent phospholipase C-gamma1 and Ras activation", EMBO J, 18, 1999, 1832-44.
Reaction
31.3.2.3 Activation of ZAP-70
Authors
Reviewers
Description
Later ZAP-70 undergoes trans-autophosphorylation at Y315 and Y319. These sites appear to be positive regulatory sites. ZAP-70 achieve its full
activation after the trans-autophosphorylation. Activated ZAP-70 phosphorylates T-cell-specific adaptors, such as LAT and SLP-76 leading to the
recruitment and activation of other kinase families and enzymes, resulting in secondary messenger generation and culminating in T cell
activation.
References
167-80.
227-36.
BL Williams, BJ Irvin, SL Sutor, CC Chini, E Yacyshyn, J Bubeck Wardenburg, M Dalton, AC Chan, RT Abraham, "Phosphorylation of Tyr319 in
ZAP-70 is required for T-cell antigen receptor-dependent phospholipase C-gamma1 and Ras activation", EMBO J, 18, 1999, 1832-44.
Reaction
31.3.3 Generation of second messenger molecules
Authors
Reviewers
Description
In addition to serving as a scaffold via auto-phosphorylation, ZAP-70 also phosphorylates a restricted set of substrates following TCR stimulation
- including LAT and SLP-76. These substrates have been recognized to play pivotal role in TCR signaling by releasing second messengers.
When phosphorylated, LAT and SLP-76 act as adaptor proteins which serve as nucleation points for the construction of a higher order
signalosome: GADS, PLC-gamma1 and GRB2 bind to the LAT on the phosphorylated tyrosine residues (steps 8 and 13). SLP-76 and SOS are
then moved to the signalosome by interacting with the SH3 domains of GRB2 and GADS via their proline rich sequences (step 9). Three SLP-76
acidic domain N-term tyrosine residues are phosphorylated by ZAP-70, once SLP-76 binds to GADS (step 10). These phospho-tyrosine residues
act as binding sites to the SH2 domains of PLC-gamma1, Vav and Itk (steps 11 and 12).
PLC-gamma1 is activated by dual phosphorylation on the tyrosine residues at positions 771, 783 and 1254 by Itk and ZAP-70 (step 14).
Phosphorylated PLC-gamma1 subsequently detaches from LAT and SLP-76 and translocates to the plasma membrane by binding to
phosphatidylinositol-4,5-bisphosphate (PIP2) via its PH domain (step 15). PLC-gamma1 goes on to hydrolyse PIP2 to second messengers DAG
and IP3. These second messengers are involved in PKC and NF-kB activation and calcium mobilization (step 16).
References
31.3.3.1 Phosphorylation of TBSMs in LAT
Authors
Reviewers
Description
The adaptor molecule LAT (Linker for the Activation of T cells) is a membrane protein that links the TCR signal to the cell activation. It has a total
10 (Y36, Y45, Y64, Y110, Y127, Y132, Y171, Y191, and Y226) conserved TBSMs (tyrosine based signaling motifs) in its cytoplasmic region.
These tyrosine residues are phosphorylated by the activated ZAP-70 upon TCR/CD3 complex engagement. Phosphorylation of LAT creates
binding sites for the Src homology 2 (SH2) domains of other proteins, including PLC-gamma1, Grb2 and Gads, and indirectly binds SOS, Vav,
SLP-76, and Itk. The residues Y171, Y191 and Y226 are responsible for Grb2 binding, Y171 and Y191 but not Y226, are necessary for Gads
binding and Y132 for the PLC-gamma1 binding.
References
CE Rudd, H Wang, "Hematopoietic adaptors in T-cell signaling: potential applications to transplantation", Am J Transplant, 3, 2003, 1204-10.
RL Wange, "LAT, the linker for activation of T cells: a bridge between T cell-specific and general signaling pathways", Sci STKE, 2000, 2000,
RE1.
W Zhang, RP Trible, M Zhu, SK Liu, CJ McGlade, LE Samelson, "Association of Grb2, Gads, and phospholipase C-gamma 1 with
phosphorylated LAT tyrosine residues. Effect of LAT tyrosine mutations on T cell angigen receptor-mediated signaling.", J Biol Chem, 275, 2000,
23355-61.
Reaction
31.3.3.2 Recruitment of Gads to LAT
Authors
Reviewers
Description
Gads is a member of the Grb2 family containing SH2 and SH3 domains with the arrangement SH3-SH2-SH3. Gads binds to the tyrosine
phosphorylated residues Y171 and Y191 of LAT through its SH2 domain. It plays a critical role in signaling from the T cell receptor by promoting
the formation of a complex between SLP-76 and LAT.
References
SK Liu, DM Berry, CJ McGlade, "The role of Gads in hematopoietic cell signalling", Oncogene, 20, 2001, 6284-90.
Reaction
31.3.3.3 Recruitment of SLP-76 to Gads
Authors
Reviewers
Description
SLP-76 is an adaptor protein that links proximal and distal T cell receptor signaling events through its function as a molecular scaffold in the
assembly of multi molecular signaling complexes. SLP-76 consists of three domains that mediate interactions with many different signaling
proteins: an N-terminal acidic domain containing three tyrosine phosphorylation sites, a large central proline-rich region, and a C-terminal SH2
domain. The function of SLP-76 is dependent on its association with Gads. SLP-76 constitutively binds through its 'RxxK' motif in the proline rich
region to the SH3 domain of Gads upon TCR activation.
References
SK Liu, DM Berry, CJ McGlade, "The role of Gads in hematopoietic cell signalling", Oncogene, 20, 2001, 6284-90.
Reaction
31.3.3.4 Phosphorylation of SLP-76
Authors
Reviewers
Description
Once SLP-76 is recruited to Gads its rapidly phosphorylated on the tyrosine residues in the N-terminal acidic domain. This domain contains
three tyrosine phosphorylation sites, Y112, Y128 and Y148. These tyrosine residues are phosphorylated by tyrosine kinase ZAP-70 and these
phosphorylated tyrosine residues provide the binding site for the SH2 domains of the incoming signaling proteins like Vav, Itk and PLC-gamma1.
References
Q Qi, A August, "Keeping the (kinase) party going: SLP-76 and ITK dance to the beat", Sci STKE, 2007, 2007, pe39.
Reaction
31.3.3.5 Recruitment of ITK to SLP-76
Authors
Reviewers
Description
ITK is a member of the Tec protein tyrosine kinase family which forms a complex with SLP-76 after TCR activation. ITK has N-terminal pleckstrin
homology (PH) domain, a Tec homology (TH) domain, a proline rich domain, a SH3 domain, an SH2 domain and a C-term kinase domain. The
SH2 domain of ITK may interact with Y145 within the N-ter acidic domain of SLP-76 and the SH3 domain of the ITK binds the proline rich region
of SLP-76. ITK plays an important role in phosphorylating and activating PLC-gamma-1, leading to the development of second-messenger
molecules.
References
Q Qi, A August, "Keeping the (kinase) party going: SLP-76 and ITK dance to the beat", Sci STKE, 2007, 2007, pe39.
Reaction
31.3.3.6 Recruitment of PLC-gamma1 to SLP-76
Authors
Reviewers
Description
PLC-gamma1 plays an important role in transducing the external signal in to second messengers by hydrolysing PIP2. PLC-gamma1 contains
an N-term PH domain, a catalytic domain 'X' followed by two SH2 domains and an SH3 domain, a C-term catalytic domain 'Y' and a C2 domain
(Ca++ binding). PLC-gamma1 interacts with both SLP-76 aswell as LAT. It interacts with its SH3 domain to the proline rich sequence of SLP-76.
This interaction aids in localizing PLC-gamma1 to the membrane. This recruitment of PLC-gamma1 to LAT and SLP-76 is essential for its TCR
induced tyrosine phosphorylation and activation.
References
MJ Kim, E Kim, SH Ryu, PG Suh, "The mechanism of phospholipase C-gamma1 regulation", Exp Mol Med, 32, 2000, 101-9.
D Yablonski, T Kadlecek, A Weiss, "Identification of a phospholipase C-gamma1 (PLC-gamma1) SH3 domain-binding site in SLP-76 required for
T-cell receptor-mediated activation of PLC-gamma1 and NFAT", Mol Cell Biol, 21, 2001, 4208-18.
Reaction
31.3.3.7 Recruitment of PLC-gamma1 to LAT
Authors
Reviewers
Description
PLC-gamma1 interacts with its SH2 domain to the pY132 residue of LAT.
References
Reaction
31.3.3.8 Phosphorylation of PLC-gamma1
Authors
Reviewers
Description
Three tyrosine residues at positions 771, 783 and 1254 in PLC-gamma1 have been identified as the sites of receptor tyrosine kinase
phosphorylation. Of these Y783 and Y1254 are required for activation of PLC-gamma1. The phosphorylation of the tyrosine residues and the
activation of PLC-gamma1 is mediated by both Syk tyrosine kinase ZAP-70 and Tec kinase ITK.
References
Reaction
31.3.3.9 Disassociation of PLC-gamma1 from LAT
Authors
Reviewers
Description
The activated PLC-gamma1 detaches from its substrate LAT and translocates to the membrane.
References
Reaction
31.3.3.10 Disassociation of PLC-gamma1 from SLP-76
Authors
Reviewers
Description
The activated PLC-gamma1 detaches from its substrate SLP-76 and translocates to the membrane.
References
Reaction
31.3.3.11 Translocation of PLC-gamma1 to PIP2
Authors
Reviewers
Description
Activated PLA-gamma1 translocates to the plasmamembrane and interacts with the inositol ring of the membrane bound phosphatidylinositol
4,5-bisphosphate (PIP2) with its PH domain.
References
Reaction
31.3.3.12 PLC-gamma1 hydrolyses PIP2
Authors
Reviewers
Description
On recruitment to plasma membrane PLC-gamma1 then hydrolyses PIP2 producing two second messengers, diacylglycerol (DAG) and inositol
1,4,5-trisphosphate (IP3). IP3 induces a transient increase in intracellular free Ca++, while DAG is a direct activator of protein kinase C (PKC
theta). These process have been implicated in many cellular physiological functions like cell proliferation, cell growth and differentiation.
Reaction
31.3.4 Downstream TCR signaling

Authors
Reviewers
Description
Changes in gene expression are required for the T cell to gain full proliferative competence and to produce effector cytokines. Three
transcription factors in particular have been found to play a key role in TCR-stimulated changes in gene expression, namely NF-kB, NFAT and
AP-1.
A key step in NF-kB activation is the stimulation and translocation of PKC theta. The critical element that effects PKC theta activation is PI3K.
This enzyme complex translocates to the plasma membrane by interacting with phospho-tyrosines on CD28 via its two SH2 domains located in
p85 subunit. The p110 subunit of PI3K phosphorylates the inositol ring of PIP2 to generate PIP3 (steps 17-18). PIP3 may also be
dephosphorylated by the phosphatase SHIP to generate PI-3,4-P2.
PIP3 and PI-3,4-P2 acts as binding sites to the PH domain of PKB/Akt and PDK1 (steps 19, 21 and 22). PKB is activated in response to PI3K
stimulation by PDK1 (step 23). PDK1 has an essential role in regulating the activation of PKC theta and recruitment of CBM complex to the
immune synapse. PKC theta is a member of novel class (DAG dependent, Ca++ independent) of PKC and the only member known to
translocate to this synapse. Prior to TCR stimulation PKC theta exists in an inactive closed conformation. Upon release of DAG, it binds to PKC
theta via the C1 domain and undergoes phosphorylation on tyrosine 90 by Lck to attain an open conformation. PKC theta is further
phosphorylated by PDK1 on threonine 538. This step is critical for PKC activity (steps 24-26).
CARMA1 translocates to the plasma membrane following the interaction of its SH3 domain with the 'PxxP' motif on PDK1. CARMA1 is
phosphorylated by PKC-theta on residue S552, leading to the oligomerization of CARMA1. This complex acts as a scaffold, recruiting Bcl10 to
the synapse by interacting with their CARD domains.
Bcl10 undergoes phosphorylation mediated by the enzyme RIP2. Activated Bcl10 then mediates the ubiquitination of NEMO by recruiting MALT1
and TRAF6. MALT1 binds to Bcl10 with its Ig-like domains and undergoes oligomerization. TRAF6 binds to the oligomerized MALT1 and also
undergoes oligomerization.
Oligomerized TRAF6 acts as a ubiquitin-protein ligase, catalyzing auto-K63-linked polyubiquitination (steps 27-33). This K-63 ubiquitinated
TRAF6 activates TAK1 kinase bound to TAB2 and also ubiquitinates NEMO/IKK-gamma in the IKK complex. TAK1 undergoes
autophosphorylation on residues T184 and T187 and gets activated. Activated TAK1 kinase phosphorylates IKK-beta on residues S177 and
S181 in the activation loop and activates the IKK kinase activity. IKK-beta phosphorylates the IkB-alpha bound to the NF-kB heterodimer, on
residues S19 and S23 and directs IkB-beta to 26S proteasome degradation (step 34-38 & 40).
The NF-kB heterodimer with a free NTS sequence finally migrates to the nucleus to regulate gene transcription (step 39).
References
31.3.4.1 Recruitment of PI3K to plasmamembrane

Authors
Reviewers
Description
In response to the TCR stimulation, phsophoinositides are phosphorylated on the 3-position of the inositol ring by PI3K to generate lipid second
messengers that serve as membrane docking sites for a variety of downstream effector proteins such as PDK1 and PKB. PI3K is a heterodimer
comprising a regulatory subunit p85 and a catalytic subunit p110 which associate constitutively and are activated upon interaction with
tyrosine-phosphorylated proteins at the plasma membrane. The p85 subunit contains two SH2 domains and an SH3 domain. p85 subunit is
involved in interaction with two phsophotyrosine residues of the adaptor protein TRIM with its two SH2 domains. This interaction is important in
recruiting the p110 subunit to the plasma membrane and activate the p110 kinase activity, which is normally inhibited in the p85-p110 complex.
References
S Koyasu, "The role of PI3K in immune cells", Nat Immunol, 4, 2003, 313-9.
U Kolsch, B Arndt, D Reinhold, JA Lindquist, N Juling, S Kliche, K Pfeffer, E Bruyns, B Schraven, L Simeoni, "Normal T-cell development and
immune functions in TRIM-deficient mice", Mol Cell Biol, 26, 2006, 3639-48.
E Bruyns, A Marie-Cardine, H Kirchgessner, K Sagolla, A Shevchenko, M Mann, F Autschbach, A Bensussan, S Meuer, B Schraven, "T cell
receptor (TCR) interacting molecule (TRIM), a novel disulfide-linked dimer associated with the TCR-CD3-zeta complex, recruits intracellular
signaling proteins to the plasma membrane", J Exp Med, 188, 1998, 561-75.
Reaction
31.3.4.2 PI3K phosphorylates PIP2 to PIP3
Authors

Reviewers
Description
PI3K enzyme bound to adaptor protein TRIM, uses phosphatidylinositol 4,5-bisphosphate (PIP2) as its substrate and phosphorylates it to
generate phosphatidylinositol 3,4,5-trisphosphate (PIP3). This PIP3 acts as a membrane anchor for the downstream proteins like PDK1 and
PKB.
References
S Koyasu, "The role of PI3K in immune cells", Nat Immunol, 4, 2003, 313-9.
Reaction
31.3.4.3 Hydrolysis of PIP3 to PI(3,4)P2

Authors
Reviewers
Description
After the generation of PIP3 by PI3K, a part of it is further dephosphorylated to generate other forms of PI which are also involved in signaling.
Two major routes for the degradation of PIP3 exists: dephosphorylation by the haematopoietic-specific SH2 domain-containing inositol 5'
phosphatase SHIP-1 and dephosphorylation by the 3' phosphoinositide phosphatase PTEN.
SHIP-1 appears to set an activation threshold on T cell signaling. SHIP-1 phosphatase activity removes the 5' phosphate of PIP3 and generate
phosphatidylinositol 3,4-bisphosphate. PI(3,4)P2 along with PIP3 preferentially binds to the PH domains of PKB and PDK1.
References
ME March, K Ravichandran, "Regulation of the immune response by SHIP", Semin Immunol, 14, 2002, 37-47.
LR Rohrschneider, JF Fuller, I Wolf, Y Liu, DM Lucas, "Structure, function, and biology of SHIP proteins", Genes Dev, 14, 2000, 505-20.
Reaction
31.3.4.4 Hydrolysis of PIP3 to PIP2
Authors

Reviewers
Description
PTEN dephosphorylates 3' position of PIP3 to generate PIP2 and negatively regulates the PI3K pathway and PKB activation.
Reaction
31.3.4.5 Translocation of PDK1 to Plasma membrane
Authors
Reviewers
Description
PDK1 is a Ser/Thr kinase with a N-term kinase domain and a C-term PH domain. PDK1 with its PH domain binds to PIP3 and PI(3,4)P2 and is
translocated to the plasma membrane. PDK1 seems to exist in an active, phosphorylated configuration under basal conditions. PDK1 pays an
important role in activating and phosphorylating the enzymes PKB and PKC theta.
References
B Vanhaesebroeck, DR Alessi, "The PI3K-PDK1 connection: more than just a road to PKB", Biochem J, 346, 2000, 561-76.
Reaction
31.3.4.6 Translocation of PKB to plasma membrane
Authors
Reviewers
Description
PKB/Akt is also a Ser/Thr kinase with PH domain that preferentially binds PIP3 and PI(3,4)P2. PKB is cytosolic in unstimulated cells and some of
it translocates to the plasma membrane upon activation of PI3K, where it becomes activated by PDK1. PKB which is recruited to the plasma
membrane in response to increased level of phosphoinositols and then its activation by PDK1, is considered to be a chief downstream target of
PI3K.
References
B Vanhaesebroeck, DR Alessi, "The PI3K-PDK1 connection: more than just a road to PKB", Biochem J, 346, 2000, 561-76.
Reaction
31.3.4.7 Phosphorylation of PKB by PDK1
Authors
Reviewers
Description
PKB consists of an N-term PH domain, a kinase domain and a C-term regulatory region. Two specific sites, one in the kinase domain (Thr 308)
and the other in the C-term regulatory region (Ser 473), need to be phosphorylated for full activation. This phosphorylation is mediated by PDK1.
The activated PKB releases from the plasma membrane and pass through the cytosol in to the nucleus. This movement of activated PKB away
from the plasma membrane is an important process because it brings the activated kinase into close proximity with its substrates.
References
D Stokoe, LR Stephens, T Copeland, PR Gaffney, CB Reese, GF Painter, AB Holmes, F McCormick, PT Hawkins, "Dual role of
phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B", Science, 277, 1997, 567-70.
DR Alessi, SR James, CP Downes, AB Holmes, PR Gaffney, CB Reese, P Cohen, "Characterization of a 3-phosphoinositide-dependent protein
kinase which phosphorylates and activates protein kinase Balpha", Curr Biol, 7, 1997, 261-9.
C Partovian, M Simons, "Regulation of protein kinase B/Akt activity and Ser473 phosphorylation by protein kinase Calpha in endothelial cells",
Cell Signal, 16, 2004, 951-7.
Reaction
31.3.4.8 Translocation of PKC theta to plasma membrane

Authors
Reviewers
Description
PKC theta is a member of the Ca++ independent and DAG dependent, novel PKC subfamily expressed mainly in T cells. It contains, N-term C2
like domain, a pseudosubstrate (PS), DAG binding (C1) domain and a C-term kinase domain. The PS sequence resembles an ideal substrate
with the exception that it contains an alanine residue instead of a substrate serine residue, is bound to the kinase domain in the resting state. As
a result, PKC theta is maintained in a closed inactive state, which is inaccessible to cellular substrates.
References
A Altman, M Villalba, "Protein kinase C-theta (PKC theta): a key enzyme in T cell life and death", J Biochem (Tokyo), 132, 2002, 841-6.
S Manicassamy, S Gupta, Z Sun, "Selective function of PKC-theta in T cells", Cell Mol Immunol, 3, 2006, 263-70.
Reaction
31.3.4.9 Change of PKC theta conformation
Authors
Reviewers
Description
PKC theta localizes at the interface between T cells and antigen presenting cells. Upon the T cell activation and release of the second
messengers Ca++ and DAG by PLC-gamma1, DAG binds to the C1 domain of the PKC theta thereby enhances the attachment to the plasma
membrane. Upon membrane translocation, PKC theta is phosphorylated at tyrosine 90 in the C2 like domain. This phosphorylation is mediated
by the tyrosine kinase Lck. These association and, most likely, other regulatory interactions, lead to a change in PKC theta conformation into an
open, active state whereby it can now access its substrates and phosphorylate them.
References
Reaction
31.3.4.10 Phosphorylation of PKC theta
Authors
Reviewers
Description
Raft localized PKC theta is further phosphorylated and activated by PDK1. The threonine residue (T538) in the kinase domain is the potential
target of PDK1. Phosphorylation of this site is critical for the PKC theta kinase activity, and its ability to activate NF-kB pathway. PKC theta is
later trans-autophopshorylated on putative phosphorylation sites (S676, S695) for the fine-tuning of its kinase activity.
References
Reaction
31.3.4.11 Translocation of CARMA1 to Plasma membrane
Authors
Reviewers
Description
CARMA1 and Bcl10 are the possible link between PKC theta and IKK activation. PDK1 is also required for PKC theta mediated activation of IKK.
CARMA1 has a N-terminal CARD motif, a coiled coiled region, a linker region, and a MAGUK-typical PDZ, SH3 and a GUK domains. The linker
region is proposed to contain a hinge region and a CARD binding domain. CARMA1 exists in an inactive conformation in which the linker region
binds to and blocks the accessibility of the CARD motif. CARMA1 is recruited to the plasma membrane by binding to the 'PxxP' motif of
membrane bound PDK1 with its SH3 domain.
References
M Thome, R Weil, "Post-translational modifications regulate distinct functions of CARMA1 and BCL10", Trends Immunol, 28, 2007, 281-8.
M Thome, "CARMA1, BCL-10 and MALT1 in lymphocyte development and activation", Nat Rev Immunol, 4, 2004, 348-59.
X Lin, D Wang, "The roles of CARMA1, Bcl10, and MALT1 in antigen receptor signaling", Semin Immunol, 16, 2004, 429-35.
Reaction
31.3.4.12 Phosphorylation of CARMA1
Authors
Reviewers
Description
Antigen receptor triggered PKC theta dependent linker phosphorylation of S552 residue is required to release this inhibition and expose the
CARD motif for downstream Bcl10 recruitment. PDK1 and maybe other unknown adapter proteins bring PKC theta and CARMA1 into close
proximity, facilitating PKC theta mediated CARMA1 phosphorylation and consequent activation.
References
D Rueda, M Thome, "Phosphorylation of CARMA1: the link(er) to NF-kappaB activation", Immunity, 23, 2005, 551-3.
Reaction
31.3.4.13 Oligomerization of CARMA1
Authors
Reviewers
Description
After the phosphorylation and activation CARMA1 undergoes oligomerization, likely through its CC domain. CARMA1 is thought to oligomerize
first as a trimer which triggers downstream oligomerization cascade that is ultimately necessary for the subsequent activation of the IKK
complex.
References
X Lin, D Wang, "The roles of CARMA1, Bcl10, and MALT1 in antigen receptor signaling", Semin Immunol, 16, 2004, 429-35.
Reaction
31.3.4.14 Interaction of Bcl10 to CARMA1
Authors
Reviewers
Description
Bcl10 is recruited to activated, oligomeric CARMA1 through a CARD-CARD interaction. Bcl10 is characterized by an N-terminal CARD motif and
a C-terminal extension of ~130 amino acids rich in serine and threonine residues that serve as targets for multiple phosphorylation events.
References
Reaction
31.3.4.15 Phosphorylation of Bcl10
Authors
Reviewers
Description
Upon interaction with CARMA1, Bcl10 undergoes phosphorylation and oligomerization. The oligomerized Bcl10 acts as a adaptor for the
incoming MALT1 and TRAF6. Phosphorylation events of Bcl10 can both positively and negatively regulate the NF-kB pathway. Phosphorylation
of Bcl10 that depends on the Ser/Thr kinase RIP2 and correlated with the physical association of Bcl10 with RIP2 has a activation effect on the
NF-kB pathway. The target sites of RIP2-mediated phosphorylation has not yet been identified.
References
Reaction
31.3.4.16 Oligomerization of Bcl10
Authors
Reviewers
Description
Association with RIP2 and its phosphorylation allows subsequent trimerization of Bcl10.
References
Reaction
31.3.4.17 Interaction and oligomerization of MALT1 to Bcl10

Authors
Reviewers
Description
Oligomerized Bcl10 facilitates the association with MALT1 to form the CBM signalosome. MALT1 possesses one death domain (DD) and 2
immunoglobulin-like domains (Ig-like) in its N-terminal region and a caspase like domain (CLD) in its C-terminal region. The region between
amino acids 107 and 119 of Bcl10 bind to the two Ig-like domains of MALT1. After binding to CARMA1 and Bcl10 complex, MALT1 also
undergoes oligomerization. Only the oligomerized forms of Bcl10 and MALT1 are capable of activating IKK.
References
L Sun, L Deng, CK Ea, ZP Xia, ZJ Chen, "The TRAF6 ubiquitin ligase and TAK1 kinase mediate IKK activation by BCL10 and MALT1 in T
lymphocytes", Mol Cell, 14, 2004, 289-301.
Reaction
31.3.4.18 Translocation of TRAF6 to CBM complex
Authors
Reviewers
Description
TRAF6, which plays central role in innate immune responses, is implicated as proximal downstream effector of MALT1. TRAF6 is a member of
the TRAF proteins. It contains an N-term RING domain, followed by several Zn finger domains and C-term MATH domain. The MALT1 oligomers
bind to TRAF6, induce TRAF6 oligomerization and thereby activate the ubiquitin ligase activity of TRAF6 to polyubiquitinate itself and NEMO.
References
Reaction
31.3.4.19 Auto-ubiquitination of TRAF6
Authors
Reviewers
Description
TRAF6 possesses ubiquitin ligase activity and undergoes K-63-linked auto-ubiquitination after its oligomerization. In the first step, ubiquitin is
activated by a E1 ubiquitin activating enzyme. The activated ubiquitin is transfered to the E2 conjugating enzyme (a heterodimer of proteins
Ubc13 and Uev1A) forming the E2-Ub thioester. Finally, in the prescence of ubiquitin-protein ligase E3 (TRAF6, a RING-domain E3), ubiquitin is
attached to the target protein (TRAF6 on residue Lysine 124) through an isopeptide bond between the C-ter of ubiquitin and the epsilon-amino
group of a lysine residue in the target protein. In contrast to K-48-linked ubiquitination that leads to the proteosome degradation of the target
protein, K-63-linked polyubiquitin chains act as a scaffold to assemble protein kinase complexes and mediate their activation through
proteosome-independent mechanisms. This K63 polyubiquitinated TRAF6 activates the TAK1 kinase complex.
References
B Lamothe, A Besse, AD Campos, WK Webster, H Wu, BG Darnay, "Site-specific Lys-63-linked tumor necrosis factor receptor-associated factor
6 auto-ubiquitination is a critical determinant of I kappa B kinase activation", J Biol Chem, 282, 2007, 4102-12.
Reaction
31.3.4.20 Activation of TAK1-TAB2 complex
Authors
Reviewers
Description
Ubiquitinated TRAF6 recruits TAB2 and activates the TAB2-associated TAK1 kianse by promoting the autophosphorylation of TAK1. TAB2
contains an N-term pseudophosphatase domain, which is indispensable for TAK1 activation, and a C-term domain that binds to and activates
TAK1. The activation of TAK1/TAB2 complex requires a ubiquitination reaction catalysed by E1, Ubc13/Uev1A (E2) and TRAF6 (E3). TAK1
undergoes autophosphorylation on residues T184 and T187 and gets activated. Activated TAK1 then phosphorylates and activates IKK beta.
References
A Adhikari, M Xu, ZJ Chen, "Ubiquitin-mediated activation of TAK1 and IKK", Oncogene, 26, 2007, 3214-26.
ZJ Chen, "Ubiquitin signalling in the NF-kappaB pathway", Nat Cell Biol, 7, 2005, 758-65.
Reaction
31.3.4.21 Activation of IKK complex
Authors

Reviewers
Description
The IkB kinase (IKK) complex serves as the master regulator for the activation of NF-kB by various stimuli. It contains two catalytic subunits, IKK
alpha and IKK beta, and a regulatory subunit, IKKgamma/NEMO. The activation of IKK complex is dependent on the phosphorylation of IKK
alpha/beta at its activation loop and the K63-linked ubiquitination of NEMO. This basic trimolecular complex is referred to as the IKK complex.
IKK subunits have a N-term kinase domain a leucine zipper (LZ) motifs, a helix-loop-helix (HLH) and a C-ter NEMO binding domain (NBD). IKK
catalytic subunits are dimerized through their LZ motifs. IKK beta is the major IKK catalytic subunit for NF-kB activation. Activated TAK1
phosphorylate IKK beta on serine residues (S177 and S181) in the activation loop and thus activate the IKK kinase activity, leading to the IkB
alpha phosphorylation and NF-kB activation.
References
H Hacker, M Karin, "Regulation and function of IKK and IKK-related kinases", Sci STKE, 2006, 2006, re13.
Reaction
31.3.4.22 Ubiquitination of NEMO by TRAF6
Authors
Reviewers
Description
During the phosphorylation of the IKK beta, the regulatory subunit NEMO undergoes K-63-linked polyubiquitination. Ubiquitinated TRAF6 trimer,
acts as a E3 ligase and induces this ubiquitination. The ubiquitin target sites in NEMO are not yet clearly identified. Residues Lysine 321 and
Lysine 326 may be few of the potent ubiquitination sites in NEMO.
References
H Hacker, M Karin, "Regulation and function of IKK and IKK-related kinases", Sci STKE, 2006, 2006, re13.
H Sebban-Benin, A Pescatore, F Fusco, V Pascuale, J Gautheron, S Yamaoka, A Moncla, MV Ursini, G Courtois, "IDENTIFICATION OF
TRAF6-DEPENDENT NEMO POLYUBIQUITINATION SITES THROUGH ANALYSIS OF A NEW NEMO MUTATION CAUSING
INCONTINENTIA PIGMENTI", Hum Mol Genet, 127, 2007.
Reaction
31.3.4.23 Activation of NF-kB complex
Authors
Reviewers
Description
NF-kB is a family of transcription factors that play pivotal roles in immune, inflammatory, and antiapoptotic responses. NF-kB/Rel family include
five members, p65 (RelA), RelB, c-Rel, p50/p105 (NF-kB1) and p52/p100 (NF-kB2), exist in unstimulated cells as homo or heterodimers. NF-kB
is sequestered in the cytosol of unstimulated cells through the interactions with a class of inhibitor proteins, called IkBs, which mask the nuclear
localization signal of NF-kB and prevent its nuclear translocation. Various stimuli induce the activation of the IkB kinase (IKK) complex, which
then phosphorylates IkBs. The phosphorylated IkBs are ubiquitinated and then degraded through the proteasome-mediated pathway. The
degradation of IkBs releases NF-kB to translocate into nucleus and induces the expression of various genes.
The phosphorylation and ubiquitination of IkB kinase complex is mediated by two distinct pathways, either the classical or alternative pathway. In
the classical NF-kB signaling pathway, the activated IKK complex, predominantly acting through IKK beta in an IKK gamma-dependent manner,
catalyzes the phosphorylation of IkBs (at sites equivalent to Ser32 and Ser36 of IkB-alpha or Ser19 and Ser22 of IkB-beta), polyubiquitination (at
sites equivalent to Lys21 and Lys22 of IkB-alpha) and subsequent degradation by the 26S proteasome. The K-48 ubiquitination is mediated by
the E2 ubiquitin ligases (or SCFs) formed by three subunits: Skp1, Cul A (Cdc53), and one of many F-box proteins.
References
JA DiDonato, M Hayakawa, DM Rothwarf, E Zandi, M Karin, "A cytokine-responsive IkappaB kinase that activates the transcription factor
NF-kappaB", Nature, 388, 1997, 548-54.
G Bonizzi, M Karin, "The two NF-kappaB activation pathways and their role in innate and adaptive immunity", Trends Immunol, 25, 2004, 280-8.
PB Shambharkar, M Blonska, BP Pappu, H Li, Y You, H Sakurai, BG Darnay, H Hara, J Penninger, X Lin, "Phosphorylation and ubiquitination of
the IkappaB kinase complex by two distinct signaling pathways", EMBO J, 26, 2007, 1794-805.
MS Hayden, S Ghosh, "Signaling to NF-kappaB", Genes Dev, 18, 2004, 2195-224.
Reaction
31.4 Cell surface interactions at the vascular wall
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
Leukocyte extravasation is a rigorously controlled process that guides white cell movement from the vascular lumen to sites of tissue
inflammation. The powerful adhesive interactions that are required for leukocytes to withstand local flow at the vessel wall is a multistep process
mediated by different adhesion molecules. Platelets adhered to injured vessel walls form strong adhesive substrates for leukocytes. For
instance, the initial tethering and rolling of leukocytes over the site of injury are mediated by reversible binding of selectins to their cognate
cell-surface glycoconjugates.
Endothelial cells are tightly connected through various proteins, which regulate the organization of the junctional complex and bind to
cytoskeletal proteins or cytoplasmic interaction partners that allow the transfer of intracellular signals. An important role for these junctional
proteins in governing the transendothelial migration of leukocytes under normal or inflammatory conditions has been established.
This pathway describes some of the key interactions that assist in the process of platelet and leukocyte interaction with the endothelium, in
response to injury.
References
SP Jackson, N Mistry, Y Yuan, "Platelets and the injured vessel wall-- "rolling into action": focus on glycoprotein Ib/V/IX and the platelet
cytoskeleton", Trends Cardiovasc Med, 10, 2000, 192-7.
A Schober, C Weber, "Mechanisms of monocyte recruitment in vascular repair after injury", Antioxid Redox Signal, 7, 2005, 1249-57.
BF Becker, B Heindl, C Kupatt, S Zahler, "Endothelial function and hemostasis", Z Kardiol, 89, 2000, 160-7.
B Furie, BC Furie, "The molecular basis of platelet and endothelial cell interaction with neutrophils and monocytes: role of P-selectin and the
P-selectin ligand, PSGL-1", Thromb Haemost, 74, 1995, 224-7.
31.4.1 Binding of GP VI:Fc Epsilon R1 gamma receptor complex with collagen

Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
References
Reaction
31.4.2 MAC1 binds JAM-C
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.

Description
Recruitment of monocytic cells to the vessel wall by platelets is mediated via CD11b/CD18 (Mac-1) and platelet JAM-C. In the case of dendritic
cells, this interaction leads to their activation and platelet phagocytosis. This process may be of importance for progression of atherosclerotic
lesions.
References
HF Langer, K Daub, G Braun, T Schonberger, AE May, M Schaller, GM Stein, K Stellos, A Bueltmann, D Siegel-Axel, HP Wendel, H Aebert, M
Roecken, P Seizer, S Santoso, S Wesselborg, P Brossart, M Gawaz, "Platelets recruit human dendritic cells via Mac-1/JAM-C interaction and
modulate dendritic cell function in vitro", Arterioscler Thromb Vasc Biol, 27, 2007, 1463-70.
Reaction
31.4.3 JAM-B binds JAM-C
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
JAM-B and -C bind each other and are strongly expressed by endothelial cells of high endothelial venules, the predominant site of leukocyte
extravasation. JAM-B and -C also bind to the leukocyte integrins VLA-4 and Mac-1 respectively.
References
RJ Ludwig, TM Zollner, S Santoso, K Hardt, J Gille, H Baatz, PS Johann, J Pfeffer, HH Radeke, MP Schon, R Kaufmann, WH Boehncke, M
Podda, "Junctional adhesion molecules (JAM)-B and -C contribute to leukocyte extravasation to the skin and mediate cutaneous inflammation",
J Invest Dermatol, 125, 2005, 969-76.
Reaction
31.4.4 VLA-4 binds JAM-B
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Description
Several key IgSF cell adhesion molecules engage integrin and in so doing impact on the multi-step paradigm of leukocyte emigration. The
interaction between JAM-B and VLA-4 is facilitated by prior engagement of JAM-B with JAM-C.
References
SA Cunningham, JM Rodriguez, MP Arrate, TM Tran, TA Brock, "JAM2 interacts with alpha4beta1. Facilitation by JAM3.", J Biol Chem, 277,
2002, 27589-92.
Reaction
31.4.5 JAM-A homodimerises
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
JAM-A is the most widely expressed member of the family, and has been shown to be expressed on endothelial and epithelial cells, on platelets,
and on a number of leukocyte subsets. In endothelial cells, JAM-A locates to the tight junctions, where it appears to engage in homophilic
binding to JAM-A on adjacent cells, an interaction that is considered to play a critical role in angiogenesis.
References
A Woodfin, CA Reichel, A Khandoga, M Corada, MB Voisin, C Scheiermann, DO Haskard, E Dejana, F Krombach, S Nourshargh, "JAM-A
mediates neutrophil transmigration in a stimulus-specific manner in vivo: evidence for sequential roles for JAM-A and PECAM-1 in neutrophil
transmigration", Blood, 110, 2007, 1848-56.
H Huang, F Cruz, G Bazzoni, "Junctional adhesion molecule-A regulates cell migration and resistance to shear stress", J Cell Physiol, 209,
2006, 122-30.
PF Bradfield, S Nourshargh, M Aurrand-Lions, BA Imhof, "JAM family and related proteins in leukocyte migration (Vestweber series)",
Reaction
31.4.6 LFA1 binds JAM-A
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
JAM-A plays a key role in leukocyte transmigration and inflammatory extravasation. Transmigration of human leukocytes has been shown to
involve heterophilic interactions of JAM-A with its integrin receptor LFA-1.
References
G Ostermann, L Fraemohs, T Baltus, A Schober, M Lietz, A Zernecke, EA Liehn, C Weber, "Involvement of JAM-A in mononuclear cell
recruitment on inflamed or atherosclerotic endothelium: inhibition by soluble JAM-A", Arterioscler Thromb Vasc Biol, 25, 2005, 729-35.
L Fraemohs, RR Koenen, G Ostermann, B Heinemann, C Weber, "The functional interaction of the beta 2 integrin lymphocyte
function-associated antigen-1 with junctional adhesion molecule-A is mediated by the I domain", J Immunol, 173, 2004, 6259-64.
G Ostermann, KS Weber, A Zernecke, A Schroder, C Weber, "JAM-1 is a ligand of the beta(2) integrin LFA-1 involved in transendothelial
migration of leukocytes", Nat Immunol, 3, 2002, 151-8.
Reaction
31.4.7 Integrin alphaX beta2 binds JAM-C
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
Although JAM-C is better known for its interaction with MAC-1, an interaction with CD11c/CD18 (known as alpha X beta 2), has also been
described.
References
S Santoso, UJ Sachs, H Kroll, M Linder, A Ruf, KT Preissner, T Chavakis, "The junctional adhesion molecule 3 (JAM-3) on human platelets is a
counterreceptor for the leukocyte integrin Mac-1", J Exp Med, 196, 2002, 679-91.
PF Bradfield, C Scheiermann, S Nourshargh, C Ody, FW Luscinskas, GE Rainger, GB Nash, M Miljkovic-Licina, M Aurrand-Lions, BA Imhof,
"JAM-C regulates unidirectional monocyte transendothelial migration in inflammation", Blood, 110, 2007, 2545-55.
Reaction
31.4.8 MERTK receptor binds ligands (Gas6 or Protein S)
Authors
Ouwehand, W.H., 2007-11-12.

Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
MerTK appears to be required for ingestion of apoptotic cells by professional phagocytes such as monocytes/macrophages, retinal pigment
epithelial cells and dendritic cells. Mer appears to be able to induce the cytoskeletal remodelling that is required for engulfment during
phagocytosis. For instance, a deletion in the MERTK gene was identified as the underlying cause for retinal dystrophy which involves an
impairment in the ingestion of shed photoreceptor cell fragments by retinal pigment epithelial cells.
The biological ligands for MerTK are two highly similar vitamin K-dependent proteins, Gas6 and protein S (PS), a negative regulator of blood
coagulation. Both proteins are composed an N-terminal region containing multiple post-translationally modified gamma-carboxyglutamic acid
residues (Gla). The Gla region possesses the ability to interact in a conformationally specific manner with negatively charged membrane
phospholipids, which is thought to mediate the binding of both Gas6 and PS to apoptotic cells. In this way, they are thought to act as recognition
bridges between apoptotic cells and the phagocyte cell that ingest them.
References
S Hafizi, B Dahlback, "Gas6 and protein S. Vitamin K-dependent ligands for the Axl receptor tyrosine kinase subfamily.", FEBS J, 273, 2006,
5231-44.
S Hafizi, B Dahlback, "Signalling and functional diversity within the Axl subfamily of receptor tyrosine kinases", Cytokine Growth Factor Rev, 17,
2006, 295-304.
HM Seitz, TD Camenisch, G Lemke, HS Earp, GK Matsushima, "Macrophages and dendritic cells use different Axl/Mertk/Tyro3 receptors in
clearance of apoptotic cells", J Immunol, 178, 2007, 5635-42.
Reaction
31.4.9 CD84 homodimerises
Authors
Ouwehand, W.H., 2007-11-12.

Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
CD84 is a homophilic receptor expressed on T cells, B cells, dendritic cells, monocytes, macrophages, eosinophils, mast cells, granulocytes, and
platelets. CD84 expression increases following activation of T cells, B cells, and dendritic cells. CD84 homophilic engagement is known to
induce platelet stimulation.
References
Q Yan, VN Malashkevich, A Fedorov, E Fedorov, E Cao, JW Lary, JL Cole, SG Nathenson, SC Almo, "Structure of CD84 provides insight into
SLAM family function", Proc Natl Acad Sci U S A, 104, 2007, 10583-8.
M Martin, X Romero, MA de la Fuente, V Tovar, N Zapater, E Esplugues, P Pizcueta, J Bosch, P Engel, "CD84 functions as a homophilic
adhesion molecule and enhances IFN-gamma secretion: adhesion is mediated by Ig-like domain 1", J Immunol, 167, 2001, 3668-76.
Reaction
31.4.10 P-selectin binds P-selectin ligand
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
PSGL-1 is expressed as a homodimer of two 120-kDa subunits that binds all three selectins, with the highest affinity for P-selectin, and is known
to be constitutively expressed on the surface of platelets and most types of leukocytes. Besides playing a critical role in the inflammatory
response by mediating leukocyte-leukocyte and leukocyte-endothelium interactions, PSGL-1 also participates in the hemostatic process by
mediating leukocyte-platelet interactions.
References
P da Costa Martins, JJ Garcia-Vallejo, JV van Thienen, M Fernandez-Borja, JM van Gils, C Beckers, AJ Horrevoets, PL Hordijk, JJ Zwaginga,
"P-selectin glycoprotein ligand-1 is expressed on endothelial cells and mediates monocyte adhesion to activated endothelium", Arterioscler
Reaction
31.4.11 JAM-B homodimerises
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
Apart from its well-established interaction with VLA-4, JAM-B is also known to homodimerize.
References
Reaction
31.4.12 JAM-C homodimerises
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
JAM-C has been detected in epithelial-cell desmosomes. JAM-C homodimers are prominently located in endothelial-cell tight junctions.
References
G Mandicourt, S Iden, K Ebnet, M Aurrand-Lions, BA Imhof, "JAM-C regulates tight junctions and integrin-mediated cell adhesion and migration",
J Biol Chem, 282, 2007, 1830-7.
Reaction
31.4.13 CD177 binds PECAM-1
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.

Description
CD177 is a 58- to 64-kDa glycosylphosphatidylinositol-anchored glycoprotein expressed exclusively by neutrophils, neutrophilic metamyelocytes,
and myelocytes, but not by any other blood cells. It has been shown that neutrophil-specific CD177 is a heterophilic binding partner of PECAM-1,
constituting a novel pathway that promotes neutrophil transmigration.
References
UJ Sachs, CL Andrei-Selmer, A Maniar, T Weiss, C Paddock, VV Orlova, EY Choi, PJ Newman, KT Preissner, T Chavakis, S Santoso, "The
neutrophil-specific antigen CD177 is a counter-receptor for platelet endothelial cell adhesion molecule-1 (CD31)", J Biol Chem, 282, 2007,
23603-12.
A Kalinowska, J Losy, "PECAM-1, a key player in neuroinflammation", Eur J Neurol, 13, 2006, 1284-90.
Reaction
31.4.14 CD47 binds SIRP
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
Integrin-associated protein (IAP or CD47) is a receptor for thrombospondin family members, a ligand for the transmembrane signaling protein
SIRP-alpha and -gamma, and a component of a supramolecular complex containing specific integrins, heterotrimeric G proteins and cholesterol.
References
AN Barclay, MH Brown, "The SIRP family of receptors and immune regulation", Nat Rev Immunol, 6, 2006, 457-64.
Reaction
31.4.15 CD48 binds CD244
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
CD2, CD48, CD84, CD244 and CD58 have a similar extracellular domain arichitecture consisting of two IgSF domains. CD244 is closely related
to CD84 in having a long cytoplasmic tail with tyrosine-based motifs (TxYxxI/V) resembling immunoreceptor tyrosine-based inhibitory motifs
(ITIMs). CD2 has a cytoplasmic domain with proline-rich regions which recruit an Src homology 3 (SH3)- containing protein called
CD2-associated protein (CD2AP). CD48 is glycosyl-phosphatidyl-inositol (GPI)-anchored to the membrane.
CD244 is known to be activated by binding to CD48 in humans.
References
SG Tangye, JH Phillips, LL Lanier, "The CD2-subset of the Ig superfamily of cell surface molecules: receptor-ligand pairs expressed by NK cells
and other immune cells", Semin Immunol, 12, 2000, 149-57.
B Messmer, P Eissmann, S Stark, C Watzl, "CD48 stimulation by 2B4 (CD244)-expressing targets activates human NK cells", J Immunol, 176,
2006, 4646-50.
Reaction
31.4.16 CD58 binds CD2
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
The crystal structure of the human CD2-CD58 complex also shows that most of the residues at the interface between these two proteins are
charged and form several inter-protein salt bridges.
References
AL Wilkins, W Yang, JJ Yang, "Structural biology of the cell adhesion protein CD2: from molecular recognition to protein folding and design",
Curr Protein Pept Sci, 4, 2003, 367-73.
MV Bayas, A Kearney, A Avramovic, PA van der Merwe, DE Leckband, "Impact of salt bridges on the equilibrium binding and adhesion of
human CD2 and CD58", J Biol Chem, 282, 2007, 5589-96.
Reaction
31.4.17 Integrin alpha 5 beta 1 binds fibronectin
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
Tenacious binding of free fibronectin to cells leads to enhanced fibronectin matrix assembly and the formation of a polymerized fibronectin
"cocoon" around the cells. This process is enhanced in the presence of CEACAM molecules.
References
C Brakebusch, R Fassler, "beta 1 integrin function in vivo: adhesion, migration and more", Cancer Metastasis Rev, 24, 2005, 403-11.
C Ordonez, AB Zhai, P Camacho-Leal, L Demarte, MM Fan, CP Stanners, "GPI-anchored CEA family glycoproteins CEA and CEACAM6
mediate their biological effects through enhanced integrin alpha5beta1-fibronectin interaction", J Cell Physiol, 210, 2007, 757-65.
Z Zhang, AO Morla, K Vuori, JS Bauer, RL Juliano, E Ruoslahti, "The alpha v beta 1 integrin functions as a fibronectin receptor but does not
support fibronectin matrix assembly and cell migration on fibronectin", J Cell Biol, 122, 1993, 235-42.
Reaction
Authors
Ouwehand, W.H., 2007-11-12.

Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
References
2006, 359-66.
16, 2005, 2694-703.
Reaction
31.4.19 protein C -> activated protein C + protein C heavy chain activation peptide
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
References
Reaction
31.4.20 OLR1 binds to oxidized LDL
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
The lectin-like oxidized low density lipoprotein receptor- 1 (Lox-1) mediates the recognition and internalization of oxidatively modified low density
lipoprotein. This interaction results in a number of pro-atherogenic cellular responses that probably play a significant role in the pathology of
atherosclerosis.
References
Q Xie, S Matsunaga, S Niimi, S Ogawa, K Tokuyasu, Y Sakakibara, S Machida, "Human lectin-like oxidized low-density lipoprotein receptor-1
functions as a dimer in living cells", DNA Cell Biol, 23, 2004, 111-7.
T Sawamura, N Kume, T Aoyama, H Moriwaki, H Hoshikawa, Y Aiba, T Tanaka, S Miwa, Y Katsura, T Kita, T Masaki, "An endothelial receptor
for oxidized low-density lipoprotein", Nature, 386, 1997, 73-7.
Reaction
31.4.21 Platelet-derived TREM-1 ligand binds to TREM-1
Authors
Ouwehand, W.H., 2007-11-12.
Reviewers
Zwaginga, JJ, 2007-11-12 16:47:00.
Description
The triggering receptor expressed on myeloid cells 1 (TREM-1) plays an important role in the innate immune response related to severe
infections and sepsis. Although the identity and occurrence of the natural TREM-1 ligands are so far unknown, the presence of a ligand for
TREM-1 on human platelets has been established.
References
P Haselmayer, L Grosse-Hovest, P von Landenberg, H Schild, MP Radsak, "TREM-1 ligand expression on platelets enhances neutrophil
activation", Blood, 110, 2007, 1029-35.
Reaction
31.4.22 Tie2 Signaling
Authors

Reviewers
Description
The Tie2/Tek receptor tyrosine kinase plays a pivotal role in vascular and hematopoietic development and is expressed exclusively on
endothelial lineage. Tie2 interacts with a group of ligands belonging to angiopoietin family and undergoes activation.
These ligands show opposing actions, angiopoietin 1 and angiopoietin 4 stimulate the Tie2 phosphorylation and angiopoietin 2 inhibits it. Upon
tyrosine phosphorylation Tie2 acts as a scaffold for various signaling proteins involved in different signal transduction cascades that can effect
survival of endothelium and angiogenic sprout formation.
References
2002, 19-27.
Authors
Reviewers
Description
Tie receptors and their angiopoietin ligands play a critical role in angiogenesis or blood vessel formation. They are considered to control
numerous signaling pathways that are involved in diverse cellular processes, such as cell migration, proliferation, survival and reorganization of
the actin cytoskeleton.
Tie (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains) represents a class of receptor tyrosine kinases
(RTKs) that are predominately expressed by vascular endothelial cells. The angiopoietins are a family of growth factors that are largely specific
for endothelium and they bind to Tie2/Tek RTKs.
Tie2 signaling initially involves the activation of Tie2 by the interaction of angiopoietin 1. Angiopoietin interacts with the Tie2 receptor with its
fibrinogen like domain (FLD). This interaction leads to the dimerization of both the receptor and the ligand, and later initiate the
trans-phosphorylation of Tie2.
References
S Davis, TH Aldrich, PF Jones, A Acheson, DL Compton, V Jain, TE Ryan, J Bruno, C Radziejewski, PC Maisonpierre, GD Yancopoulos,
"Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning", Cell, 87, 1996, 1161-9.
Y Xu, Q Yu, "Angiopoietin-1, unlike angiopoietin-2, is incorporated into the extracellular matrix via its linker peptide region", J Biol Chem, 276,
2001, 34990-8.
2002, 19-27.
Reaction
31.4.22.2 Dimerization of Tie2/Ang1 complex
Authors
Reviewers
Description
Receptor tyrosine kinase activation and signaling are typically initiated via dimerization of the receptors through homo-oligomeric ligand binding.
Angiopoietin1 may form homotrimers, but in most cases it assembles into higher-order multimers. This oligomerization is mediated by the N-ter
coiled coil domain (CCD).
The binding of Ang1 oligomers to Tie2 promotes the dimerization of Tie2, which is further assisted by the interaction between the kinase
domains of the receptors.
References
LM Shewchuk, AM Hassell, B Ellis, WD Holmes, R Davis, EL Horne, SH Kadwell, DD McKee, JT Moore, "Structure of the Tie2 RTK domain:
self-inhibition by the nucleotide binding loop, activation loop, and C-terminal tail", Structure, 8, 2000, 1105-13.
Reaction
31.4.22.3 Trans-phosphorylation of Tie2
Authors
Reviewers
Description
The dimerization of Tie2 leads to autophosphorylation and activation of its kinase domain. There are multiple tyrosine phosphorylation sites in
the Tie2 kinase domain. The phosphorylated tyrosine residues provide the interaction site for the SH2 domains of other downstream signaling
molecules like PI3K, Grb2, SHP2 etc.
References
Source reaction
This reaction was inferred from the corresponding reaction "Trans-phosphorylation of Tie2" in species Mus musculus.
Reaction
31.4.22.4 Interaction of Tie2 and p85 of PI3K
Authors
Reviewers
Description
The p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) associates with Tie2, most likely at phosphotyrosine 1102. This association leads
on to the activation of Akt/PKB, a process linked to cell survival and antiapoptosis, and that may in part account for Tie2's role in vascular growth
and maintenance.
References
Reaction
31.4.22.5 Interaction of Tie2 and Grb2
Authors
Reviewers
Description
Tie2/Tek provide mitogenic signals to endothelial cells by promoting the association of Grb2 to one of their phosphotyrosines. Grb2 is an adaptor
protein that has been linked to activation of Ras and mitogen activated protein kinase (MAPK) cell growth signaling pathways. Grb2 also binds to
the Y1102 of the kinase domain of Tie2 with one of its SH2 doamins.
References
Reaction
31.4.22.6 Interaction of SOS-1 to Tie2 bound Grb2
Authors
Reviewers
Description
Grb2 binds directly to autophosphorylated Tie2 receptor. GRB2 also contains two SH3 domains, which bring various ligands to the sites of active
signaling. One of the SH3 domains on Tie2-bound Grb2 recruits SOS1, an activating nucleotide exchange factor for Ras. This interaction of
Sos1 to Grb2 brings Sos1 towards Ras molecules leading to Ras activation. Ras is implicated in the MAP kinase cascade, a pathway in cell
growth stimulation, migration and differentiation.
References
Reaction
31.4.22.7 Sos-mediated nucleotide exchange of Ras (Tie2 receptor:Grb2:Sos)
Authors
Reviewers
Description
Sos-1 bound to Grb2:Tie2 complex promotes the exchange of inactive Ras-GDP to active Ras-GTP.
Reaction
31.4.22.8 Interaction of Tie2 and Shc1
Authors
Reviewers
Description
ShcA, an SH2-containing adapter protein, acts as a scaffold for the assembly of signaling proteins involved in the activation of the Ras-MAPK
pathway, and potentially other signaling pathways.
ShcA is one of the binding partners of endogenous Tie2 receptor on vascular endothelial cells. After Tie2 stimulation by Ang-1 interaction, ShcA
associates with Tie2 and becomes tyrosine-phosphorylated. ShcA interacts with the cytoplasmic domain of Tie2 and Y1102 of Tie2 was
identified as the primary binding site for the SH2 domain of ShcA. ShcA leads to a reduction of tyrosine phosphorylation of p85 subunit of
PI3-kinase and is involved in the inhibition of endothelial cell migration and survival.
References
E Audero, I Cascone, F Maniero, L Napione, M Arese, L Lanfrancone, F Bussolino, "Adaptor ShcA protein binds tyrosine kinase Tie2 receptor
and regulates migration and sprouting but not survival of endothelial cells", J Biol Chem, 279, 2004, 13224-33.
Reaction
31.4.22.9 Interaction of Tie2 and Dok-2
Authors
Reviewers
Description
Dok-2 is a member of a docking proteins class, termed the DOK family. The DOK family members are characterized by an N-terminal pleckstrin
homology (PH) domain followed by a central PTB domain and a proline- and tyrosine-rich C-terminal tail. Dok-2 is recruited to activated Tie2 via
its PTB domain, which results in its subsequent tyrosine phosphorylation, thereby establishing binding sites for the small GTPase-activating
protein for Ras, p120RasGAP (RasGAP) and the adapter protein Nck. The binding of DOK to the receptor leads to Nck recruitment and
subsequent phosphorylation. Binding of Pak to Nck follows. this brings about the Ang-1-dependent phosphorylation of Pak in endothelial cells.
References
Z Master, N Jones, J Tran, J Jones, RS Kerbel, DJ Dumont, "Dok-R plays a pivotal role in angiopoietin-1-dependent cell migration through
recruitment and activation of Pak", EMBO J, 20, 2001, 5919-28.
Reaction
Authors
Reviewers
Description
The major ligands for Tie2 are Ang1 and Ang2. Ang1 has been considered as the primary activating ligand of Tie2 whereas role of Ang2 remains
controversial. Ang2 acts as stimulating in some studies and inhibiting in others. The activity of Ang2 is concentration dependent. Ang2
possesses similar receptor affinity to Ang1 and they both share the same binding site on Tie2. The Ang2 fibrinogen domain is solely responsible
for receptor recognition and binding, the coiled-coil motif mediates its oligomerization.
References
E Bogdanovic, VP Nguyen, DJ Dumont, "Activation of Tie2 by angiopoietin-1 and angiopoietin-2 results in their release and receptor
internalization", J Cell Sci, 119, 2006, 3551-60.
WA Barton, D Tzvetkova, DB Nikolov, "Structure of the angiopoietin-2 receptor binding domain and identification of surfaces involved in Tie2
recognition", Structure, 13, 2005, 825-32.
Reaction
31.4.22.11 Interaction of Tie2 and Ang4
Authors
Reviewers
Description
Ang4 represents a third protein of the Ang family that binds to the Tie2 receptor. The mouse Ang3 and human Ang4 are interspecies orthologs.
Ang4 acts as an activating ligand and induces phosphorylation in Tie2.
References
2002, 19-27.
HJ Lee, CH Cho, SJ Hwang, HH Choi, KT Kim, SY Ahn, JH Kim, JL Oh, GM Lee, GY Koh, "Biological characterization of angiopoietin-3 and
angiopoietin-4", FASEB J, 18, 2004, 1200-8.
DM Valenzuela, JA Griffiths, J Rojas, TH Aldrich, PF Jones, H Zhou, J McClain, NG Copeland, DJ Gilbert, NA Jenkins, T Huang, N
Papadopoulos, PC Maisonpierre, S Davis, GD Yancopoulos, "Angiopoietins 3 and 4: diverging gene counterparts in mice and humans", Proc
Natl Acad Sci U S A, 96, 1999, 1904-9.
Reaction
31.4.23 PECAM1 interactions
Authors
Reviewers
Description
PECAM-1/CD31 is a member of the immunoglobulin superfamily (IgSF) and has been implicated to mediate the adhesion and trans-endothelial
an ITIM motif within its cytoplasmic region. PECAM-1 mediates cellular interactions by both homophilic and heterophilic interactions. The
cytoplasmic domain of PECAM-1 contains tyrosine residues which serves as docking sites for recruitment of cytosolic signaling molecules.
Under conditions of platelet activation, PECAM-1 is phosphorylated by Src kinase members. The tyrosine residues 663 and 686 are required for
recruitment of the SH2 domain containing PTPs.
References
DE Jackson, "The unfolding tale of PECAM-1", FEBS Lett, 540, 2003, 7-14.
N Gong, S Chatterjee, "Platelet endothelial cell adhesion molecule in cell signaling and thrombosis", Mol Cell Biochem, 253, 2003, 151-8.
31.4.23.1 Trans-homophilic interaction of PECAM-1
Authors
Reviewers
Description
References
JP Newton, AP Hunter, DL Simmons, CD Buckley, DJ Harvey, "CD31 (PECAM-1) exists as a dimer and is heavily N-glycosylated", Biochem
JP Newton, CD Buckley, EY Jones, DL Simmons, "Residues on both faces of the first immunoglobulin fold contribute to homophilic binding sites
of PECAM-1/CD31", J Biol Chem, 272, 1997, 20555-63.
Reaction
31.4.23.2 Heterophilic interaction of PECAM-1 and Integrin alpha-v beta-3
Authors
Reviewers
Description
Alpha v beta 3 integrin is one of the potential heterophilic ligands of PECAM-1 that is involved in down-regulation of T-cell responses. The
heterophilic interaction of alpha v beta 3 integrin on endothelial cells with PEACAM-1 on leukocytes increases the adhesive function of beta
integrins on T cells, monocytes, neutrophils and NK cells suggesting that leukocyte PEACAM-1 act as a signaling molecule.
References
CD Buckley, R Doyonnas, JP Newton, SD Blystone, EJ Brown, SM Watt, DL Simmons, "Identification of alpha v beta 3 as a heterotypic ligand
for CD31/PECAM-1", J Cell Sci, 109, 1996, 437-45.
L Piali, P Hammel, C Uherek, F Bachmann, RH Gisler, D Dunon, BA Imhof, "CD31/PECAM-1 is a ligand for alpha v beta 3 integrin involved in
adhesion of leukocytes to endothelium", J Cell Biol, 130, 1995, 451-60.
Reaction
31.4.23.3 Phoshorylation of PECAM-1 by Fyn or Lyn or c-Src
Authors
Reviewers
Description
PECAM-1 is capable of transmitting information into the cell following its engagement and becomes tyrosine-phosphorylated during the platelet
aggregation process. The Src family of tyrosine kinases (more specifically, Src, Lyn, and c-src) has been widely implicated in the
phosphorylation of PECAM-1. Conserved tyrosine residues (Tyr663 and Tyr686) within the PECAM-1 cytoplasmic ITIM motif have been shown
to become phosphorylated. Tyrosine phosphorylation of PECAM-1 prompts its association with intracellular signal transduction molecules.
References
MY Cao, M Huber, N Beauchemin, J Famiglietti, SM Albelda, A Veillette, "Regulation of mouse PECAM-1 tyrosine phosphorylation by the Src
and Csk families of protein-tyrosine kinases", J Biol Chem, 273, 1998, 15765-72.
M Cicmil, JM Thomas, T Sage, FA Barry, M Leduc, C Bon, JM Gibbins, "Collagen, convulxin, and thrombin stimulate aggregation-independent
tyrosine phosphorylation of CD31 in platelets. Evidence for the involvement of Src family kinases.", J Biol Chem, 275, 2000, 27339-47.
Reaction
31.4.23.4 Interaction of PECAM-1 and SHIP
Authors
Reviewers
Description
PECAM/CD31 is a member of the immunoglobulin superfamily (IgSF) and has been implicated to mediate the adhesion and trans-endothelial
an ITIM motif within its cytoplasmic region.
References
1999, 77-83.
Reaction
Authors
Reviewers
Description
PECAM-1 becomes tyrosine-phosphorylated during the platelet aggregation process and that this creates docking sites for the protein-tyrosine
phosphatase SHP-2. The interaction between SHP-2 and PECAM-1 is dependent upon integrin-mediated platelet/platelet interactions and
occurs via the Src homology 2 (SH2) domains of the phosphatase and highly conserved phosphatase-binding motifs encompassing
phosphotyrosines 663 and 686 within the cytoplasmic domain of PECAM-1.
References
DE Jackson, CM Ward, R Wang, PJ Newman, "The protein-tyrosine phosphatase SHP-2 binds platelet/endothelial cell adhesion molecule-1
(PECAM-1) and forms a distinct signaling complex during platelet aggregation. Evidence for a mechanistic link between PECAM-1- and
integrin-mediated cellular signaling.", J Biol Chem, 272, 1997, 6986-93.
Reaction
31.4.23.6 Interaction of PECAM-1 and PLC gamma1
Authors

Reviewers
Description
Like SHP-1 and SHP-2, PLC-gamma 1 also interacts with PECAM-1. PLC-gamma 1 binds with both the tyrosine residues (Y663 and Y686).
Unlike the N-SH2 domain, the C-SH2 domain on PLC-gamma 1 can only bind phosphotyrosine 663. The engagement of PECAM-1 with
PLC-gamma 1 may lead to PLC-gamma 1 activation and subsequent calcium influx.
References
1999, 77-83.
Reaction
Authors
Reviewers
Description
The phosphorylation of two tandem tyrosine residues (Y663 and Y686) within the cytoplasmic domain of PECAM-1 is required for the
downstream signalling events observed following PECAM-1 ligation. Both SH2 domains of SHP-1 are required in tandem to bind PECAM-1.
References
1999, 77-83.
CT Hua, JR Gamble, MA Vadas, DE Jackson, "Recruitment and activation of SHP-1 protein-tyrosine phosphatase by human platelet endothelial
cell adhesion molecule-1 (PECAM-1). Identification of immunoreceptor tyrosine-based inhibitory motif-like binding motifs and substrates.", J Biol
Chem, 273, 1998, 28332-40.
Reaction
31.4.24 Basigin interactions
Authors
Reviewers
Description
Basigin is a widely expressed transmembrane glycoprotein that belongs to the Ig superfamily and is highly enriched on the surface of epithelial
cells. Basigin is involved in intercellular interactions involved in various immunologic phenomena, differentiation, and development, but a major
function of basigin is stimulation of synthesis of several matrix metalloproteinases. Basigin also induces angiogenesis via stimulation of VEGF
production.
Basigin has an extracellular region with two Ig-like domains of which the N-term Ig-like domain is involved in interactions. It undergoes
interactions between basigin molecules on opposing cells or on neighbouring cells. It also interacts with a variety of other proteins like
caveolin-1, cyclophilins, integrins and annexin II that play important roles in cell proliferation, energy metabolism, migration, adhesion and
motion, especially in cancer metastasis.
References
JL Jiang, J Tang, "CD147 and its interacting proteins in cellular functions", Sheng Li Xue Bao, 59, 2007, 517-23.
31.4.24.1 CyP60 chaperones Basigin
Authors
Reviewers
Description
Basigin serves as a signaling receptor for extracellular cyclophilins. Its been reported that cyclophilin 60 (Cyp60), a distinct member of the
cyclophilin family is involved in the regulation of intracellular transport of basigin. The mechanism of this activity involves interaction of Cyp60
with the proline-containing region within or adjacent to the predicted transmembrane domain basigin. Cyp60 is co-localized with basigin at the
plasma membrane suggesting that Cyp60 may function as a chaperone escorting basigin through the secretory pathway.
References
T Pushkarsky, V Yurchenko, C Vanpouille, B Brichacek, I Vaisman, S Hatakeyama, KI Nakayama, B Sherry, MI Bukrinsky, "Cell surface
expression of CD147/EMMPRIN is regulated by cyclophilin 60", J Biol Chem, 280, 2005, 27866-71.
Reaction
31.4.24.2 Basigin homodimerises

Authors
Reviewers
Description
Basigin (Bsg) is a highly glycosylated transmembrane protein belonging to the Ig superfamily with two Ig domains. Bsg forms homo-oligomers on
the plasma membrane in a cis-dependent manner. The N-terminal Ig-like domain is functionally important in oligomer formation.
References
S Yoshida, M Shibata, S Yamamoto, M Hagihara, N Asai, M Takahashi, S Mizutani, T Muramatsu, K Kadomatsu, "Homo-oligomer formation by
basigin, an immunoglobulin superfamily member, via its N-terminal immunoglobulin domain", Eur J Biochem, 267, 2000, 4372-80.
Reaction
31.4.24.3 Caveolin-1 binds Basigin
Authors
Reviewers
Description
Stromal fibroblasts secrete multiple matrix metalloproteinases (MMP)1 that can promote tumor cell growth, survival, invasion, angiogenesis, and
metastasis. Basigin on the surface of carcinoma cells, stimulates production of MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), and
MMP-3 (stromelysin). Basigin has been shown to co-immunoprecipitate with caveolin-1. The second Ig domain of Basigin is required for this
association, which leads to decreased Besigin self-association on the cell surface. Therefore, caveolin-1 is a negative regulator of CD147
self-association, and its MMP-inducing activity.
References
W Tang, SB Chang, ME Hemler, "Links between CD147 function, glycosylation, and caveolin-1", Mol Biol Cell, 15, 2004, 4043-50.
W Tang, ME Hemler, "Caveolin-1 regulates matrix metalloproteinases-1 induction and CD147/EMMPRIN cell surface clustering", J Biol Chem,
279, 2004, 11112-8.
Reaction
31.4.24.4 Basigin binds CyPA
Authors
Reviewers
Description
Cyclophilin A (CyPA)1 is an intracellular protein belonging to the immunophilin family and is recognized as the major target for the potent
immunosuppressive drug cyclosporin A. CD147 is the natural cell surface receptor for CyPA. It is demonstrated that CD147 is an essential
component in the CyPA-initiated signaling cascade that culminates in ERK activation.
References
V Yurchenko, G Zybarth, M O'Connor, WW Dai, G Franchin, T Hao, H Guo, HC Hung, B Toole, P Gallay, B Sherry, M Bukrinsky, "Active site
residues of cyclophilin A are crucial for its signaling activity via CD147", J Biol Chem, 277, 2002, 22959-65.
Reaction
32 Signaling by Insulin receptor
Authors
Bevan, AP, 2003-07-31.
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Barroso, I, Stanley, FM, 0000-00-00.
Description
Insulin binding to its receptor results in receptor autophosphorylation on tyrosine residues and the tyrosine phosphorylation of insulin receptor
substrates (e.g. IRS and Shc) by the insulin receptor tyrosine kinase. This allows association of IRSs with downstream effectors such as PI-3K
via its Src homology 2 (SH2) domains leading to end point events such as Glut4 translocation. Shc when tyrosine phosphorylated associates
with Grb2 and can thus activate the Ras/MAPK pathway independent of the IRSs.
Signal transduction by the insulin receptor is not limited to its activation at the cell surface. The activated ligand-receptor complex initially at the
cell surface, is internalised into endosomes itself a process which is dependent on tyrosine autophosphorylation. Endocytosis of activated
receptors has the dual effect of concentrating receptors within endosomes and allows the insulin receptor tyrosine kinase to phosphorylate
substrates that are spatially distinct from those accessible at the plasma membrane. Acidification of the endosomal lumen, due to the presence
of proton pumps, results in dissociation of insulin from its receptor. (The endosome constitutes the major site of insulin degradation). This loss of
the ligand-receptor complex attenuates any further insulin-driven receptor re-phosphorylation events and leads to receptor dephosphorylation by
extra-lumenal endosomally-associated protein tyrosine phosphatases (PTPs). The identity of these PTPs is not clearly established yet. A
discussion of candidates will be added in the near future.
32.1 Insulin binding
Authors
Bevan, AP, 2003-07-31.
Description
Under normal physiological conditions blood glucose levels are kept under tight control by a series of regulated steps that ensure glucose
homeostasis. Upon feeding glucose levels rise and in response to this the body secretes insulin from pancreatic beta-cells into the blood. At
physiological concentrations insulin is present in the blood in its monomeric form. Binding of insulin to its receptor occurs on the receptor
alpha-subunits. There are two binding domains involved on the receptor (L1 and L2) and it is thought that the amino-terminus of insulin binds
with L1 on one of the alpha-subunits and the carboxyterminus with L2 on the other alpha-subunit.
The binding of insulin to its receptor causes a conformational change in the alpha-subunits. This in turn produces a conformational change in the
beta-subunits leading to the activation of the intrinsic insulin receptor tyrosine kinase.
References
E Van Obberghen, V Baron, L Delahaye, B Emanuelli, N Filippa, S Giorgetti-Peraldi, P Lebrun, I Mothe-Satney, P Peraldi, S Rocchi, D
Sawka-Verhelle, S Tartare-Deckert, J Giudicelli, "Surfing the insulin signaling web.", Eur J Clin Invest, 31, 2001, 966-77.
MF White, CR Kahn, "The insulin signaling system.", J Biol Chem, 269, 1994, 1-4.
B Cheatham, CR Kahn, "Insulin action and the insulin signaling network.", Endocr Rev, 16, 1995, 117-42.
Reaction
32.2 Autophosphorylation of insulin receptor
Authors
Bevan, AP, 2003-07-31.
Description
For the receptor to autophosphorylate requires a lysine at position 1030 to stabilize the gamma phosphate of ATP whilst the adenosine of ATP
itself interacts with three glycines at residues 1003 - 1008. The first tyrosine residues to be autophosphorylated are 1158, 1162 and 1163 in the
tyrosine kinase domain. This is shortly followed by tyrosine 972 in the juxtamembrane domain and tyrosines 1328 and 1330. These tyrosines fall
into the three distinct tyrosine phosphorylation domains of the beta-subunit. In total there are 13 potential tyrosines that may be phosphorylated.
The receptor phosphorylates itself in a trans rather than cis manner. That is one beta-subunit of the receptor phosphorylates the other rather
than itself.
References
MF White, CR Kahn, "The insulin signaling system.", J Biol Chem, 269, 1994, 1-4.
CR Kahn, "Banting Lecture. Insulin action, diabetogenes, and the cause of type II diabetes.", Diabetes, 43, 1994, 1066-84.
Reaction
32.3 Insulin receptor signalling cascade
Authors
Bevan, AP, 2003-07-31.
Description
Autophosphorylation of the insulin receptor triggers a series of signalling events, mediated by SHC or IRS, and resulting in activation of the
Ras/RAF and MAP kinase cascades. A second effect of the autophosphorylation of the insulin receptor is its internalisation into an endosome,
which downregulates its signalling activity.
References
PR Shepherd, DJ Withers, K Siddle, "Phosphoinositide 3-kinase: the key switch mechanism in insulin signalling.", Biochem J, 333, 1998, 471-90.
P Bevan, "Insulin signalling.", J Cell Sci, 114, 2001, 1429-30.
32.3.1 SHC-related events
Authors
Bevan, AP, 2003-07-31.
Description
SHC is one of the mediators of insulin signalling events. It is activated by phosphorylation and triggers a cascade of events involving SOS, RAF
and the MAP kinases.
32.3.1.1 SHC activation
Authors
2003-07-28.
Description
SHC interacts via its SH2 domain with the carboxyterminal phosphorylated tyrosines of the insulin receptor. As a result, SHC is tyrosine
phosphorylated (Tyr317) by the insulin receptor, later falling away from the receptor. The phosphorylation of tyrosine 317 of SHC allows GRB2 to
bind to it via its SH2 domain.
32.3.1.1.1 Binding of SHC to insulin receptor
Description
At the beginning of this reaction, 1 molecule of 'activated insulin receptor', and 1 molecule of 'SHC transforming protein' are present. At the end
of this reaction, 1 molecule of 'SHC:activated insulin receptor' is present.
This reaction takes place on the 'internal side of plasma membrane'.

Reaction
32.3.1.1.2 Phosphorylation of SHC
Description
At the beginning of this reaction, 1 molecule of 'ATP', and 1 molecule of 'SHC:activated insulin receptor' are present. At the end of this reaction,
1 molecule of 'phospho-SHC: activated insulin receptor', and 1 molecule of 'ADP' are present.
This reaction takes place on the 'internal side of plasma membrane' and is mediated by the 'transmembrane receptor protein tyrosine kinase
activity' of 'SHC:activated insulin receptor'.
Reaction
32.3.1.1.3 Dissociation of SHC-P from insulin receptor
Description
At the beginning of this reaction, 1 molecule of 'phospho-SHC: activated insulin receptor' is present. At the end of this reaction, 1 molecule of
'phospho-SHC', and 1 molecule of 'activated insulin receptor' are present.
Reaction
32.3.1.2 SHC-mediated signalling
Authors
Description
Release of phospho-SHC from the insulin receptor triggers a cascade of signalling events via SOS, RAF and the MAP kinases.
32.3.1.2.1 GRB2:SOS binds to SHC-P
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
Tyrosine receptor kinase stimulation phosphorylates Shc which recruits the SH2 domain of the adaptor protein GRB2, which is complexed with
SOS, an exchange factor for p21ras and RAC, through its SH3 domain. Besides SOS, the GRB2 SH3 domain can associate with other
intracellular targets, including GAB1. Erk and Rsk mediated phosphorylation results in dissociation of the SOS-GRB2 complex. This may explain
why Erk activation through Shc and SOS-GRB2 is transient. Inactive p21ras-GDP is found anchored to the plasma membrane by a farnesyl
residue. As Shc is phosphorylated by the the stimulated receptor near to the plasma membrane, the SOS-GRB2:Shc interaction brings the SOS
enzyme into close proximity to p21ras.
References
S Okada, JE Pessin, "Interactions between Src homology (SH) 2/SH3 adapter proteins and the guanylnucleotide exchange factor SOS are
differentially regulated by insulin and epidermal growth factor.", J Biol Chem, 271, 1996, 25533-8.
Reaction
32.3.1.2.2 SOS mediated nucleotide exchange of RAS (SHC)
Authors
Editors
Schmidt, EE, 0000-00-00.

Reviewers
Greene, LA, 2007-11-08.
Description
SOS promotes the formation of GTP-bound RAS, thus activating this protein. RAS activation results in activation of the protein kinases RAF1,
B-Raf, and MAP-ERK kinase kinase (MEKK), and the catalytic subunit of PI3K, as well as of a series of RALGEFs. The activation cycle of RAS
GTPases is regulated by their interaction with specific guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs).
GEFs promote activation by inducing the release of GDP, whereas GAPs inactivate RAS-like proteins by stimulating their intrinsic GTPase
activity. NGF-induced RAS activation via SHC-GRB2-SOS is maximal at 2 min but it is no longer detected after 5 min. Therefore, the transient
activation of RAS obtained through SHC-GRB2-SOS is insufficient for the prolonged activation of ERKs found in NGF-treated cells.
References
PA Boriack-Sjodin, SM Margarit, D Bar-Sagi, J Kuriyan, "The structural basis of the activation of Ras by Sos", Nature, 394, 1998, 337-43.
Reaction
32.3.1.2.3 RAF activation
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
Phosphorylated RAF is activated by Ras binding and stabilised in its active form by transient disassociation and reassociation of 14-3-3, as well
as further phosphorylation.
References
32.3.1.2.3.1 Transient dissociation of 14-3-3 upon Ras binding
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Heldin, CH, 2008-02-12.
Description
References
Reaction
32.3.1.2.3.2 Stabilisation of RAS:RAF by 14-3-3
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
14-3-3 has been displaced from Ser259. 14-3-3 may now bind its higher affinity Ser621 (S2) site. This stabilises an 'open' Raf-1 conformation
that is catalytically active, and bound to p21ras via both p21ras-binding domains, CRD and RBD.
References
Reaction
32.3.1.2.3.3 Stabilisation of RAF by further phosphorylation
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
Un as yet unidentified protein tyrosine kinase located in the plasma membrane phosphorylates Tyrosines 340 and 341 (Y1, Y2) of Raf-1.
Tyrosine phosphorylation serves to further stabilise the active 'open' Raf-1 conformation. (While the kinase has not been definitively identified,
the Src kinase is a plausible candidate.)
References
Reaction
32.3.1.2.4 MAP kinase cascade
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
The MAP kinase cascade describes a sequence of phosphorylation events involving serine/threonine-specific protein kinases. Used by various
signal transduction pathways, this cascade constitutes a common 'module' in the transmission of an extracellular signal into the nucleus.
32.3.1.2.4.1 MEK activation
Authors
Reviewers
Greene, LA, 2007-11-08.

Description
MEK is phosphorylated and activated by RAF.
32.3.1.2.4.1.1 Activated RAF complex binds MEK
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
MEK-1/2 have a proline rich domain (P) and critical Serine residues (MEK-1 S218/222, MEK-2 unknown, S) upon which the molecules may be
phosphorylated. MEK1/2 are found in the cytoplasm of unstimulated cells.
MEK-1/2 bind to active Raf-1 via the proline-rich domain. Active Raf-1 is able to phosphorylate target Serine and Threonine residues in the
presence of ATP.
Source reaction
This reaction was inferred from the corresponding reaction "Activated RAF complex binds MEK" in species Rattus norvegicus.
AD Catling, HJ Schaeffer, CW Reuter, GR Reddy, MJ Weber, "A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and
regulates MEK function.", Mol Cell Biol, 15, 1995, 5214-25.
Reaction
32.3.1.2.4.1.2 RAF phosphorylates MEK
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
Active Raf-1 phosphorylates MEK-1/2 on Serine residues, converting ATP to ADP. The MEK-1/2 kinase is now active.
32.3.1.2.4.1.2.1 RAF phosphorylates MEK1
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
Source reaction
This reaction was inferred from the corresponding reaction "RAF phosphorylates MEK1" in species Rattus norvegicus.
R Seger, D Seger, AA Reszka, ES Munar, H Eldar-Finkelman, G Dobrowolska, AM Jensen, JS Campbell, EH Fischer, EG Krebs,
"Overexpression of mitogen-activated protein kinase kinase (MAPKK) and its mutants in NIH 3T3 cells. Evidence that MAPKK involvement in
cellular proliferation is regulated by phosphorylation of serine residues in its kinase subdomains VII and VIII.", J Biol Chem, 269, 1994,
25699-709.
C Papin, A EychÃ¨ne, A Brunet, G PagÃ¨s, J PouyssÃ©gur, G Calothy, JV Barnier, "B-Raf protein isoforms interact with and phosphorylate
Mek-1 on serine residues 218 and 222.", Oncogene, 10, 1995, 1647-51.
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
Reaction
32.3.1.2.4.2 ERK activation
Reviewers
Greene, LA, 2007-11-08.
Description
Activated MEK phosphorylates and activates ERK.
32.3.1.2.4.2.1 ERK1 activation
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
Activated MEK1 phosphorylates and activates ERK1.

32.3.1.2.4.2.1.1 MEK1 binds ERK-1
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
In the cytoplasm activated MEK1 (Serine phosphorylated) may encounter monomeric, inactive ERK1. ERK1 in its inactive form is not
phosphorylated on a critical Threonine (T) and a critical Tyrosine (Y).
References
CF Zheng, KL Guan, "Cloning and characterization of two distinct human extracellular signal-regulated kinase activator kinases, MEK1 and
MEK2.", J Biol Chem, 268, 1993, 11435-9.
Reaction
32.3.1.2.4.2.1.2 MEK1 phosphorylates ERK-1
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
MEK1 phosphorylates the critical Tyrosine and Threonine on ERK1, converting two ATP to ADP. Phosphorylation of ERK-1 activates its kinase
activity.
References
MEK2.", J Biol Chem, 268, 1993, 11435-9.
Reaction
32.3.1.2.4.2.1.3 Dimerisation of phospho-ERK-1
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
Two phospho-ERK1 molecules dimerise and enter the nucleus, where they may phosphorylate downstream targets.
Source reaction
This reaction was inferred from the corresponding reaction "Dimerisation of phospho-ERK-2" in species Rattus norvegicus.
Reaction
32.3.1.2.4.2.1.4 Nuclear translocation of phospho-ERK-1 dimer
Reviewers
Greene, LA, 2007-11-08.
Description
In this reaction, 1 molecule of 'phospho-ERK-1 dimer' is translocated from cytosol to nucleoplasm.

Source reaction
This reaction was inferred from the corresponding reaction "Nuclear translocation of phospho-ERK-2 dimer" in species Rattus norvegicus.
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
32.3.1.2.4.2.2.1 MEK2 binds ERK-2
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
phosphorylated on a critical Threonine (T183) and a critical Tyrosine (Y185).
References
MEK2.", J Biol Chem, 268, 1993, 11435-9.
Reaction

Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
activity.
References
MEK2.", J Biol Chem, 268, 1993, 11435-9.
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
Source reaction
Reaction
Reviewers
Greene, LA, 2007-11-08.
Description

Source reaction
Reaction
32.3.2 IRS-related events
Authors
Bevan, AP, 2003-07-31.
Description
IRS is one of the mediators of insulin signalling events. It is activated by phosphorylation and triggers a cascade of events involving PI3K, SOS,
RAF and the MAP kinases. The proteins mentioned under IRS are examples of IRS family members acting as indicated. More family members
are to be confirmed and added in the future.
32.3.2.1 IRS activation
Authors
2003-07-28.
Description
Using receptor mutagenesis studies it is known that IRS1 via its PTB domain binds to the insulin receptor at the juxtamembrane region at
tyrosine 972. The interaction is further stabilized by the PH domain of IRS1 which interacts with the phospholipids of the plasma membrane. This
allows the receptor to phosphorylate IRS1 on up to 13 of its tyrosine residues. Once phosphorylated the IRS1 falls away from the receptor.
(Other IRS family members can also be phosphorylated by the insulin receptor - will be added in the near future.) Now in a tyrosine
phosphorylated and hence activated state other proteins can interact with the IRS proteins.
32.3.2.1.1 Binding of IRS to insulin receptor
Description
At the beginning of this reaction, 1 molecule of 'activated insulin receptor', and 1 molecule of 'IRS' are present. At the end of this reaction, 1
molecule of 'IRS:activated insulin receptor' is present.
Reaction
32.3.2.1.2 Phosphorylation of IRS
Description
At the beginning of this reaction, 1 molecule of 'ATP', and 1 molecule of 'IRS:activated insulin receptor' are present. At the end of this reaction, 1
molecule of 'phospho-IRS:activated insulin receptor', and 1 molecule of 'ADP' are present.
This reaction takes place on the 'internal side of plasma membrane' and is mediated by the 'transmembrane receptor protein tyrosine kinase
activity' of 'IRS:activated insulin receptor'.
Reaction
32.3.2.1.3 Dissociation of IRS-P from insulin receptor
Description
At the beginning of this reaction, 1 molecule of 'phospho-IRS:activated insulin receptor' is present. At the end of this reaction, 1 molecule of
'activated insulin receptor', and 1 molecule of 'phospho-IRS' are present.

Reaction
32.3.2.2 IRS-mediated signalling
Authors
Description
Release of phospho-IRS from the insulin receptor triggers a cascade of signalling events via PI3K, SOS, RAF and the MAP kinases.
32.3.2.2.1 PI3K Cascade
32.3.2.2.1.1 PI3K activation
Authors
2003-07-28.
Description
IRS1, IRS2 and IRS3 are all known to bind the regulatory subunit of PI3K via its SH2 domain, an interaction that itself activates the kinase
activity of the PI3K catalytic subunit.
Reaction
32.3.2.2.1.2 PKB-mediated events
Authors
Scott, J, Tatoud, R, 2005-05-09.
Description
PKB and PDK1 are activated via membrane-bound PIP3. Activated PDK1 phosphorylates PKB, which in turn phosphorylates PDE3B. The latter
hydrolyses cAMP to 5'AMP, depleting cAMP pools.
32.3.2.2.1.2.1 PDE3B signalling
32.3.2.2.1.2.1.1 Hydrolysis of cAMP to 5' AMP by Phosphorylated PDE3B
Description
At the beginning of this reaction, 1 molecule of '3',5'-Cyclic AMP' is present. At the end of this reaction, 1 molecule of 'AMP' is present.
This reaction is mediated by the 'hydrolase activity' of 'Phosphorylated PDE3B'.
References
E Degerman, P Belfrage, VC Manganiello, "Structure, localization, and regulation of cGMP-inhibited phosphodiesterase (PDE3)", J Biol Chem,
272, 1997, 6823-6.
VC Manganiello, M Taira, E Degerman, P Belfrage, "Type III cGMP-inhibited cyclic nucleotide phosphodiesterases (PDE3 gene family)", Cell
Signal, 7, 1995, 445-55.
Reaction
32.3.2.2.1.2.1.2 Phosphorylation of PDE3B
Description
At the beginning of this reaction, 2 molecules of 'ATP', and 1 molecule of 'PDE3B' are present. At the end of this reaction, 1 molecule of
'Phosphorylated PDE3B', and 2 molecules of 'ADP' are present.
This reaction is mediated by the 'kinase activity' of 'PIP3:Phosphorylated PKB complex'.
Source reaction
This reaction was inferred from the corresponding reaction "Phosphorylation of PDE3B by AKT-1" in species Mus musculus.
T Kitamura, Y Kitamura, S Kuroda, Y Hino, M Ando, K Kotani, H Konishi, H Matsuzaki, U Kikkawa, W Ogawa, M Kasuga, "Insulin-induced
phosphorylation and activation of cyclic nucleotide phosphodiesterase 3B by the serine-threonine kinase Akt", Mol Cell Biol, 19, 1999, 6286-96.
Reaction
32.3.2.2.1.2.2 mTOR signalling
32.3.2.2.1.2.2.1 Inhibition of TSC complex formation by PKB
Description
Phosphorylation of TSC2 by PKB disrupts TSC1/TSC2 heterodimer formation (PMID 15314020). TSC2 function is affected in at least two ways:
first, phosphorylation decreases the activity of TSC2; second, phosphorylation destabilizes the TSC2 protein. This destabilization is achieved by
disrupting complex formation between TSC1 and TSC2 and inducing ubiquination of the free TSC2 (PMID 12172553). Phosphorylation of
complexed TSC2 by PKB induces degradation of both TSC1 and TSC2 through the proteosome pathway (PMID 121676664). Phosphorylation of
complexed TSC2 by PKB may therefore result in the dissociation of the TSC1:TSC2 complex (PMID 12423332).
32.3.2.2.1.2.2.1.1 Phosphorylation of TSC2 by PKB
Description
At the beginning of this reaction, 3 molecules of 'ATP', and 1 molecule of 'TSC2-1' are present. At the end of this reaction, 3 molecules of 'ADP',
and 1 molecule of 'Inhibited TSC2-1-P at Ser 939, 1130 and Thr 1462' are present.
References
N Hay, N Sonenberg, "Upstream and downstream of mTOR", Genes Dev, 18, 2004, 1926-45.
Reaction
32.3.2.2.1.2.2.1.2 Phosphorylation of complexed TSC2 by PKB
Description
At the beginning of this reaction, 3 molecules of 'ATP', and 1 molecule of 'TSC1:TSC2' are present. At the end of this reaction, 3 molecules of
'ADP', and 1 molecule of 'TSC1:Inhibited TSC2-1-P' are present.
References
Reaction
32.3.2.2.1.2.2.2 GTP loading by Rheb
Description
Rheb is a GTP binding protein that exhibits GTPase activity. GDP is exchanged for GTP in the [Rheb:GDP] complex to form [Rheb:GTP], which
binds and activates the mTORC1 complex. This exchange is catalysed by an as yet unidentified guanine exchange factor (GEF) (PMIDs
15951850 and 15755954).
References
DP Siderovski, FS Willard, "The GAPs, GEFs, and GDIs of heterotrimeric G-protein alpha subunits", Int J Biol Sci, 1, 2005, 51-66.
Reaction
32.3.2.2.1.2.2.3 Formation of active mTORC1 complex

Description
mTOR forms a functional protein complex with at least two proteins: Raptor (Regulated Associated Protein of mTOR) and mLst8. This complex
is called mammalian TOR complex 1 (mTORC1). Raptor serves as a scaffolding protein to bridge the interaction between mTOR and its
substrates. mLst8 enhances the association of mTOR with Raptor. [Rheb:GTP] binds and activates mTORC1. Besides binding directly to mTOR,
Rheb can also bind to Raptor and mLst8 (PMIDs 15854902, 15755954 and 12150926).
References
K Inoki, H Ouyang, Y Li, KL Guan, "Signaling by target of rapamycin proteins in cell growth control", Microbiol Mol Biol Rev, 69, 2005, 79-100.
Reaction
32.3.2.2.1.2.2.4 mTORC1-mediated signalling
32.3.2.2.1.2.2.4.1 S6K1-mediated signalling
32.3.2.2.1.2.2.4.1.1 Activation of S6K1
Description
S6K1 contains a TOS motif. mTORC1 requires an intact TOS motif to bind and phosphorylate S6K1 (PMID 15809305).
References
SM Ali, DM Sabatini, "Structure of S6 kinase 1 determines whether raptor-mTOR or rictor-mTOR", J Biol Chem, 280, 2005, 19445-8.
Reaction
32.3.2.2.1.2.2.4.1.2 S6K1 signalling
32.3.2.2.1.2.2.4.1.2.1 Phosphorylation of Ribosomal protein S6 by activated S6K1
Description
Once phosphorylated, S6K1-P phosphorylates and activates ribosomal protein S6 (rpS6), which in turn selectively increases the translation of
5Ã¢â‚¬â„¢TOP mRNAs. These mRNAs encode exclusively for components of the translation machinery (PMID 15809305).
References
Reaction
32.3.2.2.1.2.2.4.1.2.2 Phosphorylation and inactivation of eEF2K by activated S6K1
Description
Phosphorylation of eEF2 kinase by S6K1-P results in decreased activity of this kinase. eEF2 kinase normally phosphorylates and deactivates
eEF2, preventing its binding to the ribosome.
References
Reaction
32.3.2.2.1.2.2.4.1.2.3 Phosphorylation and activation of eIF4G by activated S6K1
Description
At the beginning of this reaction, 3 molecules of 'ATP', and 1 molecule of 'eIF4G' are present. At the end of this reaction, 3 molecules of 'ADP',
and 1 molecule of 'eIF4G-P' are present.
This reaction takes place in the 'cytosol' and is mediated by the 'kinase activity' of 'S6K1-P'.
References
Reaction
32.3.2.2.1.2.2.4.1.2.4 Phosphorylation and activation of eIF4B by activated S6K1
Description
eIF4B is a physiologically relevant target of S6K1. Once phosphorylated and activated by S6K1, eIF4B specifically stimulates the ATPase and
RNA helicase activities of eIF4A.
References
Reaction
32.3.2.2.1.2.2.4.2 Release of eIF4E
Description
Raptor recruits mTOR to non-phosphorylated 4E-BP1 bound to eIF4E and positively modulates phosphorylation of 4E-BP1 by mTOR. 4E-BP1 is
further phosphorylated on multiple sites by other unknown kinases, also contributing to the dissociation of 4E-BP1 from eIF4E. Thus mTORC1
relieves the inhibitory effect of 4E-BP1 on eIF4E dependent translation initiation (PMIDs 15755954, 10872469, 11297505, 12150926 and
15809305).
32.3.2.2.1.2.2.4.2.1 Phosphorylation of 4E-BP1 by activated mTORC1
Description
At the beginning of this reaction, 2 molecules of 'ATP', and 1 molecule of 'eIF4E:4E-BP' are present. At the end of this reaction, 1 molecule of
'eIF4E:4E-BP1-P', and 2 molecules of 'ADP' are present.
This reaction is mediated by the 'kinase activity' of 'Activated mTORC1'.

References
K Hara, Y Maruki, X Long, K Yoshino, N Oshiro, S Hidayat, C Tokunaga, J Avruch, K Yonezawa, "Raptor, a binding partner of target of
rapamycin (TOR), mediates TOR", Cell, 110, 2002, 177-89.
Reaction
32.3.2.2.1.2.2.4.2.2 Dissociation of phosphorylated 4EBP1 from eIF4E
Description
At the beginning of this reaction, 1 molecule of 'eIF4E:4E-BP1-P' is present. At the end of this reaction, 1 molecule of '4E-BP1-P', and 1
molecule of 'eIF4E' are present.
References
Reaction
32.3.2.2.1.3 Activation of PKB
32.3.2.2.1.3.1 PDK1 attachment to plasma membrane
Description
At the beginning of this reaction, 1 molecule of '3-phosphoinositide dependent protein kinase-1 ', and 1 molecule of
'Phosphatidylinositol-3,4,5-trisphosphate' are present. At the end of this reaction, 1 molecule of 'PIP3:PDK complex [plasma membrane]' is
present.
Reaction
32.3.2.2.1.3.2 PKB attachment to plasma membrane
Description
At the beginning of this reaction, 1 molecule of 'PKB:PKB Regulator', and 1 molecule of 'Phosphatidylinositol-3,4,5-trisphosphate' are present. At
the end of this reaction, 1 molecule of 'PKB regulator', and 1 molecule of 'PIP3:PKB complex ' are present.
References
L Stephens, K Anderson, D Stokoe, H Erdjument-Bromage, GF Painter, AB Holmes, PR Gaffney, CB Reese, F McCormick, P Tempst, J
Coadwell, PT Hawkins, "Protein kinase B kinases that mediate phosphatidylinositol 3,4,5-trisphosphate-dependent activation of protein kinase
B", Science, 279, 1998, 710-4.
SM Maira, I Galetic, DP Brazil, S Kaech, E Ingley, M Thelen, BA Hemmings, "Carboxyl-terminal modulator protein (CTMP), a negative regulator
of PKB/Akt and v-Akt at the plasma membrane", Science, 294, 2001, 374-80.
K Du, S Herzig, RN Kulkarni, M Montminy, "TRB3: a tribbles homolog that inhibits Akt/PKB activation by insulin in liver", Science, 300, 2003,
1574-7.
SR James, CP Downes, R Gigg, SJ Grove, AB Holmes, DR Alessi, "Specific binding of the Akt-1 protein kinase to phosphatidylinositol
3,4,5-trisphosphate without subsequent activation", Biochem J, 315, 1996, 709-13.
Reaction
32.3.2.2.1.3.3 Phosphorylation of PKB by PDK1
Description
At the beginning of this reaction, 2 molecules of 'ATP', and 1 molecule of 'PIP3:PKB complex ' are present. At the end of this reaction, 1
molecule of 'PIP3:Phosphorylated PKB complex', and 2 molecules of 'ADP' are present.
This reaction takes place on the 'plasma membrane' and is mediated by the 'kinase activity' of 'PIP3:PDK complex [plasma membrane]'.
References
D Stokoe, LR Stephens, T Copeland, PR Gaffney, CB Reese, GF Painter, AB Holmes, F McCormick, PT Hawkins, "Dual role of
phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B", Science, 277, 1997, 567-70.
DR Alessi, SR James, CP Downes, AB Holmes, PR Gaffney, CB Reese, P Cohen, "Characterization of a 3-phosphoinositide-dependent protein
kinase which phosphorylates and activates protein kinase Balpha", Curr Biol, 7, 1997, 261-9.
C Partovian, M Simons, "Regulation of protein kinase B/Akt activity and Ser473 phosphorylation by protein kinase Calpha in endothelial cells",
Cell Signal, 16, 2004, 951-7.
Reaction
32.3.2.2.2 SOS-mediated signalling
Authors
Description
SOS is recruited to the plasma membrane and mediates activation of Ras.
32.3.2.2.2.1 GRB2:SOS binds IRS-P
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
Inactive p21ras-GDP is found anchored to the plasma membrane by a farnesyl residue.
Insulin stimulation results in phosphorylation of IRS1/2 on tyrosine residues (Y). GRB2 binds the phosphotyrosine residues of IRS via its SH2
domain. As IRS is phosphorylated by the insulin receptor near to the plasma membrane, the SOS-GRB2:IRS interaction brings the SOS enzyme
into close proximity to p21ras.
References
EY Skolnik, A Batzer, N Li, CH Lee, E Lowenstein, M Mohammadi, B Margolis, J Schlessinger, "The function of GRB2 in linking the insulin
receptor to Ras signaling pathways.", Science, 260, 1993, 1953-5.
Reaction
32.3.2.2.2.2 SOS mediated nucleotide exchange of RAS (IRS)
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
Source reaction
This reaction was inferred from the corresponding reaction "SOS mediated nucleotide exchange of RAS (IRS)" in species Rattus norvegicus.
EY Skolnik, A Batzer, N Li, CH Lee, E Lowenstein, M Mohammadi, B Margolis, J Schlessinger, "The function of GRB2 in linking the insulin
receptor to Ras signaling pathways.", Science, 260, 1993, 1953-5.
Reaction
32.3.2.2.2.3 RAF activation

Authors
Reviewers
Greene, LA, 2007-11-08.
Description
References
32.3.2.2.2.3.1 Transient dissociation of 14-3-3 upon Ras binding
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Heldin, CH, 2008-02-12.
Description
References
Reaction
32.3.2.2.2.3.2 Stabilisation of RAS:RAF by 14-3-3
Authors
Editors
Schmidt, EE, 0000-00-00.

Description
References
Reaction
32.3.2.2.2.3.3 Stabilisation of RAF by further phosphorylation
Authors
Editors
Schmidt, EE, 0000-00-00.

Description
References
Reaction
32.3.2.2.2.4 MAP kinase cascade
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
32.3.2.2.2.4.1 MEK activation
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
32.3.2.2.2.4.1.1 Activated RAF complex binds MEK
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
presence of ATP.
Source reaction
Reaction
32.3.2.2.2.4.1.2 RAF phosphorylates MEK
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
32.3.2.2.2.4.1.2.1 RAF phosphorylates MEK1
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
Source reaction
25699-709.
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
Reaction
32.3.2.2.2.4.2 ERK activation
Reviewers
Greene, LA, 2007-11-08.
Description
32.3.2.2.2.4.2.1 ERK1 activation
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
32.3.2.2.2.4.2.1.1 MEK1 binds ERK-1
Authors
Editors
Schmidt, EE, 0000-00-00.

Reviewers
Greene, LA, 2007-11-08.
Description
References
MEK2.", J Biol Chem, 268, 1993, 11435-9.
Reaction
32.3.2.2.2.4.2.1.2 MEK1 phosphorylates ERK-1
Authors
Editors
Schmidt, EE, 0000-00-00.

Reviewers
Greene, LA, 2007-11-08.
Description
activity.
References
MEK2.", J Biol Chem, 268, 1993, 11435-9.
Reaction
32.3.2.2.2.4.2.1.3 Dimerisation of phospho-ERK-1
Authors
Editors
Schmidt, EE, 0000-00-00.

Reviewers
Greene, LA, 2007-11-08.
Description
Source reaction
Reaction
32.3.2.2.2.4.2.1.4 Nuclear translocation of phospho-ERK-1 dimer

Reviewers
Greene, LA, 2007-11-08.
Description
Source reaction
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
32.3.2.2.2.4.2.2.1 MEK2 binds ERK-2
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
References
MEK2.", J Biol Chem, 268, 1993, 11435-9.
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
activity.
References
MEK2.", J Biol Chem, 268, 1993, 11435-9.
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
Source reaction
Reaction
Reviewers
Greene, LA, 2007-11-08.
Description

Source reaction
Reaction
32.4 Internalisation of the insulin receptor
Authors
Bevan, AP, 2003-07-31.
Description
Almost concomitantly a second effect resulting from the tyrosine phosphorylation of the insulin receptor begins to occur. The phosphorylation of
the tyrosine in the NPEY sequence found in the juxtamembrane is also a signal for endocytosis to occur. Whilst invagination of the plasma
membrane commences the receptor tyrosine kinase activity continues unabated as does substrate phosphorylation.
As the invagination continues certain proteins are concentrated in the area of invagination. In addition to the insulin receptor itself there is a
recruitment of insulin-specific protein tyrosine phosphatases (PTPs). This process takes less than one minute. (The identity of these PTPs is not
clearly established yet. A discussion of candidates will be added in the near future.)
The formation of the endosome containing the activated ligand-receptor complex is completed within two minutes following ligand presentation at
the plasma membrane and is maximal by five minutes. Endocytosis of activated receptors has the dual effect of concentrating receptors within
endosomes and allowing the insulin receptor tyrosine kinase to phosphorylate substrates that are spatially distinct from those accessible at the
plasma membrane. The endosome also contains other proteins crucial to the signal transduction process. These include a proton pump and the
insulin degrading activity. It is not certain how these proteins arrive in the endosome since it could be via the endosome maturation or fusion
pathways.
References
KV Kandror, PF Pilch, "Compartmentalization of protein traffic in insulin-sensitive cells.", Am J Physiol, 271, 1996, E1-14.
PG Drake, PC Baass, F Authier, BI Posner, JJ Bergeron, "Insulin receptor internalization and signalling.", Mol Cell Biochem, 182, 1999, 59-63.
AP Bevan, PG Drake, JJ Bergeron, BI Posner, "Intracellular Signal Transduction: The Role of Endosomes", Trends in Endocrinology and
Metabolism, 7, 1996, 13-21.
Reaction
32.5 Insulin receptor recycling
Authors
Bevan, AP, 2003-07-31.
Description
Triggered by acidification of the endosome, insulin dissociates from the receptor and is degraded. The receptor is dephosphorylated and
re-integrated into the plasma membrane, ready to be activated again by the binding of insulin molecules.
32.5.1 Endosome acidification
Authors
Bevan, AP, 2003-07-31.
Description
The effect of the proton pump is to allow entry of [H+] ions into the lumen of the endosome. The net effect of this is to lower the pH of the lumen
from pH 7.4 (the pH at the plasma membrane) to pH 6.0 (documented with studies using FITC-labeled insulin - a pH dependent fluorescence
marker).
References
WC Duckworth, RG Bennett, FG Hamel, "Insulin degradation: progress and potential.", Endocr Rev, 19, 1998, 608-24.
WC Duckworth, "Insulin degradation: mechanisms, products, and significance.", Endocr Rev, 9, 1989, 319-45.
F Authier, BI Posner, JJ Bergeron, "Endosomal proteolysis of internalized proteins.", FEBS Lett, 389, 1996, 55-60.
Reaction
32.5.2 Dissociation of insulin from insulin receptor
Authors
Bevan, AP, 2003-07-31.
Description
As the endosomal lumen acidifies the insulin dissociates from the insulin receptor making it available for degradation by the insulin degrading
activity (IDA) present in the endosomal membrane.
References
WC Duckworth, RG Bennett, FG Hamel, "Insulin degradation: progress and potential.", Endocr Rev, 19, 1998, 608-24.
WC Duckworth, "Insulin degradation: mechanisms, products, and significance.", Endocr Rev, 9, 1989, 319-45.
F Authier, BI Posner, JJ Bergeron, "Endosomal proteolysis of internalized proteins.", FEBS Lett, 389, 1996, 55-60.
Reaction
32.5.3 Insulin receptor de-phosphorylation
Authors
Bevan, AP, 2003-07-31.
Description
With insulin dissociated from its receptor the signal to sustain the receptor kinase's activity is also removed. Thus endosomally-associated
protein tyrosine phosphatases (PTPs) are able to dephosphorylate the receptor which now can not rephosphorylate themselves since insulin is
removed and the receptor is in the inactive protein conformation. (The identity of these PTPs is not clearly established yet. A discussion of
candidates will be added in the near future.)
The dephosphorylation of the receptor is also a signal for the receptor to recycle back to the plasma membrane.
References
AP Bevan, PG Drake, JJ Bergeron, BI Posner, "Intracellular Signal Transduction: The Role of Endosomes", Trends in Endocrinology and
Metabolism, 7, 1996, 13-21.
PG Drake, BI Posner, "Insulin receptor-associated protein tyrosine phosphatase(s): role in insulin action.", Mol Cell Biochem, 182, 1999, 79-89.
Reaction
32.5.4 Re-integration of insulin receptor into plasma membrane
Authors
Bevan, AP, 2003-07-31.
Description
The endosome fuses with the plasma membrane allowing the insulin receptor to re-integrate there. Any degraded insulin remnants which
remained in the endosome are also expelled (The majority having been excreted into the cytoplasm and secreted out of the cell via other
mechanisms).
The cycle is complete with the dephosphorylated receptor now back in the plasma membrane available to bind the next insulin molecule
presented to it. There is some insulin receptor degradation over time when damaged insulin receptors are not recycled but fuse instead with the
lysosomes where they are degraded. However the majority of insulin receptors are recycled back to the plasma membrane with greater than
95% efficiency.
References
JJ Bergeron, J Cruz, MN Khan, BI Posner, "Uptake of insulin and other ligands into receptor-rich endocytic components of target cells: the
endosomal apparatus.", Annu Rev Physiol, 47, 1985, 383-403.
Reaction
33 Signalling by NGF
Authors
Nasi, S, Annibali, D, 2006-10-10.
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
Neurotrophins (NGF, BDNF, NT-3, NT-4/5) play pivotal roles in survival, differentiation, and plasticity of neurons in the peripheral and central
nervous system. They are produced, and secreted in minute amounts, by a variety of tissues. They signal through two types of receptors: TRK
tyrosine kinase receptors (TRKA, TRKB, TRKC), which specifically interact with the different neurotrophins, and p75NTR, which interacts with all
neurotrophins. TRK receptors are reported in a variety of tissues in addition to neurons. p75NTRs are also widespread.
Neurotrophins and their receptors are synthesized as several different splice variants, which differ in terms of their biological activities. The nerve
growth factor (NGF) was the first growth factor to be identified and has served as a model for studying the mechanisms of action of
neurotrophins and growth factors. The mechanisms by which NGF generates diverse cellular responses have been studied extensively in the rat
pheochromocytoma cell line PC12. When exposed to NGF, PC12 cells exit the cell cycle and differentiate into sympathetic neuron-like cells.
Current data show that signalling by the other neurotrophins is similar to NGF signalling.
33.1 NGF processing
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
All neurotrophins (NTs) are generated as pre-pro-neurotrophin precursors. The signal peptide is cleaved off as NT is associated with the
endoplasmic reticulum (ER). The resulting pro-NT can form a homodimer spontaneously which then transits to the Golgi apparatus and then
onto the trans-Golgi network (TGN). Resident protein convertases (PCs) can cleave off the pro-sequence and mature NT is is targeted to
constitutively released vesicles. The pro-NT form can also be released to the extracellular region.
References
V Lessmann, K Gottmann, M Malcangio, "Neurotrophin secretion: current facts and future prospects", Prog Neurobiol, 69, 2003, 341-74.
33.1.1 The signal peptide is excised from beta-NGF pre-pro-precursor
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
Pre-pro- precursors of the neurotrophins NGF, BDNF, NT-3, NT-4/5 are synthesized in various cell types by endoplasmic reticulum (ER)
attached ribosomes, leading to sequestration of the newly formed polypeptide chain into the ER. The mouse NGF gene gives rise to two major
transcripts that contain NGF (12.5 kDa) at the C-terminus and differ by alternative splicing of an N-terminal exon, so that the large precursor (34
kDa) has 67 amino acids upstream of an internal signal peptide and the smaller precursor (27 kDa) has this signal peptide at its N-terminus. The
transcript for the large precursor predominates in the submaxillary gland, whereas the transcript for the smaller precursor predominates in
virtually all other tissues.
The signal peptide is cleaved off immediately after sequestration into the ER. Therefore, expression of either NGF transcript gives rise to an
apparently identical intracellular glycosylated precursor formed by cleavage of the primary gene product after the signal peptide.
Source reaction
This reaction was inferred from the corresponding reaction "The signal peptide is excised from pre-pro-NGF" in species Mus musculus.
MA Bothwell, EM Shooter, "Dissociation equilibrium constant of beta nerve growth factor", J Biol Chem, 252, 1977, 8532-6.
RH Edwards, MJ Selby, WC Mobley, SL Weinrich, DE Hruby, WJ Rutter, "Processing and secretion of nerve growth factor: expression in
mammalian cells with a vaccinia virus vector", Mol Cell Biol, 8, 1988, 2456-64.
Reaction
33.1.2 pro-beta-NGF dimerizes
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
The pro-neurotrophin (pro-NGF: 27 kDa) spontaneously forms stable, non-covalent dimers directly in the ER. The homodimer is associated by
noncovalent forces, with an equilibrium dissociation constant of 10 pM. The neurotrophin pro- domain is important for proper folding and
intracellular sorting. Heterodimers of different neurotrophin monomers can also be generated at the ER.
Source reaction
This reaction was inferred from the corresponding reaction "pro-NGF dimerizes" in species Mus musculus.
MA Bothwell, EM Shooter, "Dissociation equilibrium constant of beta nerve growth factor", J Biol Chem, 252, 1977, 8532-6.
Reaction
33.1.3 pro-beta-NGF homodimer transits to the golgi apparatus
Authors
Editors
Jassal, B, 2006-10-10.
Description
From the endoplasmic reticulum, the pro-neurotrophins transit to the golgi apparatus, most likely via intermediate non-clathrin-coated transport
vesicles, and finally accumulate in the trans-golgi network (TGN). In the TGN, two different types of secretory vesicles can be generated and
filled with neurotrophins: regulated and constitutive secretory vesicles.
Source reaction
This reaction was inferred from the corresponding reaction "pro-beta-NGF homodimer transits to the golgi apparatus" in species Rattus
norvegicus.
EM Barth, S Korsching, H Thoenen, "Regulation of nerve growth factor synthesis and release in organ cultures of rat iris", J Cell Biol, 99, 1984,
839-43.
Reaction
33.1.4 Part of pro-beta-NGF is processed to mature beta-NGF
Authors
Editors
Jassal, B, 2006-10-10.
Description
The pro-neurotrophins are rapidly cleaved intra-cellularly by furin or the pro-protein convertases at a highly conserved site, to produce the
mature protein of 12-14 kDa in size (mature NGF or beta-NGF: 12.5 kDa). Furin, PACE4 and PC7 belong to the constitutive secretory pathway;
NEC1/PC1, NEC2/PC2, PC4 and PC5 are instead targeted to regulated secretory granules. Furin is expressed ubiquitously in all tissues,
whereas NEC1 and NEC2 are the dominant pro-protein convertases in neurons. The mature neurotrophins can be stored within neurons and
released extra-cellularly upon stimulation.
Cells, however, appear to have a limited capacity to process pro-neurotrophins, a capacity that may be exhausted when they are produced in
excess (Matsumoto T et al, 2008). In this case, the proforms of NGF and BDNF are secreted and cleaved extracellularly by the serine protease
plasmin and by selective matrix metalloproteinases (MMPs). The signalling capacities of pro-neurotrophins and mature neurotrophins are
markedly different. The pro-neurotrophins are high affinity ligands for p75NTR and can induce p75NTR dependent apoptosis in cultured neurons
with minimal activation of TRK receptor mediated differentiation or survival. The biological action of neurotrophins may thus be regulated by
proteolytic cleavage, with proforms preferentially activating p75NTR to mediate apoptosis and mature forms activating TRK receptors to promote
survival.
It is possible that pro-neurotrophins may somehow be released during development and eliminate neurons in a p75NTR dependent fashion.
Substantial quantities of proNGF are found in the cerebrospinal fluid of adult rodents after brain injury, perhaps following NGF expression by
inflammatory cells that may not efficiently process pro-neurotrophins. When proBDNF is added as recombinant protein, activation of p75NTR by
proBDNF facilitates hippocampal long-term depression (LTD; Woo NH et al, 2005). However, it is unclear whether proBDNF plays any role in
LTD under physiological conditions.
References
NG Seidah, S Benjannet, S Pareek, D Savaria, J Hamelin, B Goulet, J Laliberte, C Lazure, M Chretien, RA Murphy, "Cellular processing of the
nerve growth factor precursor by the mammalian pro-protein convertases", Biochem J, 314, 1996, 951-60.
NH Woo, HK Teng, CJ Siao, C Chiaruttini, PT Pang, TA Milner, BL Hempstead, B Lu, "Activation of p75NTR by proBDNF facilitates hippocampal
long-term depression", Nat Neurosci, 8, 2005, 1069-77.
T Matsumoto, S Rauskolb, M Polack, J Klose, R Kolbeck, M Korte, YA Barde, "Biosynthesis and processing of endogenous BDNF: CNS
neurons store and secrete BDNF, not pro-BDNF", Nat Neurosci, 11, 2008, 131-3.
Reaction
33.1.5 Pro-beta-NGF and mature beta-NGF are secreted
Authors
Editors
Jassal, B, 2006-10-10.
Description
Both mature neurotrophin and pro-neurotrophin are released extracellularly and are biologically active. The precursor proNGF, instead of mNGF
(mature NGF), is the molecular form preferentially released by neurons in an activity-dependent manner. Neurotrophins are secreted in low
amounts from several tissues, mainly from target tissues of innervating neurons. In the nervous system, they are secreted by neurons,
astrocytes and microglia. Neurotrophin secretion can be both constitutive and regulated. Constitutive release is observed in cells lacking a
regulated pathway, and additional stimulus-dependent regulated secretion is evident in those cells where this route is available. Secretion is
regulated by a number of stimuli, including neurotrophins themselves. In neurons, regulated secretion appears to be the prevalent pathway. NGF
is secreted from the cell soma and the dendrites, while it is unclear whether it can also be secreted by axons. Constitutive secretion of NGF is
observed only from the soma and the most proximal dendrites. Similar considerations hold for the other neurotrophins as well.
References
V Lessmann, K Gottmann, M Malcangio, "Neurotrophin secretion: current facts and future prospects", Prog Neurobiol, 69, 2003, 341-74.
Reaction
33.2 p75 NTR receptor-mediated signalling
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Friedman, WJ, 2008-05-20, Chao, MV, 2008-05-28.
Description
Besides signalling through the tyrosine kinase receptors TRK A, B, and C, the mature neurotrophins NGF, BDNF, and NT3/4 signal through their
common receptor p75NTR. NGF binding to p75NTR activates a number of downstream signalling events controlling survival, death, proliferation,
and axonogenesis, according to the cellular context. p75NTR is devoid of enzymatic activity, and signals by recruiting other proteins to its own
intracellular domain. p75 interacting proteins include NRIF, TRAF2, 4, and 6, NRAGE, necdin, SC1, NADE, RhoA, Rac, ARMS, RIP2, FAP and
PLAIDD. Here we annotate only the proteins for which a clear involvement in p75NTR signalling was demonstrated.
A peculiarity of p75NTR is the ability to bind the pro-neurotrophins proNGF and proBDNF. Proneurotrophins do not associate with TRK
receptors, whereas they efficiently signal cell death by apoptosis through p75NTR. The biological action of neurotrophins is thus regulated by
proteolytic cleavage, with proforms preferentially activating p75NTR, mediating apoptosis, and mature forms activating TRK receptors, to
promote survival. Moreover, the two receptors are utilised to differentially modulate neuronal plasticity. For instance, proBDNF-p75NTR
signalling facilitates LTD, long term depression, in the hippocampus (Woo NH, et al, 2005), while BDNF-TRKB signalling promotes LTP (long
term potentiation). Many biological observations indicate a functional interaction between p75NTR and TRKA signaling pathways.
References
NH Woo, HK Teng, CJ Siao, C Chiaruttini, PT Pang, TA Milner, BL Hempstead, B Lu, "Activation of p75NTR by proBDNF facilitates hippocampal
long-term depression", Nat Neurosci, 8, 2005, 1069-77.
JC Arevalo, SH Wu, "Neurotrophin signaling: many exciting surprises!", Cell Mol Life Sci, 63, 2006, 1523-37.
LF Reichardt, "Neurotrophin-regulated signalling pathways", Philos Trans R Soc Lond B Biol Sci, 361, 2006, 1545-64.
MV Chao, "Neurotrophins and their receptors: a convergence point for many signalling pathways", Nat Rev Neurosci, 4, 2003, 299-309.
33.2.1 NFG and proNGF binds to p75NTR

Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
When the co-receptor sortilin is present at the cell surface, proNGF preferentially interacts with a p75NTR:sortilin complex. Thus, proNGF, which
does not bind TRKA, discriminates between TRKA and p75NTR, in cells that express both receptors. The same is true for proBDNF.
Pro-neurotrophin binding to p75NTR:sortilin activates an apoptotic cascade, which may be involved in cell death after injury, and in
neurodegenerative diseases such as Alzheimer's dementia.
33.2.1.1 Binding of pro-NGF to p75NTR:sortilin
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
Sortilin is a membrane protein that acts as co-receptor for p75NTR. Superior cervical ganglion (SCG) neurons, vascular smooth muscle (SM-11)
cells, oligodendrocytes and CNS neurons (including basal forebrain neurons) (Volosin et al, 2006) express significant levels of sortilin and
p75NTR. Schwann cells, instead, do not express sortilin. It is expressed during embryogenesis in areas where NGF and proNGF have
well-characterized effects. It is important for proNGF signalling, but has little or no role on mature NGF initiated signalling. ProNGF preferentially
binds to a p75NTR:sortilin complex, whereas mature NGF preferentially binds p75NTR alone. ProNGF binds to p75NTR with a dissociation
constant (Kd) ~15-20 nM, and to sortilin with a Kd ~5 nM. In the presence of sortilin, proNGF binds to p75NTR with a Kd 0.2 nM. In contrast,
mature NGF binds to p75NTR with a Kd of 1-2 nM, whereas it binds sortilin very weakly (Kd ~ 90 nM). Therefore, in the presence of sortilin,
p75NTR binds more strongly to proNGF than to NGF, and proNGF signalling predominates. In the absence of sortilin, NGF binding is stronger
than proNGF, and it is the mature NGF signalling that prevails. proNGF interacts with sortilin via its pro-domain, whereas the interaction with
p75NTR is mediated by the mature domain.
References
A Nykjaer, R Lee, KK Teng, P Jansen, P Madsen, MS Nielsen, C Jacobsen, M Kliemannel, E Schwarz, TE Willnow, BL Hempstead, CM
Petersen, "Sortilin is essential for proNGF-induced neuronal cell death", Nature, 427, 2004, 843-8.
R Lee, P Kermani, KK Teng, BL Hempstead, "Regulation of cell survival by secreted proneurotrophins", Science, 294, 2001, 1945-8.
M Volosin, W Song, RD Almeida, DR Kaplan, BL Hempstead, WJ Friedman, "Interaction of survival and death signaling in basal forebrain
neurons: roles of neurotrophins and proneurotrophins", J Neurosci, 26, 2006, 7756-66.
Reaction
33.2.1.2 NGF homodimer binds to p75NTR
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
p75NTR exists in a multimeric form both in presence or absence of NGF. In the NGF:p75NTR complex, a single p75 molecule is asymmetrically
bound to a NGF homodimer, along the homodimeric interface of NGF. This causes an allosteric conformational change, which disables the NGF
symmetry-related second p75 binding site. Therefore, it is possible that NGF has to perturb or alter the preformed p75 dimer orientation in order
to initiate intracellular signalling. NGF:p75NTR complexes are not so long living as the NGF:TRKA complexes. This is due, at least in part, to the
fact that TRKA homodimers are internalized, and continue signalling in endosomes.
Contrary to what is commonly believed, NGF bind to p75NTR and TRKA, individually, with a similar equilibrium binding constant (Kd ~ 1-2 nM).
As a matter of fact, the association constant for NGF binding to p75NTR (k+1 = 8x10 to power of 6 M-1 s-1) is faster than for TRKA (k+1 = 8x10
to power of 5 M-1 s-1). On the other hand, the off rate of the NGF:TRKA complex ( k-1 = 7.2x10 to power of -5 s-1) is much slower than the
NGF:p75NTR complex (k-1 = 1x10 to power of -3 s-1) . p75NTR and TRK receptors functionally interact, but the precise means by which this
occurs has remained unresolved. This could result from a direct physical interaction or be explained by convergent signalling of these two
receptors. Co-expression of both p75NTR and TRKA at the cell surface appears to result in the formation of a "high-affinity" binding site that has
an accelerated rate of NGF association and a 30- to 100-fold higher affinity for NGF (Kd ~ 1-3 x 10 to power of -11 M) than either receptor alone.
The high-affinity binding sites appear to constitute 10%â€"15% of the total NGF binding sites. The nature of such high affinity binding sites is still
unclear. They could be due to a multimeric complex of p75:TRKA proteins. Alternatively, NGF might first rapidly bind to p75NTR and then be
presented to TRKA in a conformation that lowers its TRKA association rate. Some authors even question the existence of these high affinity
sites. Structural data on NGF complexes with p75NTR and TRKA extracellular domains suggest that formation of a ternary complex
TRKA:NGF:p75NTR in a 1:2:1 ration is theoretically possible, although unlikely. However, biochemical data so far failed to show that this
complex forms.
References
XL He, KC Garcia, "Structure of nerve growth factor complexed with the shared neurotrophin receptor p75", Science, 304, 2004, 870-5.
D Mahadeo, L Kaplan, MV Chao, BL Hempstead, "High affinity nerve growth factor binding displays a faster rate of association than p140trk
binding. Implications for multi-subunit polypeptide receptors", J Biol Chem, 269, 1994, 6884-91.
T Wehrman, X He, B Raab, A Dukipatti, H Blau, KC Garcia, "Structural and mechanistic insights into nerve growth factor interactions with the
TrkA and p75 receptors", Neuron, 53, 2007, 25-38.
Reaction
33.2.2 Cell death signalling via NRAGE, NRIF and NADE
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers

Description
p75NTR is a key regulator of neuronal apoptosis, both during development and after injury. Apoptosis is triggered by binding of either mature
neurotrophin or proneurotrophin (proNGF, proBDNF). ProNGF is at least 10 times more potent than mature NGF in inducing apoptosis. TRKA
signalling protects neurons from apoptosis. The molecular mechanisms involved in p75NTR-apoptosis are not well understood. The death
signalling requires activation of c-JUN N-terminal Kinase (JNK), as well as transcriptional events. JNK activation appears to involve the receptor
interacting proteins TRAF6, NRAGE, and Rac. The transcription events are thought to be regulated by another p75-interacting protein, NRIF.
Two other p75-interacting proteins, NADE and Necdin, have been implicated in apoptosis, but their role is less clear.
References
S Rabizadeh, DE Bredesen, "Ten years on: mediation of cell death by the common neurotrophin receptor p75(NTR)", Cytokine Growth Factor
Rev, 14, 2003, 225-39.
33.2.2.1 NRAGE signals death through JNK
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
Once bound by either NGF or proNGF, p75NTR interacts with NRAGE, thus leading to phosphorylation and activation of JUN Kinase (JNK). JNK
controls apoptosis in two ways: it induces transcription of pro-apoptotic genes, and directly activates the cell death machinery.
References
AH Salehi, S Xanthoudakis, PA Barker, "NRAGE, a p75 neurotrophin receptor-interacting protein, induces caspase activation and cell death
through a JNK-dependent mitochondrial pathway", J Biol Chem, 277, 2002, 48043-50.
33.2.2.1.1 A ligand:p75NTR complex binds to NRAGE

Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
NRAGE (neurotrophin receptor-interacting MAGE homolog), a member of the MAGE family of proteins, is a cytoplasmic protein that mediates
neurotrophin-induced cell death. NRAGE binding is stimulated following NGF or proNGF binding to p75. Some studies indicate that NRAGE
expression is limited to proliferative neural populations, whereas others indicate its presence in differentiated neurons in hippocampus. Another
MAGE protein, Necdin, was reported to interact with p75 and affect cell death.
Source reaction
This reaction was inferred from the corresponding reaction "A ligand:p75NTR complex binds to Nrage" in species Mus musculus.
AH Salehi, PP Roux, CJ Kubu, C Zeindler, A Bhakar, LL Tannis, JM Verdi, PA Barker, "NRAGE, a novel MAGE protein, interacts with the p75
neurotrophin receptor and facilitates nerve growth factor-dependent apoptosis", Neuron, 27, 2000, 279-88.
Reaction
33.2.2.1.2 NRAGE sequesters CHE1 in the cytoplasm
Authors

Editors
Jassal, B, 2008-05-20.
Reviewers
Description
CHE1, also named AATF, is an Apoptosis Antagonizing Transcription Factor in cortical neurons. NRAGE binds to CHE1, inhibiting its nuclear
localization by sequestering it in the cytoplasm, and, consequently, antagonizes its anti-apoptotic function.
Source reaction
This reaction was inferred from the corresponding reaction "Nrage sequesters Che1 in the cytoplasm" in species Mus musculus.
MG Di Certo, N Corbi, T Bruno, S Iezzi, F De Nicola, A Desantis, MT Ciotti, E Mattei, A Floridi, M Fanciulli, C Passananti, "NRAGE associates
with the anti-apoptotic factor Che-1 and regulates its degradation to induce cell death", J Cell Sci, 120, 2007, 1852-8.
Reaction
33.2.2.1.3 NRAGE activates JNK
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
The NGF:p75:NRAGE complex promotes threonine and tyrosine phosphorylation, and activation of JNK, by an unknown mechanism.
Source reaction
This reaction was inferred from the corresponding reaction "Nrage activates Jnk1" in species Rattus norvegicus.
AH Salehi, S Xanthoudakis, PA Barker, "NRAGE, a p75 neurotrophin receptor-interacting protein, induces caspase activation and cell death
through a JNK-dependent mitochondrial pathway", J Biol Chem, 277, 2002, 48043-50.
Reaction
33.2.2.1.4 p75NTR indirectly activates RAC and Cdc42 via a guanyl-nucleotide exchange factor
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers

Description
Following NGF binding, p75NTR activates the RAC (Ras-related C3 botulinum toxin substrate) GTPase.
References
AW Harrington, JY Kim, SO Yoon, "Activation of Rac GTPase by p75 is necessary for c-jun N-terminal kinase-mediated apoptosis", J Neurosci,
22, 2002, 156-66.
Reaction
33.2.2.1.5 GTP-bound RAC contributes to JNK activation
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
RAC activation was described as essential for p75NTR to induce JNK and apoptosis in cortical oligodendrocytes. The simultaneous activation of
TRKA counteracts the apoptotic action of p75, by modulating the kinetics of p75-mediated RAC activation.
References
CE Bazenet, MA Mota, LL Rubin, "The small GTP-binding protein Cdc42 is required for nerve growth factor withdrawal-induced neuronal death",
Reaction
33.2.2.1.6 JNK phosphorylates BIM, BAD and other targets
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
Once activated, JNK phosphorylates targets in cytoplasm, including BIM and BAD that promote the release of cytochrome c and activation of
caspases 9, 6 and 3.
References
EB Becker, J Howell, Y Kodama, PA Barker, A Bonni, "Characterization of the c-Jun N-terminal kinase-BimEL signaling pathway in neuronal
apoptosis", J Neurosci, 24, 2004, 8762-70.
AL Bhakar, JL Howell, CE Paul, AH Salehi, EB Becker, F Said, A Bonni, PA Barker, "Apoptosis induced by p75NTR overexpression requires Jun
kinase-dependent phosphorylation of Bad", J Neurosci, 23, 2003, 11373-81.
Reaction
33.2.2.1.7 Active JNK moves to the nucleus and phosphorylates different transcription factors
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
The different molecules listed (NRAGE, NRIF, NADE, TRAF6) mediate, through unclear mechanisms, JNK activation by threonine and tyrosine
phosphorylation. While active JNK does move to the nucleus and phosphorylates and activate transcription factors such as c-JUN and ATF2,
these have not been implicated in p75-mediated cell death, but rather the direct activation of the cell death machinery by JNK has been
implicated. p75 activates the intrinsic caspase pathway (involving mitochondrial release of cytochrome c, Apaf-1, and caspases-9) rather than
the extrinsic (caspase-8) pathway activated by most other death receptors.
References
CM Troy, JE Friedman, WJ Friedman, "Mechanisms of p75-mediated death of hippocampal neurons. Role of caspases", J Biol Chem, 277,
2002, 34295-302.
Reaction
33.2.2.2 NRIF signals cell death from the nucleus
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
NRIF (nuclear receptor-interacting factor) is a DNA binding protein that is essential for p75-mediated apoptosis in retina and sympathetic
neurons. Neurotrophin or proneurotrophin binding to p75TR induces nuclear translocation of NRIF, which involves gamma-secretase cleavage of
p75NTR ICD (Intra Cellular Domain). Once in the nucleus, NRIF mediates apoptosis, probably by acting as transcription factor.
References
A Nykjaer, TE Willnow, CM Petersen, "p75NTR--live or let die", Curr Opin Neurobiol, 15, 2005, 49-57.
33.2.2.2.1 NRIF binds to p75NTR
Authors

Editors
Jassal, B, 2008-05-20.
Reviewers
Description
NRIF is a ubiquitously expressed zinc finger protein of the Kruppel family that may transduce cell death signals during development and
functions in association with TRAF6 to induce activation of JNK. NRIF-induced cell death through p75NTR requires p53 and NRIF nuclear
translocation, which is modulated by TRAF6-mediated polyubiquitination of NRIF at lysine 63.
Source reaction
This reaction was inferred from the corresponding reaction "NRIF binds to p75NTR" in species Mus musculus.
E Casademunt, BD Carter, I Benzel, JM Frade, G Dechant, YA Barde, "The zinc finger protein NRIF interacts with the neurotrophin receptor
p75(NTR) and participates in programmed cell death", EMBO J, 18, 1999, 6050-61.
Reaction
33.2.2.2.2 TRAF6 binds to p75NTR:NRIF
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
Upon neurotrophin stimulation, p75NTR interacts with the ubiquitin 3 ligase TRAF6 (TNF receptor-associated factor 6). It is unclear whether
TRAF6 binds to p75NTR directly, or whether it needs to be recruited through an adaptor protein such as MyD88.Recruitment of NRIF and
TRAF6 to p75NTR is followed by an interaction between the two cytoplasmic proteins, It is possible that the NRIF:TRAF6 interaction promotes
formation of a multimeric signalling complex. TRAF6 appears to promote NRIF release from p75NTR
References
JJ Gentry, NJ Rutkoski, TL Burke, BD Carter, "A functional interaction between the p75 neurotrophin receptor interacting factors, TRAF6 and
NRIF", J Biol Chem, 279, 2004, 16646-56.
Reaction
33.2.2.2.3 NRIF and TRAF6 may activate JNK
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers

Description
NRIF and TRAF6 appear to cooperate in JNK activation. TRAF6 is involved both in JNK activation and in NF-kB activation. Although the
NRIF:TRAF6 interaction enhances by threefold the TRAF6-mediated activation of JNK, it only modestly affects TRAF6-mediated activation of
NF-kB.
Source reaction
This reaction was inferred from the corresponding reaction "NRIF and TRAF6 may activate JNK" in species Mus musculus.
EC Yeiser, NJ Rutkoski, A Naito, J Inoue, BD Carter, "Neurotrophin signaling through the p75 receptor is deficient in traf6-/- mice", J Neurosci,
24, 2004, 10521-9.
Reaction
33.2.2.2.4 gamma-secretase cleaves p75NTR, releasing NRIF and TRAF6
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers

Description
Neurotrophin or proneurotrophin signalling promotes p75NTR cleavage by gamma-secretase, allowing the release of p75 ICD and NRIF. This
mechanism was shown in sympathetic neurons.
Gamma-secretase can be activated in a number of ways, including signalling via p75NTR. The phorbol esther PMA induces p75 cleavage,
followed by NRIF nuclear translocation, after 30 min. Neurotrophin binding to p75, instead, triggers the same events only after 12 h.
References
RS Kenchappa, N Zampieri, MV Chao, PA Barker, HK Teng, BL Hempstead, BD Carter, "Ligand-dependent cleavage of the P75 neurotrophin
receptor is necessary for NRIF nuclear translocation and apoptosis in sympathetic neurons", Neuron, 50, 2006, 219-32.
Reaction
33.2.2.2.5 TRAF6 polyubiquitinates NRIF
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
TRAF6 attaches a lysine 63-linked polyubiquitin chain to lysine 19 of NRIF. Mutation of NRIF lysine 19 prevents p75-mediated apoptosis.
p75NTR cleavage by gamma-secretase is required for NRIF ubiquitination.
References
T Geetha, RS Kenchappa, MW Wooten, BD Carter, "TRAF6-mediated ubiquitination regulates nuclear translocation of NRIF, the p75 receptor
interactor", EMBO J, 24, 2005, 3859-68.
Reaction
33.2.2.2.6 Polyubiquitinated NRIF binds to p62 (Sequestosome)
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
Binding of NRIF to p62 (Sequestosome) is suspected to modulate NRIF transcriptional activity.
References
T Geetha, RS Kenchappa, MW Wooten, BD Carter, "TRAF6-mediated ubiquitination regulates nuclear translocation of NRIF, the p75 receptor
interactor", EMBO J, 24, 2005, 3859-68.
Reaction
33.2.2.2.7 Polyubiquitinated NRIF migrates to the nucleus
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
NRIF polyubiquitination is necessary for nuclear translocation. The carboxyl terminus of NRIF mediates nuclear localization, whereas the amino
terminus prevents it. Once in the nucleus, NRIF regulates gene expression, acting as a transcriptional repressor.
References
RS Kenchappa, N Zampieri, MV Chao, PA Barker, HK Teng, BL Hempstead, BD Carter, "Ligand-dependent cleavage of the P75 neurotrophin
receptor is necessary for NRIF nuclear translocation and apoptosis in sympathetic neurons", Neuron, 50, 2006, 219-32.
Reaction
33.2.2.3 NADE modulates death signalling
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
NADE protein (p75NTR-associated cell death executor) may induce cell death upon NGF binding, but not BDNF, NT3, or NT4/5 binding, to
p75NTR. The NADE-dependent apoptosis is modulated by the 14-3-3-epsilon protein (Kimura MT et al, 2001).
References
MT Kimura, S Irie, S Shoji-Hoshino, J Mukai, D Nadano, M Oshimura, TA Sato, "14-3-3 is involved in p75 neurotrophin receptor-mediated signal
transduction", J Biol Chem, 276, 2001, 17291-300.
33.2.2.3.1 p75NTR binds to NADE
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers

Description
The NADE protein interacts with p75NTR to mediate cell death. The interaction is mediated by NADE NES (nuclear export signal), also
responsible for self-association of NADE (Mukai J et al, 2002).
References
J Mukai, S Shoji, MT Kimura, S Okubo, H Sano, P Suvanto, Y Li, S Irie, TA Sato, "Structure-function analysis of NADE: identification of regions
that mediate nerve growth factor-induced apoptosis", J Biol Chem, 277, 2002, 13973-82.
J Mukai, T Hachiya, S Shoji-Hoshino, MT Kimura, D Nadano, P Suvanto, T Hanaoka, Y Li, S Irie, LA Greene, TA Sato, "NADE, a
p75NTR-associated cell death executor, is involved in signal transduction mediated by the common neurotrophin receptor p75NTR", J Biol
Chem, 275, 2000, 17566-70.
Reaction
33.2.2.3.2 p75NTR:NADE promotes caspase2/3 activation
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
Once bound to the NGF:p75NTR complex, NADE contributes to cell death signalling by promoting activation of caspases 2 and 3. It is unclear
whether JNK activation is involved. The apoptotic function of NADE was observed in oligodendrocytes.
References
J Mukai, S Shoji, MT Kimura, S Okubo, H Sano, P Suvanto, Y Li, S Irie, TA Sato, "Structure-function analysis of NADE: identification of regions
that mediate nerve growth factor-induced apoptosis", J Biol Chem, 277, 2002, 13973-82.
Reaction
33.2.2.3.3 14-3-3epsilon attentuates NADE-related apoptosis
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
NADE forms a complex with the 14-3-3epsilon isoform. The last one interacts with caspase 3 through its C terminal region. The
NADE:4-3-3epsilon complex negatively regulates p75NTR-mediated apoptosis, probably by down regulating caspase activity.
References
MT Kimura, S Irie, S Shoji-Hoshino, J Mukai, D Nadano, M Oshimura, TA Sato, "14-3-3 is involved in p75 neurotrophin receptor-mediated signal
transduction", J Biol Chem, 276, 2001, 17291-300.
Reaction
33.2.3 p75NTR negatively regulates cell cycle via SC1
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
SC1 (Schwann Cell factor 1; also called PR domain zinc finger protein 4, PRDM4) interacts with an NGF:p75NTR complex and signals cell cycle
arrest by regulating the levels of cyclin E.
References
N Lopez-Sanchez, JM Frade, "Control of the cell cycle by neurotrophins: lessons from the p75 neurotrophin receptor", Histol Histopathol, 17,
2002, 1227-37.
33.2.3.1 PRDM4 (SC1) binds to p75NTR
Authors

Editors
Jassal, B, 2008-05-20.
Reviewers
Description
PRDM4, usually named SC1, for Schwann Cell factor 1, is a zinc finger protein that functions as a repressor of transcription. It is present in many
tissues, and abundant in brain. It interacts with the NGF:p75NTR complex to signal cell cycle arrest. It is unclear whether it already forms a
complex with p75NTR before NGF binding to p75.
Source reaction
This reaction was inferred from the corresponding reaction "Prdm4 (Sc1) binds to p75NTR" in species Rattus norvegicus.
A Chittka, MV Chao, "Identification of a zinc finger protein whose subcellular distribution is regulated by serum and nerve growth factor", Proc
Natl Acad Sci U S A, 96, 1999, 10705-10.
Reaction
33.2.3.2 PRDM4 translocates to the nucleus
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
PRDM4 is found in the cytoplasm. Following binding of NGF to p75NTR, after 1 hour of NGF treatment, PRDM4 is redistributed from the
cytoplasm to the nucleus. The relocalisation of PRDM4 appears to be specific for NGF, as it is not affected by BDNF or NT3.
Source reaction
This reaction was inferred from the corresponding reaction "Prdm4 translocates to the nucleus" in species Rattus norvegicus.
A Chittka, MV Chao, "Identification of a zinc finger protein whose subcellular distribution is regulated by serum and nerve growth factor", Proc
Natl Acad Sci U S A, 96, 1999, 10705-10.
Reaction
33.2.3.3 PRDM4 inhibits cyclin E transcription
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers

Description
Within the nucleus, PRDM4 is found in a complex with HDACs (histone deacetylases) 1, 2, and 3. It interacts with the regulatory regions of the
cyclin E gene, strongly inhibiting transcription. It was also shown to weakly affect cyclib B transcription. PRDM4 is believed to interact with
regulatory regions of other genes, which are unknown.
Source reaction
This reaction was inferred from the corresponding reaction "Prdm4 inhibits cyclin E transcription" in species Rattus norvegicus.
A Chittka, JC Arevalo, M Rodriguez-Guzman, P Perez, MV Chao, M Sendtner, "The p75NTR-interacting protein SC1 inhibits cell cycle
progression by transcriptional repression of cyclin E", J Cell Biol, 164, 2004, 985-96.
Reaction
33.2.4 p75NTR signals via NF-kB
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
The NF-kB pathway is an important pro-survival signalling pathway activated by mature NGF, but not BDNF or NT-3, through p75NTR. It is
unclear whether TRKA activity also affects NF-kB activation.
References
S Memet, "NF-kappaB functions in the nervous system: from development to disease", Biochem Pharmacol, 72, 2006, 1180-95.
33.2.4.1 p75NTR recruits signalling complexes
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
NF-kB activation involves recruitment at the cell membrane of several proteins such as RIP2, MYD88, IRAK1, TRAF6, p62 and atypical PKC by
the NGF:p75NTR complex.
33.2.4.1.1 p75NTR interacts with RIP2
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers

Description
The RIP2 (receptor-interacting protein-2) kinase is a mediator of NGF-dependent NF-kB activity. It contains a serine/threonine kinase domain
and a caspase recruitment domain (CARD) at the C terminus. It binds to the death domain of p75NTR via its CARD domain in an
NGF-dependent manner. RIP2 may also bind TRAF proteins, suggesting the existence of complexes of TRAF and RIP2 proteins with the p75
receptor. RIP2 is also able to interact with p62. It is highly expressed in Schwann cells.
References
G Khursigara, J Bertin, H Yano, H Moffett, PS DiStefano, MV Chao, "A prosurvival function for the p75 receptor death domain mediated via the
caspase recruitment domain receptor-interacting protein 2", J Neurosci, 21, 2001, 5854-63.
Reaction
33.2.4.1.2 p75NTR interacts with IRAK:MYD88
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
The serine/threonine kinase IRAK (interleukin-1 receptor-associated kinase) is necessary for NF-kB activation. Under basal conditions, IRAK is
not bound to the p75 receptor, and stays inactive in the cytoplasm. It associates with p75NTR following neurotrophin binding. MYD88 functions
as an adapter, by recruiting IRAK to the p75 receptor. Upon stimulation with NGF, a MYD88: IRAK1 complex quickly forms that is recruited to
p75NTR.
Source reaction
This reaction was inferred from the corresponding reaction "p75NTR interacts with Irak:Myd88" in species Rattus norvegicus.
V Mamidipudi, X Li, MW Wooten, "Identification of interleukin 1 receptor-associated kinase as a conserved component in the p75-neurotrophin
receptor activation of nuclear factor-kappa B", J Biol Chem, 277, 2002, 28010-8.
Reaction
33.2.4.1.3 IRAK is activated
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
Upon recruitment to p75NTR, Interleukin-1 receptor-associated kinase (IRAK) is rapidly phosphorylated and activated by an unknown
mechanism and protein.
Source reaction
This reaction was inferred from the corresponding reaction "Irak is activated" in species Rattus norvegicus.
V Mamidipudi, X Li, MW Wooten, "Identification of interleukin 1 receptor-associated kinase as a conserved component in the p75-neurotrophin
receptor activation of nuclear factor-kappa B", J Biol Chem, 277, 2002, 28010-8.
Reaction
33.2.4.1.4 IRAK interacts with TRAF6
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
Neurotrophin binding to p75NTR leads to recruitment of TRAF6. This protein is an E3 ubiquitin ligase, which, together with an E2 Ubiquitin
conjugating enzyme, mediates the assembly of lysine 63-linked polyubiquitin chains and their attachment to a lysine of a substrate protein.
Activation of IRAK1 promotes recruitment of TRAF6. TRAF6 is able to bind to p75NTR (juxtamembrane region, residues 113-128), IRAK1
(N-terminal residues 1-198 and C-terminal residues 523-618), and MYD88. It might be recruited through the MYD88:IRAK1 complex.
References
G Khursigara, JR Orlinick, MV Chao, "Association of the p75 neurotrophin receptor with TRAF6", J Biol Chem, 274, 1999, 2597-600.
Reaction
33.2.4.1.5 MYD88 dissociates
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
After recruiting IRAK, MYD88 leaves the receptor complex. The amount of MYD88 that associates with p75 peaks at 1 min of NGF treatment
and declines thereafter.
References
K Burns, F Martinon, C Esslinger, H Pahl, P Schneider, JL Bodmer, F Di Marco, L French, J Tschopp, "MyD88, an adapter protein involved in
interleukin-1 signaling", J Biol Chem, 273, 1998, 12203-9.
Reaction
33.2.4.1.6 p62 is recruited and forms a complex with TRAF6
Authors

Editors
Jassal, B, 2008-05-20.
Reviewers
Description
NGF binding to p75NTR induces recruitment of the atypical PKC interacting protein, p62, necessary for coupling IKKbeta with p75NTR. The
kinase activity of IRAK1 is necessary for p62 recruitment. IRAK1 interaction with TRAF6 precedes (1 min) its interaction with p62 (5 min). p62
has two protein interaction domains, named UBA and PB1. The UBA domain binds non-covalently to polyubiquitin chains. The PB1 domain has
structural homology with the UbL (ubiquitin like) domain, and is able to interact with the 26S proteasome subunit Rpt1. Other protein interaction
domains also exist within p62, suggesting that it may have a role in the formation of multimeric signalling complexes.p62 forms a complex with
TRAF6, which involves the two domains PB1 at the p62 C-terminal end, and UBA, at the N-terminus.
References
MW Wooten, ML Seibenhener, V Mamidipudi, MT Diaz-Meco, PA Barker, J Moscat, "The atypical protein kinase C-interacting protein p62 is a
scaffold for NF-kappaB activation by nerve growth factor", J Biol Chem, 276, 2001, 7709-12.
L Sanz, MT Diaz-Meco, H Nakano, J Moscat, "The atypical PKC-interacting protein p62 channels NF-kappaB activation by the IL-1-TRAF6
pathway", EMBO J, 19, 2000, 1576-86.
Reaction
33.2.4.1.7 The TRAF6:p62 complex regulates trafficking of TRKA and other substrates
Authors

Editors
Jassal, B, 2008-05-20.
Reviewers
Description
The p62:TRAF6 complex regulates trafficking of several substrates, presumably in the following way. TRAF6 ubiquitinates the substrate, while
p62 may serve as a shuttling factor that directs for turnover poly-ubiquitinated proteins interacting with the UBA domain. A known substrate is the
NGF receptor TRKA. In this case, the p62:TRAF6 complex serves as a molecular bridge linking the two NGF receptors TRKA and p75. TRKA is
lysine 63-polyubiquitinated on lysine 485 by the action of TRAF-6 and the E2 ubiquitin-conjugating enzyme UbcH7. Internalization, trafficking
and sorting of TRKA are known to be regulated by the TRF6:p62 complex.
Reaction
33.2.4.1.8 TRAF6 is auto-ubiquitinated
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers

Description
The activity of TRAF6 is regulated by autoubiquitination. This process, in turn, is regulated by p62. Cells devoid of p62 exhibit low basal TRAF6
polyubiquitination. When p62 is expressed, auto-ubiquitination of TRAF6 is enhanced. The details curated in this event represent the
ubiquitination of TRAF6, even as ubiquitin is shown as 1 stoichiometrically.
References
K Yang, J Zhu, S Sun, Y Tang, B Zhang, L Diao, C Wang, "The coiled-coil domain of TRAF6 is essential for its auto-ubiquitination", Biochem
Reaction
33.2.4.1.9 p62 recruits an atypical PKC
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
The atypical protein kinase C-iota isoform (aPKC-i) is recruited to the p75NTR receptor complex by p62 and becomes active. p62 recruits aPKC
both via TRAF6 and RIP2.
References
MW Wooten, ML Seibenhener, V Mamidipudi, MT Diaz-Meco, PA Barker, J Moscat, "The atypical protein kinase C-interacting protein p62 is a
scaffold for NF-kappaB activation by nerve growth factor", J Biol Chem, 276, 2001, 7709-12.
Reaction
33.2.4.1.10 IKK-beta is recruited
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
NGF stimulation results in recruitment of IKK-beta (Inhibitor of nuclear factor kappa-B kinase subunit beta) to the p75NTR receptor complex.
IKK-beta recruitment involves p62.
Reaction
33.2.4.2 NF-kB is activated and signals survival

Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
Upon activation in response to NGF, NF-kB moves to the nucleus, where it turns on genes that promote survival, and triggers the expression of
HES1/5 to modulate dendritic growth.
References
MP Mattson, "NF-kappaB in the survival and plasticity of neurons", Neurochem Res, 30, 2005, 883-93.
33.2.4.2.1 IKKbeta is activated
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
Atypical PKC isoforms phosphorylate the beta subunit of the IKK complex (on Serines 177 and 181) thereby serving as an IKK kinase. TRAF6
and p62 as well appear to have a role in IKK activation. TRAF6 mediates the assembly of K63-linked poly-Ub chains required for IKK activation.
The ubiquitin binding property of p62 may also be relevant in regulating IKK activation.
References
MJ Lallena, MT Diaz-Meco, G Bren, CV Paya, J Moscat, "Activation of IkappaB kinase beta by protein kinase C isoforms", Mol Cell Biol, 19,
1999, 2180-8.
Reaction
33.2.4.2.2 IKKbeta phosphorylates IkB causing NF-kB to dissociate
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
IkB is an inhibitory protein that sequesters NF-kB in the cytoplasm, by masking a nuclear localization signal, located just at the C-terminal end in
each of the NF-kB subunits. A key event in NF-kB activation involves phosphorylation of IkB by an IkB kinase (IKK). NGF stimulates the activity
of the IkB kinase IKK-beta, and, possibly, IKK-alpha as well. Once IkB is phosphorylated, the IkB:NF-kB complex dissociates.
References
JA DiDonato, M Hayakawa, DM Rothwarf, E Zandi, M Karin, "A cytokine-responsive IkappaB kinase that activates the transcription factor
NF-kappaB", Nature, 388, 1997, 548-54.
Reaction
33.2.4.2.3 IkB is ubiquitinated and degraded
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
Once dissociated from NF-kB, the phosphorylated IkB protein is ubiquitinated at lysines 21 and 22, and degraded by the proteosome.
Reaction
33.2.4.2.4 NF-kB migrates to the nucleus and turns on transcription

Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
Once dissociated from IkB, NF-kB moves to the nucleus. Once in the nucleus, NF-kB binds DNA at promoters of target genes. This entails
transcription of several genes including the two HLH transcriptional regulators HES1 and HES5. HES1 and HES5 transcription can also be
activated via NOTCH signalling. Increased production of HES1 and HES5 reduces the number of primary dendrites and promotes dendrite
elongation.
References
P Salama-Cohen, MA ArÃ©valo, J Meier, R Grantyn, A RodrÃ-guez-TÃ©bar, "NGF controls dendrite development in hippocampal neurons by
binding to p75NTR and modulating the cellular targets of Notch", Mol Biol Cell, 16, 2005, 339-47.
Reaction
33.2.5 Ceramide signalling
Authors

Editors
Jassal, B, 2008-05-20.
Reviewers
Description
In certain cell types, ligand binding to p75NTR leads to ceramide production, which can mediate either cell survival (e.g. in noecotical subplate
neurons) or apoptosis (e.g. in oligodendrocytes). Low levels of ceramide are also able to stimulate axonal outgrowth in hippocampal neurons.
References
EJ Huang, LF Reichardt, "Trk receptors: roles in neuronal signal transduction", Annu Rev Biochem, 72, 2003, 609-42.
33.2.5.1 Sphingomyelinase is activated by the NGF:p75NTR complex
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
NGF binding to p75NTR activates N-SMase (Neutral sphingomyelinase), and possibly A-SMase (acid sphingomyelinase), an enzyme that
converts sphingomyelin to ceramide. The mode and mechanism of interaction between p75 and N-SMase have not been determined but is
thought to involve the recruitment of Mg2+ to the active site of the enzyme.
Source reaction
This reaction was inferred from the corresponding reaction "Sphingomyelinase is activated by the NGF:p75NTR complex" in species Rattus
norvegicus.
RT Dobrowsky, GM Jenkins, YA Hannun, "Neurotrophins induce sphingomyelin hydrolysis. Modulation by co-expression of p75NTR with Trk
receptors.", J Biol Chem, 270, 1995, 22135-42.
Reaction
33.2.5.2 Production of ceramide which can activate JNK and other targets
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
Sphingomyielinase promotes the conversion of sphingomyelin to ceramide. Ceramide can activate JNK and other targets. The molecular details
of the p75NTR-activated ceramide signalling cascade are only partially understood.
Source reaction
This reaction was inferred from the corresponding reaction "Production of ceramide which can activate JNK and other targets" in species Rattus
norvegicus.
RT Dobrowsky, MH Werner, AM Castellino, MV Chao, YA Hannun, "Activation of the sphingomyelin cycle through the low-affinity neurotrophin
receptor", Science, 265, 1994, 1596-9.
Reaction
33.2.6 p75NTR regulates axonogenesis
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
p75NTR modulates axonal growth by regulating the activity of small GTPases like RHOA and RHOB, that control the state of actin
polymerization. The best studied is RHOA. In its active, GTP-bound form, RHOA rigidifies the actin cytoskeleton, thereby inhibiting axonal
elongation and causing growth cone collapse. Depending on the ligand that binds to it, p75NTR can either promote or inhibit axonal growth,
Neurotrophin binding leads to inhibition of RHOA activity and axonal growth. Axonal growth inhibition is caused by myelin molecules named
MDGIs (myelin-derived growth inhibitors), such as NOGO, MAG, OMGP. MDGIs bind to a complex made up of p75NTR and the NOGO
receptor, causing RHOA activation and axonal growth inhibition.
References
XF Zhou, HY Li, "Roles of glial p75NTR in axonal regeneration", J Neurosci Res, 85, 2007, 1601-5.
33.2.6.1 Axonal growth stimulation
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
Complex formation between p75NTR and RHOA can leads to inhibition of RHOA activity and axonal growth.
References
T Yamashita, M Fujitani, S Yamagishi, K Hata, F Mimura, "Multiple signals regulate axon regeneration through the Nogo receptor complex", Mol
Neurobiol, 32, 2005, 105-11.
33.2.6.1.1 p75NTR and RHOA-GDI interact
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers

Description
p75NTR directly complexes with RHO-GDI (RHO-GDP Dissociation Inhibitor). RHO-GDI inhibits the dissociation of GDP and the subsequent
binding of GTP to RHOA, thus preventing formation of active RHOA. Once bound to p75NTR, RHOA-GDI is less active. p75NTR acts on RHOB
in a similar mechanism.
References
NH Keep, M Barnes, I Barsukov, R Badii, LY Lian, AW Segal, PC Moody, GC Roberts, "A modulator of rho family G proteins, rhoGDI, binds
these G proteins via an immunoglobulin-like domain and a flexible N-terminal arm", Structure, 5, 1997, 623-33.
T Yamashita, M Tohyama, "The p75 receptor acts as a displacement factor that releases Rho from Rho-GDI", Nat Neurosci, 6, 2003, 461-7.
Reaction
33.2.6.1.2 NGF binding to p75NTR inactivates RHOA
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
Neurotrophin (NGF or BDNF) binding to p75NTR increases RHO-GDI activity, possibly by loosening the grip of p75NTR on RHO-GDI, thus
leading to axonal growth.
References
S Gehler, G Gallo, E Veien, PC Letourneau, "p75 neurotrophin receptor signaling regulates growth cone filopodial dynamics through modulating
RhoA activity", J Neurosci, 24, 2004, 4363-72.
Reaction
33.2.6.1.3 p75NTR reduces RHO-GDI activity
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
By binding RHO-GDI, p75NTR facilitates the release of RHOA from RHO-GDI, thus reducing RHO-GDI activity. This step allows for RHOA
activation by guanine nucleotide exchange factors and for membrane association of the GTP-bound form of RHOA . p75NTR is able to activate
RHOB as well.
References
T Yamashita, KL Tucker, YA Barde, "Neurotrophin binding to the p75 receptor modulates Rho activity and axonal outgrowth", Neuron, 24, 1999,
585-93.
Reaction
33.2.6.1.4 Nucleotide exchange on RHOA
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
In its active, GTP-bound form, RHOA rigidifies the actin cytoskeleton, thereby inhibiting axonal elongation and causing growth cone collapse.
References
Reaction
33.2.6.2 Axonal growth inhibition (RHOA activation)
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
p75NTR can also form a receptor complex with the Nogo receptor (NgR). Such complexes mediates axonal outgrowth inhibitory signals of
MDGIs (myelin-derived growth-inhibitors), such as Nogo66, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein
(OMGP).
References
MT Filbin, "Myelin-associated inhibitors of axonal regeneration in the adult mammalian CNS", Nat Rev Neurosci, 4, 2003, 703-13.
33.2.6.2.1 p75NTR interacts with the NOGO receptor
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers

Description
The p75NTR extracellular domain interacts with NOGO receptor (NgR), a glycosyl phosphatidylinositol (GPI)-anchored protein present as a
homomultimer at the cell surface. As NgR lacks an intracellular domain, it utilizes p75NTR as a co-receptor for intracellular signalling.
References
AE Fournier, T GrandPre, SM Strittmatter, "Identification of a receptor mediating Nogo-66 inhibition of axonal regeneration", Nature, 409, 2001,
341-6.
Reaction
33.2.6.2.2 p75NTR:NgR complex interacts with the axonal inhibitor LINGO1
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
The NgR1:p75NTR complex also interacts with LINGO1, a nervous system-specific transmembrane protein. LINGO1 is a potent axonal inhibitor
of oligodendrocyte differentiation and myelination, and is regulated by NGF and its receptor TRKA .
References
S Mi, X Lee, Z Shao, G Thill, B Ji, J Relton, M Levesque, N Allaire, S Perrin, B Sands, T Crowell, RL Cate, JM McCoy, RB Pepinsky, "LINGO-1
is a component of the Nogo-66 receptor/p75 signaling complex", Nat Neurosci, 7, 2004, 221-8.
Reaction
33.2.6.2.3 Myelin components can interact with p75NTR:NgR:LINGO1
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
A group of myelin components named MDGIs (myelin-derived growth-inhibitors), bind to NgR and inhibit neurite outgrowth. Examples of such
components are NOGO, OMGP (oligodendrocyte myelin glycoprotein) and MAG (myelin-associated glycoprotein). The amino-terminal region of
NgR, covering eight leucine-rich repeats (LRR) and the LRR carboxy-terminal domain (LRRCT) is sufficient to interact with MAG, OMGP and
NOGO. Their binding to NgR enhances the NgR-p75 interaction.These inhibitors bind to a receptor complex made up of the NOGO receptor,
NgR, and p75NTR. Such complexes then activate RHOA, thereby inhibiting axonal growth.
References
ST Wong, JR Henley, KC Kanning, KH Huang, M Bothwell, MM Poo, "A p75(NTR) and Nogo receptor complex mediates repulsive signaling by
myelin-associated glycoprotein", Nat Neurosci, 5, 2002, 1302-8.
Reaction
33.2.6.2.4 The p75NTR:NgR:MDGI complex reduces RHOA-GDI activity, displacing RHOA
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
The p75NTR:RHO-GDI complex appears to be strengthened in presence of a GMDI (MAG, NOGO or OMGP) bound to a NgR:p75 complex.
When in a complex with MDGIs and NgR, p75NTR restrains the activity of RHO-GDI (GDP Dissociation Inhibitor). This facilitates the
displacement of RHO-GDI from RHOA.
References
Reaction
33.2.6.2.5 RhoA is activated by nucleotide exchange and inhibits axonal growth
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
RhoA is activated by guanine nucleotide exchange factors (RhoGEF), exchanging GDP for GTP. RHOA, activated following binding of MDGIs to
the NgR:p75NTR complex, rigidifies the actin cytoskeleton, thereby inhibiting axonal elongation and causing growth cone collapse.
References
Reaction
33.2.7 Regulated proteolysis of p75NTR

Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
p75NTR undergoes a process of regulated intramembrane proteolysis (RIP) similar to other transmembrane proteins such as NOTCH,
beta-amyloid precursor protein (APP), and ERBB4. Each of these proteins is subjected to two sequential cleavages. The first one occurs in the
extracellular part of the protein and is mediated by the metalloproteinase alpha-secretase which causes shedding of the extracellular domain.
The second cleavage occurs in the intramembrane region and is mediated by gamma-secretase and causes release of the intracellular domain,
ICD, and of a small peptide. The ICD often traffics to the nucleus and, in some instances (e.g. NOTCH), was found to act as transcriptional
regulator. Whether the p75NTR ICD does translocate to the nucleus to regulate gene expression in a way similar to the NOTCH receptor
remains to be seen. The alpha- and gamma-secretase mediated cleavage of p75 appears to be regulated by neurotrophin (NGF, BDNF) binding
to TRKA or TRKB. p75NTR processing also occurs in response to MAG in cerebellar granule neurons.
References
FC Bronfman, "Metalloproteases and gamma-secretase: new membrane partners regulating p75 neurotrophin receptor signaling?", J
Neurochem, 103, 2007, 91-100.
S Urra, CA Escudero, P Ramos, F Lisbona, E Allende, P Covarrubias, JI Parraguez, N Zampieri, MV Chao, W Annaert, FC Bronfman, "TrkA
receptor activation by nerve growth factor induces shedding of the p75 neurotrophin receptor followed by endosomal
gamma-secretase-mediated release of the p75 intracellular domain", J Biol Chem, 282, 2007, 7606-15.
33.2.7.1 alpha-secretase cleaves the p75NTR extracellular domain
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
alpha-secretase (ADAM17) is a metalloprotese that has the ability to cleave the p75NTR extracellular domain, in proximity of the transmembrane
region. The cleaved extracellular domain is shed from the cell membrane, whereas the rest of the protein, the C-terminal fragment, stays
anchored to the membrane. The released extracellular domain represents a binding protein for many potential ligands, including neurotrophins,
pro-neurotrophin precursors, beta-amyloid. Shedding of the p75NTR extracellular region can be both constituve and stimulated. The constitutive
shedding is dependent on signalling via the p38 MAP kinase. Shedding can be stimulated by the phorbol ester PMA, acting through protein
kinase C and ERK activation, and by a tyrosine phosphatase inhibitor. Activation of TRKA by NGF (or TRKB by BDNF) also induces release of
the p75NTR extracellular domain. The alpha-secretase cleavage is required for the subsequent cleavage by gamma-secretase.
References
KM Jung, S Tan, N Landman, K Petrova, S Murray, R Lewis, PK Kim, DS Kim, SH Ryu, MV Chao, TW Kim, "Regulated intramembrane
proteolysis of the p75 neurotrophin receptor modulates its association with the TrkA receptor", J Biol Chem, 278, 2003, 42161-9.
N Zampieri, CF Xu, TA Neubert, MV Chao, "Cleavage of p75 neurotrophin receptor by alpha-secretase and gamma-secretase requires specific
receptor domains", J Biol Chem, 280, 2005, 14563-71.
KC Kanning, M Hudson, PS Amieux, JC Wiley, M Bothwell, LC Schecterson, "Proteolytic processing of the p75 neurotrophin receptor and two
homologs generates C-terminal fragments with signaling capability", J Neurosci, 23, 2003, 5425-36.
Reaction
33.2.7.2 The p75NTR C-terminal fragment enters endosomes
Authors

Editors
Jassal, B, 2008-05-20.
Reviewers
Description
Most of the p75NTR C-terminal fragment resulting from alpha-secretase cleavage enters the early/recycling endosomal compartment.
References
Reaction
33.2.7.3 gamma-secretase cleaves the p75NTR transmembrane domain
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers

Description
Within early endosomes, p75NTR C-Terminal Fragment undergoes processing by gamma-secretase, a complex composed of a presenilin
homodimer (PSEN1 or PSEN2), nicastrin (NCSTN), APH1 (APH1A or APH1B) and PEN2. Such a minimal complex is sufficient for secretase
activity, although other components may exist. The p75NTR cleavage by gamma-secretase gives rise to a 20-kD ICD (IntraCellular Domain)
fragment, and to a small peptide, the significance of which is unknown but that is analogous to the A-beta peptides generated from amyloid
precursor protein. p75NTR ICD may have cytoplasmic and nuclear signalling functions and it is unstable.
References
Reaction
33.2.7.4 p75NTR ICD signals to NF-kB
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers

Description
The p75NTR ICD has the potential to bind many intracellular proteins, including TRAF6, SC1, NADE, NRAGE, and RHOA. It may bring these
proteins to function in different cellular compartments. p75NTR ICD was shown to activate NF-kB via TRAF6.
References
Reaction
33.2.7.5 Part of the ICD migrates to the nucleus
Authors
Editors
Jassal, B, 2008-05-20.
Reviewers
Description
A portion of the p75NTR ICD produced by gamma-secretase cleavage moves to the nucleus where it may participate in transcriptional
regulation.
References
Reaction
33.3 Activation of TRKA receptors
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
Trk receptors can either be activated by neurotrophins or by two G-protein-coupled receptors (GPCRs) although the biological relevance of
GPCRs remains to be shown.
References
LF Reichardt, "Neurotrophin-regulated signalling pathways", Philos Trans R Soc Lond B Biol Sci, 361, 2006, 1545-64.
33.3.1 TRKA activation by NGF
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
Neurotrophin functions are mediated by binding of the secreted neurotrophin homodimers to their common neurotrophin receptor p75NTR, and
to their cognate tropomyosin related kinase (TRK) receptor. NGF binds to TRKA, BDNF and NT4 bind to TRKB, NT3 binds to TRKC. A
tri-molecular signalling complex (NGF-p75NTR-TRKA) might also be possible.
References
PA Barker, RA Murphy, "The nerve growth factor receptor: a multicomponent system that mediates the actions of the neurotrophin family of
proteins", Mol Cell Biochem, 110, 1992, 1-15.
F Lamballe, R Klein, M Barbacid, "The trk family of oncogenes and neurotrophin receptors", Princess Takamatsu Symp, 22, 1991, 153-70.
33.3.1.1 beta-NGF dimer binds to TrkA receptor
Authors
Editors
Jassal, B, 2006-10-10.
Description
Neurotrophin dimer binding to TRK receptors causes receptor dimerization. Although the dissociation constants of NGF for TRK and p75NTR
are very similar, the binding kinetics are quite different: NGF associates with and dissociates from p75NTR much more rapidly than from TRKA.
p75NTR regulates the affinity and specificity of TRK receptor activation by neurotrophins is regulated. Its presence is required to observe high
affinity binding to TRK receptors, since it increases the rate of neurotrophin association with TRK proteins. The major ligand binding domain in
TRK receptors is the membrane-proximal Ig-C2-like domain (named Ig2 domain or domain 5), although other regions in in the TRK extracellular
domains are also important for ligand binding.
The N termini of neurotrophins are important in controlling binding specificity, and the structure of this region is reorganized upon binding to a
TRK receptor. In some neurons, TRK receptors are localized to intracellular vesicles in the absence of signals. Electrical activity, cAMP, and
Ca2+ stimulate TRK insertion into the cell surface by exocytosis of cytoplasmic membrane vesicles containing TRK. At axon terminals, TRK
receptors undergo ligand-dependent endocytosis upon ligand binding. The internalized neurotrophin-TRK complex is then sorted and enters
either recycling or retrograde transport pathways.
References
C Wiesmann, MH Ultsch, SH Bass, AM de Vos, "Crystal structure of nerve growth factor in complex with the ligand-binding domain of the TrkA
receptor", Nature, 401, 1999, 184-8.
R Klein, SQ Jing, V Nanduri, E O'Rourke, M Barbacid, "The trk proto-oncogene encodes a receptor for nerve growth factor", Cell, 65, 1991,
189-97.
DR Kaplan, BL Hempstead, D Martin-Zanca, MV Chao, LF Parada, "The trk proto-oncogene product: a signal transducing receptor for nerve
growth factor", Science, 252, 1991, 554-8.
Reaction
33.3.1.2 The bound receptor dimerizes
Authors
Editors
Jassal, B, 2006-10-10.
Description
The binding of neurotrotrophin to TrkA receptors induce their dimerization to form receptor homodimers.
References
C Wiesmann, MH Ultsch, SH Bass, AM de Vos, "Crystal structure of nerve growth factor in complex with the ligand-binding domain of the TrkA
receptor", Nature, 401, 1999, 184-8.
R Klein, SQ Jing, V Nanduri, E O'Rourke, M Barbacid, "The trk proto-oncogene encodes a receptor for nerve growth factor", Cell, 65, 1991,
189-97.
Reaction
33.3.1.3 TrkA receptor autophosphorylates
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
NGF binding induces a conformational change in TRKA, which entails the activation of the receptor kinase domain. TRK receptor activation
results in phosphorylation of several of ten evolutionary conserved tyrosines present in the cytoplasmic domain of each receptor.
Phosphorylation of the three tyrosines in the activation loop of the kinase domain (Y670, Y674, and Y675 in TRKA) enhances tyrosine kinase
activity. Phosphorylation of TRKA Y490 and Y785 creates docking sites for proteins containing SH2 or PTB domains: Y490 is the docking site for
SHC, FRS2 and IRS1/2, Y785 interacts with PLC-gamma-1. Three other tyrosine residues are important for signalling but it is not clear how. It is
possible that they play a structural role in the receptor. Therefore, full activity of TRKA receptor requires eight tyrosine residues.
Human TRKA comes in two isoforms, named TRKA- I (790 a.a long) and TRKA- II (796 a.a. long). The tyrosine phosphorylation site numbering
refers to TRKA- I. The site numbering in TRK-II is equal to TRK- I numbering + 6 (that is: Y490 in TRK- I corresponds to Y496 in TRK- II, and so
on).The same modifications occur at the homologous sites of rat TrkA, which also comes in the two isoforms I and II.
Source reaction
This reaction was inferred from the corresponding reaction "Trk receptor autophosphorylates" in species Rattus norvegicus.
RM Stephens, DM Loeb, TD Copeland, T Pawson, LA Greene, DR Kaplan, "Trk receptors use redundant signal transduction pathways involving
SHC and PLC-gamma 1 to mediate NGF responses", Neuron, 12, 1994, 691-705.
ME Cunningham, RM Stephens, DR Kaplan, LA Greene, "Autophosphorylation of activation loop tyrosines regulates signaling by the TRK nerve
growth factor receptor", J Biol Chem, 272, 1997, 10957-67.
Reaction
33.3.1.4 Activated TrkA receptor internalizes to endosomes
Description
TRKA at the plasma membrane typically results in rapid endocytosis and subsequent passage of the receptors through a network of endosomal
compartments.
Reaction
33.3.2 NGF-independant TRKA activation
Authors
Editors
Jassal, B, 2006-10-10.
Description
TRK receptors can also be activated by at least two G-protein-coupled receptors (GPCR), the adenosine A2a receptor and the PACAP type I
receptor, without involvement of neurotrophins. Activity of both receptors is mediated by G proteins that activate adenyl cyclase. How this leads
to TRKA activation has not been fully elucidated, although a SRC-family tyrosine kinase and intracellular Ca2+ appear to play a role. TRKA
activation through GPCRs occurs with slow kinetics (over 1 hr adenosine or PACAP treatment is required) in an intracellular location (probably
the Golgi apparatus), and requires transcriptional and protein synthesis events that may influence the processing and activation of the receptors.
GPCR-mediated transactivation of TRK receptors causes the preferential activation of AKT versus ERKs. This leads to a cell survival response.
References
F Jeanneteau, MV Chao, "Promoting neurotrophic effects by GPCR ligands", Novartis Found Symp, 276, 2006, 181-9; discussion 18.
33.3.2.1 By adenosine A2a receptor
Authors
Editors
Jassal, B, 2006-10-10.
Description
Adenosine, acting through A2A receptors can induce TrkA activation. The mechanism of this activation is not understood.
Source reaction
This reaction was inferred from the corresponding reaction "By adenosine A2a receptor" in species Rattus norvegicus.
FS Lee, MV Chao, "Activation of Trk neurotrophin receptors in the absence of neurotrophins", Proc Natl Acad Sci U S A, 98, 2001, 3555-60.
Reaction
33.3.2.2 By PACAP type 1 receptor
Authors
Editors
Jassal, B, 2006-10-10.
Description
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that acts through type 1 PACAP receptor, a G protein-coupled
receptor. PACAP exerts its trophic effects using TrkA receptors and utilizing tyrosine kinase signaling pathways. The mechanism of activation is
still poorly understood.
Source reaction
This reaction was inferred from the corresponding reaction "By PACAP type 1 receptor" in species Rattus norvegicus.
FS Lee, R Rajagopal, AH Kim, PC Chang, MV Chao, "Activation of Trk neurotrophin receptor signaling by pituitary adenylate cyclase-activating
polypeptides", J Biol Chem, 277, 2002, 9096-102.
Reaction
33.4 TRKA signalling from the plasma membrane
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
Trk receptors signal from the plasma membrane and from intracellular membranes, particularly from early endosomes. Signalling from the
plasma membrane is fast but transient; signalling from endosomes is slower but long lasting. Signalling from the plasma membrane is annotated
here. TRK signalling leads to proliferation in some cell types and neuronal differentiation in others. Proliferation is the likely outcome of short
term signalling, as observed following stimulation of EGFR (EGF receptor). Long term signalling via TRK receptors, instead, was clearly shown
to be required for neuronal differentiation in response to neurotrophins.
References
MV Sofroniew, CL Howe, WC Mobley, "Nerve growth factor signaling, neuroprotection, and neural repair", Annu Rev Neurosci, 24, 2001,
1217-81.
WJ Friedman, LA Greene, "Neurotrophin signaling via Trks and p75", Exp Cell Res, 253, 1999, 131-42.
DR Kaplan, FD Miller, "Neurotrophin signal transduction in the nervous system", Curr Opin Neurobiol, 10, 2000, 381-91.
33.4.1 Signalling to ERKs
Authors

Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
Neurotrophins utilize multiple pathways to activate ERKs (ERK1 and ERK2), a subgroup of the large MAP kinase (MAPK) family, from the
plasma membrane. The major signalling pathways to ERKs are via RAS, ocurring from caveolae in the plasma membrane or from
clathrin-coated vesicles, and via RAP1, taking place in early endosomes. Whereas RAS activation by NGF is transient, RAP1 activation by NGF
is sustained for hours.
References
SS Grewal, RD York, PJ Stork, "Extracellular-signal-regulated kinase signalling in neurons", Curr Opin Neurobiol, 9, 1999, 544-53.
33.4.1.1 Signalling to RAS
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
Signalling through Shc adaptor proteins appears to be identical for both NGF and EGF. It leads to a fast, but transient, MAPK/ERK activation,
which is insufficient to explain the prolonged activation of MAPK found in NGF-treated cells.
References
MV Sofroniew, CL Howe, WC Mobley, "Nerve growth factor signaling, neuroprotection, and neural repair", Annu Rev Neurosci, 24, 2001,
1217-81.
33.4.1.1.1 Shc binds to the activated TrkA receptor
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
Shc proteins (Shc 1, 2, 3) are signalling adapters, able to interact with phosphorylated Y496 of TRKA. Shc2 and Shc3 appear to be the primary
Shc adaptor proteins in neurons as they are expressed in both the developing and adult nervous system. Shc1 is expressed embryonically but
not in the adult brain, whereas Shc3 expression is lower in the embryonic brain and increases post-natally. Pi-Y496 of TrkA can also be bound
by FRS2. The competitive binding between Frs2 and Shc at this phospho-tyrosine residue contributes to a cellular switch between cell cycle
progression (Shc recruitment) and cell cycle arrest/differentiation (Frs2 recruitment).
Reaction
33.4.1.1.2 Shc, complexed with TrkA, is tyrosine-phosphorylated
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
Phosphorylation of Shc adapter proteins, and the concomitant recruitment of GRB2/SOS, results in the RAS-dependent, transient activation of
ERKs, which is correlated with mitogenic and proliferative cell signalling. Prolonged activation of ERKs is instead regulated by a parallel
pathway, involving CRK/C3G-dependent activation of the RAS-like GTPase RAP-1, and takes place in early endosomes.
Reaction
33.4.1.1.3 Phospho-Shc dissociates from the TrkA receptor
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
Once Shc is phosphorylated, it dissociates from the receptor.
Source reaction
This reaction was inferred from the corresponding reaction "Phospho-Shc dissociates from the Trk receptor" in species Rattus norvegicus.
T Basu, PH Warne, J Downward, "Role of Shc in the activation of Ras in response to epidermal growth factor and nerve growth factor",
Oncogene, 9, 1994, 3483-91.
Reaction
33.4.1.1.4 GRB2:SOS binds to SHC-P
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
Tyrosine receptor kinase stimulation phosphorylates Shc which recruits the SH2 domain of the adaptor protein GRB2, which is complexed with
SOS, an exchange factor for p21ras and RAC, through its SH3 domain. Besides SOS, the GRB2 SH3 domain can associate with other
intracellular targets, including GAB1. Erk and Rsk mediated phosphorylation results in dissociation of the SOS-GRB2 complex. This may explain
why Erk activation through Shc and SOS-GRB2 is transient. Inactive p21ras-GDP is found anchored to the plasma membrane by a farnesyl
residue. As Shc is phosphorylated by the the stimulated receptor near to the plasma membrane, the SOS-GRB2:Shc interaction brings the SOS
enzyme into close proximity to p21ras.
References
Reaction
33.4.1.1.5 SOS mediated nucleotide exchange of RAS (SHC)
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
References
PA Boriack-Sjodin, SM Margarit, D Bar-Sagi, J Kuriyan, "The structural basis of the activation of Ras by Sos", Nature, 394, 1998, 337-43.
Reaction
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
References
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Heldin, CH, 2008-02-12.
Description
References
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
References
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
References
Reaction
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
Authors
Reviewers
Greene, LA, 2007-11-08.

Description
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
presence of ATP.
Source reaction
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
Source reaction
25699-709.
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
Reaction
Reviewers
Greene, LA, 2007-11-08.
Description
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description

33.4.1.1.7.2.1.1 MEK1 binds ERK-1
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
References
MEK2.", J Biol Chem, 268, 1993, 11435-9.
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
activity.
References
MEK2.", J Biol Chem, 268, 1993, 11435-9.
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
Source reaction
Reaction
Reviewers
Greene, LA, 2007-11-08.
Description

Source reaction
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
33.4.1.1.7.2.2.1 MEK2 binds ERK-2
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
References
MEK2.", J Biol Chem, 268, 1993, 11435-9.
Reaction

Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
activity.
References
MEK2.", J Biol Chem, 268, 1993, 11435-9.
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
Source reaction
Reaction
Reviewers
Greene, LA, 2007-11-08.
Description

Source reaction
Reaction
33.4.1.1.8 p38MAPK events
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
NGF induces sustained activation of p38, a member of the MAPK family (Morooka T, Nishida E, 1998). Both p38 and the ERKs appear to be
involved in neurite outgrowth and differentiation caused by NGF in PC12 cells. As a matter of fact, PC12 cell differentiation appears to involve
activation of both ERK/MAPK and p38. Both ERK/MAPK and p38 pathways contribute to the phosphorylation of the transcription factor CREB
and the activation of immediate-early genes (Xing J, 1998). p38 activation by NGF may occur by at least two mechanisms, involving SRC or
MEK kinases.
References
J Xing, JM Kornhauser, Z Xia, EA Thiele, ME Greenberg, "Nerve growth factor activates extracellular signal-regulated kinase and p38
mitogen-activated protein kinase pathways to stimulate CREB serine 133 phosphorylation", Mol Cell Biol, 18, 1998, 1946-55.
T Morooka, E Nishida, "Requirement of p38 mitogen-activated protein kinase for neuronal differentiation in PC12 cells", J Biol Chem, 273, 1998,
24285-8.
T Joneson, D Bar-Sagi, "Ras effectors and their role in mitogenesis and oncogenesis", J Mol Med, 75, 1997, 587-93.
33.4.1.1.8.1 Ral-GDS binds to Ras-GTP
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
Besides the RAF kinase, RAS can activate several ral guanine nucleotide dissociation stimulators (RALGDSs). Binding of RALGDS with RAS
competes with RAF binding to RAS.
References
F Hofer, S Fields, C Schneider, GS Martin, "Activated Ras interacts with the Ral guanine nucleotide dissociation stimulator", Proc Natl Acad Sci
U S A, 91, 1994, 11089-93.
Reaction
33.4.1.1.8.2 Guanine nucleotide exchange on Ral
Authors

Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
The Ras-related protein, Ral becomes activated once GDP is replaced by GTP.
Reaction
33.4.1.1.8.3 Activation of SRC by Ral-GTP
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.

Description
The active form of Ral, Ral-GTP, activates Src tyrosine kinase.
Reaction
33.4.1.1.8.4 Binding and activation of MAP Kinase
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
The NGF activation of p38 MAP kinase is transient, being maximal at 10 min and declining to near control levels by 30 min. T180 and Y182 are
two sites that become newly phosphorylated as p38 MAPK becomes activated.
Reaction
33.4.1.1.8.5 MAP kinase activates MAPKAPK2, MAPKAPK3 and MSK1
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
p38 MAPK is known to activate the Ser/Thr protein kinase MAPKAP kinase 2 and a closely related kinase, MAPKAP kinase 3, through
phosphorylation at multiple sites. According to some authors, NGF does not induce any significant activation of MAPKAPK2 activity in PC12
cells. Potential p38 signaling effectors include transcription factors, such as cAMP-response element-binding protein and MEF2, cytoskeleton
modulators, and a number of protein kinases. After activation, MAPKAP kinase 2 and 3 move to the nucleus.
References
R Ben-Levy, IA Leighton, YN Doza, P Attwood, N Morrice, CJ Marshall, P Cohen, "Identification of novel phosphorylation sites required for
activation of MAPKAP kinase-2", EMBO J, 14, 1995, 5920-30.
Reaction
33.4.1.2 Signalling to p38 via RIT and RIN
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
RIT and RIN are two small guanine nucleotide binding proteins that share more than 50% sequence identity with RAS, including highly
conserved core effector domains. Unlike RAS, the C termini of RIT and RIN lack a typical prenylation motif (CAAX, XXCC, or CXC) required for
the association of RAS proteins with the plasma membrane. RIT is expressed in all tissues, whereas RIN is neuron-specific. They have similar
signalling properties and are activated by NGF through unknown exchange factors. They signal to ERKs and p38 MAP kinase. They mainly lead
to p38 activation via the BRAF-MEK kinase cascade.
References
GW Reuther, CJ Der, "The Ras branch of small GTPases: Ras family members don't fall far from the tree", Curr Opin Cell Biol, 12, 2000,
157-65.
33.4.1.2.1 TrkA recruits RIT and RIN
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.

Description
TrkA can bind the Ras subfamly members RIT and RIN.
Reaction
33.4.1.2.2 RIT/RIN are activated
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
RIT and RIN are activated by neurotrophins through unknown exchange factors. Activation reaches a maximal level between 5 and 15 min after
NGF stimulation and remains elevated for at least 2 h. RIN, which is neuron-specific, might function as a component of a neuron specific B-RAF
signalosome complex, in which RIN provides spatial and/or substrate specificity to the B-RAF-MEK kinase cascade to direct stimulation of p38
signaling.
Reaction
33.4.1.2.3 RIT/RIN-GTP binds B-Raf
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
Once activated by RIT or RIN, B-RAF activates, through MEK, the p38 MAP kinase. Whereas RIN appears to activate p38 (specifically the
p38-alpha isoform) but not the ERKs, RIN was described to activate ERK1/ ERK2 as well, although to a much lower extent than p38. RIN
signaling gives rise to sustained activation of p38 MAP kinase.
Reaction
Authors
Reviewers
Greene, LA, 2007-11-08.

Description
References
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Heldin, CH, 2008-02-12.
Description
References
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.

Description
References
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.

Description
References
Reaction
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
presence of ATP.
Source reaction
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
Source reaction
25699-709.
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
Reaction
Reviewers
Greene, LA, 2007-11-08.
Description
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
33.4.1.2.5.2.1.1 MEK1 binds ERK-1
Authors
Editors
Schmidt, EE, 0000-00-00.

Reviewers
Greene, LA, 2007-11-08.
Description
References
MEK2.", J Biol Chem, 268, 1993, 11435-9.
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.

Reviewers
Greene, LA, 2007-11-08.
Description
activity.
References
MEK2.", J Biol Chem, 268, 1993, 11435-9.
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.

Reviewers
Greene, LA, 2007-11-08.
Description
Source reaction
Reaction

Reviewers
Greene, LA, 2007-11-08.
Description
Source reaction
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
33.4.1.2.5.2.2.1 MEK2 binds ERK-2
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
References
MEK2.", J Biol Chem, 268, 1993, 11435-9.
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
activity.
References
MEK2.", J Biol Chem, 268, 1993, 11435-9.
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
Source reaction
Reaction
Reviewers
Greene, LA, 2007-11-08.
Description

Source reaction
Reaction
33.4.1.3 Prolonged ERK activation events
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
After NGF binding, activated Trk receptors provide multiple docking sites for adaptor proteins and enzymes. Two docking proteins, the
Ankyrin-Rich Membrane Spanning protein (ARMS/Kidins220) and Fibroblast growth factor receptor substrate 2 (Frs2), target signaling molecules
in response to NGF stimulation and link receptor activation with the MAP kinase (also called the Extracellular signal-Regulated Kinase cascade,
ERK) cascade, an essential process for growth factor-induced cell proliferation and differentiation.
A feature of NGF signaling is the sustained activation of the MAPK cascade. This is achieved by the small G protein, Rap1 which binds to and
activates B-Raf, an activator of the MAPK cascade. Rap1 is a member of the Ras family of G proteins and like all G proteins, Rap1 is in an
inactive state when bound to GDP and is active when bound to GTP. A specific GEF (guanine nucleotide exchange factor) called C3G can
activate Rap1 by exchanging GDP for GTP.
References
PJ Stork, "Does Rap1 deserve a bad Rap?", Trends Biochem Sci, 28, 2003, 267-75.
DR Kaplan, FD Miller, "Signal transduction by the neurotrophin receptors", Curr Opin Cell Biol, 9, 1997, 213-21.
33.4.1.3.1 Frs2-mediated activation
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
The adaptor protein Frs2 (Fibroblast growth factor receptor substrate 2) can mediate the prolonged activation of the MAPK (ERK) cascade.
References
33.4.1.3.1.1 Frs2 binds to active TrkA receptor
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
FRS2 binds to TRKA through the same motif (NPXY) around the TRKA phospho-tyrosine residue Y496 to which SHC proteins bind. The
competition between SHC proteins and FRS2 for binding to NGF-activated TRKA may provide a novel mechanism by which proliferation and
differentiation may be regulated in response to neurotrophin stimulation. Tyrosine phosphorylation of FRS2 occurs within 2 min of NGF
stimulation.
References
SH Ong, GR Guy, YR Hadari, S Laks, N Gotoh, J Schlessinger, I Lax, "FRS2 proteins recruit intracellular signaling pathways by binding to
diverse targets on fibroblast growth factor and nerve growth factor receptors", Mol Cell Biol, 20, 2000, 979-89.
Reaction
33.4.1.3.1.2 Frs2 is phosphorylated by active TrkA receptor
Authors

Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
Activated TrkA induces the tyrosine phosphorylation of the lipid-anchored docking protein, FRS2. FRS2 is an adapter protein that links NGF
receptors to downstream signaling pathways. It is involved in the activation of MAP kinases.
References
H Kouhara, YR Hadari, T Spivak-Kroizman, J Schilling, D Bar-Sagi, I Lax, J Schlessinger, "A lipid-anchored Grb2-binding protein that links
FGF-receptor activation to the Ras/MAPK signaling pathway", Cell, 89, 1997, 693-702.
Reaction
33.4.1.3.1.3 Phospho-Frs2 binds CrkL
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.

Description
Besides CRK, FRS2 also binds GRB2, the cyclin-dependent kinase substrate p13(SUC1), and the SH3 domain of SRC. There is also evidence
for a C3G/CRK/SHP2/GAB2 complex, which is trafficked to the endosome, where C3G interacts with RAP1, triggering sustained RAP1
activation and prolonged B-RAF/MEK1/MAPK signalling. Crk-L is the predominant CRK isoform that interacts with C3G in several cell types; it is
abundant in PC12 cells. PC12 cells also express high levels of Crk-II and low, but detectable, levels of Crk-I. Activation of Elk-1 by NGF was
potently increased by cotransfection of exogenous Crk-II and Crk-L, but only weakly by Crk-I. In the absence of NGF, the expression of CRK
isoforms activated Elk-1 minimally.
Reaction
33.4.1.3.1.4 Phospho-Frs2:CrkL engages C3G
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
C3G is a guanine nucleotide exchange factor for Rap1, which is recruited by Crk adaptor proteins.
References
BS Knudsen, SM Feller, H Hanafusa, "Four proline-rich sequences of the guanine-nucleotide exchange factor C3G bind with unique specificity to
the first Src homology 3 domain of Crk", J Biol Chem, 269, 1994, 32781-7.
Reaction
33.4.1.3.1.5 (Frs2)C3G stimulates nucleotide exchange on Rap1
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
Rap1 is a small G protein, necessary for prolonged ERK activity in PC12 cells. In such cells, NGF triggers a program of neuronal differentiation
through the activation of a Rap1:B-RAF:ERK module Rap1 is activated by NGF, but not by epidermal growth factor (EGF), although both growth
factors cause transient activation of RAS. Activation of Rap1 by NGF requires internalization of TRKA to intracellular vesicles, mostly
endosomes, containing Rap1, B-RAF, MEK and ERKs. Rap1 does not co-localize with RAS. Therefore, the ability of Rap1 to bind RAF-1 without
activating it might sequester RAF-1 from RAS. Activation of GEFs that couple to Rap1 as well as RAS might provide a mechanism to limit
signals to RAS.
Source reaction
This reaction was inferred from the corresponding reaction "C3G stimulates nucleotide exchange on Rap1" in species Rattus norvegicus.
RD York, H Yao, T Dillon, CL Ellig, SP Eckert, EW McCleskey, PJ Stork, "Rap1 mediates sustained MAP kinase activation induced by nerve
growth factor", Nature, 392, 1998, 622-6.
Reaction
33.4.1.3.1.6 (Frs2)Rap1-GTP binds to and activates B-Raf
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
Rap1 binds to B-RAF; as a consequence, B-RAF is recruited to endosomes. The binding event of Rap1 to B-RAF is thought to be very similar to
the binding of RAS to RAF-1. In neuronal cells that express B-Raf, NGF induced activation of Rap1 promotes a sustained activation of ERKs and
is required for the induction of electrical excitability and a subset of neuron-specific genes. As regards morphological differentiation (e. g. neurite
outgrowth in PC12 cells), things are more complex. The transient activation of ERKs via RAS is not sufficient for neurite outgrowth in the
absence of additional signals. On the contrary, constitutive activation of Rap1 is sufficient to trigger neurite outgrowth, but it is not necessary for
this response.
Clearly, morphological differentiation of PC12 cells involves the activation of multiple pathways by NGF. Rap1 activates B-Raf, but inhibits
RAF-1. Consequently, Rap1 could have two opposing functions: to limit ERK activation in B-RAF-negative cells and to increase ERK activation
in B-Raf-positive cells.
Source reaction
This reaction was inferred from the corresponding reaction "Rap1-GTP binds and activates B-Raf" in species Rattus norvegicus.
Reaction
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
Authors
Reviewers
Greene, LA, 2007-11-08.
Description

Authors
Editors
Schmidt, EE, 0000-00-00.
Description
presence of ATP.
Source reaction
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
Source reaction
25699-709.
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
Reaction
Reviewers
Greene, LA, 2007-11-08.
Description
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
33.4.1.3.1.7.2.1.1 MEK1 binds ERK-1
Authors
Editors
Schmidt, EE, 0000-00-00.

Reviewers
Greene, LA, 2007-11-08.
Description
References
MEK2.", J Biol Chem, 268, 1993, 11435-9.
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.

Reviewers
Greene, LA, 2007-11-08.
Description
activity.
References
MEK2.", J Biol Chem, 268, 1993, 11435-9.
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.

Reviewers
Greene, LA, 2007-11-08.
Description
Source reaction
Reaction

Reviewers
Greene, LA, 2007-11-08.
Description
Source reaction
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
33.4.1.3.1.7.2.2.1 MEK2 binds ERK-2
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
References
MEK2.", J Biol Chem, 268, 1993, 11435-9.
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
activity.
References
MEK2.", J Biol Chem, 268, 1993, 11435-9.
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
Source reaction
Reaction
Reviewers
Greene, LA, 2007-11-08.
Description

Source reaction
Reaction
33.4.1.3.2 ARMS-mediated activation
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
ARMS (Ankyrin-Rich Membrane Spanning/Kidins 220) is a 220kD tetraspanning adaptor protein which becomes rapidly tyrosine phosphorylated
by active Trk receptors. ARMS is another adaptor protein which is involved in the activation of Rap1 and the subsequent prolonged activation of
the MAPK cascade.
References
33.4.1.3.2.1 ARMS:Crk complex binds to active TrkA receptor
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
Ankyrin-Rich Membrane Spanning protein (ARMS or Kidins220) is a specific target of Trk receptor tyrosine phosphorylation. The
ARMS/Kidins220:Crk complex is an upstream component of the C3G-Rap1-MAP kinase cascade and is SH3 dependent.
Source reaction
This reaction was inferred from the corresponding reaction "ARMS:Crk binds to active Trk receptor" in species Rattus norvegicus.
JC Arevalo, H Yano, KK Teng, MV Chao, "A unique pathway for sustained neurotrophin signaling through an ankyrin-rich membrane-spanning
protein", EMBO J, 23, 2004, 2358-68.
Reaction
33.4.1.3.2.2 ARMS is phosphorylated by active TrkA receptor
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
Phosphorylation of ARMS by Trk receptor (on tyrosine 1096) enables ARMS to recruit Crk via it's SH2 domain and freeing the SH3 domain. The
SH3 domain of Crk is then free to bind C3G for MAP kinase activation.
References
JC Arevalo, DB Pereira, H Yano, KK Teng, MV Chao, "Identification of a switch in neurotrophin signaling by selective tyrosine phosphorylation",
J Biol Chem, 281, 2006, 1001-7.
Reaction
33.4.1.3.2.3 Crk's SH3 domain engages C3G
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
C3G is a guanine nucleotide exchange factor for Rap1, which is recruited by Crk adaptor proteins.
References
J Biol Chem, 281, 2006, 1001-7.
Reaction
33.4.1.3.2.4 C3G stimulates nucleotide exchange on Rap1
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
Rap1 is a small G protein, necessary for prolonged ERK activity in PC12 cells. In such cells, NGF triggers a program of neuronal differentiation
through the activation of a Rap1:B-RAF:ERK module Rap1 is activated by NGF, but not by epidermal growth factor (EGF), although both growth
factors cause transient activation of RAS. Activation of Rap1 by NGF requires internalization of TRKA to intracellular vesicles, mostly
endosomes, containing Rap1, B-RAF, MEK and ERKs. Rap1 does not co-localize with RAS. Therefore, the ability of Rap1 to bind RAF-1 without
activating it might sequester RAF-1 from RAS. Activation of GEFs that couple to Rap1 as well as RAS might provide a mechanism to limit
signals to RAS.
References
J Biol Chem, 281, 2006, 1001-7.
Reaction
33.4.1.3.2.5 (ARMS)Rap1-GTP binds and activates B-Raf
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
Rap1 binds to B-RAF; as a consequence, B-RAF is recruited to endosomes. The binding event of Rap1 to B-RAF is thought to be very similar to
the binding of RAS to RAF-1. In neuronal cells that express B-Raf, NGF induced activation of Rap1 promotes a sustained activation of ERKs and
is required for the induction of electrical excitability and a subset of neuron-specific genes. As regards morphological differentiation (e. g. neurite
outgrowth in PC12 cells), things are more complex. The transient activation of ERKs via RAS is not sufficient for neurite outgrowth in the
absence of additional signals. On the contrary, constitutive activation of Rap1 is sufficient to trigger neurite outgrowth, but it is not necessary for
this response.
Clearly, morphological differentiation of PC12 cells involves the activation of multiple pathways by NGF. Rap1 activates B-Raf, but inhibits
RAF-1. Consequently, Rap1 could have two opposing functions: to limit ERK activation in B-RAF-negative cells and to increase ERK activation
in B-Raf-positive cells.
Source reaction
This reaction was inferred from the corresponding reaction "Rap1-GTP binds and activates B-Raf" in species Rattus norvegicus.
Reaction
Authors
Reviewers
Greene, LA, 2007-11-08.

Description
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
presence of ATP.
Source reaction
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
Source reaction
25699-709.
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Description
Reaction
Reviewers
Greene, LA, 2007-11-08.
Description
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
33.4.1.3.2.6.2.1.1 MEK1 binds ERK-1
Authors
Editors
Schmidt, EE, 0000-00-00.

Reviewers
Greene, LA, 2007-11-08.
Description
References
MEK2.", J Biol Chem, 268, 1993, 11435-9.
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.

Reviewers
Greene, LA, 2007-11-08.
Description
activity.
References
MEK2.", J Biol Chem, 268, 1993, 11435-9.
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.

Reviewers
Greene, LA, 2007-11-08.
Description
Source reaction
Reaction

Reviewers
Greene, LA, 2007-11-08.
Description
Source reaction
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
33.4.1.3.2.6.2.2.1 MEK2 binds ERK-2
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
References
MEK2.", J Biol Chem, 268, 1993, 11435-9.
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
activity.
References
MEK2.", J Biol Chem, 268, 1993, 11435-9.
Reaction
Authors
Editors
Schmidt, EE, 0000-00-00.
Reviewers
Greene, LA, 2007-11-08.
Description
Source reaction
Reaction
Reviewers
Greene, LA, 2007-11-08.
Description

Source reaction
Reaction
33.4.2 PLC-gamma1 signalling
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
The activation of phosphlipase C-gamma (PLC-gamma) and subsequent mobilization of calcium from intracellular stores are essential for
neurotrophin secretion. PLC-gamma is activated through the phosphorylation by TrkA receptor kinase and this form hydrolyses PIP2 to generate
inositol tris-phosphate (IP3) and diacylglycerol (DAG). IP3 promotes the release of Ca2+ from internal stores and this results in activation of
enzymes such as protein kinase C and Ca2+ calmodulin-regulated protein kinases.
References
G Carpenter, Q Ji, "Phospholipase C-gamma as a signal-transducing element", Exp Cell Res, 253, 1999, 15-24.
33.4.2.1 Binding of PLCG1 to active TrkA receptor
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
The PLC-gamma 1 docking site in Trk receptor (Y785) is important for initiation and maintenance of hippocampal LTP (long term potentiation);
this residue in TrkA receptor also binds to CHK tyrosine kinase, which participates in MAPK pathway activation and is involved in PC12 cells
neurite outgrowth in response to NGF. PLC-gamma 1 activation results in long term induction of a sodium channel gene (PN1).
Reaction
33.4.2.2 TrkA phosphorylates PLCG1
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
Mutational analysis of tyrosine residues, highly conserved in the cytoplasmic domain of all Trk receptors, reveal that the activation of
PLC-gamma is necessary to mobilize Ca2+ from intracellular stores, the key mechanism for regulated NT secretion.
Reaction
33.4.2.3 Active PLCG1 dissociates from TrkA receptor
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
Once phosphorylated, PLC-gamma dissociates from the receptor
Reaction
33.4.2.4 Active PLCG1 hydrolyses PIP2
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
Inositol 1,4,5-triphosphate (IP3) is a second messenger produced by phospholipase C (PLC) metabolism of phosphoinositol 4,5-bisphosphate
(PIP2).
Reaction
33.4.2.5 DAG stimulates protein kinase C-delta
Reviewers
Greene, LA, 2007-11-08.
Description
Diacylglycerol (DAG) activates protein kinase C-delta (PKC-delta), which stimulates ERK1/2 and triggers neurite outgrowth. It probably acts
between RAF and MEK. PKC-delta contributes to growth factor specificity and response to neuronal cells by promoting cell-type-specific
differences in growth factor signalling. DAG can also activate PKC-epsilon in the same manner.
Reaction
33.4.2.6 IP3 binds with the IP3 receptor, opening the Ca2+ channel
Authors

Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.
Description
The IP3 receptor (IP3R) is an IP3-gated calcium channel. It is a large, homotetrameric protein, similar to other calcium channel proteins such as
ryanodine. The four subunits form a 'four-leafed clover' structure arranged around the central calcium channel. Binding of ligands such as IP3
results in conformational changes in the receptor's structure that leads to channel opening.
Reaction
33.4.2.7 Release of calcium from intracellular stores by IP3 receptor activation
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.

Description
IP3 promotes the release f intracellular calcium.
Reaction
33.4.3 PI3K/AKT signalling
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
PI3K/AKT signalling is a major regulator of neuron survival. It blocks cell death by both impinging on the cytoplasmic cell death machinery and
by regulating the expression of genes involved in cell death and survival. In addition, it may also use metabolic pathways to regulate cell
survival.The PI3K/AKT pathway also affects axon diameter and branching (Marcus et al, 2002) and regulates small G proteins like RhoA
(Vanhaesebroeck, B and Waterman, MD, 1999), which control the behaviour of the F-actin cytoskeleton. Moreover, through its connection with
the TOR pathway, it promotes translation of a subset of mRNAs.
References
A Markus, J Zhong, WD Snider, "Raf and akt mediate distinct aspects of sensory axon growth", Neuron, 35, 2002, 65-76.
B Vanhaesebroeck, MD Waterfield, "Signaling by distinct classes of phosphoinositide 3-kinases", Exp Cell Res, 253, 1999, 239-54.
33.4.3.1 Active TRKA binds IRS1/2
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
IRS1 and IRS2 bind directly to TRK receptors phosphorylated at Y490, through their phosphotyrosine- binding (PTB) domains.
Source reaction
This reaction was inferred from the corresponding reaction "Active NTRK1 binds IRS1/2" in species Mus musculus.
C Miranda, A Greco, C Miele, MA Pierotti, E Van Obberghen, "IRS-1 and IRS-2 are recruited by TrkA receptor and oncogenic TRK-T1", J Cell
Physiol, 186, 2001, 35-46.
Reaction
33.4.3.2 TRKA phosphorylates IRS
Authors
Reviewers
Greene, LA, 2007-11-08.

Description
IRS1 and IRS2 are tyrosine phosphorylated at multiple YXXM motifs by the active TRKA kinase.
Source reaction
This reaction was inferred from the corresponding reaction "NTRK1 phosphorylates IRS" in species Mus musculus.
C Miranda, A Greco, C Miele, MA Pierotti, E Van Obberghen, "IRS-1 and IRS-2 are recruited by TrkA receptor and oncogenic TRK-T1", J Cell
Physiol, 186, 2001, 35-46.
Reaction
33.4.3.3 Active IRS recruits PI3K to the plasma membrane and activates it
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
The PI3K regulatory subunit p85 binds to IRS1 or IRS2, tyrosine-phosphorylated at YXXM motifs, through its SH2 domain.
As the p85 subunt is constitutively associated with the p110 catalytic subunit, the outcome is that the whole PI3K complex is recruited to the
membrane. The interaction at the plasma membrane of the p85 regulatory subunit with the p110 catalytic subunit of PI3K
(phosphatidylinositol-4,5-bisphosphate 3-kinase) causes a conformational change, resulting in activation of the catalytic subunit.
Reaction
33.4.3.4 PI3K produces PIP3 and other phosphatidyl inositides
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
PI3-kinase phosphorylates several phosphatidyl-inositides (phospholipids) at the plasma membrane: the most relevant is PtdIns(3,4,5)P3, also
named PIP3.
References
MP Scheid, PA Marignani, JR Woodgett, "Multiple phosphoinositide 3-kinase-dependent steps in activation of protein kinase B", Mol Cell Biol,
22, 2002, 6247-60.
Reaction
33.4.3.5 PIP3 binds to RhoA and activates it
Authors
Reviewers
Greene, LA, 2007-11-08.

Description
Several guanine exchange factors (GEFs) for the Rho family of GTPases contain PH domains that bind to PIP3. RhoA protein activation is a
mechanism whereby PI3K acts independently of AKT.
Reaction
33.4.3.6 PIP3 recruits PDK1 and AKT to the membrane
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
Phosphatidyl-inositides generated by PI3K recruit phosphatidylinositide-dependent protein kinase 1 (PDK1) and AKT (also known as protein
kinase B) to the membrane, through their PH (pleckstrin-homology) domains. The binding of PIP3 to the PH domain of AKT is the rate-limiting
step in AKT activation. In mammals there are three AKT isoforms (AKT1-3) encoded by three separate genes. The three isoforms share a high
degree of amino acid identity and have indistinguishable substrate specificity in vitro. However, isoform-preferred substrates in vivo cannot be
ruled out. The relative expression of the three isoforms differs in different mammalian tissues: AKT1 is the predominant isoform in the majority of
tissues, AKT2 is the predominant isoform in insulin-responsive tissues, and AKT3 is the predominant isoform in brain and testes. (Note: all data
in the pathway refer to AKT1, which was the most studied.)
References
MP Scheid, PA Marignani, JR Woodgett, "Multiple phosphoinositide 3-kinase-dependent steps in activation of protein kinase B", Mol Cell Biol,
22, 2002, 6247-60.
Reaction
33.4.3.7 PDK1 phosphorylates AKT at T308
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
Once AKT is localized at the plasma membrane, it is phosphorylated at two critical residues for its full activation. These residues are a threonine
(T308 in AKT1) in the activation loop within the catalytic domain, and a serine (S473 in AKT1), in a hydrophobic motif (HM) within the carboxy
terminal, non catalytic region. PDK1 is the activation loop kinase; this kinase can also directly phosphorylate p70S6K. As detailed in the following
event, the HM kinase, previously termed PDK2, has been identified as the mammalian TOR (Target Of Rapamycin; Sarbassov et al., 2005).
References
BM Burgering, PJ Coffer, "Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction", Nature, 376, 1995, 599-602.
M Delcommenne, C Tan, V Gray, L Rue, J Woodgett, S Dedhar, "Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase
kinase 3 and protein kinase B/AKT by the integrin-linked kinase", Proc Natl Acad Sci U S A, 95, 1998, 11211-6.
Reaction
33.4.3.8 TORC2 (mTOR) phosphorylates AKT at S473
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
S473 phosphorylation in AKT is carried out by mTOR (mammalian Target of Rapamycin). This kinase is found in two structurally and functionally
distinct protein complexes, named TOR complex 1 (TORC1) and TOR complex 2 (TORC2). It is TORC2 complex, which is is composed of
mTOR, RICTOR, SIN1 (also named MAPKAP1) and LST8, that phosphorylates AKT at S473 (Sarbassov et al., 2005). This complex also
regulates actin cytoskeletal reorganization (Jacinto et al., 2004; Sarbassov et al., 2004). TORC1, on the other hand, is a major regulator of
ribosomal biogenesis and protein synthesis (Hay and Sonenberg, 2004). TORC1 regulates these processes largely by the
phosphorylation/inactivation of the repressors of mRNA translation 4E binding proteins (4E BPs) and by the phosphorylation/activation of
ribosomal S6 kinase (S6K1). TORC1 is also the principal regulator of autophagy.
References
DD Sarbassov, DA Guertin, SM Ali, DM Sabatini, "Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex", Science, 307, 2005,
1098-101.
E Jacinto, R Loewith, A Schmidt, S Lin, MA Ruegg, A Hall, MN Hall, "Mammalian TOR complex 2 controls the actin cytoskeleton and is
rapamycin insensitive", Nat Cell Biol, 6, 2004, 1122-8.
DD Sarbassov, SM Ali, DH Kim, DA Guertin, RR Latek, H Erdjument-Bromage, P Tempst, DM Sabatini, "Rictor, a novel binding partner of
mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton", Curr Biol, 14, 2004, 1296-302.
Reaction
33.4.3.9 AKT phosphorylates targets in the cytosol
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
Following activation, AKT can phosphorylate an array of target proteins in the cytoplasm, many of which are involved in cell survival control.
Phosphorylation of TSC2 feeds positively to the TOR kinase, which, in turn, contributes to AKT activation (positive feedback loop).
33.4.3.9.1 AKT phosphorylates BAD
Authors

Reviewers
Greene, LA, 2007-11-08.
Description
AKT phosphorylates the BCL-2 family member BAD at serine 136, blocking the BAD-induced death (of neurons).
References
L del Peso, M Gonzalez-Garcia, C Page, R Herrera, G Nunez, "Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt",
Science, 278, 1997, 687-9.
Reaction
33.4.3.9.2 AKT phosphorylates GSK3
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
GSK3 (glycogen synthase kinase-3) participates in the Wnt signaling pathway. It is implicated in the hormonal control of several regulatory
proteins including glycogen synthase, and the transcription factors MYB and JUN. GSK3 phosphorylates JUN at sites proximal to its
DNA-binding domain, thereby reducing its affinity for DNA. GSK3 is inhibited when phosphorylated by AKT1.
Source reaction
This reaction was inferred from the corresponding reaction "Akt1 phosphorylates GSK3" in species Rattus norvegicus.
DA Cross, DR Alessi, P Cohen, M Andjelkovich, BA Hemmings, "Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase
B", Nature, 378, 1995, 785-9.
Reaction
33.4.3.9.3 AKT phosphorylates caspase-9
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
AKT can phosphorylate the apoptotic protease caspase-9, inhibiting it.
References
MH Cardone, N Roy, HR Stennicke, GS Salvesen, TF Franke, E Stanbridge, S Frisch, JC Reed, "Regulation of cell death protease caspase-9 by
phosphorylation", Science, 282, 1998, 1318-21.
Reaction
33.4.3.9.4 AKT phosphorylates MDM2
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
AKT-mediated phosphorylation of ubiquitin-protein ligase E3 MDM2 promotes nuclear localization and inhibits interaction between MDM2 and
p19ARF, thereby decreasing p53 stability. This leads to a decreased expression of p53 target genes, such as BAX, that promote apoptosis.
References
BP Zhou, Y Liao, W Xia, Y Zou, B Spohn, MC Hung, "HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2 phosphorylation", Nat Cell
Biol, 3, 2001, 973-82.
Reaction
33.4.3.9.5 AKT phosphorylates IKKalpha
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
AKT mediates IKKalpha (Inhibitor of nuclear factor kappa B kinase subunit alpha) phosphorylation at threonine 23, which is required for NF-kB
activation. NF-kB promoted gene transcription enhances neuronal survival.
References
ON Ozes, LD Mayo, JA Gustin, SR Pfeffer, LM Pfeffer, DB Donner, "NF-kappaB activation by tumour necrosis factor requires the Akt
serine-threonine kinase", Nature, 401, 1999, 82-5.
Reaction
33.4.3.9.6 AKT phosphorylates p21Cip1 and p27Kip1
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
Phosphorylation of p27Kip1 at T157 and of p21Cip1 at T145 by AKT leads to their retention in the cytoplasm, segregating these
cyclin-dependent kinase (CDK) inhibitors from cyclin-CDK complexes.
References
G Viglietto, ML Motti, P Bruni, RM Melillo, A D'Alessio, D Califano, F Vinci, G Chiappetta, P Tsichlis, A Bellacosa, A Fusco, M Santoro,
"Cytoplasmic relocalization and inhibition of the cyclin-dependent kinase inhibitor p27(Kip1) by PKB/Akt-mediated phosphorylation in breast
cancer", Nat Med, 8, 2002, 1136-44.
Reaction
33.4.3.9.7 AKT phosphorylates TSC2, inhibiting it

Authors
Reviewers
Greene, LA, 2007-11-08.
Description
AKT phosphorylates and inhibit TSC2 (tuberin), a suppressor of the TOR kinase pathway, which senses nutrient levels in the environment. TSC2
forms a TSC1-TSC2 protein complex that is a GAP (GTPase activating protein) for the RHEB G-protein. RHEB, in turn, activates the TOR
kinase. Thus an active AKT1 activates the TOR kinase, both of which are positive signals for cell growth (an increase in cell mass) and division.
The TOR kinase regulates two major processes: translation of selected mRNAs in the cell and autophagy. In the presence of high nutrient levels
TOR is active and phosphorylates the 4EBP protein releasing the eukaryotic initiation factor 4E (eIF4E), which is essential for cap-dependent
initiation of translation and promoting growth of the cell (PMID: 15314020). TOR also phosphorylates the S6 kinase, which is implicated in
ribosome biogenesis as well as in the modification of the S6 ribosomal protein. AKT can also activate mTOR by another mechanism, involving
phosphorylation of PRAS40, an inhibitor of mTOR activity.
References
K Inoki, Y Li, T Zhu, J Wu, KL Guan, "TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling", Nat Cell Biol, 4, 2002,
648-57.
BD Manning, AR Tee, MN Logsdon, J Blenis, LC Cantley, "Identification of the tuberous sclerosis complex-2 tumor suppressor gene product
tuberin as a target of the phosphoinositide 3-kinase/akt pathway", Mol Cell, 10, 2002, 151-62.
Reaction
33.4.3.9.8 AKT phosphorylates PRAS40
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
PRAS40 (proline-rich Akt/PKB substrate 40 kDa) is a substrate of AKT, the phosphorylation of which leads to the binding of this protein to
14-3-3. PRAS40 binds to mTOR complexes, mediating AKT signals to mTOR. Interaction of PRAS40 with the mTOR kinase domain is induced
under conditions that inhibit mTOR signalling, such as growth factor deprivation. Binding of PRAS40 inhibits mTOR. PRAS40 phosphorylation by
AKT and association with the cytosolic anchor protein 14-3-3, lead to mTOR stimulation (Vander Haar E, et al, 2007). Although it was originally
identified in the context of insulin signalling, it was later shown that PRAS40 may also play a role in nerve growth factor-mediated
neuroprotection (Saito A, et al, 2004).
References
E Vander Haar, SI Lee, S Bandhakavi, TJ Griffin, DH Kim, "Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40", Nat Cell
Biol, 9, 2007, 316-23.
KS Kovacina, GY Park, SS Bae, AW Guzzetta, E Schaefer, MJ Birnbaum, RA Roth, "Identification of a proline-rich Akt substrate as a 14-3-3
binding partner", J Biol Chem, 278, 2003, 10189-94.
A Saito, P Narasimhan, T Hayashi, S Okuno, M Ferrand-Drake, PH Chan, "Neuroprotective role of a proline-rich Akt substrate in apoptotic
neuronal cell death after stroke: relationships with nerve growth factor", J Neurosci, 24, 2004, 1584-93.
Reaction
33.4.3.10 AKT translocates to the nucleus
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
AKT, phosphorylated at threonine (AKT1 308; AKT2 309; AKT3 305) and serine (AKT1 473; AKT2 474; AKT3 472), translocates to the nucleus,
reaching a maximum after 15 min and returning to a basal level after 45 min of NGF stimulation. Control of the amount of nuclear AKT is
achieved through the action of the phosphatase PP2A.
Source reaction
This reaction was inferred from the corresponding reaction "Akt translocates to the nucleus" in species Rattus norvegicus.
P Borgatti, AM Martelli, G Tabellini, A Bellacosa, S Capitani, LM Neri, "Threonine 308 phosphorylated form of Akt translocates to the nucleus of
PC12 cells under nerve growth factor stimulation and associates with the nuclear matrix protein nucleolin", J Cell Physiol, 196, 2003, 79-88.
Reaction
33.4.3.11 AKT phosphorylates targets in the nucleus

Authors
Reviewers
Greene, LA, 2007-11-08.
Description
After translocation into the nucleus, AKT can phosphorylate a number of targets there such as CREB, forkhead transcription factors, SRK and
NUR77.
33.4.3.11.1 AKT phosphorylates CREB
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
AKT phosphorylates CREB (cAMP response element-binding protein) at serine 133 and activates gene expression via a CREB-dependent
mechanism, thus promoting cell survival.
References
K Du, M Montminy, "CREB is a regulatory target for the protein kinase Akt/PKB", J Biol Chem, 273, 1998, 32377-9.
Reaction
33.4.3.11.2 AKT can phosphorylate forkhead box transcription factors
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
Cell survival and growth are also promoted by AKT phosphorylation of Forkhead box (FOX) transcription factors, most notably FoxO1, FoxO3a
and FoxO4. Once phosphorylated by AKT, these factors are removed from the nucleus, associate with 14-3-3 proteins, and are retained in the
cytoplasm, thus producing a change in their transcriptional activity. For instance, unphosphorylated FoxO3a in the nucleus triggers apoptosis,
most likely by inducing the expression of critical genes, such as the Fas ligand gene. In another example, AKT phosphorylation of FOXO4
prevents FOXO4-mediated upregulation of p27Kip1.
References
G Rena, S Guo, SC Cichy, TG Unterman, P Cohen, "Phosphorylation of the transcription factor forkhead family member FKHR by protein kinase
B", J Biol Chem, 274, 1999, 17179-83.
A Brunet, A Bonni, MJ Zigmond, MZ Lin, P Juo, LS Hu, MJ Anderson, KC Arden, J Blenis, ME Greenberg, "Akt promotes cell survival by
phosphorylating and inhibiting a Forkhead transcription factor", Cell, 96, 1999, 857-68.
H Matsuzaki, A Ichino, T Hayashi, T Yamamoto, U Kikkawa, "Regulation of intracellular localization and transcriptional activity of FOXO4 by
protein kinase B through phosphorylation at the motif sites conserved among the FOXO family", J Biochem (Tokyo), 138, 2005, 485-91.
Reaction
33.4.3.11.2.1 AKT phosphorylates FOXO1A

Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
One or more of the isoforms of AKT catalyzes the phosphorylation of FOXO1A protein at three sites, threonine-24, serine-256, and serine-319
(Zhang et al. 2002, 2006). This reaction occurs in the nucleoplasm, and thus is dependent on the phosphorylation and nuclear import of AKT in
response to upstream regulatory factors (Burgering and Kops 2002).
References
X Zhang, S Zhang, H Yamane, R Wahl, A Ali, JA Lofgren, RL Kendall, "Kinetic mechanism of AKT/PKB enzyme family", J Biol Chem, 281, 2006,
13949-56.
2002, 45276-84.
BM Burgering, GJ Kops, "Cell cycle and death control: long live Forkheads", Trends Biochem Sci, 27, 2002, 352-60.
Reaction
33.4.3.11.3 AKT can phosphorylate SRK

Authors
Reviewers
Greene, LA, 2007-11-08.
Description
Ribosomal protein S6 kinase beta-2 (RSK) activation is a highly conserved mitogenic response, and the activities of RSK are stimulated by
multiple serine/threonine phosphorylations by different upstream kinases, one of which is AKT.
References
H Koh, K Jee, B Lee, J Kim, D Kim, YH Yun, JW Kim, HS Choi, J Chung, "Cloning and characterization of a nuclear S6 kinase, S6
kinase-related kinase (SRK); a novel nuclear target of Akt", Oncogene, 18, 1999, 5115-9.
Reaction
33.4.3.11.4 AKT can phosphorylate NUR77
Authors
Reviewers
Greene, LA, 2007-11-08.

Description
AKT inhibits DNA binding of NUR77 and inhibits its pro-apoptotic function (PMID 11438550). However, The relevance of AKT for NUR77
phosphorylation has recently been questioned: according to recent work, NUR77 is phosphorylated by RSK (and MSK) rather than by AKT
(PMID 16223362).
References
Y Pekarsky, C Hallas, A Palamarchuk, A Koval, F Bullrich, Y Hirata, R Bichi, J Letofsky, CM Croce, "Akt phosphorylates and regulates the
orphan nuclear receptor Nur77", Proc Natl Acad Sci U S A, 98, 2001, 3690-4.
Reaction
33.4.3.12 Negative regulation of the PI3K/AKT network
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
The PI3K/AKT network is negatively regulated by phosphatases that dephosphorylate PIP3, thus hampering AKT activation.
33.4.3.12.1 PTEN dephosphorylates PIP3

Authors
Reviewers
Greene, LA, 2007-11-08.
Description
The PI3K network is negatively regulated by phospholipid phosphatases that dephosphorylate PIP3, thus hampering AKT activation. The tumour
supressor PTEN is the primary phospholipid phosphatase.
References
T Maehama, JE Dixon, "The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol
3,4,5-trisphosphate", J Biol Chem, 273, 1998, 13375-8.
Reaction
33.4.3.12.2 CTMP and/or TRB3 inhibit AKT phosphorylation
Authors
Reviewers
Greene, LA, 2007-11-08.

Description
The phosphorylation of AKT at threonine and serine can be inhibited by direct binding of two different proteins, C-terminal modulator protein
(CTMP), which binds to the carboxy-terminal tail of AKT, or Tribbles homolog 3 (TRB3), which binds tot he catalytic domain of AKT.
References
SM Maira, I Galetic, DP Brazil, S Kaech, E Ingley, M Thelen, BA Hemmings, "Carboxyl-terminal modulator protein (CTMP), a negative regulator
of PKB/Akt and v-Akt at the plasma membrane", Science, 294, 2001, 374-80.
K Du, S Herzig, RN Kulkarni, M Montminy, "TRB3: a tribbles homolog that inhibits Akt/PKB activation by insulin in liver", Science, 300, 2003,
1574-7.
Reaction
33.4.3.12.3 PHLPP dephosphorylates S473 in AKT
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
The PH domain leucine-rich repeat-containing protein phosphatase (PHLPP) can specifically dephosphorylate the serine residue and inactivate
AKT.
References
T Gao, F Furnari, AC Newton, "PHLPP: a phosphatase that directly dephosphorylates Akt, promotes apoptosis, and suppresses tumor growth",
Mol Cell, 18, 2005, 13-24.
Reaction
33.4.4 Signalling to STAT3
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
Neurotrophin-induced increase in Signal transducer and activator of transcription 3 (STAT3; acute-phase response factor) activation appears to
underly several downstream functions of neurotrophin signalling, such as transcription of immediate early genes, proliferation arrest, and neurite
outgrowth.
33.4.4.1 STAT3 activation
Authors

Reviewers
Greene, LA, 2007-11-08.
Description
Activation of TRKA by NGF triggers STAT3 phosphorylation at Ser-727, and enhances the DNA binding and transcriptional activities of STAT3.
Ser-727 phosphorylation of STAT3 begins within 5 min, and the levels of Ser(P) STAT3 remain elevated up to 30 min of NGF stimulation. Ser(P)
STAT3 was localized to the cytoplasm, nuclei, and growth cones of neurites. Although the mechanisms by which STAT3 is activated by
neurotrophins remaines unknown, phosphorylation of STAT3 at serine 727 might function as a convergent point for several signaling pathways
triggered by Trk activation. Inhibition of STAT3 expression was found to attenuate NGF-induced transcription of immediate early genes, to
suppress NGF-induced cyclin D1 expression, and to decrease BDNF-promoted neurite outgrowth in hippocampal neurons.
Reaction
33.4.5 Signalling to ERK5
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
The location of neurotrophin stimulation appears to determine the nature of the transcriptional response through differential uses of individual
MAP kinases. The ERK5 pathway has a unique function in retrograde signalling; in contrast, ERK1/2, which mediate nuclear responses following
direct cell body stimulation, does not transmit a retrograde signal. Following neurotrophin stimulation of distal axons, phosphorylated TRK
receptors are endocytosed and transported to the cell bodies, where MEK5 phosphorylates ERK5, leading to ERK5 nuclear translocation,
phosphorylation of transcription factors, and neuronal survival. In contrast, neurotrophin stimulation of the cell bodies causes concurrent
activation and nuclear transport of ERK1/2 as well as ERK5. Several distinctive features of the ERK5 pathway might be important for retrograde
signalling. The ERK5 cascade does not depend on activation of the G-protein RAS. Instead, this pathway may use other G-proteins such as
RAP that are associated with vesicles, or may not require any G-protein intermediate. Another distinctive feature is that the MEK5 isoform, which
is expressed in the nervous system, exhibits a punctate staining pattern, suggesting a vesicular localization. ERK5 itself significantly differs from
ERK1/2, and its substrate specificity also differs from ERK1/2. For instance, ERK5 directly activates transcription factors, including MEF2, that
are not phosphorylated by ERK1/2. Conversely, ERK1/2, but not ERK5, activate transcription factors such as ELK1 and MITF.
33.4.5.1 ERK5 is activated
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
Extracellular signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase family. ERK5 is twice the size of the ERK1/2,
containing a conserved amino terminal kinase domain that is 53% identical to ERK1/2, and a unique carboxyterminal region which contains
potential binding sites for signalling molecules such as CRK, PI3 kinase and SRC. The second proline-rich region may interact with actin,
targeting the kinase to a specific location in the cell. In contrast to ERK1 and ERK2, which are activated by neurotrophins (NTs), cAMP, and
neuronal activity in neurons, ERK5 appears to be activated only by neurotrophins. The small GTPase Rap1 and the MEKK2 or MEKK3 kinases
are critical upstream signaling molecules mediating neurotrophin stimulation of ERK5 in neurons. MEKK2 or MEKK3 activate MEK5, which
appears to be localised in intracellular vesicles. MEK5 then activates ERK5. Once phosphorylated, ERK5 translocates to the nucleus.
Source reaction
This reaction was inferred from the corresponding reaction "ERK5 is activated" in species Rattus norvegicus.
FL Watson, HM Heerssen, A Bhattacharyya, L Klesse, MZ Lin, RA Segal, "Neurotrophins use the Erk5 pathway to mediate a retrograde survival
response", Nat Neurosci, 4, 2001, 981-8.
Reaction
33.4.5.2 ERK5 translocates to the nucleus
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
On phosphorylation, ERK5 translocates to the nucleus.
Reaction
33.5 Retrograde neurotrophin signalling
Authors
Editors
Jassal, B, 2006-10-10.
Reviewers
Greene, LA, 2007-11-08.

Description
Neurotrophin-TRK complexes can be internalized and enter signalling vesicles, which travel retrogradely over long distances from distal nerve
terminals to neuronal cell bodies. Such retrograde signalling by neurotrophin-TRK complexes regulates survival, synaptogenesis and
maintenance of proper neural connectivity. The neurotrophin-TRK complex may use three distinct internalization pathways. Although
Clathrin-mediated endocytosys appears to be the major internalization route, it is controversial whether it also represents the dominant pathway
for retrograde transport and signalling. Pyncher-mediated endocytosis might be more relevant in this regard. Moreover, also caveolin-mediated
endocytosis may play a role in NGF-TrkA internalization.
Retrograde transport of TRKs is microtubule-dependent: TRKs remain activated and bound to neurotrophins during retrograde transport. The
current view is reflected in the signalling endosome model. It is a specialized vesicle containing ligand (NGF, BDNF) bound to its activated TRK
receptor, together with activated downstream signalling proteins, transported by motor proteins (dyneins) from nerve terminals to remote cell
bodies, where the receptors trigger signalling cascades.
33.5.1 Formation of clathrin-coated vesicle
Authors
Editors
Jassal, B, 2006-10-10.
Description
Both BDNF and NGF treatment recruits clathrin and AP2 (adaptor protein 2) proteins to the plasma membrane. Clathrin is the major protein of
the polyhedral coat of vesicles. The AP2 complex mediates both the recruitment of clathrin to membranes and the recognition of sorting signals
within the cytosolic tails of transmembrane cargo molecules.
Source reaction
This reaction was inferred from the corresponding reaction "Formation of clathrin-coated vesicle" in species Rattus norvegicus.
EC Beattie, CL Howe, A Wilde, FM Brodsky, WC Mobley, "NGF signals through TrkA to increase clathrin at the plasma membrane and enhance
clathrin-mediated membrane trafficking", J Neurosci, 20, 2000, 7325-33.
Reaction
33.5.2 Endocytosis (internalization) of clathrin-coated vesicle
Authors
Editors
Jassal, B, 2006-10-10.
Description
Dynamin is a microtubule-associated force-producing protein involved in producing microtubule bundles and able to bind and hydrolyze GTP. It
is involved in vesicle trafficking processes and is necessary for endocytosis.
Source reaction
This reaction was inferred from the corresponding reaction "Endocytosis of clathrin-coated vesicle" in species Rattus norvegicus.
Y Zhang, DB Moheban, BR Conway, A Bhattacharyya, RA Segal, "Cell surface Trk receptors mediate NGF-induced survival while internalized
receptors regulate NGF-induced differentiation", J Neurosci, 20, 2000, 5671-8.
Reaction
33.5.3 Axonal transport of NGF:Trk complexes
Authors

Editors
Jassal, B, 2006-10-10.
Description
Of the internalized NGF:TRK complexes, many undergo recycling and/or proteolysis. Only a small fraction is retrogradely transported. Vesicles
containing neurotrophin, activated receptors and downstream kinases are transported through axons by the action of dynein, which produces a
force towards the end of microtubules.
References
H Yano, FS Lee, H Kong, J Chuang, J Arevalo, P Perez, C Sung, MV Chao, "Association of Trk neurotrophin receptors with components of the
cytoplasmic dynein motor", J Neurosci, 21, 2001, RC125.
Reaction
33.6 Nuclear Events (kinase and transcription factor activation)
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
An important function of the kinase cascade triggered by neurotrophins is to induce the phosphorylation and activation of transcription factors in
the nucleus to initiate new programs of gene expression. Transcription factors directly activated by neurotrophin signalling are responsible for
induction of immediate-early genes, many of which are transcription factors. These in turn are involved in the induction of delayed-early genes.
References
RA Segal, ME Greenberg, "Intracellular signaling pathways activated by neurotrophic factors", Annu Rev Neurosci, 19, 1996, 463-89.
33.6.1 ERK/MAPK targets
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
ERK/MAPK kinases have a number of targets within the nucleus, usually transcription factors or other kinases. The best known targets, ELK1,
ETS1, ATF2, MITF, MAPKAPK2, MSK1, RSK1/2/3 and MEF2 are annotated here.
33.6.1.1 ERK1/2 activates ELK1
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
Following translocation to the nucleus, ERK1/2 directly phosphorylates key effectors, including the ubiquitous transcription factors ELK1 (Ets like
protein 1). At least five residues in the C terminal domain of ELK1 are phosphorylated upon growth factor stimulation. ELK1 can form a ternary
complex with the serum response factor (SRF) and consensus sequences, such as serum response elements (SRE), on DNA, thus stimulating
transcription of a set of immediate early genes like c fos.
Source reaction
This reaction was inferred from the corresponding reaction "ERK1/2 activates ELK1" in species Mus musculus.
H Gille, M Kortenjann, O Thomae, C Moomaw, C Slaughter, MH Cobb, PE Shaw, "ERK phosphorylation potentiates Elk-1-mediated ternary
complex formation and transactivation", EMBO J, 14, 1995, 951-62.
Reaction
33.6.1.2 ERK1/2 phosphorylates MSK1
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
MSK1 (Ribosomal protein S6 kinase alpha-5) is a serine/threonine kinase that is localised in the nucleus. It contains two protein kinase domains
in a single polypeptide. It can be activated 5-fold by ERK1/2 through phosphorylation at four key residues.
References
M Deak, AD Clifton, LM Lucocq, DR Alessi, "Mitogen- and stress-activated protein kinase-1 (MSK1) is directly activated by MAPK and
SAPK2/p38, and may mediate activation of CREB", EMBO J, 17, 1998, 4426-41.
Reaction
33.6.1.3 p38MAPK phosphorylates MSK1
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
MSK1 (Ribosomal protein S6 kinase alpha-5) is a serine/threonine kinase that is localised in the nucleus. It contains two protein kinase domains
in a single polypeptide. It can be activated 5-fold by p38MAPK through phosphorylation at four key residues.
References
Reaction
33.6.1.4 ERK1/2/5 activate RSK1/2/3
Authors

Reviewers
Greene, LA, 2007-11-08.
Description
The p90 ribosomal S6 kinases (RSK1-4) comprise a family of serine/threonine kinases that lie at the terminus of the ERK pathway. RSK family
members are unusual among serine/threonine kinases in that they contain two distinct kinase domains, both of which are catalytically functional .
The C-terminal kinase domain is believed to be involved in autophosphorylation, a critical step in RSK activation, whereas the N-terminal kinase
domain, which is homologous to members of the AGC superfamily of kinases, is responsible for the phosphorylation of all known exogenous
substrates of RSK.
RSKs can be activated by the ERKs (ERK1, 2, 5) in the cytoplasm as well as in the nucleus, they both have cytoplasmic and nuclear substrates,
and they are able to move from nucleus to cytoplasm. Efficient RSK activation by ERKs requires its interaction through a docking site located
near the RSK C terminus. The mechanism of RSK activation has been studied mainly with regard to ERK1 and ERK2. RSK activation leads to
the phosphorylation of four essential residues Ser239, Ser381, Ser398, and Thr590, and two additional sites, Thr377 and Ser749 (the amino
acid numbering refers to RSK1). ERK is thought to play at least two roles in RSK1 activation. First, activated ERK phosphorylates RSK1 on
Thr590, and possibly on Thr377 and Ser381, and second, ERK brings RSK1 into close proximity to membrane-associated kinases that may
phosphorylate RSK1 on Ser381 and Ser398.
Moreover, RSKs and ERK1/2 form a complex that transiently dissociates upon growth factor signalling. Complex dissociation requires
phosphorylation of RSK1 serine 749, a growth factor regulated phosphorylation site located near the ERK docking site. Serine 749 is
phosphorylated by the N-terminal kinase domain of RSK1 itself. ERK1/2 docking to RSK2 and RSK3 is also regulated in a similar way. The
length of RSK activation following growth factor stimulation depends on the duration of the RSK/ERK complex, which, in turn, differs among the
different RSK isoforms. RSK1 and RSK2 readily dissociate from ERK1/2 following growth factor stimulation stimulation, but RSK3 remains
associated with active ERK1/2 longer, and also remains active longer than RSK1 and RSK2.
References
A Ranganathan, GW Pearson, CA Chrestensen, TW Sturgill, MH Cobb, "The MAP kinase ERK5 binds to and phosphorylates p90 RSK", Arch
Reaction
33.6.1.5 ERK5 activates the transcription factor MEF2

Authors
Reviewers
Greene, LA, 2007-11-08.
Description
The MEF2 (Myocyte-specific enhancer factor 2) proteins constitute a family of transcription factors: MEF2A, MEF2B, MEF2C, and MEF2D.
MEF2A and MEF2C are known substrates of ERK5, and their transactivating activity can be stimulated by ERK5 via direct phosphorylation.
MEF2A and MEF2C are expressed in developing and adult brain including cortex and cerebellum.
Source reaction
This reaction was inferred from the corresponding reaction "ERK5 activates the transcription factor MEF2" in species Rattus norvegicus.
L Liu, JE Cavanaugh, Y Wang, H Sakagami, Z Mao, Z Xia, "ERK5 activation of MEF2-mediated gene expression plays a critical role in
BDNF-promoted survival of developing but not mature cortical neurons", Proc Natl Acad Sci U S A, 100, 2003, 8532-7.
Reaction
33.6.1.6 ERKs are inactivated by protein phosphatase 2A
Authors

Reviewers
Greene, LA, 2007-11-08.
Description
ERKs are inactivated by the protein phosphatase 2A (PP2A). The PP2A holoenzyme is a heterotrimer that consists of a core dimer, composed
of a scaffold (A) and a catalytic (C) subunit that associates with a variety of regulatory (B) subunits. The B subunits have been divided into gene
families named B (or PR55), B0 (or B56 or PR61) and B00 (or PR72). Each family comprises several members. B56 family members of PP2A in
particular, increase ERK dephosphorylation, without affecting its activation by MEK.
Induction of PP2A is involved in the extracellular signal-regulated kinase (ERK) signalling pathway, in which it provides a feedback control, as
well as in a broad range of other cellular processes, including transcriptional regulation and control of the cell cycle.This diversity of functions is
conferred by a diversity of regulatory subunits, the combination of which can give rise to over 50 different forms of PP2A. For example, five
distinct mammalian genes encode members of the B56 family, called B56a, b, g, d and e, generating at least eight isoforms. Whether a specific
holoenzyme dephosphorylates ERK and whether this activity is controlled during mitogenic stimulation is unknown.
References
C Letourneux, G Rocher, F Porteu, "B56-containing PP2A dephosphorylate ERK and their activity is controlled by the early gene IEX-1 and
ERK", EMBO J, 25, 2006, 727-38.
Reaction
33.6.1.7 ERKs are inactivated
Reviewers
Greene, LA, 2007-11-08.

Description
MAP Kinases are inactivated by a family of protein named MAP Kinase Phosphatases (MKPs). They act through dephosphorylation of threonine
and/or tyrosine residues within the signature sequence -pTXpY- located in the activation loop of MAP kinases (pT=phosphothreonine and
pY=phosphotyrosine). MKPs are divided into three major categories depending on their preference for dephosphorylating; tyrosine,
serine/threonine and both the tyrosine and threonine (dual specificity phoshatases or DUSPs). The tyrosine-specific MKPs include PTP-SL,
STEP and HePTP, serine/threonine-specific MKPs are PP2A and PP2C, and many DUSPs acting on MAPKs are known. Activated MAP kinases
trigger activation of transcription of MKP genes. Therefore, MKPs provide a negative feedback regulatory mechanism on MAPK signaling, by
inactivating MAPKs via dephosphorylation, in the cytoplasm and the nucleus. Some MKPs are more specific for ERKs, others for JNK or
p38MAPK.
References
I Amit, A Citri, T Shay, Y Lu, M Katz, F Zhang, G Tarcic, D Siwak, J Lahad, J Jacob-Hirsch, N Amariglio, N Vaisman, E Segal, G Rechavi, U
Alon, GB Mills, E Domany, Y Yarden, "A module of negative feedback regulators defines growth factor signaling", Nat Genet, 39, 2007, 503-12.
A Farooq, MM Zhou, "Structure and regulation of MAPK phosphatases", Cell Signal, 16, 2004, 769-79.
33.6.1.7.1 ERKs are inactivated by protein phosphatase 2A
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
ERKs are inactivated by the protein phosphatase 2A (PP2A). The PP2A holoenzyme is a heterotrimer that consists of a core dimer, composed
of a scaffold (A) and a catalytic (C) subunit that associates with a variety of regulatory (B) subunits. The B subunits have been divided into gene
families named B (or PR55), B0 (or B56 or PR61) and B00 (or PR72). Each family comprises several members. B56 family members of PP2A in
particular, increase ERK dephosphorylation, without affecting its activation by MEK.
Induction of PP2A is involved in the extracellular signal-regulated kinase (ERK) signalling pathway, in which it provides a feedback control, as
well as in a broad range of other cellular processes, including transcriptional regulation and control of the cell cycle.This diversity of functions is
conferred by a diversity of regulatory subunits, the combination of which can give rise to over 50 different forms of PP2A. For example, five
distinct mammalian genes encode members of the B56 family, called B56a, b, g, d and e, generating at least eight isoforms. Whether a specific
holoenzyme dephosphorylates ERK and whether this activity is controlled during mitogenic stimulation is unknown.
References
C Letourneux, G Rocher, F Porteu, "B56-containing PP2A dephosphorylate ERK and their activity is controlled by the early gene IEX-1 and
ERK", EMBO J, 25, 2006, 727-38.
Reaction
33.6.1.7.2 ERKs are inactivated by dual-specific phosphatases (DUSPs)
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
Over 10 dual specificity phosphatases (DUSPs) active an MAP kinases are known. Among them, some possess good ERK docking sites and so
are more specific for the ERKS (DUSP 3, 4, 6, 7), others are more specific for p38MAPK (DUSP1 and 10), while others do not seem to
discriminate. It is noteworthy that transcription of DUSP genes is induced by growth factor signaling itself, so that these phosphatases provide
feedback attenuation of signaling. Moreover, differential activation of DUSPs by different stimuli is thought to contribute to pathway specificity.
References
I Amit, A Citri, T Shay, Y Lu, M Katz, F Zhang, G Tarcic, D Siwak, J Lahad, J Jacob-Hirsch, N Amariglio, N Vaisman, E Segal, G Rechavi, U
Alon, GB Mills, E Domany, Y Yarden, "A module of negative feedback regulators defines growth factor signaling", Nat Genet, 39, 2007, 503-12.
Reaction
33.6.2 CREB phosphorylation
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
Nerve growth factor (NGF) activates multiple signalling pathways that mediate the phosphorylation of CREB at the critical regulatory site, serine
133. CREB phosphorylation at serine 133 is a crucial event in neurotrophin signalling, being mediated by ERK/RSK, ERK/MSK1 and
p38/MAPKAPK2 pathways. Several kinases, such as MSK1, RSK1/2/3 (MAPKAPK1A/B/C), and MAPKAPK2, are able to directly phosphorylate
CREB at S133. MSK1 is also able to activate ATF (Cyclic-AMP-dependent transcription factor). However, the NGF-induced CREB
phosphorylation appears to correlate better with activation of MSK1 rather than RSK1/2/3, or MAPKAPK2. In retrograde signalling, activation of
CREB occurs within 20 minutes after neurotrophin stimulation of distal axons.
33.6.2.1 MSK1 activates CREB
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
MSK1 is required for the mitogen-induced phosphorylation of the transcription factor, cAMP response element-binding protein (CREB).
References
Reaction
33.6.2.2 MSK1 activates ATF1
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
Cyclic-AMP-dependent transcription factor 1 (ATF1) can be phosphorylated at Serine 63 by MSK1, thus activating it.
Reaction
33.6.2.3 RSK1/2/3 phosphorylates CREB at Serine 133
Authors
Reviewers
Greene, LA, 2007-11-08.
Description
CREB is phosphorylated at Serine 133 by RSK1/2/3.
References
D De Cesare, S Jacquot, A Hanauer, P Sassone-Corsi, "Rsk-2 activity is necessary for epidermal growth factor-induced phosphorylation of
CREB protein and transcription of c-fos gene", Proc Natl Acad Sci U S A, 95, 1998, 12202-7.
Reaction
33.6.2.4 MAPKAPK2 phosphorylates CREB at Serine 133
Authors

Reviewers
Greene, LA, 2007-11-08.
Description
p38 MAPK activation leads to CREB Serine 133 phosphorylation through the activation of MAPKAP kinase 2 or the closely related MAPKAP
kinase 3.
Reaction
34 Signaling by Notch
Authors
Jassal, B, 2004-12-15.
Reviewers
Joutel, A, 0000-00-00.
Description
The Notch Signaling Pathway (NSP) is a highly conserved pathway for cell-cell communication. NSP is involved in the regulation of cellular
differentiation, proliferation, and specification. For example, it is utilised by continually renewing adult tissues such as blood, skin, and gut
epithelium not only to maintain stem cells in a proliferative, pluripotent, and undifferentiated state but also to direct the cellular progeny to adopt
different developmental cell fates. Analogously, it is used during embryonic development to create fine-grained patterns of differentiated cells,
notably during neurogenesis where the NSP controls patches such as that of the vertebrate inner ear where individual hair cells are surrounded
by supporting cells.
This process is known as lateral inhibition: a molecular mechanism whereby individual cells within a field are stochastically selected to adopt
particular cell fates and the NSP inhibits their direct neighbours from doing the same. The NSP has been adopted by several other biological
systems for binary cell fate choice. In addition, the NSP is also used during vertebrate segmentation to divide the growing embryo into regular
blocks called somites which eventually form the vertebrae. The core of this process relies on regular pulses of Notch signaling generated from a
molecular oscillator in the presomatic mesoderm.
The Notch receptor is synthesized in the rough endoplasmic reticulum as a single polypeptide precursor. Newly synthesized Notch receptor is
proteolytically cleaved in the trans-golgi network, creating a heterodimeric mature receptor comprising of non-covalently associated extracellular
and transmembrane subunits. This assembly travels to the cell surface ready to interact with specific ligands. Following ligand activation and
further proteolytic cleavage, an intracellular domain is released and translocates to the nucleus where it regulates gene expression.
References
G Weinmaster, "Notch signal transduction: a real rip and more", Curr Opin Genet Dev, 10, 2000, 363-9.
JS Mumm, R Kopan, "Notch signaling: from the outside in", Dev Biol, 228, 2000, 151-65.
S Artavanis-Tsakonas, MD Rand, RJ Lake, "Notch signaling: cell fate control and signal integration in development", Science, 284, 1999, 770-6.
F Schweisguth, "Notch signaling activity", Curr Biol, 14, 2004, R129-38.
34.1 Transport of Notch receptor precursor to golgi
Authors
Jassal, B, 2004-12-15.
Description
Higher vertebrates possess multiple Notch receptors whereas there is only one Notch receptor homologue in Drosophila melanogaster. In
humans, 4 receptors have been identified (human Notch 1-4; hN1-4). They are closely related to each other, and are synthesized as single
polypeptide precursors in the endoplasmic reticulum. From here, they travel through the trans-golgi network (TGN) where they are cleaved to the
heterodimeric mature form.
References
CM Blaumueller, H Qi, P Zagouras, S Artavanis-Tsakonas, "Intracellular cleavage of Notch leads to a heterodimeric receptor on the plasma
membrane", Cell, 90, 1997, 281-91.
34.1.1 Notch 1 precursor transport to golgi
Description
In this reaction, 1 molecule of 'Notch 1 receptor precursor' is translocated from endoplasmic reticulum lumen to Golgi lumen.
This reaction takes place in the 'membrane'.
References
G Weinmaster, VJ Roberts, G Lemke, "A homolog of Drosophila Notch expressed during mammalian development", Development, 113, 1991,
199-205.
membrane", Cell, 90, 1997, 281-91.
Reaction

Description
References
G Weinmaster, VJ Roberts, G Lemke, "Notch2: a second mammalian Notch gene", Development, 116, 1992, 931-41.
membrane", Cell, 90, 1997, 281-91.
Reaction
Description
References
membrane", Cell, 90, 1997, 281-91.
M Lardelli, J Dahlstrand, U Lendahl, "The novel Notch homologue mouse Notch 3 lacks specific epidermal growth factor-repeats and is
expressed in proliferating neuroepithelium", Mech Dev, 46, 1994, 123-36.
Reaction
Description
References
H Uyttendaele, G Marazzi, G Wu, Q Yan, D Sassoon, J Kitajewski, "Notch4/int-3, a mammary proto-oncogene, is an endothelial cell-specific
mammalian Notch gene", Development, 122, 1996, 2251-9.
membrane", Cell, 90, 1997, 281-91.
Reaction
34.2 Maturation of Notch precursor via proteolytic cleavage
Authors
Jassal, B, 2004-12-15.
Description
The Notch receptor is synthesized as a precursor polypeptide (approx. 300 kDa) in the endoplasmic reticulum. To produce a mature Notch
receptor, it must be cleaved to result in a heterodimer. The enzyme responsible is a furin-like convertase which cleaves the full-length precursor
into a transmembrane fragment (NTM) of approximate size 110kDa and an extracellular fragment (NEC) of approximate size 180kDa. The
mature Notch receptor is reassembled as a heterodimer, the two parts are thought to be held together by either disulfide bonds or calcium ions.
Evidence for either method is apparent, but there is still much debate over this issue. This process takes place in the trans-golgi network.
References
F Logeat, O LeBail, S Jarriault, NG Seidah, "The Notch1 receptor is cleaved constitutively by a furin-like convertase", Proc Natl Acad Sci U S A,
95, 1998, 8108-12.
MD Rand, LM Grimm, S Artavanis-Tsakonas, V Patriub, SC Blacklow, J Sklar, "Calcium depletion dissociates and activates heterodimeric notch
receptors", Mol Cell Biol, 20, 2000, 1825-35.
YM Chan, YN Jan, "Roles for proteolysis and trafficking in notch maturation and signal transduction", Cell, 94, 1998, 423-6.
membrane", Cell, 90, 1997, 281-91.
34.2.1 Notch 1 precursor cleaved to form a heterodimer
Description
At the beginning of this reaction, 1 molecule of 'Notch 1 receptor precursor' is present. At the end of this reaction, 1 molecule of 'NTM-NEC 1
heterodimer' is present.
References
95, 1998, 8108-12.
membrane", Cell, 90, 1997, 281-91.
Reaction
Description
References
95, 1998, 8108-12.
membrane", Cell, 90, 1997, 281-91.
Reaction
Description
References
95, 1998, 8108-12.
membrane", Cell, 90, 1997, 281-91.
Reaction
Description
References
95, 1998, 8108-12.
membrane", Cell, 90, 1997, 281-91.
Reaction
34.3 Mature Notch receptor trafficks to plasma membrane
Authors
Jassal, B, 2004-12-17.
Description
The mature Notch receptor translocates to the plasma membrane.
References
J Frisen, U Lendahl, "Oh no, Notch again!", Bioessays, 23, 2001, 3-7.
membrane", Cell, 90, 1997, 281-91.
34.3.1 Notch 1 heterodimer trafficks to the plasma membrane
Description
In this reaction, 1 molecule of 'NTM-NEC 1 heterodimer' is translocated from Golgi lumen to plasma membrane.
References
membrane", Cell, 90, 1997, 281-91.
Reaction
Description
References
membrane", Cell, 90, 1997, 281-91.
Reaction
Description
References
membrane", Cell, 90, 1997, 281-91.
Reaction
Description
References
membrane", Cell, 90, 1997, 281-91.
Reaction
34.4 Notch receptor binds with a ligand
Authors
Jassal, B, 2004-12-15.
Description
Ligands for human Notch receptors are proteins belonging to two subclasses of the DSL family (Delta and Serrate-Lag2). Human versions have
two members from the Serrate family (Jagged 1 and 2) and three members from the Delta family (Dll-1, Dll-3 and Dll-4). These are single pass
transmembrane proteins, analogous to the ligands found in Drosophila. They comprise of three main parts; a large extracellular domain, a small
intracellular domain and an amino-terminal DSL region. The DSL region is thought to interact with the EC region on the Notch receptor, initiating
a subsequent proteolytic cleavage, although this has not been demonstrated experimentally.
References
B Luo, JC Aster, RP Hasserjian, F Kuo, J Sklar, "Isolation and functional analysis of a cDNA for human Jagged2, a gene encoding a ligand for
the Notch1 receptor", Mol Cell Biol, 17, 1997, 6057-67.
GE Gray, RS Mann, E Mitsiadis, D Henrique, ML Carcangiu, A Banks, J Leiman, D Ward, D Ish-Horowitz, S Artavanis-Tsakonas, "Human
ligands of the Notch receptor", Am J Pathol, 154, 1999, 785-94.
CE Lindsell, CJ Shawber, J Boulter, G Weinmaster, "Jagged: a mammalian ligand that activates Notch1", Cell, 80, 1995, 909-17.
34.4.1 Notch 1 heterodimer binds with a Notch ligand in the extracellular space
Description
At the beginning of this reaction, 1 molecule of 'Notch Ligand', and 1 molecule of 'NTM-NEC 1 heterodimer' are present. At the end of this
reaction, 1 molecule of 'NTM-NEC1-Notch ligand complex' is present.
References
K Shimizu, S Chiba, N Hosoya, K Kumano, T Saito, M Kurokawa, Y Kanda, Y Hamada, H Hirai, "Binding of Delta1, Jagged1, and Jagged2 to
Notch2 rapidly induces cleavage, nuclear translocation, and hyperphosphorylation of Notch2", Mol Cell Biol, 20, 2000, 6913-22.
E Six, D Ndiaye, Y Laabi, N Gupta-Rossi, F Logeat, "The Notch ligand Delta1 is sequentially cleaved by an ADAM protease and
gamma-secretase", Proc Natl Acad Sci U S A, 100, 2003, 7638-43.
Reaction
Description
At the beginning of this reaction, 1 molecule of 'NTM-NEC 2 heterodimer', and 1 molecule of 'Notch Ligand' are present. At the end of this
References
Reaction
Description
References
Reaction
Description

References
Reaction
34.5 Receptor-ligand binding initiates the second proteolytic cleavage of

Notch receptor
Authors
Jassal, B, 2004-12-17.
Description
Some transmembrane proteins such as the Notch receptor can be cleaved to release a cytosolic domain that translocates to the nucleus to
control gene transcription. This is an example of a process called regulated intramembrane proteolysis (Rip). Rip is a control mechanism that is
conserved from bacteria to humans and influences process from cellular differentiation to lipid metabolism.
Once the ligand binds with the receptor, a proposed conformational change in the Notch receptor occurs which relieves an inhibition on the S2
protease cleavage site proximal to the plasma membrane. The S2 cleavage is catalyzed by an ADAM metalloprotease called TACE (TNF alpha
converting enzyme). This enzyme is a homolog of the Drosophila Kuzbanian gene product which performs the same task. Cleavage at the S2
site generates a transient intermediate peptide termed NEXT (Notch EXtracellular Truncation). The remainder of the Notch receptor is still bound
with the ligand at this point.
References
JS Mumm, R Kopan, DJ Pan, WJ Ray, "Ligand-induced "ectodomain shedding" regulates gamma-secretase-like proteolytic activation of
Notch1.", 2000.
J Ye, RB Rawson, "Regulated intramembrane proteolysis: a control mechanism conserved from bacteria to humans", Cell, 100, 2000, 391-8.
C Brou, F Logeat, N Gupta, C Bessia, O LeBail, JR Doedens, A Cumano, P Roux, RA Black, A Israel, "A novel proteolytic cleavage involved in
Notch signaling: the role of the disintegrin-metalloprotease TACE", Mol Cell, 5, 2000, 207-16.
34.5.1 Notch 1-ligand complex is cleaved to produce NEXT1
Description
At the beginning of this reaction, 1 molecule of 'NTM-NEC1-Notch ligand complex' is present. At the end of this reaction, 1 molecule of 'Notch 1
NEXT fragment', and 1 molecule of 'Notch 1 ligand-bound fragment-Notch ligand complex' are present.
This reaction takes place in the 'extracellular region' and is mediated by the 'metallopeptidase activity' of 'ADAM 10 metalloprotease (Zn
cofactor)'.
References
Reaction
Description
ligand-bound fragment-Notch ligand complex', and 1 molecule of 'Notch 2 NEXT fragment' are present.
cofactor)'.
References
Reaction
Description
ligand-bound fragment-Notch ligand complex', and 1 molecule of 'Notch 3 NEXT fragment' are present.
cofactor)'.
References
Reaction
Description
NEXT fragment', and 1 molecule of 'Notch 4 ligand-bound fragment-Notch ligand complex' are present.
cofactor)'.
References
Reaction
34.6 A third proteolytic cleavage releases NICD
Authors
Jassal, B, 2005-01-07.
Description
The third proteolytic cleavage releases the Notch IntraCellular Domain (NICD) from the NEXT fragment. The catalyst for this cleavage is a
membrane protease complex called gamma-secretase (GS). GS cleaves type I membrane proteins such as the Notch receptor and amyloid
beta-protein precursor (APP, implicated in Alzheimer's Disease). GS is made up of 4 components; Presenilin, nicastrin, APH-1 and PEN-2.
Presenilins are homodimeric, multipass transmembrane proteins and are believed to be the catalytic core of GS. All aspartyl proteases have two
catalytic aspartates and presenilin contains these residues. The others are essential cofactors for the correct function of GS.
References
EH Schroeter, JA Kisslinger, R Kopan, "Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain", Nature, 393, 1998,
382-6.
PC Fraering, W Ye, JM Strub, G Dolios, MJ LaVoie, BL Ostaszewski, Dorsselaer van, R Wang, DJ Selkoe, MS Wolfe, "Purification and
characterization of the human gamma-secretase complex", Biochemistry, 43, 2004, 9774-89.
T Iwatsubo, "The gamma-secretase complex: machinery for intramembrane proteolysis", Curr Opin Neurobiol, 14, 2004, 379-83.
ME Fortini, "Gamma-secretase-mediated proteolysis in cell-surface-receptor signalling", Nat Rev Mol Cell Biol, 3, 2002, 673-84.
B De Strooper, W Annaert, P Cupers, P Saftig, K Craessaerts, JS Mumm, EH Schroeter, V Schrijvers, MS Wolfe, WJ Ray, A Goate, R Kopan, "A
presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain", Nature, 398, 1999, 518-22.
34.6.1 NEXT1 is cleaved to produce NICD1
Description
At the beginning of this reaction, 1 molecule of 'Notch 1 NEXT fragment' is present. At the end of this reaction, 1 molecule of 'Transmembrane
remnant 1', and 1 molecule of 'NICD 1 fragment' are present.
This reaction takes place on the 'plasma membrane' and is mediated by the 'aspartic-type endopeptidase activity' of 'gamma-secretase
complex'.
References
SS Huppert, A Le, EH Schroeter, JS Mumm, MT Saxena, LA Milner, R Kopan, "Embryonic lethality in mice homozygous for a
processing-deficient allele of Notch1", Nature, 405, 2000, 966-70.
382-6.
Reaction
Description
At the beginning of this reaction, 1 molecule of 'Notch 2 NEXT fragment' is present. At the end of this reaction, 1 molecule of 'NICD 2 fragment',
and 1 molecule of 'Transmembrane remnant 2' are present.
complex'.
References
382-6.
Reaction
Description
complex'.
References
382-6.
Reaction
Description
complex'.
References
382-6.
Reaction
34.7 NICD trafficks to nucleus
Authors
Jassal, B, 2005-01-10.
Description
NICD translocates to the nucleus and assembles into a ternary complex with the CSL (human CBF1/ fly Suppressor of Hairless/ worm Lag1)
DNA-binding protein and the Mastermind co-activator. This complex activates the transcription of downstream gene targets like HES
(Hairy/Enhancer of Split) and HES-related (HRT, also named CHF, HEY, HESR, gridlock) genes.
34.7.1 NICD1 trafficks to the nucleus
Authors
Ferrer, J, 2008-05-23.
Editors
Reviewers
Jensen, J, 2008-05-12.
Description
In this reaction, 1 molecule of 'NICD 1 fragment' is translocated from cytosol to nucleoplasm.
Source reaction
This reaction was inferred from the corresponding reaction "Drosophila NICD trafficks to nucleus" in species Drosophila melanogaster.
G Struhl, A Adachi, "Nuclear access and action of notch in vivo", Cell, 93, 1998, 649-60.
M Lecourtois, F Schweisguth, "Indirect evidence for Delta-dependent intracellular processing of notch in Drosophila embryos", Curr Biol, 8, 1998,
771-4.
Reaction
Description

Source reaction
771-4.
Reaction
Description
Source reaction
771-4.
Reaction
Description
Source reaction
771-4.
Reaction
35 Signaling by Rho GTPases
Authors
Van Aelst, L, 2007-04-28.
Editors
Reviewers
Bernards, A, 2007-04-28.
Description
The Rho family of small guanine nucleotide binding proteins is one of five generally recognized branches of the Ras superfamily. Like most Ras
superfamily members, typical Rho proteins function as binary switches controlling a variety of biological processes. They perform this function by
cycling between active GTP-bound and inactive GDP-bound conformations. Mammalian Rho GTPases include RhoA, RhoB and RhoC (Rho
proteins), Rac1 3 (Rac proteins), Cdc42, TC10, TCL, Wrch1, Chp/Wrch2, RhoD and RhoG, to name some. The family also includes RhoH and
Rnd1-3, which lack GTPase activity and are predicted to exist in a constitutively active state.
Members of the Rho family have been identified in all eukaryotes. Including the atypical RHOBTB1-3 and RHOT1-2 proteins, 24 Rho family
members have been identified in mammals (Jaffe and Hall, 2005; Bernards, 2005; Ridley, 2006). Among Rho GTPases, RhoA, Rac1 and Cdc42
have been most extensively studied. These proteins are best known for their ability to induce dynamic rearrangements of the plasma
membrane-associated actin cytoskeleton (Aspenstrom et al, 2004; Murphy et al, 1999; Govek et al, 2005). Beyond this function, Rho GTPases
also regulate actomyosin contractility and microtubule dynamics. Rho mediated effects on transcription and membrane trafficking are believed to
be secondary to these functions. At the more macroscopic level, Rho GTPases have been implicated in many important cell biological
processes, including cell growth control, cytokinesis, cell motility, cell cell and cell extracellular matrix adhesion, cell transformation and invasion,
and development (Govek et al., 2005). The illustration below lists Rho GTPase effectors implicated in actin and microtubule dynamics (courtesy:
Govek et al., 2005, Genes and Development, CSHL Press). Detailed annotations of various biological processes regulated by Rho GTPases will
be available in future releases.
References
A Bernards, "GAPs galore! A survey of putative Ras superfamily GTPase activating proteins in man and Drosophila", Biochim Biophys Acta,
1603, 2003, 47-82.
A Schmidt, A Hall, "Guanine nucleotide exchange factors for Rho GTPases: turning on the switch", Genes Dev, 16, 2002, 1587-609.
EE Govek, SE Newey, L Van Aelst, "The role of the Rho GTPases in neuronal development", Genes Dev, 19, 2005, 1-49.
AB Jaffe, A Hall, "Rho GTPases: biochemistry and biology", Annu Rev Cell Dev Biol, 21, 2005, 247-69.
P Aspenstrom, A Fransson, J Saras, "Rho GTPases have diverse effects on the organization of the actin filament system", Biochem J, 377,
2004, 327-37.
GA Murphy, PA Solski, SA Jillian, P Perez de la Ossa, P D'Eustachio, CJ Der, MG Rush, "Cellular functions of TC10, a Rho family GTPase:
regulation of morphology, signal transduction and cell growth", Oncogene, 18, 1999, 3831-45.
A Bernards, "Ras superfamily and interacting proteins database", Methods Enzymol, 407, 2005, 1-9.
35.1 Rho GTPase cycle
Authors
Van Aelst, L, 2007-04-28.
Editors
Reviewers
Bernards, A, 2007-04-28.
Description
The cycling of Rho GTPases is tightly controlled by three classes of protein. These are (1) guanine nucleotide dissociation inhibitors or GDIs,
which maintain Rho proteins in an inactive state in the cytoplasm, (2) guanine nucleotide exchange factors or GEFs, which destabilize the
interaction between Rho proteins and their bound nucleotide, the net result of which is the exchange of bound GDP for the more abundant GTP,
and (3) GTPase Activating Proteins or GAPs, which stimulate the low intrinsic GTP hydrolysis activity of Rho family members, thus promoting
their inactivation. GDIs, GEFs, and GAPs are themselves subject to tight regulation, and the overall level of Rho activity reflects the balance of
their activities.
In their active GTP-bound state, Rho family members have the ability to interact with a large variety of so-called effector proteins. By changing
the subcellular localization of effectors, by altering their enzymatic properties, or by directing the formation of specific effector complexes,
members of the Rho family mediate their various effects.
This Rho GTPase cycle is diagrammed in the figure below. External or internal cues promote the release of Rho GTPases from the inhibitory
complex (1) which allows them to associate with the plasma membrane (2) where they are activated by GEFs (3) and can signal to effector
proteins. Then, GAPs inactivate the GTPases by accelerating the intrinsic GTPase activity, leading to the GDP bound form (4). Once again, the
GDI molecules stabilize the inactive GDP bound form in the cytoplasm, waiting for further instructions (5). (Figure and text from Tcherkezian and
Lamarche Vane, 2007).
References
L Van Aelst, C D'Souza-Schorey, "Rho GTPases and signaling networks", Genes Dev, 11, 1997, 2295-322.
J Tcherkezian, N Lamarche-Vane, "Current knowledge of the large RhoGAP family of proteins", Biol Cell, 99, 2007, 67-86.
35.1.1 GEFs activate Rho GTPase:GDP
Authors
Van Aelst, L, 2007-04-28.
Editors
Reviewers
Bernards, A, 2007-04-28.
Description
Guanine nucleotide exchange factors (GEFs) activate GTPases by enhancing the exchange of bound GDP for GTP. Much evidence points to
GEFs being critical mediators of Rho GTPase activation (Schmidt and Hall, 2002). Many GEFs are known to be highly specific to specific
GTPases, like Fgd1/Cdc42, p115RhoGEF/Rho (Hart et al., 1996, Zheng et al., 1996). Others may have a broader spectrum and activate several:
Vav1 for Rac, Rho, and Cdc42 (Hart et al, 1994). Detailed annotations of processes involved in the actions of individual GEFs and their
regulation will be made available in future releases.
References
NG Pasteris, A Cadle, LJ Logie, ME Porteous, CE Schwartz, RE Stevenson, TW Glover, RS Wilroy, JL Gorski, "Isolation and characterization of
the faciogenital dysplasia (Aarskog-Scott syndrome) gene: a putative Rho/Rac guanine nucleotide exchange factor", Cell, 79, 1994, 669-78.
MJ Hart, A Eva, D Zangrilli, SA Aaronson, T Evans, RA Cerione, Y Zheng, "Cellular transformation and guanine nucleotide exchange activity are
catalyzed by a common domain on the dbl oncogene product", J Biol Chem, 269, 1994, 62-5.
A Schmidt, A Hall, "Guanine nucleotide exchange factors for Rho GTPases: turning on the switch", Genes Dev, 16, 2002, 1587-609.
Y Zheng, DJ Fischer, MF Santos, G Tigyi, NG Pasteris, JL Gorski, Y Xu, "The faciogenital dysplasia gene product FGD1 functions as a
Cdc42Hs-specific guanine-nucleotide exchange factor", J Biol Chem, 271, 1996, 33169-72.
F Ramos-Morales, F Romero, F Schweighoffer, G Bismuth, J Camonis, M Tortolero, S Fischer, "The proline-rich region of Vav binds to Grb2 and
Grb3-3", Oncogene, 11, 1995, 1665-9.
MJ Hart, S Sharma, N elMasry, RG Qiu, P McCabe, P Polakis, G Bollag, "Identification of a novel guanine nucleotide exchange factor for the
Rho GTPase", J Biol Chem, 271, 1996, 25452-8.
T Reid, A Bathoorn, MR Ahmadian, JG Collard, "Identification and characterization of hPEM-2, a guanine nucleotide exchange factor specific for
Cdc42", J Biol Chem, 274, 1999, 33587-93.
Y Ren, R Li, Y Zheng, H Busch, "Cloning and characterization of GEF-H1, a microtubule-associated guanine nucleotide exchange factor for Rac
and Rho GTPases", J Biol Chem, 273, 1998, 34954-60.
Reaction
35.1.2 Rho GTPase:GTP activates downstream effectors
Authors
Van Aelst, L, 2007-04-28.
Editors
Reviewers
Bernards, A, 2007-04-28.
Description
To transduce signals, the activated, GTP-bound Rho GTPases interact with specific effector molecules. It has been observed that GEFs
contribute to the signaling specificity of their downstream target GTPase via association with scaffolding molecules that link them and the
GTPase to specific GTPase effectors (Govek et al., 2005). Some of the effector molecules implicated in actin and microtubule dynamics include
diaphanous-related formins, Toca 1, WIP, WASP, Pak, p35/Cdk5, Wave, Nap125, MLCK, MLC, IRSp53. Detailed annotations of the
downstream events stimulated by activated, GTP bound Rho GTPases will be available in future releases.
References
EE Govek, SE Newey, L Van Aelst, "The role of the Rho GTPases in neuronal development", Genes Dev, 19, 2005, 1-49.
DW Leung, MK Rosen, "The nucleotide switch in Cdc42 modulates coupling between the GTPase-binding and allosteric equilibria of
Wiskott-Aldrich syndrome protein", Proc Natl Acad Sci U S A, 102, 2005, 5685-90.
H Miki, H Yamaguchi, S Suetsugu, T Takenawa, "IRSp53 is an essential intermediate between Rac and WAVE in the regulation of membrane
ruffling", Nature, 408, 2000, 732-5.
YC Tan, H Wu, WN Wang, Y Zheng, ZX Wang, "Characterization of the interactions between the small GTPase RhoA and its guanine nucleotide
exchange factors", Anal Biochem, 310, 2002, 156-62.
H Zhou, RH Kramer, "Integrin engagement differentially modulates epithelial cell motility by RhoA/ROCK and PAK1", J Biol Chem, 280, 2005,
10624-35.
N Abdul-Manan, B Aghazadeh, GA Liu, A Majumdar, O Ouerfelli, KA Siminovitch, MK Rosen, "Structure of Cdc42 in complex with the
GTPase-binding domain of the 'Wiskott-Aldrich syndrome' protein", Nature, 399, 1999, 379-83.
D Yarar, JA D'Alessio, RL Jeng, MD Welch, "Motility determinants in WASP family proteins", Mol Biol Cell, 13, 2002, 4045-59.
S Govind, R Kozma, C Monfries, L Lim, S Ahmed, "Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing the 58-kD
insulin receptor substrate to filamentous actin", J Cell Biol, 152, 2001, 579-94.
HY Ho, R Rohatgi, AM Lebensohn, Ma Le, J Li, SP Gygi, MW Kirschner, "Toca-1 mediates Cdc42-dependent actin nucleation by activating the
N-WASP-WIP complex", Cell, 118, 2004, 203-16.
M Soltau, D Richter, HJ Kreienkamp, "The insulin receptor substrate IRSp53 links postsynaptic shank1 to the small G-protein cdc42", Mol Cell
Neurosci, 21, 2002, 575-83.
Y Zheng, "Dbl family guanine nucleotide exchange factors", Trends Biochem Sci, 26, 2001, 724-32.
Reaction
35.1.3 GAPs inactivate Rho GTPase:GTP by hydrolysis
Authors
Van Aelst, L, 2007-04-28.
Editors
Reviewers
Bernards, A, 2007-04-28.
Description
The human genome includes approximately 70 genes that are predicted to encode Rho-specific GTPase Activating Proteins (RhoGAPs). As in
the case of GEFs, some RhoGAPs are believed to be highly specific, whereas others are more promiscuous with respect to their target
GTPases. Increasing evidence suggests that GAPs are also regulated by external cues in addition to being signal terminators leading to Rho
GTPase inactivation. These proteins play important role in many Rho mediated signaling pathways.
Some known GAPs include p190 A, cdGAP, ARAP3, MgcRacGAP, Chimaerin, Nadrin, TCGAP, DLC 1, 2, ArhGAP6, Myosin IXA. These and
other GAPs have been implicated in many processes, such as exocytosis, endocytosis, cytokinesis, cell differentiation, migration, neuronal
morphogenesis, angiogenesis and tumor suppression. Detailed annotations of the biological role of GAPs in Rho mediated signaling will be
available in future releases.
References
LC Santy, JE Casanova, "GTPase signaling: bridging the GAP between ARF and Rho", Curr Biol, 12, 2002, R360-2.
BZ Yuan, MJ Miller, CL Keck, DB Zimonjic, SS Thorgeirsson, NC Popescu, "Cloning, characterization, and chromosomal localization of a gene
frequently deleted in human liver cancer (DLC-1) homologous to rat RhoGAP", Cancer Res, 58, 1998, 2196-9.
S Krugmann, KE Anderson, SH Ridley, N Risso, A McGregor, J Coadwell, K Davidson, A Eguinoa, CD Ellson, P Lipp, M Manifava, N Ktistakis,
G Painter, JW Thuring, MA Cooper, ZY Lim, AB Holmes, SK Dove, RH Michell, A Grewal, A Nazarian, H Erdjument-Bromage, P Tempst, LR
Stephens, PT Hawkins, "Identification of ARAP3, a novel PI3K effector regulating both Arf and Rho GTPases, by selective capture on
phosphoinositide affinity matrices", Mol Cell, 9, 2002, 95-108.
CA Lancaster, PM Taylor-Harris, AJ Self, S Brill, HE van Erp, A Hall, "Characterization of rhoGAP. A GTPase-activating protein for rho-related
small GTPases.", J Biol Chem, 269, 1994, 1137-42.
J Tcherkezian, N Lamarche-Vane, "Current knowledge of the large RhoGAP family of proteins", Biol Cell, 99, 2007, 67-86.
N Richnau, P Aspenstrom, "Rich, a rho GTPase-activating protein domain-containing protein involved in signaling by Cdc42 and Rac1", J Biol
Chem, 276, 2001, 35060-70.
E Fainstein, C Marcelle, A Rosner, E Canaani, RP Gale, O Dreazen, SD Smith, CM Croce, "A new fused transcript in Philadelphia chromosome
positive acute lymphocytic leukaemia", Nature, 330, 1987, 386-8.
Reaction
35.1.4 GDIs block activation of Rho GTPase:GDP
Authors
Van Aelst, L, 2007-04-28.
Editors
Reviewers
Bernards, A, 2007-04-28.
Description
GDP dissociation inhibitors or GDIs confer an additional but important layer of Rho GTPase regulation along with GEFs and GAPs. GDIs mainly
inhibit the dissociation of bound guanine nucleotide (usually GDP) from their partner GTPases. So far, three human GDIs with proven biological
functions have been found: RhoGDI/GDIalpha/GDI1, hematopoietic cell selective Ly/D4GDI/GDIbeta/GDI2, and Rho GDIgamma/GDI3
(DerMardirossian and Bokoch, 2005). Three specific biochemical functions of GDIs have been established: inhibiting the dissociation of GDP
from Rho proteins, maintaining the GTPases in an inactive form, and preventing GTPase activation by GEFs (Olofsson, 1999). Detailed
annotations of GDIs will be available in future releases.
References
YQ Gosser, TK Nomanbhoy, B Aghazadeh, D Manor, C Combs, RA Cerione, MK Rosen, "C-terminal binding domain of Rho GDP-dissociation
inhibitor directs N-terminal inhibitory peptide to GTPases", Nature, 387, 1997, 814-9.
CN Adra, D Manor, JL Ko, S Zhu, T Horiuchi, L Van Aelst, RA Cerione, B Lim, "RhoGDIgamma: a GDP-dissociation inhibitor for Rho proteins
with preferential expression in brain and pancreas", Proc Natl Acad Sci U S A, 94, 1997, 4279-84.
B Olofsson, "Rho guanine dissociation inhibitors: pivotal molecules in cellular signalling", Cell Signal, 11, 1999, 545-54.
L Seetharam, N Gotoh, Y Maru, G Neufeld, S Yamaguchi, M Shibuya, "A unique signal transduction from FLT tyrosine kinase, a receptor for
vascular endothelial growth factor VEGF", Oncogene, 10, 1995, 135-47.
GR Hoffman, N Nassar, RA Cerione, "Structure of the Rho family GTP-binding protein Cdc42 in complex with the multifunctional regulator
RhoGDI", Cell, 100, 2000, 345-56.
MJ Hart, Y Maru, D Leonard, ON Witte, T Evans, RA Cerione, "A GDP dissociation inhibitor that serves as a GTPase inhibitor for the Ras-like
protein CDC42Hs", Science, 258, 1992, 812-5.
K Scheffzek, I Stephan, ON Jensen, D Illenberger, P Gierschik, "The Rac-RhoGDI complex and the structural basis for the regulation of Rho
proteins by RhoGDI", Nat Struct Biol, 7, 2000, 122-6.
C DerMardirossian, GM Bokoch, "GDIs: central regulatory molecules in Rho GTPase activation", Trends Cell Biol, 15, 2005, 356-63.
J Sun, JT Barbieri, "ExoS Rho GTPase-activating protein activity stimulates reorganization of the actin cytoskeleton through Rho GTPase
guanine nucleotide disassociation inhibitor", J Biol Chem, 279, 2004, 42936-44.
E Dransart, B Olofsson, J Cherfils, "RhoGDIs revisited: novel roles in Rho regulation", Traffic, 6, 2005, 957-66.
D Bachner, Z Sedlacek, B Korn, H Hameister, A Poustka, "Expression patterns of two human genes coding for different rab GDP-dissociation
inhibitors (GDIs), extremely conserved proteins involved in cellular transport", Hum Mol Genet, 4, 1995, 701-8.
Reaction
35.1.5 Dissociation of Rho GTP:GDP from GDI complex
Authors
Van Aelst, L, 2007-04-28.
Editors
Reviewers
Bernards, A, 2007-04-28.
Description
GDIs sequester the inactive GTPases, preventing the dissociation of GDP and interactions with regulatory and effector molecules. They maintain
Rho GTPases as soluble cytosolic proteins by forming high affinity complexes. In these complexes, the geranylgeranyl membrane targeting
moiety present at the C terminus of the Rho GTPases is shielded from the solvent by its insertion into the hydrophobic pocket formed by the
immunoglobulin like beta sandwich of the GDI (DerMardirossian and Bokoch, 2005).
Rho proteins, when released from the sequestering cytosolic GDIs, insert into the lipid bilayer of the plasma membrane with their isoprenylated
C termini. The membrane bound GEFs activate these free RhoGTPases and thereby trigger the downstream signaling events via respective
effector proteins on the membrane (Robbe et al., 2003). Detailed annotation of the activities of farnesyltransferase / geranylgeranyltransferases
on prenylation of Rho GTPases thereby enabling their subsequent localization to plasma membrane will be available in future releases.
References
YQ Gosser, TK Nomanbhoy, B Aghazadeh, D Manor, C Combs, RA Cerione, MK Rosen, "C-terminal binding domain of Rho GDP-dissociation
inhibitor directs N-terminal inhibitory peptide to GTPases", Nature, 387, 1997, 814-9.
GR Hoffman, N Nassar, RA Cerione, "Structure of the Rho family GTP-binding protein Cdc42 in complex with the multifunctional regulator
RhoGDI", Cell, 100, 2000, 345-56.
K Robbe, A Otto-Bruc, P Chardin, B Antonny, "Dissociation of GDP dissociation inhibitor and membrane translocation are required for efficient
activation of Rac by the Dbl homology-pleckstrin homology region of Tiam", J Biol Chem, 278, 2003, 4756-62.
C DerMardirossian, GM Bokoch, "GDIs: central regulatory molecules in Rho GTPase activation", Trends Cell Biol, 15, 2005, 356-63.
K Scheffzek, I Stephan, ON Jensen, D Illenberger, P Gierschik, "The Rac-RhoGDI complex and the structural basis for the regulation of Rho
proteins by RhoGDI", Nat Struct Biol, 7, 2000, 122-6.
Reaction
36 Signaling by TGF beta
Authors
Heldin, CH, Moustakas, A, Huminiecki, L, Jassal, B, 2006-02-02.
Editors
Jassal, B, 2006-04-18.
Reviewers
Heldin, CH, 2006-04-18.
Description
The TGFÃƒÅ¸/BMP pathway incorporates several signalling pathways that share most, but not all, components of a central signal transduction
engine. The general signalling scheme is rather simple: upon binding of a ligand, an activated plasma membrane receptor complex is formed,
which passes on the signal towards the nucleus through a phosphorylated receptor SMAD (R-SMAD). In the nucleus, the activated R-SMAD
promotes transcription in a complex with a closely-related helper molecule termed the CO-SMAD. However, this simple linear pathway expands
into a network when various regulatory components and mechanisms are taken into account. The signalling pathway includes a great variety of
different TGFÃƒÅ¸/BMP superfamily ligands and receptors, several types of the R-SMAD, and functionally critical negative feedback loops. The
R-SMAD/CO-SMAD can interact with a great number of transcriptional co-activators/co-repressors to regulate positively or negatively effector
genes, so that the interpretation of a signal depends on the cell-type and cross talk with other signalling pathways such as Notch, MAPK and
Wnt. The pathway plays a number of different biological roles in the control of embryonic and adult cell proliferation and differentiation, and it is
implicated in a great number of human diseases.
36.1 Latent TGF-beta1 is cleaved by furin
Authors
Reviewers
Heldin, CH, 2006-04-18.
Description
In the Golgi apparatus, TGF-beta 1 is activated by furin protease cleavage of the N-terminal pro-peptide portion. This leads to the formation of
the N-terminal disulphide-linked dimeric pro-peptides, also known as latency-associated proteins (LAPs) and the C-terminal mature
disulphide-linked dimeric TGF-beta 1. However, the N- and C-terminal polypeptides do not physically separate. Rather they stay in one complex.
In addition, the LAP forms disulphide links with separate secreted proteins, the Latent TGF-beta binding proteins (LTBPs). Together, the
LTBPs-linked to the LAP and the non-covalently linked mature TGF-beta1 remain together and form the large latent complex (LLC)
References
CM Dubois, MH Laprise, F Blanchette, LE Gentry, R Leduc, "Processing of transforming growth factor beta 1 precursor by human furin
convertase", J Biol Chem, 270, 1995, 10618-24.
JP Annes, JS Munger, DB Rifkin, "Making sense of latent TGFbeta activation", J Cell Sci, 116, 2003, 217-24.
Reaction
36.2 Secretion and activation of the latent large complex of TGF-beta1
Authors
Reviewers
Heldin, CH, 2006-04-18.
Description
The large latent complex of TGF-beta1 is secreted by exocytosis to the extracellular region. TGF-beta 1 in the LLC cannot interact with the
receptors and for this reason we say that it requires "activation". This means release from the LLC. This release is achieved by many
mechanisms: proteolytic cleavage of the LTBPs, thrombospondin-1 binding to the LLC, integrin alphaV-beta6 binding to the LLC, reactive
oxygen species and low pH. The release of mature dimeric TGF-beta1 is essentially a mechanical process that demands cleavage and opening
of the LLC structure so that the caged mature C-terminal TGF-beta1 polypeptide is released to reach the receptor.
References
J Keski-Oja, K Koli, H von Melchner, "TGF-beta activation by traction?", Trends Cell Biol, 14, 2004, 657-9.
JP Annes, JS Munger, DB Rifkin, "Making sense of latent TGFbeta activation", J Cell Sci, 116, 2003, 217-24.
Reaction
36.3 Dimeric TGF-beta1 binds to the receptor
Authors

Reviewers
Heldin, CH, 2006-04-18.
Description
The mature dimeric TGF-beta 1 binds with high affinity to its signaling receptor, the type II receptor serine/threonine kinase. The type II receptor
is known to form dimeric complexes even in the absence of TGF-beta 1.
Reaction
36.4 Type II receptor recruits type I receptor
Authors
Reviewers
Heldin, CH, 2006-04-18.
Description
The protein complex of dimeric TGF-beta 1 with the type II receptor dimer recruits the low affinity receptor, type I receptor, which is also known
to pre-exist in a dimeric form; thus forming a hetero-tetrameric receptor bound to the dimeric ligand on the extracellular face of the plasma
membrane.
References
P Franzen, P ten Dijke, H Ichijo, H Yamashita, P Schulz, CH Heldin, K Miyazono, "Cloning of a TGF beta type I receptor that forms a
heteromeric complex with the TGF beta type II receptor", Cell, 75, 1993, 681-92.
A Moustakas, HY Lin, YI Henis, J Plamondon, MD O'Connor-McCourt, HF Lodish, "The transforming growth factor beta receptors types I, II, and
III form hetero-oligomeric complexes in the presence of ligand", J Biol Chem, 268, 1993, 22215-8.
JL Wrana, L Attisano, J Carcamo, A Zentella, J Doody, M Laiho, XF Wang, J Massague, "TGF beta signals through a heteromeric protein kinase
receptor complex", Cell, 71, 1992, 1003-14.
Reaction
36.5 Type II receptor phosphorylates type I receptor
Authors
Reviewers
Heldin, CH, 2006-04-18.
Description
Formation of the hetero-tetrameric TGF-beta 1 receptor complex induces receptor rotation, so that their cytoplasmic kinase domains face each
other in a catalytically favourable configuration. The constitutively active type II receptor kinase (which auto-phosphorylates in the absence of
ligand), trans-phosphorylates specific serine residues at the conserved Gly-Ser-rich juxtapositioned domain of the type I receptor.
References
JL Wrana, L Attisano, R Wieser, F Ventura, J Massague, "Mechanism of activation of the TGF-beta receptor", Nature, 370, 1994, 341-7.
S Souchelnytskyi, P ten Dijke, K Miyazono, CH Heldin, "Phosphorylation of Ser165 in TGF-beta type I receptor modulates TGF-beta1-induced
cellular responses", EMBO J, 15, 1996, 6231-40.
Reaction
36.6 An anchoring protein, SARA, recruits R-SMAD
Authors
Reviewers
Heldin, CH, 2006-04-18.
Description
The activated TGF-beta receptor complex is internalized by clathrin-mediated endocytosis into early endosomes. SARA resides in the
membrane of early endosomes. Crystallographic studies suggest that dimeric SARA in the early endosome co-ordinates two R-SMAD molecules
per one receptor complex.
References
T Tsukazaki, TA Chiang, AF Davison, L Attisano, JL Wrana, "SARA, a FYVE domain protein that recruits Smad2 to the TGFbeta receptor", Cell,
95, 1998, 779-91.
Reaction
36.7 Ubiquitin-dependent degradation controls basal levels of R-SMAD
Authors

Reviewers
Heldin, CH, 2006-04-18.
Description
r-SMAD levels are kept low within cells. This is achieved by constant ubiquitination of r-SMADs by Smurf ubiquitin ligases and ensuing
proteasomal degradation.
References
Y Zhang, C Chang, DJ Gehling, A Hemmati-Brivanlou, R Derynck, "Regulation of Smad degradation and activity by Smurf2, an E3 ubiquitin
ligase", Proc Natl Acad Sci U S A, 98, 2001, 974-9.
Reaction
36.8 Activated type I receptor phosphorylates R-SMAD directly
Authors
Reviewers
Heldin, CH, 2006-04-18.

Description
Activated type I receptor kinase directly phosphorylates two of the C-terminal serine residues of the R-SMAD. Binding of the R-SMAD to the L45
loop of the type I receptor is critical for this event.
References
S Souchelnytskyi, L Ronnstrand, CH Heldin, P ten Dijke, "Phosphorylation of Smad signaling proteins by receptor serine/threonine kinases",
Methods Mol Biol, 124, 2001, 107-20.
Reaction
36.9 Phospho-R-SMAD dissociates from the receptor complex
Authors
Reviewers
Heldin, CH, 2006-04-18.
Description
Upon phosphorylation of the R-SMAD, the conformation of the C-terminal (MH2) domain of the R-SMAD changes, lowering its affinity to the type
I receptor and endofin. As a result, the phosphorylated-R-SMAD dissociates from the activated receptor complex.
References
A Nakao, S Souchelnytskyi, M Kawabata, A Ishisaki, E Oeda, K Tamaki, J Hanai, CH Heldin, K Miyazono, P ten Dijke, "TGF-beta
receptor-mediated signalling through Smad2, Smad3 and Smad4", EMBO J, 16, 1997, 5353-62.
M Macias-Silva, S Abdollah, PA Hoodless, R Pirone, L Attisano, JL Wrana, "MADR2 is a substrate of the TGFbeta receptor and its
phosphorylation is required for nuclear accumulation and signaling", Cell, 87, 1996, 1215-24.
S Souchelnytskyi, K Tamaki, U Engstrom, C Wernstedt, P ten Dijke, CH Heldin, "Phosphorylation of Ser465 and Ser467 in the C terminus of
Smad2 mediates interaction with Smad4 and is required for transforming growth factor-beta signaling", J Biol Chem, 272, 1997, 28107-15.
Reaction
36.10 Phospho-R-SMAD forms a complex with CO-SMAD
Authors
Reviewers
Heldin, CH, 2006-04-18.
Description
The phosphorylated C-terminal tail of r-SMAD induces a conformation change of the MH2 domain, which now acquires high affinity towards
Co-SMAD (common mediator of signal transduction in TGF-beta/BMP signalling). The r-SMAD:Co-SMAD complex most likely is a trimer of two
r-SMADs with one Co-SMAD. It is important to notice that the Co-SMAD itself cannot be phosphorylated as it lacks the C-terminal serine motif.
References
A Nakao, S Souchelnytskyi, M Kawabata, A Ishisaki, E Oeda, K Tamaki, J Hanai, CH Heldin, K Miyazono, P ten Dijke, "TGF-beta
receptor-mediated signalling through Smad2, Smad3 and Smad4", EMBO J, 16, 1997, 5353-62.
Reaction
36.11 The phospho-R-SMAD:CO-SMAD transfers to the nucleus
Authors
Reviewers
Heldin, CH, 2006-04-18.
Description
The phosphorylated-R-SMAD:CO-SMAD complex rapidly translocates to the nucleus where it binds directly to DNA and interacts with a plethora
of transcription co-factors. Regulation of target gene expression can be either positive or negative. A classic example of a target gene of the
pathway are the genes encoding for I-SMADs. Thus, TGF-beta/SMAD signaling induces the expression of the negative regulators of the
pathway (negative feedback loop).
References
Z Xiao, R Latek, HF Lodish, "An extended bipartite nuclear localization signal in Smad4 is required for its nuclear import and transcriptional
activity", Oncogene, 22, 2003, 1057-69.
L Xu, YG Chen, J Massague, "The nuclear import function of Smad2 is masked by SARA and unmasked by TGFbeta-dependent
phosphorylation", Nat Cell Biol, 2, 2000, 559-62.
A Kurisaki, S Kose, Y Yoneda, CH Heldin, A Moustakas, "Transforming growth factor-beta induces nuclear import of Smad3 in an importin-beta1
and Ran-dependent manner", Mol Biol Cell, 12, 2001, 1079-91.
Reaction
36.12 Ubiquitin-dependent degradation of the SMAD complex terminates

TGF-beta signaling
Authors
Reviewers
Heldin, CH, 2006-04-18.
Description
The nuclear r-SMAD:Co-SMAD complex recruits ubiquitin conjugating enzymes that ubiquitinate the complex and eventually lead to its
proteasomal degradation. This provides an end point to the signaling pathway.
References
CH Heldin, P ten Dijke, "SMAD destruction turns off signalling", Nat Cell Biol, 1, 1999, E195-7.
RS Lo, J Massague, "Ubiquitin-dependent degradation of TGF-beta-activated smad2", Nat Cell Biol, 1, 1999, 472-8.
Reaction
36.13 SKI complexes with the SMAD complex, suppressing TGF-beta

signaling
Authors
Reviewers
Heldin, CH, 2006-04-18.
Description
SKI resides in both the nucleus and the cytoplasm. Cytoplasmic SKI binds to the R-SMAD:C-SMAD complex and disrupts it (the mechanism is
not clear). Nuclear SKI binds to the active R-SMAD:CO-SMAD complex while bound to chromatin and recruits co-repressors that inhibit
transcription mediated by the active SMAD complex.
References
Y Sun, X Liu, EN Eaton, WS Lane, HF Lodish, RA Weinberg, "Interaction of the Ski oncoprotein with Smad3 regulates TGF-beta signaling", Mol
Cell, 4, 1999, 499-509.
Reaction
36.14 I-SMAD competes with R-SMAD for type I receptor
Authors

Reviewers
Heldin, CH, 2006-04-18.
Description
I-SMADs reside in the nucleus presumably to be sequestered from the TGF-beta receptor complex and thus avoid inappropriate silencing of the
signaling pathway. Upon activation of the signaling pathway, I-SMADs exit the nucleus and are recruited to the signaling TGF-beta receptor
complex. I-SMADs directly bind to the so-called L45 loop of the type I receptor, the site of binding of R-SMADs. Thus, I-SMADs competitively
inhibit the activation/phosphorylation of R-SMADs.
References
A Nakao, M Afrakhte, A Moren, T Nakayama, JL Christian, R Heuchel, S Itoh, M Kawabata, NE Heldin, CH Heldin, P ten Dijke, "Identification of
Smad7, a TGFbeta-inducible antagonist of TGF-beta signalling", Nature, 389, 1997, 631-5.
H Hayashi, S Abdollah, Y Qiu, J Cai, YY Xu, BW Grinnell, MA Richardson, JN Topper, Jr Gimbrone MA, JL Wrana, D Falb, "The MAD-related
protein Smad7 associates with the TGFbeta receptor and functions as an antagonist of TGFbeta signaling", Cell, 89, 1997, 1165-73.
Reaction
36.15 I-SMAD recruits type I receptor phosphatase and ubiquitin ligases to

mediate receptor downregulation
Authors
Reviewers
Heldin, CH, 2006-04-18.
Description
I-SMADs are adaptor proteins that bind with phosphatases and/or ubiquitin ligases (e.g. Smurfs) as well as type I receptors. These enzymes
de-phosphorylate or ubiquitinate the type I receptor, thus leading to receptor inactivation and ultimate degradation in lysosomes upon
endocytosis (the latter events are not shown in the Reactome pathway).
37 Signaling by VEGF
Authors
Editors
Reviewers
Claesson-Welsh, L, 2008-02-28.
Description
In normal development vascular endothelial growth factors (VEGFs) are crucial regulators of vascular development during embryogenesis
(vasculogenesis) and blood-vessel formation in the adult (angiogenesis). In tumor progression, activation of VEGF pathways promotes tumor
vascularization, facilitating tumor growth and metastasis. Abnormal VEGF function is also associated with inflammatory diseases including
atherosclerosis, and hyperthyroidism. The members of the VEGF and VEGF-receptor protein families have distinct but overlapping
ligand-receptor specificities, cell-type expression, and function. VEGF-receptor activation in turn regulates a network of signaling processes in
the body that promote endothelial cell growth, migration and survival (Hicklin and Ellis, 2005; Shibuya and Claesson-Welsh, 2006).
Molecular features of the VGF signaling cascades are outlined in the figure below (from Olsson et al. 2006; Nature Publishing Group). Tyrosine
residues in the intracellular domains of VEGF receptors 1, 2,and 3 are indicated by dark blue boxes; residues susceptible to phosphorylation are
numbered. A circled R indicates that phosphorylation is regulated by cell state (VEGFR2), by ligand binding (VEGFR1), or by heterodimerization
(VEGFR3). Specific phosphorylation sites (boxed numbers) bind signaling molecules (dark blue ovals), whose interaction with other cytosolic
signaling molecules (light blue ovals) leads to specific cellular (pale blue boxes) and tissue-level (pink boxes) responses in vivo. Signaling
cascades whose molecular details are unclear are indicated by dashed arrows. DAG, diacylglycerol; EC, endothelial cell; eNOS, endothelial
nitric oxide synthase; FAK, focal adhesion kinase; HPC, hematopoietic progenitor cell; HSP27, heat-shock protein-27; MAPK, mitogen-activated
protein kinase; MEK, MAPK and ERK kinase; PI3K, phosphatidylinositol 3' kinase; PKC, protein kinase C; PLCgamma, phospholipase
C-gamma; Shb, SH2 and beta-cells; TSAd, T-cell-specific adaptor.
In the current release, the first events in these cascades - the interactions between VEGF proteins and their receptors - are annotated. Details of
signaling events and their biological outcome, concisely illustrated in the image below, will be available in future versions of this pathway.
References
MJ Cross, J Dixelius, T Matsumoto, L Claesson-Welsh, "VEGF-receptor signal transduction", Trends Biochem Sci, 28, 2003, 488-94.
AK Olsson, A Dimberg, J Kreuger, L Claesson-Welsh, "VEGF receptor signalling - in control of vascular function", Nat Rev Mol Cell Biol, 7, 2006,
359-71.
DJ Hicklin, LM Ellis, "Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis", J Clin Oncol, 23, 2005, 1011-27.
M Shibuya, L Claesson-Welsh, "Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis", Exp Cell Res,
312, 2006, 549-60.
T Matsumoto, H Mugishima, "Signal transduction via vascular endothelial growth factor (VEGF) receptors and their roles in atherogenesis", J
Atheroscler Thromb, 13, 2006, 130-5.
37.1 VEGF ligand-receptor interactions
Authors
Editors
Reviewers
Description
The VEGF family is encoded by seven genes (VEGF-A, B, C, D, E: PLGF (Placenta Growth Factor)-1, 2). Six isoforms of VEGF-A protein,
containing 121, 145, 165, 183, 189, and 206 amino acid residues, and two isoforms of VEGF-B (167 and 186 residues) are specified by
alternatively spliced mRNAs. The active form of each of these proteins is a homodimer.
The specificities of the three VEGF tyrosine kinase receptors, VEGFR-1, VEGFR-2 and VEGFR-3, for these ligands are shown in the figure
(Hicklin and Ellis 2005). All VEGF-A isoforms bind both VEGFR-1 and VEGFR-2; PLGF-1 and -2, and VEGF-B isoforms bind only VEGFR-1;
VEGF-E binds VEGFR-2; and VEGF-C and -D bind both VEGFR-2 and -3. VEGF-D undergoes a complex series of post-translational
modifications that results in secreted forms with increased activity toward VEGFR-3 and VEGFR-2.
Two co-receptor proteins in the cell membrane, neuropilin (NRP)-1 and NRP-2, interact with VEGFR proteins to increase the affinity of the latter
for their ligands (Neufeld et al.,2002). They differ from VEGFR proteins in not having intracellular signaling domains.
References
359-71.
M Shibuya, "Differential roles of vascular endothelial growth factor receptor-1 and receptor-2 in angiogenesis", J Biochem Mol Biol, 39, 2006,
469-78.
312, 2006, 549-60.
G Neufeld, O Kessler, Y Herzog, "The interaction of Neuropilin-1 and Neuropilin-2 with tyrosine-kinase receptors for VEGF", Adv Exp Med Biol,
515, 2002, 81-90.
37.1.1 Homodimerization of VEGF proteins
Authors
Editors
Reviewers
Description
VEGF proteins bind their receptors as homodimers. Heterodimers with PLGF and among different VEGF proteins have been observed but have
no known function.
References
359-71.
Reaction
37.1.2 VEGF binds to VEGFR leading to receptor dimerization
Authors
Editors
Reviewers
Description
The binding of VEGF ligands to VEGFR receptors in the cell membrane induces dimerization and activation of the latter, initiating intracellular
signaling cascades that result in proliferation, survival, migration and increased permeability of vascular endothelial cells (Matsumoto and
Mugishima, 2006). The receptors predominantly form homodimers but heterodimers between VEGFR-1 and -2 have been observed. Although
both VEGFR-1 and -2 are expressed in the vascular endothelium, the angiogenic activities of VEGFs are transduced mainly through VEGFR-2 in
vivo.
References
359-71.
37.1.2.1 VEGF-A,B,PLGF bind to VEGFR1 leading to receptor dimerization
Authors
Editors
Reviewers
Description
VEGFR-1 binds VEGF-A, VEGF-B, and PLGF homodimers. This interaction is required for normal angiogenesis and hematopoiesis, although
many of the detailed molecular steps from binding to these physiological consequences remain unclear (Hickins and Ellis, 2005). VEGFR-1 is
made up of 1338 aa and has three regions: an extracellular region consisting of 7 immunoglobin-like domains, a transmembrane (TM) domain
and a cytosolic tyrosine kinase (TK) domain. An alternatively spliced form, soluble VEGFR-1 (sVEGFR1), also binds VEGF proteins and may
serve in the body to down-regulate VEGF activation of membrane-bound receptors. Overexpression of sVEGFR1 (VEGF121) is associated with
preeclampsia, a major disorder of pregnancy (Shibuya and Claesson-Welsh 2006; Levine et al. 2004).
References
RJ Levine, SE Maynard, C Qian, KH Lim, LJ England, KF Yu, EF Schisterman, R Thadhani, BP Sachs, FH Epstein, BM Sibai, VP Sukhatme, SA
Karumanchi, "Circulating angiogenic factors and the risk of preeclampsia", N Engl J Med, 350, 2004, 672-83.
YA Muller, B Li, HW Christinger, JA Wells, BC Cunningham, AM de Vos, "Vascular endothelial growth factor: crystal structure and functional
mapping of the kinase domain receptor binding site", Proc Natl Acad Sci U S A, 94, 1997, 7192-7.
M Autiero, A Luttun, M Tjwa, P Carmeliet, "Placental growth factor and its receptor, vascular endothelial growth factor receptor-1: novel targets
for stimulation of ischemic tissue revascularization and inhibition of angiogenic and inflammatory disorders", J Thromb Haemost, 1, 2003,
1356-70.
DW Leung, G Cachianes, WJ Kuang, DV Goeddel, N Ferrara, "Vascular endothelial growth factor is a secreted angiogenic mitogen", Science,
246, 1989, 1306-9.
B Olofsson, K Pajusola, A Kaipainen, G von Euler, V Joukov, O Saksela, A Orpana, RF Pettersson, K Alitalo, U Eriksson, "Vascular endothelial
growth factor B, a novel growth factor for endothelial cells", Proc Natl Acad Sci U S A, 93, 1996, 2576-81.
312, 2006, 549-60.
T Takahashi, S Yamaguchi, K Chida, M Shibuya, "A single autophosphorylation site on KDR/Flk-1 is essential for VEGF-A-dependent activation
of PLC-gamma and DNA synthesis in vascular endothelial cells", EMBO J, 20, 2001, 2768-78.
N Ito, C Wernstedt, U Engstrom, L Claesson-Welsh, "Identification of vascular endothelial growth factor receptor-1 tyrosine phosphorylation sites
and binding of SH2 domain-containing molecules", J Biol Chem, 273, 1998, 23410-8.
Reaction
37.1.2.2 VEGF-A,C,D,E bind to VEGFR2 leading to receptor dimerization
Authors
Editors
Reviewers
Description
VEGFR-2 binds VEGF-A, -C, -D, and -E homodimers. VEGFR-2 is the primary mediator of the physiological effects of VEGF-A in angiogenesis,
including microvascular permeability, endothelial cell proliferation, invasion, migration, and survival. In endothelial cells, these effects are
mediated via activation of a phospholipase gamma-protein kinase C-Raf-MAPK signaling pathway for proliferation and PI3K and focal adhesion
kinase for survival and migration. VEGFR-2 is the important receptor among VEGFR protiens and its activation and signaling may be positively
or negatively regulated by co-expression and activation of various factors and other VEGF receptors like VEGFR-1 (Hicklin and Ellis 2005).The
regulatory events of this receptor will be annotated in subsequent modules.
References
YA Muller, B Li, HW Christinger, JA Wells, BC Cunningham, AM de Vos, "Vascular endothelial growth factor: crystal structure and functional
mapping of the kinase domain receptor binding site", Proc Natl Acad Sci U S A, 94, 1997, 7192-7.
BI Terman, M Dougher-Vermazen, ME Carrion, D Dimitrov, DC Armellino, D Gospodarowicz, P Bohlen, "Identification of the KDR tyrosine
kinase as a receptor for vascular endothelial cell growth factor", Biochem Biophys Res Commun, 187, 1992, 1579-86.
L Seetharam, N Gotoh, Y Maru, G Neufeld, S Yamaguchi, M Shibuya, "A unique signal transduction from FLT tyrosine kinase, a receptor for
vascular endothelial growth factor VEGF", Oncogene, 10, 1995, 135-47.
YJ Tsai, RK Lee, SP Lin, YH Chen, "Identification of a novel platelet-derived growth factor-like gene, fallotein, in the human reproductive tract",
MG Achen, M Jeltsch, E Kukk, T Makinen, A Vitali, AF Wilks, K Alitalo, SA Stacker, "Vascular endothelial growth factor D (VEGF-D) is a ligand
for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4)", Proc Natl Acad Sci U S A, 95, 1998, 548-53.
V Joukov, K Pajusola, A Kaipainen, D Chilov, I Lahtinen, E Kukk, O Saksela, N Kalkkinen, K Alitalo, "A novel vascular endothelial growth factor,
VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases", EMBO J, 15, 1996, 290-98.
312, 2006, 549-60.
J Dixelius, T Makinen, M Wirzenius, MJ Karkkainen, C Wernstedt, K Alitalo, L Claesson-Welsh, "Ligand-induced vascular endothelial growth
factor receptor-3 (VEGFR-3) heterodimerization with VEGFR-2 in primary lymphatic endothelial cells regulates tyrosine phosphorylation sites", J
Biol Chem, 278, 2003, 40973-9.
T Takahashi, S Yamaguchi, K Chida, M Shibuya, "A single autophosphorylation site on KDR/Flk-1 is essential for VEGF-A-dependent activation
of PLC-gamma and DNA synthesis in vascular endothelial cells", EMBO J, 20, 2001, 2768-78.
Reaction
37.1.2.3 VEGF-C,D bind to VEGFR3 leading to receptor dimerization
Authors
Editors
Reviewers
Description
VEGFR-3 preferentially binds VEGF-C and -D. Mutations of the VEGFR-3 tyrosine kinase domain are seen in human lymphedema. VEGFR-3
expression has been correlated with transient lymphangiogenesis in wound healing and may modulate VEGFR-2 signaling in maintaining
vascular integrity (Hicklin and Ellis 2005).
References
T Makinen, T Veikkola, S Mustjoki, T Karpanen, B Catimel, EC Nice, L Wise, A Mercer, H Kowalski, D Kerjaschki, SA Stacker, MG Achen, K
Alitalo, "Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3", EMBO J,
20, 2001, 4762-73.
MG Achen, M Jeltsch, E Kukk, T Makinen, A Vitali, AF Wilks, K Alitalo, SA Stacker, "Vascular endothelial growth factor D (VEGF-D) is a ligand
for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4)", Proc Natl Acad Sci U S A, 95, 1998, 548-53.
V Joukov, K Pajusola, A Kaipainen, D Chilov, I Lahtinen, E Kukk, O Saksela, N Kalkkinen, K Alitalo, "A novel vascular endothelial growth factor,
VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases", EMBO J, 15, 1996, 290-98.
J Dixelius, T Makinen, M Wirzenius, MJ Karkkainen, C Wernstedt, K Alitalo, L Claesson-Welsh, "Ligand-induced vascular endothelial growth
factor receptor-3 (VEGFR-3) heterodimerization with VEGFR-2 in primary lymphatic endothelial cells regulates tyrosine phosphorylation sites", J
Biol Chem, 278, 2003, 40973-9.
J Lee, A Gray, J Yuan, SM Luoh, H Avraham, WI Wood, "Vascular endothelial growth factor-related protein: a ligand and specific activator of the
tyrosine kinase receptor Flt4", Proc Natl Acad Sci U S A, 93, 1996, 1988-92.
Reaction
37.1.3 Neurophilin interactions with VEGF and VEGFR
Authors
Editors
Reviewers
Description
The plasma membrane-associated Neuropilin receptors NRP-1 and -2 bind some of the VEGF proteins and associate with VEGF receptor
proteins. NRP-1 binds VEGF-A165, -B, and PLGF-2; NRP-2 also binds VEGF-A165 and PLGF-2, as well as VEGF-A145 and -C. The Neurolipin
receptors appear to act as cofactors for the VEGF receptors, increasing their affinities for specific VEGF ligands, although the importance of this
function in vivo remains unclear (Neufeld et al. 2002). Detailed annotation of NRP interactions with VEGF proteins will be covered in future
releases.
References
G Neufeld, O Kessler, Y Herzog, "The interaction of Neuropilin-1 and Neuropilin-2 with tyrosine-kinase receptors for VEGF", Adv Exp Med Biol,
515, 2002, 81-90.
37.1.3.1 NRP-1 forms a ternary complex with VEGF165 and VEGFR1

Authors
Editors
Reviewers
Description
Plasma membrane-associated Neuropilin-1 (NRP1) binds vascular endothelial growth factor (VEGF) family members. NRP1 has three distinct
extracellular domains, a1a2, b1b2, and c but lacks a distinct intracellular domain. VEGF165 mediates the formation of complexes containing
VEGFR-2 and NRP-1, enhancing VEGF165-receptor binding on the endothelial cell membrane (Soker et al. 2002). The role of heparin, a critical
component of NRP-1 interactions with VEGF proteins, will annotated in detail in future.
References
Y Shintani, S Takashima, Y Asano, H Kato, Y Liao, S Yamazaki, O Tsukamoto, O Seguchi, H Yamamoto, T Fukushima, K Sugahara, M
Kitakaze, M Hori, "Glycosaminoglycan modification of neuropilin-1 modulates VEGFR2 signaling", EMBO J, 25, 2006, 3045-55.
G Fuh, KC Garcia, AM de Vos, "The interaction of neuropilin-1 with vascular endothelial growth factor and its receptor flt-1", J Biol Chem, 275,
2000, 26690-5.
H Oh, H Takagi, A Otani, S Koyama, S Kemmochi, A Uemura, Y Honda, "Selective induction of neuropilin-1 by vascular endothelial growth
factor (VEGF): a mechanism contributing to VEGF-induced angiogenesis", Proc Natl Acad Sci U S A, 99, 2002, 383-8.
S Soker, HQ Miao, M Nomi, S Takashima, M Klagsbrun, "VEGF165 mediates formation of complexes containing VEGFR-2 and neuropilin-1 that
enhance VEGF165-receptor binding", J Cell Biochem, 85, 2002, 357-68.
R Mamluk, Z Gechtman, ME Kutcher, N Gasiunas, J Gallagher, M Klagsbrun, "Neuropilin-1 binds vascular endothelial growth factor 165,
placenta growth factor-2, and heparin via its b1b2 domain", J Biol Chem, 277, 2002, 24818-25.
Reaction
37.1.3.2 NRP-2 associates with VEGFR1 forming complexes on cell surface
Authors
Editors
Reviewers
Description
NRP-2 associates with VEGFR-1 on the plasma membrane. As NRP-2 lacks an intracellular domain, this association may be the means by
which NRP-2 participates in VEGF-induced signaling. This interaction requires VEGF to bridge between NRP and the receptor.
References
Z Gluzman-Poltorak, T Cohen, Y Herzog, G Neufeld, "Neuropilin-2 is a receptor for the vascular endothelial growth factor (VEGF) forms
VEGF-145 and VEGF-165 [corrected]", J Biol Chem, 275, 2000, 18040-5.
Z Gluzman-Poltorak, T Cohen, M Shibuya, G Neufeld, "Vascular endothelial growth factor receptor-1 and neuropilin-2 form complexes", J Biol
Chem, 276, 2001, 18688-94.
Reaction
38 Signaling by Wnt
Authors
Kimelman, D, 2007-04-03.
Editors
Matthews, L, 2007-04-03.
Description
The beta-catenin destruction complex plays a key role in the canonical Wnt signaling pathway. In the absence of Wnt signaling, this complex
controls the levels of cytoplamic beta-catenin. Beta-catenin associates with and is phosphorylated by the destruction complex. Phosphorylated
beta-catenin is recognized and ubiquitinated by the SCF-beta TrCP ubiquitin ligase complex and is subsequently degraded by the proteasome
(reviewed in Kimelman and Xu, 2006).
References
D Kimelman, W Xu, "beta-catenin destruction complex: insights and questions from a structural perspective", Oncogene, 25, 2006, 7482-91.
38.1 Degradation of beta-catenin by the destruction complex
Authors
Kimelman, D, 2007-04-03.
Editors
Matthews, L, 2007-04-03.
Reviewers
Pagano, M, 2007-04-27.
Description
The beta-catenin destruction complex plays a key role in the canonical Wnt signaling pathway. In the absence of Wnt signaling, this complex
controls the levels of cytoplamic beta-catenin. Beta-catenin associates with and is phosphorylated by the destruction complex. Phosphorylated
beta-catenin is recognized and ubiquitinated by the SCF-beta TrCP ubiquitin ligase complex and is subsequently degraded by the proteasome
(reviewed in Kimelman and Xu, 2006).
References
38.1.1 Assembly of the destruction complex
Authors
Kimelman, D, 2007-04-03.
Editors
Matthews, L, 2007-04-03.
Reviewers
Pagano, M, 2007-04-27.
Description
The exact composition of the destruction complex is not known. A number of components appear to form a core complex, while others may
associate with the complex transiently when a Wnt signal is present (reviewed in Kimelman and Xu, 2006). The core components include Axin,
glycogen synthase kinase 3 (GSK-3), Casein kinase 1 (CKI) alpha, beta-catenin, Protein phosphatase 2A (PP2A) and Adenomatous Polyposis
Coli (APC). CK1 epsilon, Diversin and PP1 may also be components of the complex.
References
R Dajani, E Fraser, SM Roe, M Yeo, VM Good, V Thompson, TC Dale, LH Pearl, "Structural basis for recruitment of glycogen synthase kinase
3beta to the axin-APC scaffold complex", EMBO J, 22, 2003, 494-501.
JM Seeling, JR Miller, R Gil, RT Moon, R White, DM Virshup, "Regulation of beta-catenin signaling by the B56 subunit of protein phosphatase
2A", Science, 283, 1999, 2089-91.
Reaction
38.1.2 Association of beta-catenin with the destruction complex
Editors
Matthews, L, 2007-04-03.
Reviewers
Pagano, M, 2007-04-27.
Description
Beta-catenin associates with the destruction complex through an interaction with Axin and or APC. This association may also involve interactions
with the 15 aa repeats in APC (Spink et al., 2001) or the third APC 20aa repeat and its N-terminal flanking residues (Ha et al., 2004, Xing et al.,
2004; Liu et al., 2006).
References
Y Xing, WK Clements, I Le Trong, TR Hinds, R Stenkamp, D Kimelman, W Xu, "Crystal structure of a beta-catenin/APC complex reveals a
critical role for APC phosphorylation in APC function", Mol Cell, 15, 2004, 523-33.
NC Ha, T Tonozuka, JL Stamos, HJ Choi, WI Weis, "Mechanism of phosphorylation-dependent binding of APC to beta-catenin and its role in
beta-catenin degradation", Mol Cell, 15, 2004, 511-21.
K Eklof Spink, SG Fridman, WI Weis, "Molecular mechanisms of beta-catenin recognition by adenomatous polyposis coli revealed by the
structure of an APC-beta-catenin complex", EMBO J, 20, 2001, 6203-12.
J Liu, Y Xing, TR Hinds, J Zheng, W Xu, "The third 20 amino acid repeat is the tightest binding site of APC for beta-catenin", J Mol Biol, 360,
2006, 133-44.
Reaction
38.1.3 Beta-catenin phosphorylation cascade
Authors
Kimelman, D, 2007-04-03.
Editors
Matthews, L, 2007-04-19.
Reviewers
Pagano, M, 2007-04-27.
Description
Degradation of beta-catenin is initiated following amino-terminal serine/threonine phosphorylation. Phosphorylation of B-catenin at S45 by CK1
alpha primes the subsequent sequential GSK-3-mediated phosphorylation at Thr41, Ser37 and Ser33 (Amit et al., 2002 ; Lui et al., 2002).
References
S Amit, A Hatzubai, Y Birman, JS Andersen, E Ben-Shushan, M Mann, Y Ben-Neriah, I Alkalay, "Axin-mediated CKI phosphorylation of
beta-catenin at Ser 45: a molecular switch for the Wnt pathway", Genes Dev, 16, 2002, 1066-76.
C Liu, Y Li, M Semenov, C Han, GH Baeg, Y Tan, Z Zhang, X Lin, X He, "Control of beta-catenin phosphorylation/degradation by a dual-kinase
mechanism", Cell, 108, 2002, 837-47.
38.1.3.1 Phosphorylation of beta-catenin at Ser45 by CK1 alpha
Authors
Kimelman, D, 2007-04-03.
Editors
Matthews, L, 2007-04-03.
Reviewers
Pagano, M, 2007-04-27.
Description
CK1a binds to Axin and phosphorylates beta-catenin at Ser45 priming GSK3 mediated phosphorylation at the more N-terminal residues (Amit et
al., 2002; Liu et al., 2002; Yanagawa et al., 2002).
References
S Amit, A Hatzubai, Y Birman, JS Andersen, E Ben-Shushan, M Mann, Y Ben-Neriah, I Alkalay, "Axin-mediated CKI phosphorylation of
beta-catenin at Ser 45: a molecular switch for the Wnt pathway", Genes Dev, 16, 2002, 1066-76.
Reaction
38.1.3.2 Phosphorylation of phospho-(Ser45 ) at Thr 41 by GSK-3
Authors
Kimelman, D, 2007-04-03.
Editors
Matthews, L, 2007-04-03.
Reviewers
Pagano, M, 2007-04-27.
Description
Following CKI-mediated phosphorylation at Ser45, beta-catenin is phosphorylated by GSK3 at Thr41.
References
mechanism", Cell, 108, 2002, 837-47.
Reaction
38.1.3.3 Phosphoryation of phospho- (Ser45, Thr41) beta-catenin at Ser37 by GSK-3
Authors
Kimelman, D, 2007-04-03.
Editors
Matthews, L, 2007-04-03.
Reviewers
Pagano, M, 2007-04-27.
Description
Phospho-(Ser45, Thr41) beta-catenin is phosphorylated by GSK3 at Ser37.
References
mechanism", Cell, 108, 2002, 837-47.
Reaction
38.1.3.4 Phosphorylation of phospho-(Ser45,Thr41,Ser37) at Ser33 by GSK-3
Authors
Kimelman, D, 2007-04-03.
Editors
Matthews, L, 2007-04-03.
Reviewers
Pagano, M, 2007-04-27.
Description
Beta-catenin is then phosphorylated at Ser33. Phosphorylated S37 and S33 together with neighboring residues constitute the recognition motif
for beta-TrCP.
References
mechanism", Cell, 108, 2002, 837-47.
Reaction
38.1.4 Phosphorylation of APC component of the destruction complex
Authors
Kimelman, D, 2007-04-03.
Editors
Matthews, L, 2007-04-03.
Reviewers
Pagano, M, 2007-04-27.
Description
APC is phosphorylated on the 20 aa repeats by CK1 and potentially GSK-3. This significantly increases the binding affinity of the GSK-3 20 aa
repeats for beta-catenin, causing one of them to bind b-catenin in the same region as beta-catenin binds Axin, thus displacing beta-catenin from
Axin ( Step 5 above) (Reviewed in Kimelman, 2006).
References
L Tickenbrock, K Kossmeier, H Rehmann, C Herrmann, O Muller, "Differences between the interaction of beta-catenin with non-phosphorylated
and single-mimicked phosphorylated 20-amino acid residue repeats of the APC protein", J Mol Biol, 327, 2003, 359-67.
K Eklof Spink, SG Fridman, WI Weis, "Molecular mechanisms of beta-catenin recognition by adenomatous polyposis coli revealed by the
structure of an APC-beta-catenin complex", EMBO J, 20, 2001, 6203-12.
2006, 133-44.
Reaction
38.1.5 Dissociation of beta-catenin from Axin and association of beta catenin with phospho-(20 aa) APC in
the detruction complex
Editors
Matthews, L, 2007-04-03.
Reviewers
Pagano, M, 2007-04-27.
Description
The phosphorylation of the 20 aa repeats in APC results in an increase in affinity for beta-catenin (Ha et al., 2004, Xing et al., 2004; Liu et al.,
2006). The binding site of phospho -(20 aa) APC on beta-catenin, overlaps the binding site of Axin on beta catenin. In addition, phosphorylated
APC prevents the association of Axin with beta-catenin (Ha et al., 2004, Xing et al., 2004). In this model, phosphorylated APC may compete with
Axin for beta-catenin binding, resulting in dissociation of the Axin:beta-catenin interaction in the destruction complex (see Kimelman and Xu
2006).
References
NC Ha, T Tonozuka, JL Stamos, HJ Choi, WI Weis, "Mechanism of phosphorylation-dependent binding of APC to beta-catenin and its role in
beta-catenin degradation", Mol Cell, 15, 2004, 511-21.
2006, 133-44.
Reaction
38.1.6 Association of beta-catenin with the SCF-beta-TrCP1 ubiquitin ligase complex
Editors
Matthews, L, 2007-04-03.
Reviewers
Pagano, M, 2007-04-27.
Description
B-TrCP associates with phosphorylated beta-catenin through the B-TrCP WD40 repeat region. Currently, it is unclear whether the ubiquitin
ligase binds beta-catenin after it leaves the complex. It is equally possible that it binds beta-catenin while beta-catenin is still bound to Axin.
References
JT Winston, P Strack, P Beer-Romero, CY Chu, SJ Elledge, JW Harper, "The SCFbeta-TRCP-ubiquitin ligase complex associates specifically
with phosphorylated destruction motifs in IkappaBalpha and beta-catenin and stimulates IkappaBalpha ubiquitination in vitro", Genes Dev, 13,
1999, 270-83.
E Latres, DS Chiaur, M Pagano, "The human F box protein beta-Trcp associates with the Cul1/Skp1 complex and regulates the stability of
beta-catenin", Oncogene, 18, 1999, 849-54.
Reaction
38.1.7 Multi-ubiquitination of phospho-beta-catenin by SCF-beta-TrCP1
Authors
Kimelman, D, 2007-04-03.
Editors
Matthews, L, 2007-04-03.
Reviewers
Pagano, M, 2007-04-27.
Description
Beta-catenin is ubiquitinated by the SCF-B-TrCP1 complex.

References
G Wu, G Xu, BA Schulman, PD Jeffrey, JW Harper, NP Pavletich, "Structure of a beta-TrCP1-Skp1-beta-catenin complex: destruction motif
binding and lysine specificity of the SCF(beta-TrCP1) ubiquitin ligase", Mol Cell, 11, 2003, 1445-56.
Reaction
38.1.8 Degradation of ubiquitinated -beta catenin by the proteasome
Editors
Matthews, L, 2007-04-19.
Reviewers
Pagano, M, 2007-04-27.
Description
Ubiquitinated beta-catenin is degraded by the proteasome.
References
G Calviello, F Resci, S Serini, E Piccioni, A Toesca, A Boninsegna, G Monego, FO Ranelletti, P Palozza, "Docosahexaenoic Acid Induces
Proteasome-dependent Degradation of {beta}-catenin, Down-regulation of Survivin and Apoptosis in Human Colorectal Cancer Cells not
expressing COX-2", Carcinogenesis, 2006.
Reaction
39 Synaptic Transmission
Authors
Editors
Description
The human brain contains at least 100 billion neurons, each with the ability to influence many other cells. Clearly, highly sophisticated and
efficient mechanisms are needed to enable communication among this astronomical number of elements. This communication occurs across
synapses, the functional connection between neurons. Synapses can be divided into two general classes: electrical synapses and chemical
synapses. Electrical synapses permit direct, passive flow of electrical current from one neuron to another. The current flows through gap
junctions, specialized membrane channels that connect the two cells. Chemical synapses enable cell-to-cell communication using
neurotransmitter release. Neurotransmitters are chemical agents released by presynaptic neurons that trigger a secondary current flow in
postsynaptic neurons by activating specific receptor molecules. Neurotransmitter secretion is triggered by the influx of Ca2+ through
voltage-gated channels, which gives rise to a transient increase in Ca2+ concentration within the presynaptic terminal. The rise in Ca2+
concentration causes synaptic vesiclesâ€"the presynaptic organelles that store neurotransmittersâ€"to fuse with the presynaptic plasma
membrane and release their contents into the space between the pre- and postsynaptic cells.
References
D PURVES, DJ AUGUSTINE, D FITZPATRICK, LC KATZ, AS LaMANTIA, JO McNamara, JM WILLIAMS, "Neuroscience 2nd Edition", Sinauer
Associates, Inc., P.O. Box 407 23 Plumtree Road, Sunderland, MA 01375 U.S.A., 2001.
39.1 Transmission across Chemical Synapses
Authors
Mahajan, SS, 2008-01-14.
Editors
Mahajan, SS, 2008-01-14.

Description
Chemical synapses are specialized junctions that are used for communication between neurons, neurons and muscle or gland cells. The
synapse involves a pre-synaptic neuron and a post-synaptic neuron, muscle cell or glad cell. The pre and the post-synaptic cell are separated by
a gap of 20nm called the synaptic cleft. The signals pass in a unidirection from pre-synaptic to post-synaptic. The pre-synaptic neuron
communicates via the release of neurotransmitter which bind the receptors on the post-synaptic cell.
References
D PURVES, DJ AUGUSTINE, D FITZPATRICK, LC KATZ, AS LaMANTIA, JO McNamara, JM WILLIAMS, "Neuroscience 2nd Edition", Sinauer
Associates, Inc., P.O. Box 407 23 Plumtree Road, Sunderland, MA 01375 U.S.A., 2001.
39.1.1 Depolarization of the Presynaptic Terminal Triggers the Opening of Calcium Channels
Authors
Mahajan, SS, 2008-01-14.
Editors
Mahajan, SS, 2008-01-14.
Description
The action potential travels down the axon and reaches the pre-synaptic terminal depolarizing the membrane in the pre-synaptic terminal. The
depolarization causes the voltage-gated Ca2+ channels to open allowing the influx of Ca2+ that signals the release of neurotransmitter into the
synaptic cleft.
39.1.1.1 Ca2+ influx through voltage gated Ca2+ channels
Authors
Mahajan, SS, 2008-01-14.
Description
Ca2+ influx from the extracellular space into the presynaptic neuron through the Voltage Gated Ca2+ Channels (VGCC), is dependant on the
arrival of an action potential at the synaptic bulb. The vesicle fusion and subsequent release of glutamate into the synapse is triggered by this
influx of Ca2+. The VGCCs involved here could be of the N, P/Q or R type.
References
S Diriong, P Lory, ME Williams, SB Ellis, MM Harpold, S Taviaux, "Chromosomal localization of the human genes for alpha 1A, alpha 1B, and
alpha 1E voltage-dependent Ca2+ channel subunits", Genomics, 30, 1995, 605-9.
Reaction
39.1.2 Neurotransmitter Release Cycle
Authors
Mahajan, SS, 2008-01-14.
Editors
Mahajan, SS, 2008-01-14.
Description
Neurotransmitter is stored in the synaptic vesicle in the pre-synaptic terminal prior to its release in the synaptic cleft upon depolarization of the
pre-synaptic membrane. The release of the neurotransmitter is a multi-step process that is controlled by electrical signals passing through the
axons in form of action potential. Neurotransmitters include glutamate, acetylcholine, nor-epinephrine, dopamine and seratonin. Each of the
neurotransmitter cycle is independently described.
References
RH Edwards, "The neurotransmitter cycle and quantal size", Neuron, 55, 2007, 835-58.
39.1.2.1 Glutamate Neurotransmitter Release Cycle
Authors
Mahajan, SS, 2008-01-14.
Description
Communication at the synapse involves the release of glutamate from the presynaptic neuron and its binding to glutamate receptors on the
postsynaptic cell to generate a series of events that lead to propagation of the synaptic transmission. This process begins with the formation of
synaptic vesicles in the presynaptic neuron, proceeds to the loading of glutamate into the vesicles, and concludes with the release of glutamate
into the synaptic cleft.
The glutamate life cycle in the neuron begins with the loading of the nascent synaptic vesicles with cytosolic glutamate with the help the
transporter protein, VGLUT1, located in the synaptic vesicular membrane. Glutamate loaded vesicles are formed in the cytoplasm and then
transported to a site close to the plasma membrane where the vesicle is docked with the help of several proteins. One of the key players in the
docking process in Munc 18, which interacts with syntaxin (in the plasma membrane), MINT (Munc18 interacting molecule), and DOC2. These
interactions along with the secondary interactions are needed for docking the synaptic vesicle to the plasma membrane.
The docked synaptic vesicle is not ready for release until it undergoes molecular changes to prime it for fusion with the plasma membrane.
Munc13 is one of the main players in the priming process. Munc 13 interacts with RIM (Rab3A interacting molecule) located in the synaptic
vesicle. Munc 13 also interacts with DOC2. The precise molecular mechanisms of the interactions that result in docking versus priming are not
clear and the docking and priming process have been combined in this annotation of this pathway. Once primed the synaptic vesicle is ready for
release.
Synaptic transmission involves an action potential that is generated in the presynaptic cell which induces the opening of voltage gated Ca2+
channels (VGCC) located in the plasma membrane of the presynaptic neuron. Typically N, P/Q and R type of VGCCs are involved in the
neurotransmitter release. Ca2+ influx through these channels results in the rise of intracellular Ca2+ concentration. In the microdomain of
glutamatergic synapses, the Ca2+ concentration could rise between 10-25 micro molar. Synaptotagmin, a Ca2+-binding protein located in the
synaptic vesicular membrane, responds to the rise in the Ca2+ levels in the microdomain and induces a synaptic vesicle membrane curvature
that favors vesicle fusion. Fusion of the synaptic vesicle with the plasma membrane is characterized by the formation of a trimeric trans-SNARE
complex that involves VAMP2 from the synaptic vesicle membrane, and syntaxin and SNAP-25 from plasma membrane. Vesicle fusion
incorporates the synaptic vesicle membrane into the plasma membrane, releasing the vesicle contents (glutamate) into the synaptic cleft.
Postfusion the synaptic vesicle membrane proteins (VAMP2, Rab3A, VGLUT1, and synaptotagmin) are also found in the plasma membrane.
References
S Martens, MM Kozlov, HT McMahon, "How synaptotagmin promotes membrane fusion", Science, 316, 2007, 1205-8.
S Diriong, P Lory, ME Williams, SB Ellis, MM Harpold, S Taviaux, "Chromosomal localization of the human genes for alpha 1A, alpha 1B, and
alpha 1E voltage-dependent Ca2+ channel subunits", Genomics, 30, 1995, 605-9.
YS Andrews-Zwilling, H Kawabe, K Reim, F Varoqueaux, N Brose, "Binding to Rab3A-interacting molecule RIM regulates the presynaptic
recruitment of Munc13-1 and ubMunc13-2", J Biol Chem, 281, 2006, 19720-31.
J Tang, A Maximov, OH Shin, H Dai, J Rizo, TC Sudhof, "A complexin/synaptotagmin 1 switch controls fast synaptic vesicle exocytosis", Cell,
126, 2006, 1175-87.
I Augustin, C Rosenmund, TC Sudhof, N Brose, "Munc13-1 is essential for fusion competence of glutamatergic synaptic vesicles", Nature, 400,
1999, 457-61.
K Riento, T Galli, S Jansson, C Ehnholm, E Lehtonen, VM Olkkonen, "Interaction of Munc-18-2 with syntaxin 3 controls the association of apical
SNAREs in epithelial cells", J Cell Sci, 111, 1998, 2681-8.
"Identification of a vesicular glutamate transporter that defines a glutamatergic phenotype in neurons", Nature, 141, 143.
39.1.2.1.1 L-Glutamate [cytosolic] from leucine catabolism
Authors
Mahajan, SS, 2008-01-14.
Description
References
Reaction
39.1.2.1.2 L-Glutamine transport into neurons
Authors
Mahajan, SS, 2008-01-14.
Description
Glutamine uptake in neurons is carried out by Na+-dependant system A neutral amino acid transporter.
References
FA Chaudhry, D Schmitz, RJ Reimer, P Larsson, AT Gray, R Nicoll, M Kavanaugh, RH Edwards, "Glutamine uptake by neurons: interaction of
protons with system a transporters", J Neurosci, 22, 2002, 62-72.
M Melone, H Varoqui, JD Erickson, F Conti, "Localization of the Na(+)-coupled neutral amino acid transporter 2 in the cerebral cortex",
Neuroscience, 140, 2006, 281-92.
Reaction
39.1.2.1.3 Transport of L-Glutamine (cytosol) to mitochondrial matrix
Authors
Mahajan, SS, 2008-01-14.
Description
Glutamine in neurons is transported into mitochondrial matrix by an unknown transporter. Because this enzyme is not yet identified, it is
represented as a black box event.
Reaction
39.1.2.1.4 L-Glutamate [mitochondrial] from leucine catabolism
Authors
Mahajan, SS, 2008-01-14.
Description
References
Reaction
39.1.2.1.5 L-Glutamine conversion to L-Glutamate
Authors
Mahajan, SS, 2008-01-14.
Description
Glutamine taken up by the system A transporter located on the plasmamembrane of neurons from the extracellular space is converted to
glutamate by phosphate activate glutaminase.
Recenlty, it has been demonstrated in slices and neurons from hippocampal cultures in rat that neurons can store and produce glutamate for
prolonged period without the transport of glutamine from glia (Kam and Nicoll 2007).
References
K Kam, R Nicoll, "Excitatory synaptic transmission persists independently of the glutamate-glutamine cycle", J Neurosci, 27, 2007, 9192-200.
M Romero-Gomez, M Jover, D Diaz-Gomez, LC de Teran, R Rodrigo, I Camacho, M Echevarria, V Felipo, JD Bautista, "Phosphate-activated
glutaminase activity is enhanced in brain, intestine and kidneys of rats following portacaval anastomosis", World J Gastroenterol, 12, 2006,
2406-11.
T Eid, J Hammer, E RundÃ©n-Pran, B Roberg, MJ Thomas, K Osen, S Davanger, P Laake, IA Torgner, TS Lee, JH Kim, DD Spencer, OP
Ottersen, NC de Lanerolle, "Increased expression of phosphate-activated glutaminase in hippocampal neurons in human mesial temporal lobe
epilepsy", Acta Neuropathol, 113, 2007, 137-52.
Reaction
39.1.2.1.6 Tranport of L-Glutamate (mitochondrial matrix) to cytosol
Authors
Mahajan, SS, 2008-01-14.
Description
Glutamate from the mitochondrial matrix is transported back into the cytosol, to be loaded into synaptic vesicles, by an unknown transporter.
Sloute carrier 25 a mitochondrial glutamate transporter is known to transport glutamate, however, it is unclear if this protein in involved in the
transport of glutamate in neurons.
Reaction
39.1.2.1.7 Re-acidification of the synaptic vesicle
Authors
Mahajan, SS, 2008-01-14.
Reaction
39.1.2.1.8 L-Glutamate loading of synaptic vesicle
Authors
Mahajan, SS, 2008-01-14.
Description
Nascent synaptic vesicles are loaded with glutamate by VGLUT1 to form glutamate containing synaptic vesicles. This process occurs while the
synaptic vesicle is in the cytosol.
References
RP Seal, O Akil, E Yi, CM Weber, L Grant, J Yoo, A Clause, K Kandler, JL Noebels, E Glowatzki, LR Lustig, RH Edwards, "Sensorineural
deafness and seizures in mice lacking vesicular glutamate transporter 3", Neuron, 57, 2008, 263-75.
"Identification of a vesicular glutamate transporter that defines a glutamatergic phenotype in neurons", Nature, 141, 143.
Reaction
39.1.2.1.9 Synaptic vesicle docking and priming
Authors
Mahajan, SS, 2008-01-14.
Description
Docking occurs once the synaptic vesicle has moved from the cytoplasm to a region apposed to the plasma membrane. The vesicle is held in
close apposition to the plasma membrane by several proteins that bridge the synaptic vesicle to the plasma membrane. Some of these proteins
are in the plasma membrane while others are in the synaptic vesicle. Vesicle fusion is preceded by a priming event where molecular interactions
between the docked vesicle and the plasma membrane undergo changes. The molecules in the docking and the priming process are known,
however, the exact sequence and the precise molecular changes involved in docking and priming are not well dissected. In this reaction the
process of docking and priming has been condensed. It is known that Munc18 along with its interactors is critical for membrane docking and
fusion events while Munc 13 along with its interacting proteins is central to priming. Munc 13 could act as a positive regulator for the priming
recation. Finally the primed fusion complex is clamped in the pre-fusion form by a Complexin. Complexins are Ca2+ independent cytosolic
proteins that bind to partly or fully assembled SNARE complexes. Complexins play both a positive and a negative role in the release process.
References
I Dulubova, M Khvotchev, S Liu, I Huryeva, TC SÃ¼dhof, J Rizo, "Munc18-1 binds directly to the neuronal SNARE complex", Proc Natl Acad Sci
U S A, 104, 2007, 2697-702.
M Khvotchev, I Dulubova, J Sun, H Dai, J Rizo, TC SÃ¼dhof, "Dual modes of Munc18-1/SNARE interactions are coupled by functionally critical
binding to syntaxin-1 N terminus", J Neurosci, 27, 2007, 12147-55.
I Augustin, C Rosenmund, TC Sudhof, N Brose, "Munc13-1 is essential for fusion competence of glutamatergic synaptic vesicles", Nature, 400,
1999, 457-61.
K Riento, T Galli, S Jansson, C Ehnholm, E Lehtonen, VM Olkkonen, "Interaction of Munc-18-2 with syntaxin 3 controls the association of apical
SNAREs in epithelial cells", J Cell Sci, 111, 1998, 2681-8.
RF Toonen, KJ de Vries, R Zalm, TC SÃ¼dhof, M Verhage, "Munc18-1 stabilizes syntaxin 1, but is not essential for syntaxin 1 targeting and
SNARE complex formation", J Neurochem, 93, 2005, 1393-400.
Reaction
39.1.2.1.10 release of L-Glutamate at the synapse
Authors
Mahajan, SS, 2008-01-14.
Description
Once vesicles are docked, primed and ready to be released fusion of the synaptic vesicle with the plasma membrane can be triggered by an
influx of Ca2+ through the voltage gated Ca2+ channels (N, P/Q and R type). Ca2+ influx initiates a cascade of events in which the Ca2+
sensing protein, synaptotagmin-1 (sty-1) is central. Sty-1 promotes the membrane fusion between the synaptic vesicle and the plasma
membrane by Ca2+ dependant induction of membrane curvature. Synaptotagmin competes with SNARE complex binding in a Ca2+ dependent
manner thereby displacing complexin-1 and causing membrane curvature and fusion of the synaptic vesicle with the plasma membrane. The
fusion is characterized by the formation of a trans SNARE complex in which SNAP 25, syntaxin and synaptobrevin along with VGLUT1, the
glutamate transporter, synaptotagmin, and Rab3a either become a part of the plasma membrane or membrane delimited in the vesicular
membrane. Vesicle fusion ultimately results in the release of the glutamate into the synaptic cleft.
References
JW Barclay, TJ Craig, RJ Fisher, LF Ciufo, GJ Evans, A Morgan, RD Burgoyne, "Phosphorylation of Munc18 by protein kinase C regulates the
kinetics of exocytosis", J Biol Chem, 278, 2003, 10538-45.
S Martens, MM Kozlov, HT McMahon, "How synaptotagmin promotes membrane fusion", Science, 316, 2007, 1205-8.
J Tang, A Maximov, OH Shin, H Dai, J Rizo, TC Sudhof, "A complexin/synaptotagmin 1 switch controls fast synaptic vesicle exocytosis", Cell,
126, 2006, 1175-87.
A Stein, A Radhakrishnan, D Riedel, D Fasshauer, R Jahn, "Synaptotagmin activates membrane fusion through a Ca2+-dependent trans
interaction with phospholipids", Nat Struct Mol Biol, 14, 2007, 904-11.
Reaction
39.1.2.1.11 L-Glutamate uptake by neurons
Authors
Mahajan, SS, 2008-01-14.
Description
Excess L-Glutamate released by the pre-synaptic neuron in the synaptic cleft is cleared by high affinity transporters called the excitatory amino
acid transporters (EAATs) to terminate synaptic actions of the neurotransmitter and to recycle these molecules. Five types of EAATs have been
identified EAAT1-EAAT5 in the mammalian CNS. EAAT1 and EAAT2 are mainly expressed by astrocytes whereas EAAT3 and EAAT4 are
predominantly neuronal. EAAT3 are expressed throughout the CNS however, EAAT4 is predominantly localized to purkinje cells. EAAT5 are
expressed rod photoreceptor and bipolar cells of retina. Astrocytic EAATs are expressed in astrocytes in close apposition to the synapses and
neuronal EAATs are expressed in the extra-synaptic or peri-synaptic locations on the neurons. Astrocytic EAATs are responsible for majority of
the glutamate uptake, neuronal transporters are responsible for glutamate clearance in specialized synapses in cerebellum where the spatial
relationship between the glutamate receptors and EAATs is altered and glutamate receptors are expressed in the peri-synaptic region.
References
P Meera, PD Dodson, MH Karakossian, TS Otis, "Expression of GFP-tagged neuronal glutamate transporters in cerebellar Purkinje neurons",
Neuropharmacology, 49, 2005, 883-9.
Reaction
39.1.3 Neurotransmitter Clearance In The Synaptic Cleft
Authors
Mahajan, SS, 2008-01-14.
Editors
Mahajan, SS, 2008-01-14.
Description
Neurotransmitter released in the synaptic cleft binds to specific receptors on the post-synaptic cell and the excess of the neurotransmitter is
cleared to prevent over activation of the post-synaptic cell. The neurotransmitter is cleared by either re-uptake by the pre-synaptic neuron,
diffusion in the perisynaptic area, uptake by astrocytes surrounding the synaptic cleft or enzymatic degradation of the neurotransmitter.
This topic will be annotated in a future release.
39.1.4 Neurotransmitter uptake and Metabolism In Glial Cells
Authors
Mahajan, SS, 2008-01-14.
Editors
Mahajan, SS, 2008-01-14.

Description
Neuotransmitter uptake by astrocytes is mediated by specific transporter located on the astrocytic membrane. The imported neurotransmitter in
the astrocytes is metabolized and transported back to the neuron via specialized transporters.
39.1.4.1 Astrocytic Glutamate-Glutamine Uptake And Metabolism
Authors
Mahajan, SS, 2008-01-14.
Description
In astrocytic glutamate-glutamine cycle, the excess glutamate released by the pre-synaptic neuron in the synaptic cleft is transported into the
astrocyte by a family glutamate transporters called the excitatory amino acid transporters 1 and 2, EAAT1 and EAAT2. Astrocytes carrying these
transporters exist in close apposition to the synapse to clear excess glutamate to prevent excessive activation of neurons and hence neuronal
death. Glutamate in astrocytes is converted to glutamine by glutamine synthetase. Glutamine is then transported into the extracellular space by
system N transporters. The glutamate in the extracellular space is available for neuronal uptake.
39.1.4.1.1 glutamate uptake by astrocytes
Authors
Mahajan, SS, 2008-01-14.
Description
Excess L-Glutamate released by the pre-synaptic neuron in the synaptic cleft is cleared by high affinity transporters called the excitatory amino
acid transporters (EAATs) to terminate synaptic actions of the neurotransmitter and to recycle these molecules. Five types of EAATs have been
identified EAAT1-EAAT5 in the mammalian CNS. EAAT1 and EAAT2 are mainly expressed by astrocytes whereas EAAT3 and EAAT4 are
predominantly neuronal. EAAT3 are expressed throughout the CNS however, EAAT4 is predominantly localized to purkinje cells. EAAT5 are
expressed rod photoreceptor and bipolar cells of retina. Astrocytic EAATs are expressed in astrocytes in close apposition to the synapses and
neuronal EAATs are expressed in the extra-synaptic or peri-synaptic locations on the neurons. Astrocytic EAATs are responsible for majority of
the glutamate uptake, neuronal transporters are responsible for glutamate clearance in specialized synapses in cerebellum where the spatial
relationship between the glutamate receptors and EAATs is altered and glutamate receptors are expressed in the peri-synaptic region.
References
NJ Maragakis, J Dietrich, V Wong, H Xue, M Mayer-Proschel, MS Rao, JD Rothstein, "Glutamate transporter expression and function in human
glial progenitors", Glia, 45, 2004, 133-43.
Reaction
39.1.4.1.2 Glutamate conversion to glutamine
Authors
Mahajan, SS, 2008-01-14.
Description
Glutamate in the astrocytes is converted to glutamine by glutamine synthetase. Glutamine synthetase activity in the brain is confined to neuroglia
and is absent from neurons. Neurons, however, generate glutamate by expressing glutaminase activity that catalyses the conversion of
glutamine to glutamate.
References
CS Gibbs, KE Campbell, RH Wilson, "Sequence of a human glutamine synthetase cDNA", Nucleic Acids Res, 15, 1987, 6293.
Reaction
39.1.4.1.3 Glutamine transport from astrocytes
Authors
Mahajan, SS, 2008-01-14.
Description
Glutamine from the astrocytes is exported to the extracellular compartment via the system N amino acid transporter. The system N transporter is
Na+ dependant transporter that has substrate specificity to aspargine, glutamine and histidine.
References
M Gegelashvili, A Rodriguez-Kern, I Pirozhkova, J Zhang, L Sung, G Gegelashvili, "High-affinity glutamate transporter GLAST/EAAT1 regulates
cell surface expression of glutamine/neutral amino acid transporter ASCT2 in human fetal astrocytes", Neurochem Int, 48, 2006, 611-5.
Reaction
40 Telomere Maintenance
Authors
Blackburn, EH, Seidel, J, 2006-03-09.
Reviewers
Price, C, 2006-07-13.
Description
Telomeres are protein-DNA complexes at the ends of linear chromosomes that are important for genome stability. Telomeric DNA in humans, as
in many eukaryotic organisms, consists of tandem repeats (Blackburn and Gall 1978; Moyzis et al. 1988; Meyne et al. 1989). The repeats at
human telomeres are composed of TTAGGG sequences and stretch for several kilobase pairs. Another feature of telomeric DNA in many
eukaryotes is a G-rich 3' single strand overhang, which in humans is estimated to be approximately 50-300 bases long (Makarov et al. 1997;
Wright et al. 1997; Huffman et al. 2000). Telomeric DNA isolated from humans and several other organisms can form a lasso-type structure
called a t-loop in which the 3' single-strand end is presumed to invade the double stranded telomeric DNA repeat tract (Griffith et al. 1999).
Telomeric DNA is bound by multiple protein factors that play important roles in regulating telomere length and in protecting the chromosome end
from recombination, non-homologous end-joining, DNA damage signaling, and unregulated nucleolytic attack (reviewed in de Lange 2005).
DNA attrition can occur at telomeres, which can impact cell viability. Attrition can occur owing to the "end-replication problem", a consequence of
the mechanism of lagging-strand synthesis (Watson 1972; Olovnikov 1973). Besides incomplete replication, nucleolytic processing also likely
contributes to telomere attrition (Huffman et al. 2000). If telomeres become critically shortened, replicative senescence can result (Harley et al.
1990). Thus, in order to undergo multiple divisions, cells need a mechanism to replenish the sequence at their chromosome ends.
The primary means for maintaining the sequence at chromosome ends in many eukaryotic organisms, including humans, is based on
telomerase (Greider and Blackburn, 1985; Morin 1989). Telomerase is a ribonucleoprotein complex minimally composed of a conserved protein
subunit containing a reverse transcriptase domain (telomerase reverse transcriptase, TERT) (Lingner et al. 1997; Nakamura et al. 1997) and a
template-containing RNA (telomerase RNA component, TERC, TR, TER) (Greider and Blackburn, 1987; Feng et al 1995). Telomerase uses the
RNA template to direct addition of multiple tandem repeats to the 3' G-rich single strand overhang. Besides extension by telomerase,
maintenance of telomeric DNA involves additional activities, including C-strand synthesis, which fills in the opposing strand, and nucleolytic
processing, which likely contributes to the generation of the 3' overhang.
References
JD Watson, "Origin of concatemeric T7 DNA", Nat New Biol, 239, 1972, 197-201.
EH Blackburn, JG Gall, "A tandemly repeated sequence at the termini of the extrachromosomal ribosomal RNA genes in Tetrahymena", J Mol
Biol, 120, 1978, 33-53.
CB Harley, AB Futcher, CW Greider, "Telomeres shorten during ageing of human fibroblasts", Nature, 345, 1990, 458-60.
VL Makarov, Y Hirose, JP Langmore, "Long G tails at both ends of human chromosomes suggest a C strand degradation mechanism for
telomere shortening", Cell, 88, 1997, 657-66.
TM Nakamura, GB Morin, KB Chapman, SL Weinrich, WH Andrews, J Lingner, CB Harley, TR Cech, "Telomerase catalytic subunit homologs
from fission yeast and human", Science, 277, 1997, 955-9.
RK Moyzis, JM Buckingham, LS Cram, M Dani, LL Deaven, MD Jones, J Meyne, RL Ratliff, JR Wu, "A highly conserved repetitive DNA
sequence, (TTAGGG)n, present at the telomeres of human chromosomes", Proc Natl Acad Sci U S A, 85, 1988, 6622-6.
J Feng, WD Funk, SS Wang, SL Weinrich, AA Avilion, CP Chiu, RR Adams, E Chang, RC Allsopp, J Yu, "The RNA component of human
telomerase", Science, 269, 1995, 1236-41.
GB Morin, "The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats", Cell, 59, 1989, 521-9.
CW Greider, EH Blackburn, "A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis", Nature, 337,
1989, 331-7.
T de Lange, "Shelterin: the protein complex that shapes and safeguards human telomeres", Genes Dev, 19, 2005, 2100-10.
LR Vega, MK Mateyak, VA Zakian, "Getting to the end: telomerase access in yeast and humans", Nat Rev Mol Cell Biol, 4, 2003, 948-59.
J Lingner, TR Hughes, A Shevchenko, M Mann, V Lundblad, TR Cech, "Reverse transcriptase motifs in the catalytic subunit of telomerase",
Science, 276, 1997, 561-7.
WE Wright, VM Tesmer, KE Huffman, SD Levene, JW Shay, "Normal human chromosomes have long G-rich telomeric overhangs at one end",
Genes Dev, 11, 1997, 2801-9.
AM Olovnikov, "A theory of marginotomy. The incomplete copying of template margin in enzymic synthesis of polynucleotides and biological
significance of the phenomenon.", J Theor Biol, 41, 1973, 181-90.
CW Greider, EH Blackburn, "Identification of a specific telomere terminal transferase activity in Tetrahymena extracts", Cell, 43, 1985, 405-13.
J Meyne, RL Ratliff, RK Moyzis, "Conservation of the human telomere sequence (TTAGGG)n among vertebrates", Proc Natl Acad Sci U S A, 86,
1989, 7049-53.
A Smogorzewska, T de Lange, "Regulation of telomerase by telomeric proteins", Annu Rev Biochem, 73, 2004, 177-208.
40.1 Extension of Telomeres
Reviewers
Price, C, 2006-07-13.
Description
Telomerase acts as reverse transcriptase in the elongation of telomeres.
40.1.1 Telomere Extension By Telomerase
Authors
Reviewers
Price, C, 2006-07-13.
Description
Humans, like most eukaryotic organisms, add direct repeats to the telomere using a specialized DNA polymerase called telomerase. Telomerase
is a ribonucleoprotein (RNP) complex minimally composed of a conserved protein subunit containing a reverse transcriptase domain (human
telomerase reverse transcriptase, hTERT) and a template-containing RNA (human telomerase RNA component, hTERC, or hTR, hTER). The
primer for telomerase is the G-rich single-strand overhang at the chromosome end.
Telomerase can perform multiple rounds of repeat synthesis. The reaction cycle has been inferred from in vitro studies of telomerase from
multiple organisms and can be described as having four events: 1) DNA primer recognition, 2) RNA template alignment, 3) elongation, and 4)
translocation. Telomeric DNA is recognized in part by a presumed "anchor site" in hTERT, which preferentially binds G-rich DNA, and this
interaction can affect elongation and translocation steps. This interaction occurs 5' of the alignment of the RNA template with the end nucleotides
of the chromosome. RNA alignment positions the template adjacent to the chromosome terminus. During elongation, the template directs
sequential addition of nucleotides to the telomere end. After synthesis of a repeat is completed, relative movement of telomerase and the primer,
termed translocation, repositions telomerase at the end of the newly added sequence to allow initiation of another round of repeat addition.
References
LR Vega, MK Mateyak, VA Zakian, "Getting to the end: telomerase access in yeast and humans", Nat Rev Mol Cell Biol, 4, 2003, 948-59.
40.1.1.1 Biogenesis And Assembly Of The Telomerase RNP

Authors
Editors
Reviewers
Price, C, 2006-07-13.
Description
hTERC is transcribed as a precursor and is processed at its 3' end to yield a 451 nucleotide RNA (Zaug et al. 1996). The accumulation of
hTERC that has undergone this processing event requires a conserved region of sequence termed the box H/ACA motif (Mitchell et al. 1999a).
This motif is bound by a complex containing dyskerin, and mutations in dyskerin affect the processing and accumulation of hTERC (Mitchell et
al. 1999b; Mitchell and Collins 2000; Fu and Collins 2003).
The core components hTERC and hTERT undergo trafficking in the cell that may be important for telomerase function. hTERC has been found
localized in multiple nuclear structures, including Cajal bodies, nucleoli, and at telomeres (Mitchell et al. 1999a; Jady et al. 2004; Zhu et al. 2004;
Jady et al. 2006; Tomlinson et al. 2006). hTERT is also reported localize in Cajal bodies, nucleoli, and to associate with telomeres (Etheridge et
al. 2002; Wong et al. 2002; Yang et al. 2002; Zhu et al. 2004; Tomlinson et al. 2006). Some of the factors that regulate trafficking of these two
core components of telomerase have been identified, such as nucleolin (Khurts et al. 2004), SMN (Bachand et al. 2002), and 14-3-3 (Seimiya et
al. 2000). Cytological studies of HeLa cells suggest that the localization of the telomerase core components can change through the cell-cycle
(Jady et al. 2006; Tomlinson et al. 2006). Despite these studies, it is not clear in which compartment hTERT and hTERC assemble to form
functional telomerase RNP.
The assembly of telomerase involves the chaperone proteins p23 and Hsp90, which stably associate with telomerase in vitro (Holt et al. 1999;
Forsythe et al. 2001; Keppler et al. 2006). A number of other proteins interact with the telomerase RNP, but it is not clear if they play a role in
telomerase assembly. Interestingly, assembled human telomerase RNP can multimerize, though the function of multimerization remains unclear
(Beattie et al. 2001; Wenz et al. 2001; Arai et al. 2002).
References
GS Karlin, AD Smith, "Approaches to the superior calix: renal displacement technique and review of options", J Urol, 142, 1989, 774-7.
P Reichenbach, M Hoss, CM Azzalin, M Nabholz, P Bucher, J Lingner, "A human homolog of yeast Est1 associates with telomerase and uncaps
chromosome ends when overexpressed", Curr Biol, 13, 2003, 568-74.
F Bachand, FM Boisvert, J Cote, S Richard, C Autexier, "The product of the survival of motor neuron (SMN) gene is a human
telomerase-associated protein", Mol Biol Cell, 13, 2002, 3192-202.
Y Yang, Y Chen, C Zhang, H Huang, SM Weissman, "Nucleolar localization of hTERT protein is associated with telomerase function", Exp Cell
Res, 277, 2002, 201-9.
JM Wong, L Kusdra, K Collins, "Subnuclear shuttling of human telomerase induced by transformation and DNA damage", Nat Cell Biol, 4, 2002,
731-6.
SE Holt, DL Aisner, J Baur, VM Tesmer, M Dy, M Ouellette, JB Trager, GB Morin, DO Toft, JW Shay, WE Wright, MA White, "Functional
requirement of p23 and Hsp90 in telomerase complexes", Genes Dev, 13, 1999, 817-26.
JR Mitchell, K Collins, "Human telomerase activation requires two independent interactions between telomerase RNA and telomerase reverse
transcriptase", Mol Cell, 6, 2000, 361-71.
HL Forsythe, JL Jarvis, JW Turner, LW Elmore, SE Holt, "Stable association of hsp90 and p23, but Not hsp70, with active human telomerase", J
Biol Chem, 276, 2001, 15571-4.
S Khurts, K Masutomi, L Delgermaa, K Arai, N Oishi, H Mizuno, N Hayashi, WC Hahn, S Murakami, "Nucleolin interacts with telomerase", J Biol
Chem, 279, 2004, 51508-15.
TL Beattie, W Zhou, MO Robinson, L Harrington, "Functional multimerization of the human telomerase reverse transcriptase", Mol Cell Biol, 21,
2001, 6151-60.
KT Etheridge, SS Banik, BN Armbruster, Y Zhu, RM Terns, MP Terns, CM Counter, "The nucleolar localization domain of the catalytic subunit of
human telomerase", J Biol Chem, 277, 2002, 24764-70.
H Seimiya, H Sawada, Y Muramatsu, M Shimizu, K Ohko, K Yamane, T Tsuruo, "Involvement of 14-3-3 proteins in nuclear localization of
telomerase", EMBO J, 19, 2000, 2652-61.
K Arai, K Masutomi, S Khurts, S Kaneko, K Kobayashi, S Murakami, "Two independent regions of human telomerase reverse transcriptase are
important for its oligomerization and telomerase activity", J Biol Chem, 277, 2002, 8538-44.
BR Keppler, AT Grady, MB Jarstfer, "The biochemical role of the heat shock protein 90 chaperone complex in establishing human telomerase
activity", J Biol Chem, 281, 2006, 19840-8.
BE Jady, E Bertrand, T Kiss, "Human telomerase RNA and box H/ACA scaRNAs share a common Cajal body-specific localization signal", J Cell
Biol, 164, 2004, 647-52.
AJ Zaug, J Linger, TR Cech, "Method for determining RNA 3' ends and application to human telomerase RNA", Nucleic Acids Res, 24, 1996,
532-3.
JR Mitchell, J Cheng, K Collins, "A box H/ACA small nucleolar RNA-like domain at the human telomerase RNA 3' end", Mol Cell Biol, 19, 1999,
567-76.
BE Snow, N Erdmann, J Cruickshank, H Goldman, RM Gill, MO Robinson, L Harrington, "Functional conservation of the telomerase protein
Est1p in humans", Curr Biol, 13, 2003, 698-704.
C Wenz, B Enenkel, M Amacker, C Kelleher, K Damm, J Lingner, "Human telomerase contains two cooperating telomerase RNA molecules",
EMBO J, 20, 2001, 3526-34.
Y Zhu, RL Tomlinson, AA Lukowiak, RM Terns, MP Terns, "Telomerase RNA accumulates in Cajal bodies in human cancer cells", Mol Biol Cell,
15, 2004, 81-90.
BE Jady, P Richard, E Bertrand, T Kiss, "Cell cycle-dependent recruitment of telomerase RNA and Cajal bodies to human telomeres", Mol Biol
Cell, 17, 2006, 944-54.
RL Tomlinson, TD Ziegler, T Supakorndej, RM Terns, MP Terns, "Cell cycle-regulated trafficking of human telomerase to telomeres", Mol Biol
Cell, 17, 2006, 955-65.
JR Mitchell, E Wood, K Collins, "A telomerase component is defective in the human disease dyskeratosis congenita", Nature, 402, 1999, 551-5.
Reaction
40.1.1.2 Recruitment of Telomerase RNP to the Telomeric Chromosome End
Authors
Reviewers
Price, C, 2006-07-13.
Description
Studies in yeast and humans indicate that recruitment of telomerase to a telomere may be influenced by multiple variables, including regulatory
protein factors, hTERT domains, telomere length, and the cell cycle. First, in yeast, the telomerase associated factor Est1 and the single-strand
DNA binding protein Cdc13 play roles in telomerase recruitment (Pennock et al. 2001; Bianchi et al. 2004). Analogous proteins exist in human
cells (Est1A, Est1B, Est1C, and POT1, respectively); however, how or whether these proteins are directly involved in telomerase recruitment
remains to be elucidated. Second, N-terminal residues of hTERT within the DAT (dissociate the activities of telomerase) domain may have a role
in binding single stranded telomeric DNA as the "anchor site" (Lee et al. 1993; Moriarty et al. 2005). Third, a cis-acting mechanism in yeast and
humans that regulates telomere length maintenance may modulate telomerase access to the telomere (reviewed in Blackburn 2001;
Smogorzewska and de Lange, 2004). Long telomeres, which have more associated protein factors, are in a state that is acted on by telomerase
less frequently than that of short telomeres, which have fewer associated factors. Whether short telomeres actively recruit telomerase remains to
be determined. Last, the recruitment of telomerase to telomeres shows cell-cycle regulation (Taggart et al. 2002; Smith et al. 2003; Fisher et al.
2004; Jady et al. 2006; Tomlinson et al. 2006). Further studies will be needed to determine the details of how human telomerase is recruited to a
telomere.
References
CD Smith, DL Smith, JL DeRisi, EH Blackburn, "Telomeric protein distributions and remodeling through the cell cycle in Saccharomyces
cerevisiae", Mol Biol Cell, 14, 2003, 556-70.
A Bianchi, S Negrini, D Shore, "Delivery of yeast telomerase to a DNA break depends on the recruitment functions of Cdc13 and Est1", Mol Cell,
16, 2004, 139-46.
EH Blackburn, "Switching and signaling at the telomere", Cell, 106, 2001, 661-73.
MS Lee, EH Blackburn, "Sequence-specific DNA primer effects on telomerase polymerization activity", Mol Cell Biol, 13, 1993, 6586-99.
AK Taggart, SC Teng, VA Zakian, "Est1p as a cell cycle-regulated activator of telomere-bound telomerase", Science, 297, 2002, 1023-6.
BN Armbruster, CM Linardic, T Veldman, NP Bansal, DL Downie, CM Counter, "Rescue of an hTERT mutant defective in telomere elongation by
fusion with hPot1", Mol Cell Biol, 24, 2004, 3552-61.
EH Blackburn, "The end of the (DNA) line", Nat Struct Biol, 7, 2000, 847-50.
E Pennock, K Buckley, V Lundblad, "Cdc13 delivers separate complexes to the telomere for end protection and replication", Cell, 104, 2001,
387-96.
T Keranen, J Jolkkonen, P Klosterskov-Jensen, GP Menge, "Oxcarbazepine does not interact with cimetidine in healthy volunteers", Acta Neurol
Scand, 85, 1992, 239-42.
TJ Moriarty, DT Marie-Egyptienne, C Autexier, "Regulation of 5' template usage and incorporation of noncognate nucleotides by human
telomerase", RNA, 11, 2005, 1448-60.
Cell, 17, 2006, 944-54.
Cell, 17, 2006, 955-65.
Reaction
40.1.1.3 Alignment Of The RNA Template On The Telomeric Chromosome End
Authors
Reviewers
Price, C, 2006-07-13.
Description
In vitro studies of telomerase complexes derived from multiple organisms indicate that at least two types of interactions are important for
telomerase RNP catalytic site alignment at the 3' G-rich single-strand telomere end. In one interaction, an alignment region in hTERC base-pairs
with the 3' G-rich single-strand telomeric DNA end to form an RNA-DNA hybrid, which positions the template adjacent to the 3' end of the
telomere. In a second interaction, a portion of hTERT is proposed to interact with the DNA 5' of the telomerase RNA/DNA primer hybrid
(Harrington and Greider 1991; Morin 1991; Moriarty et al. 2005), which is important for the catalytic rate (Lee and Blackburn, 1993) and
presumably allows telomerase to maintain contact with the chromosome during the translocation step. How the anchor site binding and template
hybridization are coordinated is not known.
References
LA Harrington, CW Greider, "Telomerase primer specificity and chromosome healing", Nature, 353, 1991, 451-4.
MS Lee, EH Blackburn, "Sequence-specific DNA primer effects on telomerase polymerization activity", Mol Cell Biol, 13, 1993, 6586-99.
GB Morin, "Recognition of a chromosome truncation site associated with alpha-thalassaemia by human telomerase", Nature, 353, 1991, 454-6.
TJ Moriarty, DT Marie-Egyptienne, C Autexier, "Regulation of 5' template usage and incorporation of noncognate nucleotides by human
telomerase", RNA, 11, 2005, 1448-60.
Reaction
40.1.1.4 Elongation Of The Telomeric Chromosome End
Authors
Reviewers
Price, C, 2006-07-13.
Description
The template of hTERC directs the sequential addition of nucleotides to the 3' telomeric DNA end. Following addition of a nucleotide, the
template and catalytic site must move relative to one another within the telomerase RNP to place the appropriate template residue in the active
site. As base-pairing and nucleotide addition occur at one end of the template, base pair melting occurs at the other (Collins and Greider 1993;
Wang and Blackburn, 1997; Hammond and Cech 1998; Benjamin et al. 2000; Forstemann and Lingner 2005). This un-pairing is thought to
reduce the energy used for mediating the subsequent translocation step. Nucleotide addition can occur up until the template boundary which in
hTERC is defined by a helix called P1b (Chen and Greider 2003).
References
PW Hammond, TR Cech, "Euplotes telomerase: evidence for limited base-pairing during primer elongation and dGTP as an effector of
translocation", Biochemistry, 37, 1998, 5162-72.
K Forstemann, J Lingner, "Telomerase limits the extent of base pairing between template RNA and telomeric DNA", EMBO Rep, 6, 2005, 361-6.
H Wang, EH Blackburn, "De novo telomere addition by Tetrahymena telomerase in vitro", EMBO J, 16, 1997, 866-79.
JL Chen, CW Greider, "Template boundary definition in mammalian telomerase", Genes Dev, 17, 2003, 2747-52.
S Benjamin, N Baran, H Manor, "Interference footprinting analysis of telomerase elongation complexes", Mol Cell Biol, 20, 2000, 4224-37.
K Collins, CW Greider, "Tetrahymena telomerase catalyzes nucleolytic cleavage and nonprocessive elongation", Genes Dev, 7, 1993, 1364-76.
Reaction
40.1.1.5 Translocation Of Telomerase RNP And Alignment Of RNA Template (TERC) To Extended Single Stranded Telomeric
Chromosome-End
Authors
Reviewers
Price, C, 2006-07-13.
Description
The human telomerase RNP can catalyze multiple rounds of repeat addition on the same telomeric substrate in vitro. Before initiating synthesis
of another repeat, telomerase undergoes a translocation step to reposition itself on the telomere. Base pairs in the DNA/RNA hybrid are
unannealed, the RNA template is repositioned relative to the active site, and the template base-pairs at the 3' end of the newly synthesized DNA.
The anchor site interaction with DNA 5' of the RNA-DNA duplex is thought to maintain the interaction of telomerase with DNA during the
translocation step.
References
Reaction
40.1.1.6 Elongation of Extended Telomeric Chromosome End
Authors
Reviewers
Price, C, 2006-07-13.
Description
The elongation reaction proceeds as follows: The template of hTERC directs the sequential addition of nucleotides to the 3' telomeric DNA end.
Following addition of a nucleotide, the template and catalytic site must move relative to one another within the telomerase RNP to place the
appropriate template residue in the active site. As base-pairing and nucleotide addition occur at one end of the template, base pair melting
occurs at the other (Collins and Greider 1993; Wang and Blackburn, 1997; Hammond and Cech 1998; Benjamin et al. 2000; Forstemann and
Lingner 2005). This un-pairing is thought to reduce the energy used for mediating the subsequent translocation step. Nucleotide addition can
occur up until the template boundary which in hTERC is defined by a helix called P1b (Chen and Greider 2003).
References
PW Hammond, TR Cech, "Euplotes telomerase: evidence for limited base-pairing during primer elongation and dGTP as an effector of
translocation", Biochemistry, 37, 1998, 5162-72.
K Forstemann, J Lingner, "Telomerase limits the extent of base pairing between template RNA and telomeric DNA", EMBO Rep, 6, 2005, 361-6.
H Wang, EH Blackburn, "De novo telomere addition by Tetrahymena telomerase in vitro", EMBO J, 16, 1997, 866-79.
JL Chen, CW Greider, "Template boundary definition in mammalian telomerase", Genes Dev, 17, 2003, 2747-52.
S Benjamin, N Baran, H Manor, "Interference footprinting analysis of telomerase elongation complexes", Mol Cell Biol, 20, 2000, 4224-37.
K Collins, CW Greider, "Tetrahymena telomerase catalyzes nucleolytic cleavage and nonprocessive elongation", Genes Dev, 7, 1993, 1364-76.
Reaction
40.1.1.7 Disassociation of Telomerase RNP and the Chromosome End
Authors
Reviewers
Price, C, 2006-07-13.
Description
In vitro, telomerase can disassociate from the primer following addition of each nucleotide or during the translocation step. The regulation of
telomerase disassociation from the telomere in vivo is not well-characterized. One factor that may be involved is a helicase termed hPIF1, which
can unanneal the telomerase RNA/telomeric DNA hybrid (Boule et al., 2005; Zhang et al., 2006).
References
DH Zhang, B Zhou, Y Huang, LX Xu, JQ Zhou, "The human Pif1 helicase, a potential Escherichia coli RecD homologue, inhibits telomerase
activity", Nucleic Acids Res, 34, 2006, 1393-404.
JB Boule, LR Vega, VA Zakian, "The yeast Pif1p helicase removes telomerase from telomeric DNA", Nature, 438, 2005, 57-61.
T de Lange, "Protection of mammalian telomeres", Oncogene, 21, 2002, 532-40.
JZ Ye, JR Donigian, M van Overbeek, D Loayza, Y Luo, AN Krutchinsky, BT Chait, T de Lange, "TIN2 binds TRF1 and TRF2 simultaneously and
stabilizes the TRF2 complex on telomeres", J Biol Chem, 279, 2004, 47264-71.
XD Zhu, L Niedernhofer, B Kuster, M Mann, JH Hoeijmakers, T de Lange, "ERCC1/XPF removes the 3' overhang from uncapped telomeres and
represses formation of telomeric DNA-containing double minute chromosomes", Mol Cell, 12, 2003, 1489-98.
Reaction
40.1.2 Telomere C-strand (Lagging Strand) Synthesis
Authors
Reviewers
Price, C, 2006-07-13.
Description
References
40.1.2.1 Telomere C-strand synthesis initiation
Authors
Reviewers
Price, C, 2006-07-13.
Description
additional 20 nucleotides of DNA. There have been reports that TRF1 inhibits this activity at telomeres, though the mechanism and physiological
relevance of this inhibition remain to be elucidated.
References
EJ Smucker, JJ Turchi, "TRF1 inhibits telomere C-strand DNA synthesis in vitro", Biochemistry, 40, 2001, 2426-32.
40.1.2.1.1 The primase component of DNA polymerase:primase synthesizes a 6-10 nucleotide RNA primer on the G strand of the
telomere
Authors
Reviewers
Price, C, 2006-07-13.
Description
The complementary strand is synthesized by the polymerase primase complex using conventional RNA priming.
References
Reaction
40.1.2.1.2 The polymerase component of DNA polymerase alpha:primase synthesizes a 20-nucleotide primer on the G strand of the
telomere
Authors

Reviewers
Price, C, 2006-07-13.
Description
The complementary strand is synthesized by the polymerase primase complex using conventional RNA priming.
Reaction
40.1.2.2 Polymerase switching on the C-strand of the telomere
Authors
Reviewers
Price, C, 2006-07-13.
Description
After the primers are synthesized on the G-Rich strand, Replication Factor C binds to the 3'-end of the initiator DNA to trigger polymerase
switching. The non-processive nature of pol alpha catalytic activity and the tight binding of Replication Factor C to the primer-template junction
presumably lead to the turnover of the pol alpha:primase complex. After the Pol alpha-primase primase complex is displaced from the primer,
the proliferating cell nuclear antigen (PCNA) binds to form a "sliding clamp" structure. Replication Factor C then dissociates, and DNA
polymerase delta binds and catalyzes the processive synthesis of DNA.
References
40.1.2.2.1 RFC binding displaces Pol Alpha on the C-strand of the telomere
Authors
Reviewers
Price, C, 2006-07-13.
Description
References
Reaction
40.1.2.2.2 Loading of PCNA - Sliding Clamp Formation on the C-strand of the telomere
Authors

Reviewers
Price, C, 2006-07-13.
Description
References
Reaction
40.1.2.2.3 RFC dissociates after sliding clamp formation on the C-strand of the telomere
Authors
Reviewers
Price, C, 2006-07-13.
Description
DNA.
References
Reaction
40.1.2.2.4 Formation of Processive Complex on the C-strand of the telomere
Authors
Reviewers
Price, C, 2006-07-13.
Description
References
Natl Acad Sci U S A, 87, 1990, 5672-6.
Reaction
40.1.2.3 Processive synthesis on the C-strand of the telomere
Authors
Reviewers
Price, C, 2006-07-13.
Description
Once polymerase switching from pol alpha to pol delta is complete the processive synthesis of a short run of DNA called an Okazaki fragment
begins. DNA synthesis is discontinuous and as the extending Okazaki fragment reaches the RNA primer, this primer is folded into a
single-stranded flap, which is removed by endonucleases. The process of extension is completed by the ligation of adjacent Okazaki fragments.
References
40.1.2.3.1 Formation of C-strand Okazaki fragments
Authors
Reviewers
Price, C, 2006-07-13.
Description
References
Virol, 66, 1992, 6634-40.
10923-34.
Natl Acad Sci U S A, 87, 1990, 5672-6.
Reaction
40.1.2.3.2 Formation of the Flap Intermediate on the C-strand
Authors
Reviewers
Price, C, 2006-07-13.
Description
References
2001, 456-61.
Reaction
40.1.2.3.3 Removal of the Flap Intermediate from the C-strand
Authors
Reviewers
Price, C, 2006-07-13.
Description
References
A, 92, 1995, 7642-6.
40.1.2.3.3.1 RPA binds to the Flap on the C-strand
Authors
Reviewers
Price, C, 2006-07-13.
Description
References
2001, 456-61.
Reaction
40.1.2.3.3.2 Recruitment of Dna2 endonuclease to the C strand

Authors
Reviewers
Price, C, 2006-07-13.
Description
References
2001, 456-61.
Reaction
40.1.2.3.3.3 Removal of RNA primer and dissociation of RPA and Dna2 from the C-strand
Authors
Reviewers
Price, C, 2006-07-13.
Description
References
2001, 456-61.
vivo.", J Biol Chem, 275, 2000, 16518-29.
Reaction
40.1.2.3.3.4 Removal of remaining Flap from the C-strand
Authors
Reviewers
Price, C, 2006-07-13.
Description
References
Biol Chem, 275, 2000, 20949-55.
233-40.
Reaction
40.1.2.3.4 Joining of adjacent Okazaki fragments of the C-strand
Authors
Reviewers
Price, C, 2006-07-13.
Description
double-stranded DNA at the telomere.
References
Reaction
40.1.2.4 Disassociation of Processive Complex and Completed Telomere End
Authors
Reviewers
Price, C, 2006-07-13.
Description
At some point in the extension process a sufficient number of regulatory factors that repress telomere extension become bound to the extending
telomere. These factors include the TRF1 complexes, TRF2 complexes, telomerase, other factors, and the telomere itself. As repeats are added
to the G-rich strand, and once lagging strand synthesis completes the duplex, new binding sites become available for these repressive factors.
Once a balance is reached between telomere extension and the telomere repression factors, extension ceases. In this state extension
machinery disassociates, leaving the telomere to be folded into a stable conformation.
This module details a single transit through the telomere extension process, detailing the addition of two repeats, and the corresponding
synthesis of a section of lagging strand. An actual round of in vivo telomere extension would require thousands of telomere repeat additions, and
it is the repressive effect of the factors bound to these repeats that turns off telomere extension.
Reaction
40.2 Packaging Of Telomere Ends
Authors
Reviewers
Price, C, 2006-07-13.
Description
Multiple steps, including C-strand resection, telomerase-mediated elongation, and C-strand synthesis are involved in processing and maintaining
the telomere. Though this module posits a linear transit for the steps, in humans it is not well understood how these steps are coordinated and
what other events may be involved.
Telomeric DNA can form higher order structures. Electron microscopy of telomeric DNA isolated from human cells provided evidence for
lariat-type structures termed telomeric loops, or t-loops (Griffith et al., 1999). t-loops are proposed to result from the invasion of the 3' G-rich
single strand overhang into the double stranded telomeric TTAGGG repeat tract. The function of the t-loop is presumed to be the masking of the
3' telomeric overhang. Multiple protein factors can bind telomeric DNA and likely contribute to dynamic, higher order structures.
References
JD Griffith, L Comeau, S Rosenfield, RM Stansel, A Bianchi, H Moss, T de Lange, "Mammalian telomeres end in a large duplex loop", Cell, 97,
1999, 503-14.
40.2.1 Incorporation Of Extended And Processed Telomere End Into Associated Protein Structure
Authors
Reviewers
Price, C, 2006-07-13.
Description
In addition to telomerase-mediated elongation and C-strand synthesis, other DNA processing steps are likely involved in telomere maintenance.
In humans, nucleolytic activity is proposed to be involved in generating the G-rich 3' single strand overhang. In addition, differences in the
structure of the overhang at telomeres that have undergone leading vs. lagging strand replication suggest that DNA processing may be different
at these telomeres (Chai et al. 2006).
Many proteins associate with telomeric DNA. One complex that binds telomeres is called shelterin. Shelterin is a six-protein complex composed
of TRF1 and TRF2, which can bind double-stranded telomeric DNA, POT1, which can bind single-stranded telomeric DNA, and three other
factors, RAP1, TIN2, and TPP1 (reviewed in de Lange 2006 "Telomeres"). Human telomeric DNA is also bound by nucleosomes (Makarov et al.
1993; Nikitina and Woodcock 2004). A number of other proteins, including some that play roles in the DNA damage response, can be found at
telomeres (Zhu et al. 2000; Verdun et al. 2005).
Studies in yeast and humans indicate that the association of many proteins with telomeres is regulated through the cell cycle (Zhu et al. 2000;
Taggart et al. 2002; Fisher et al. 2004; Takata et al. 2004; Takata et al. 2005; Verdun et al. 2005). For instance, TRF1, MRE11, POT1, ATM, and
NBS1 display cell cycle regulated chromatin immunoprecipitation of telomeric DNA (Zhu et al. 2000; Verdun et al. 2005), and cytologically
observable hTERT and hTERC localize to a subset of telomeres only in S-phase (Jady et al. 2006; Tomlinson et al. 2006). These data indicate
that telomeres are dynamically remodeled through the cell cycle.
References
H Takata, Y Kanoh, N Gunge, K Shirahige, A Matsuura, "Reciprocal association of the budding yeast ATM-related proteins Tel1 and Mec1 with
telomeres in vivo", Mol Cell, 14, 2004, 515-22.
H Takata, Y Tanaka, A Matsuura, "Late S phase-specific recruitment of Mre11 complex triggers hierarchical assembly of telomere replication
proteins in Saccharomyces cerevisiae", Mol Cell, 17, 2005, 573-83.
T Nikitina, CL Woodcock, "Closed chromatin loops at the ends of chromosomes", J Cell Biol, 166, 2004, 161-5.
RE Verdun, L Crabbe, C Haggblom, J Karlseder, "Functional human telomeres are recognized as DNA damage in G2 of the cell cycle", Mol Cell,
20, 2005, 551-61.
T de Lange, "Shelterin: the protein complex that shapes and safeguards human telomeres", Genes Dev, 19, 2005, 2100-10.
XD Zhu, B Kuster, M Mann, JH Petrini, T de Lange, "Cell-cycle-regulated association of RAD50/MRE11/NBS1 with TRF2 and human
telomeres", Nat Genet, 25, 2000, 347-52.
JK Bedoyan, S Lejnine, VL Makarov, JP Langmore, "Condensation of rat telomere-specific nucleosomal arrays containing unusually short DNA
repeats and histone H1", J Biol Chem, 271, 1996, 18485-93.
W Chai, Q Du, JW Shay, WE Wright, "Human telomeres have different overhang sizes at leading versus lagging strands", Mol Cell, 21, 2006,
427-35.
Cell, 17, 2006, 944-54.
Cell, 17, 2006, 955-65.
TS Fisher, AK Taggart, VA Zakian, "Cell cycle-dependent regulation of yeast telomerase by Ku", Nat Struct Mol Biol, 11, 2004, 1198-205.
Reaction
40.2.2 Incorporation Of Extended And Processed Telomere End Into Higher Order T-Loop And Associated
Protein Structure
Authors
Reviewers
Price, C, 2006-07-13.
Description
In addition to telomerase-mediated elongation and C-strand synthesis, other DNA processing steps are likely involved in telomere maintenance.
In humans, nucleolytic activity is proposed to be involved in generating the G-rich 3' single strand overhang. In addition, differences in the
structure of the overhang at telomeres that have undergone leading vs. lagging strand replication suggest that DNA processing may be different
at these telomeres (Chai et al. 2006).
Electron microscopy studies of purified human telomeric DNA have provided evidence for telomeric loops, or t-loops (Griffith et al. 1999). t-loops
are proposed to result from invasion of the 3' G-rich single strand overhang into the double stranded portion of the telomeric TTAGGG repeat
tract. The strand displaced by invasion forms a structure called a D loop. The function of the t-loop is presumed to be the protection of the 3'
telomeric end. In vitro, the double strand telomeric DNA binding protein TRF2 can increase the frequency of t-loop formation. The prevalence of
the t-loops in vivo is not known.
Many proteins associate with telomeric DNA. One complex that binds telomeres is called shelterin. Shelterin is a six-protein complex composed
of TRF1 and TRF2, which can bind double-stranded telomeric DNA, POT1, which can bind single-stranded telomeric DNA, and three other
factors, RAP1, TIN2, and TPP1 (reviewed in de Lange 2006 "Telomeres"). Human telomeric DNA is also bound by nucleosomes (Makarov et al.
1993; Nikitina and Woodcock 2004). A number of other proteins, including some that play roles in the DNA damage response, can be found at
telomeres (Zhu et al. 2000; Verdun et al. 2005).
Studies in yeast and humans indicate that the association of many proteins with telomeres is regulated through the cell cycle (Smith et al. 1993;
Zhu et al. 2000; Taggart et al. 2002; Fisher et al. 2004; Takata et al. 2004; Takata et al. 2005; Verdun et al. 2005). For instance, TRF1, MRE11,
POT1, ATM, and NBS1 display cell cycle regulated chromatin immunoprecipitation of telomeric DNA (Zhu et al. 2000; Verdun et al. 2005), and
cytologically observable hTERT and hTERC localize to a subset of telomeres only in S-phase (Jady et al. 2006; Tomlinson et al. 2006). These
data indicate that telomeres are dynamically remodeled through the cell cycle.
References
H Takata, Y Kanoh, N Gunge, K Shirahige, A Matsuura, "Reciprocal association of the budding yeast ATM-related proteins Tel1 and Mec1 with
telomeres in vivo", Mol Cell, 14, 2004, 515-22.
H Takata, Y Tanaka, A Matsuura, "Late S phase-specific recruitment of Mre11 complex triggers hierarchical assembly of telomere replication
proteins in Saccharomyces cerevisiae", Mol Cell, 17, 2005, 573-83.
T Nikitina, CL Woodcock, "Closed chromatin loops at the ends of chromosomes", J Cell Biol, 166, 2004, 161-5.
RE Verdun, L Crabbe, C Haggblom, J Karlseder, "Functional human telomeres are recognized as DNA damage in G2 of the cell cycle", Mol Cell,
20, 2005, 551-61.
XD Zhu, B Kuster, M Mann, JH Petrini, T de Lange, "Cell-cycle-regulated association of RAD50/MRE11/NBS1 with TRF2 and human
telomeres", Nat Genet, 25, 2000, 347-52.
JK Bedoyan, S Lejnine, VL Makarov, JP Langmore, "Condensation of rat telomere-specific nucleosomal arrays containing unusually short DNA
repeats and histone H1", J Biol Chem, 271, 1996, 18485-93.
W Chai, Q Du, JW Shay, WE Wright, "Human telomeres have different overhang sizes at leading versus lagging strands", Mol Cell, 21, 2006,
427-35.
Cell, 17, 2006, 944-54.
Cell, 17, 2006, 955-65.
TS Fisher, AK Taggart, VA Zakian, "Cell cycle-dependent regulation of yeast telomerase by Ku", Nat Struct Mol Biol, 11, 2004, 1198-205.
Reaction
41 Transcription
Authors
Comai, L, Conaway, J, Conaway, R, Gustafsson, C, Hernandez, N, Hu, P, Larsson, N-G, Proudfoot, NJ, Reinberg, D, Timmers, H. T. M.,
2003-09-11.
Editors
Gillespie, ME, Gopinathrao, G, Joshi-Tope, G, 0000-00-00.
Reviewers
Paule, M, Zhao, X, Willis, I, 0000-00-00.
Description
Transcription by RNA Polymerase I, RNA polymerase II and RNA Polymerase III as well as transcription from mitochondrial promoters are
covered in Reactome as of this release.
41.1 RNA Polymerase I Transcription
Authors
Comai, L, 2003-07-03.
Editors
Reviewers
Paule, M, Zhao, X, 0000-00-00.
Description
The rRNA genes are transcribed by RNA polymerase I, one of three eukaryotic nuclear RNA polymerases. The polymerase is a multisubunit
complex, composed of two large subunits (the most conserved portions include the catalytic site that shares similarity with other eukaryotic and
bacterial multisubunit RNA polymerases) and a number of smaller subunits. Under a number of experimental conditions the core is competent to
mediate ribonucleic acid synthesis, in vivo however, it requires additional factors to select the appropriate template. In humans the RNA
transcript (45S) is approximately 13,000 nucleotides long. Before leaving the nucleus as assembled ribosomal particles, the 45S rRNA is
cleaved to give one copy each of the 28S rRNA, the 18S rRNA, and the 5.8S rRNA. Equal quantities of the three rRNAs are produced by initially
transcribing them as one transcript.
41.1.1 RNA Polymerase I Promoter Clearance
Authors
Comai, L, 2003-07-03.
Editors
Description
Promoter clearance is one of the rate-limiting steps in Polymerase I transcription. This step is composed of three phases, promoter opening,
transcription initiation and promoter escape.
41.1.1.1 RNA Polymerase I Promoter Opening
Authors
Comai, L, 2003-07-03.
Editors
Description
The activity of the upstream binding factor (UBF-1) plays an important role in the regulation of rRNA synthesis. Studies reveal that
phosphorylation of UBF-1 is required for its interaction with the RNA polymerase I complex, suggesting that phosphorylation of UBF-1 bound to
the rDNA promoter during promoter opening modulates the assembly of the transcription initiation complex.
41.1.1.1.1 UBF-1 Binds rDNA Promoter
Authors
Comai, L, 2003-07-03.
Editors
Description
UBF-1 binds directly to the CORE and UCE elements of the ribosomal DNA promoter. This binding is mediated by the HMG boxes (primarily
HMG box1). Phosphorylation may play a role in the modulation of UBF's DNA binding activity, as well as in subsequent steps. UBF is thought to
bind DNA in a conformation specific manner (as opposed to a sequence specific manner). The binding of UBF to the minor groove of DNA
induces strong DNA bending.
References
HM Jantzen, A Admon, SP Bell, R Tjian, "Nucleolar transcription factor hUBF contains a DNA-binding motif with homology to HMG proteins.",
Nature, 344, 1990, 830-6.
Reaction
41.1.1.1.2 Phosphorylation of UBF-1:rDNA Promoter
Authors
Comai, L, 2003-07-03.
Editors
Description
Phosphorylation of UBF-1, bound to the promoter, activates UBF-1 and recruits SL1, and eventually polymerase. This phosphorylation of UBF-1
by Erk1, has been shown to both weaken the binding of UBF-1 to DNA and to activate transcription (the authors of the paper showing these data
suggest that loosening the binding of UBF-1 with the promoter may somehow promote transcription initiation). Though not definitively worked out
phosphorylation of UBF-1 by Erk1 plays a role in the activation of the UBF-1:rDNA complex.
References
VY Stefanovsky, G Pelletier, R Hannan, T Gagnon-Kugler, LI Rothblum, T Moss, "An immediate response of ribosomal transcription to growth
factor stimulation in mammals is mediated by ERK phosphorylation of UBF.", Mol Cell, 8, 2001, 1063-73.
Reaction
41.1.1.2 RNA Polymerase I Transcription Initiation
Authors
Comai, L, 2003-07-03.
Editors
Description
During initiation the double-stranded DNA must be melted and transcription begins. SL1 forms and interacts with UBF-1 and the rDNA promoter.
It is this platform that will recruit active RNA polymerase I to the SL1:phosphorlated UBF-1:rDNA promoter complex.
Mammalian rRNA genes are preceded by a terminator element that is recognized by the SL1 complex. This SL1 modulated acetylation of the
basal Pol I transcription machinery has functional consequences suggesting that the reversible acetylation may be one way to regulate rDNA
transcription.
41.1.1.2.1 Formation of SL1
Description
Human SL1 is a four subunit complex composed of the TATA-binding protein (TBP) and three TBP-associated factors (TAFs): TAF(1)110,
TAF(1)63, and TAF(1)48. Note that none of these three TAFs for Pol I show any homology to the Pol II or Pol III TAFs. TAFs SL1 is a species
specific factor.
References
L Comai, N Tanese, R Tjian, "The TATA-binding protein and associated factors are integral components of the RNA polymerase I transcription
factor, SL1.", Cell, 68, 1992, 965-76.
SP Bell, CS Pikaard, RH Reeder, R Tjian, "Molecular mechanisms governing species-specific transcription of ribosomal RNA.", Cell, 59, 1989,
489-97.
L Comai, JC Zomerdijk, H Beckmann, S Zhou, A Admon, R Tjian, "Reconstitution of transcription factor SL1: exclusive binding of TBP by SL1 or
TFIID subunits.", Science, 266, 1995, 1966-72.
Reaction
41.1.1.2.2 Acetylation of SL1
Authors
Comai, L, 2003-07-03.
Editors
Description
Acetylation of the TAFI63 subunit of SL1 by PCAF stimulates the association of TAFI63 with DNA and stimulates pol I transcription in vitro.
Conversely, deacetylation by the NAD+-dependent deacetylase Sir2 represses pol I transcription.
References
V Muth, S Nadaud, I Grummt, R Voit, "Acetylation of TAF(I)68, a subunit of TIF-IB/SL1, activates RNA polymerase I transcription.", EMBO J, 20,
2001, 1353-62.
Reaction
41.1.1.2.3 Recruitment of Acetylated SL1 to phosUBF-1:rDNA Promoter
Authors
Comai, L, 2003-07-03.
Editors
Description
Human SL1 does not bind to DNA itself, rather it is recruited to the rDNA promoter through a physical interaction with UBF-1. Phosphorylation of
UBF-1 within the carboxy-terminal region is required for SL1 binding. SL1 consists of TATA-binding protein (TBP) and three associated factors
(TAFIs). SL1 has no sequence-specific DNA binding activity its recruitment to the promoter being mediated by specific interactions with UBF.
Once bound the SL1 complex makes direct contact with the DNA promoter and guides promoter-specific initiation.
Studies to identify the mechanistic relationship between SL1 and UBF-1 have indicated that the interaction between UBF-1 and SL1 is regulated
by tumor suppressor proteins such as Rb and P53, although it has also been proposed that Rb prevents UBF-1 from binding to DNA itself.
References
S Ciarmatori, PH Scott, JE Sutcliffe, A McLees, HM Alzuherri, JH Dannenberg, H te Riele, I Grummt, R Voit, RJ White, "Overlapping functions of
the pRb family in the regulation of rRNA synthesis.", Mol Cell Biol, 21, 2001, 5806-14.
KM Hannan, RD Hannan, SD Smith, LS Jefferson, M Lun, LI Rothblum, "Rb and p130 regulate RNA polymerase I transcription: Rb disrupts the
interaction between UBF and SL-1.", Oncogene, 19, 2000, 4988-99.
H Beckmann, JL Chen, T O'Brien, R Tjian, "Coactivator and promoter-selective properties of RNA polymerase I TAFs.", Science, 270, 1996,
1506-9.
W Zhai, L Comai, "Repression of RNA polymerase I transcription by the tumor suppressor p53.", Mol Cell Biol, 20, 2000, 5930-8.
JC Tuan, W Zhai, L Comai, "Recruitment of TATA-binding protein-TAFI complex SL1 to the human ribosomal DNA promoter is mediated by the
carboxy-terminal activation domain of upstream binding factor (UBF) and is regulated by UBF phosphorylation.", Mol Cell Biol, 19, 1999, 2872-9.
Reaction
41.1.1.2.4 Assembly of RNA Polymerase I Holoenzyme (human)
Description
At the beginning of this reaction, 1 molecule of 'DNA-directed RNA polymerases I, II, and III 17.1 kDa polypeptide ', 1 molecule of 'DNA-directed
RNA polymerase I 135 kDa polypeptide ', 1 molecule of 'DNA-directed RNA polymerases I, II, and III 7.0 kDa polypeptide ', 1 molecule of
'DNA-directed RNA polymerase I 16 kDa polypeptide ', 1 molecule of 'DNA-directed RNA polymerase I 40 kDa polypeptide ', and 1 molecule of
'DNA-directed RNA polymerase I largest subunit ' are present. At the end of this reaction, 1 molecule of 'RNA Polymerase I Holoenzyme
(Human)' is present.
This reaction takes place in the 'nucleolus'.
Source reaction
This reaction was inferred from the corresponding reaction "Assembly of RNA Polymerase I Holoenzyme (mouse)" in species Mus musculus.
RD Hannan, WM Hempel, A Cavanaugh, T Arino, SI Dimitrov, T Moss, L Rothblum, "Affinity purification of mammalian RNA polymerase I.
Identification of an associated kinase.", J Biol Chem, 273, 1998, 1257-67.
P Seither, I Grummt, "Molecular cloning of RPA2, the gene encoding the second largest subunit of mouse RNA polymerase I.", Genomics, 37,
1997, 135-9.
CZ Song, K Hanada, K Yano, Y Maeda, K Yamamoto, M Muramatsu, "High conservation of subunit composition of RNA polymerase I(A)
between yeast and mouse and the molecular cloning of mouse RNA polymerase I 40-kDa subunit RPA40.", J Biol Chem, 269, 1994, 26976-81.
P Seither, JF Coy, A Pouska, I Grummt, "Molecular cloning and characterization of the cDNA encoding the largest subunit of mouse RNA
polymerase I.", Mol Gen Genet, 255, 1997, 180-6.
RD Hannan, A Cavanaugh, WM Hempel, T Moss, L Rothblum, "Identification of a mammalian RNA polymerase I holoenzyme containing
components of the DNA repair/replication system.", Nucleic Acids Res, 27, 1999, 3720-7.
Reaction
41.1.1.2.5 Binding of Rrn3 to RNA Polymerase I
Authors
Editors
Description
After the assembly of the RNA Polymerase I Holoenzyme, Rrn3 binding occurs.
Reaction
41.1.1.2.6 Recruitment of Active RNA Polymerase I to SL1:phos.UBF-1:rDNA Promoter
Authors
Comai, L, 2003-07-03.
Editors
Description
Composed of Acetylated SL1, phosphorylated UBF-1 bound the rDNA promoter as well as the active RNA polymerase holoenzyme, rrn3 and
TFIIH the transcription initiation complex is complete. The assembly picture is incomplete, as the point at which TFIIH joins the complex is
unknown, though by the time that this complex is formed TFIIH is present (it has been included at this step for completeness). This forms the
transcriptionally active enzyme, that is capable of initiating transcription from the rDNA promoter.
References
X Yuan, J Zhao, H Zentgraf, U Hoffmann-Rohrer, I Grummt, "Multiple interactions between RNA polymerase I, TIF-IA and TAF(I) subunits
regulate preinitiation complex assembly at the ribosomal gene promoter.", EMBO Rep, 3, 2002, 1082-7.
Reaction
41.1.1.3 RNA Polymerase I Promoter Escape
Authors
Comai, L, 2003-07-03.
Editors
Description
As the active RNA Polymerase I complex leaves the promoter Rrn3 dissociates from the complex. RNA polymerase I Promoter Clearance is
complete and Chain Elongation begins.
41.1.1.3.1 Loss of Rrn3 from RNA Polymerase I promoter escape complex
Authors
Comai, L, 2003-07-03.
Editors
Description
Upon transcription initiation it is thought that RRN3 is inactivated and dissociates from the Loss of Rrn3 from the RNA Polymerase I promoter
escape complex. SL1 and UBF are thought to remain bound to the promoter for multiple rounds of transcription initiation
References
I Hirschler-Laszkiewicz, AH Cavanaugh, A Mirza, M Lun, Q Hu, T Smink, LI Rothblum, "Rrn3 becomes inactivated in the process of ribosomal
DNA transcription.", J Biol Chem, 278, 2003, 18953-9.
Reaction
41.1.2 RNA Polymerase I Chain Elongation
41.1.2.1 Elongation of pre-rRNA transcript
Description
At the beginning of this reaction, 1 molecule of 'elongating pre-rRNA transcript', and 1 molecule of 'NTP' are present. At the end of this reaction,
1 molecule of 'elongating pre-rRNA transcript' is present.
This reaction takes place in the 'nucleolus' and is mediated by the 'DNA-directed RNA polymerase activity' of 'RNA Polymerase I promoter
escape complex'.
Reaction
41.1.3 RNA Polymerase I Transcription Termination
Authors
Comai, L, 2003-07-03.
Editors
Description
Termination of transcription by RNA polymerase I is a 4 step process. Initially TTF-1 binds the template rDNA. This complex pauses polymerase
I allowing PTRF to interact with the quaternary complex releasing both pre-rRNA and Pol I from the template and TTF-1.
41.1.3.1 TTF-I binds to the Sal Box
Authors
Comai, L, 2003-07-03.
Editors
Description
The Transcription termination factor (TTF-1) binds an 18 base pair sequence element found in multiple copies in the nontranscribed spacer
downstream of the 18S rRNA coding region. This element is the termination signal for ribosomal gene transcription. It is bound by TTF-I, which
mediates the pausing of the elongating transcription complex.
References
I Grummt, H Rosenbauer, I Niedermeyer, U Maier, A Ohrlein, "A repeated 18 bp sequence motif in the mouse rDNA spacer mediates binding of
a nuclear factor and transcription termination.", Cell, 45, 1986, 837-46.
R Evers, I Grummt, "Molecular coevolution of mammalian ribosomal gene terminator sequences and the transcription termination factor TTF-I.",
Reaction
41.1.3.2 Polymerase I Transcription Complex/Nascent Pre rRNA Complex pauses at the TTF-I:Sal Box
Authors
Comai, L, 2003-07-03.
Editors
Description
The Polymerase I promoter escape complex/with the now complete nascent pre rRNA transcript pauses at the TTF-I bound Sal Box.
Reaction
41.1.3.3 PTRF Binds the Polymerase I Transcription Complex/Nascent Pre rRNA Complex paused at the TTF-I:Sal Box
Authors
Comai, L, 2003-07-03.
Editors
Description
Dissociation of paused ternary complexes requires the Polymerase I-transcript release factor (PTRF) a leucine zipper protein. PTRF is capable
of dissociating ternary Pol I transcription complexes, interacting with both TTF-I and Pol I to mediate the release of both Pol I and nascent
transcripts from the template.
References
P Jansa, SW Mason, U Hoffmann-Rohrer, I Grummt, "Cloning and functional characterization of PTRF, a novel protein which induces
dissociation of paused ternary transcription complexes.", EMBO J, 17, 1998, 2855-64.
Reaction
41.1.3.4 Dissociation of PTRF:Polymerase I/Nascent Pre rRNA Complex:TTF-I:Sal Box
Authors
Comai, L, 2003-07-03.
Editors
Description
PTRF binds the quaternary complex and mediates the dissociation of paused complex. PTRF interacts with the RNA polymerase I largest
subunit (p194), TTF-I and the U-rich 3' end of the nascent pre-rRNA.
References
P Jansa, SW Mason, U Hoffmann-Rohrer, I Grummt, "Cloning and functional characterization of PTRF, a novel protein which induces
dissociation of paused ternary transcription complexes.", EMBO J, 17, 1998, 2855-64.
Reaction
41.2 RNA Polymerase II Transcription
41.2.1 RNA Polymerase II Transcription Pre-Initiation
Authors
Reinberg, D, 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
References
41.2.1.1 Recognition and Binding of Core Promoter Elements by TFIID
Authors
Reinberg, D, 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
References
Reaction
41.2.1.2 Binding of TFIIA and TFIIB to the pol II promoter:TFIID complex
Authors
Reinberg, D, 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
References
Reaction
41.2.1.3 Recruitment of RNA Polymerase II Holoenzyme by TFIIF to the pol II promoter:TFIID:TFIIA:TFIIB complex
Authors
Reinberg, D, 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
References
Reaction
41.2.1.4 Binding of TFIIE to the growing preinitiation complex
Authors
Reinberg, D, 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
CTD by TFIIH.
References
Reaction
41.2.1.5 Formation of the closed pre-initiation complex
Authors
Reinberg, D, 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
References
Reaction
41.2.2 RNA Polymerase II Promoter Opening: First Transition
Authors
Timmers, H. T. M., 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
start site.
References
1992, 450-3.
7468-80.
145-56.
Reaction
41.2.3 RNA Polymerase II Transcription Initiation
Authors
Timmers, H. T. M., 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
initiation complex.
References
1479-90.
14990-7.
7468-80.
22, 2002, 762-73.
41.2.3.1 NTP Binds Active Site of RNA Polymerase II
Description
At the beginning of this reaction, 1 molecule of 'pol II open pre-initiation complex', and 2 molecules of 'NTP' are present. At the end of this
reaction, 1 molecule of 'Pol II initiation complex' is present.

Reaction
41.2.3.2 Nucleophillic Attack by 3'-hydroxyl Oxygen of nascent transcript on the Alpha Phosphate of NTP
Description
At the beginning of this reaction, 1 molecule of 'Pol II initiation complex' is present. At the end of this reaction, 1 molecule of 'Pol II Initiation
Reaction
41.2.3.3 Newly Formed Phosphodiester Bond Stabilized and PPi Released
Description
At the beginning of this reaction, 1 molecule of 'Pol II Initiation complex with phosphodiester-PPi intermediate' is present. At the end of this
reaction, 1 molecule of 'pyrophosphate', and 1 molecule of 'pol II transcription complex' are present.
Reaction
41.2.4 RNA Polymerase II Promoter Escape
Authors
Timmers, H. T. M., 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
References
7468-80.
41.2.4.1 Addition of the third nucleotide on the nascent transcript
Authors
Timmers, H. T. M., 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
positions -9 to +3.
References
7468-80.
22, 2002, 762-73.
Reaction
41.2.4.2 Addition of the fourth nucleotide on the Nascent Transcript: Second Transition
Authors
Timmers, H. T. M., 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
positions -9 to +4.
References
7468-80.
22, 2002, 762-73.
Reaction
41.2.4.3 Addition of Nucleotides 5 through 9 on the growing Transcript
Authors
Timmers, H. T. M., 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
References
1479-90.
7468-80.
Acad Sci U S A, 94, 1997, 9006-10.
Reaction
41.2.4.4 Addition of nucleotides 10 and 11 on the growing transcript: Third Transition
Authors
Timmers, H. T. M., 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
References
1479-90.
7468-80.
Reaction
41.2.4.5 Addition of nucleotides between position +11 and +30
Authors
Timmers, H. T. M., 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
References
1479-90.
Cell Biol, 21, 2001, 5815-25.
Acids Res, 29, 2001, 2706-14.
Reaction
41.2.5 RNA Pol II CTD phosphorylation and interaction with CE
Authors
Description
To facilitate co-transcriptional capping, and thereby restrict the cap structure to RNAs made by RNA polymerase II, the capping enzymes bind
directly to the RNA polymerase II. The C-terminal domain of the largest Pol II subunit contains several phosphorylation sites on its heptapeptide
repeats. The capping enzyme guanylyltransferase and the methyltransferase bind specifically to CTD phosphorylated at Serine 5 within the
CTD. Kinase subunit of TFIIH, Cdk7, catalyzes this phosphorylation event that occurs near the promoter. In addition, it has been shown that
binding of capping enzyme to the Serine-5 phosphorylated CTD stimulates guanylyltransferase activity in vitro.
41.2.5.1 Extrusion of 5'-end of 30 nt long transcript through the pore in Pol II complex
Description
At the beginning of this reaction, 1 molecule of 'Pol II transcription complex containing transcript to +30' is present. At the end of this reaction, 1
molecule of 'Pol II transcription complex containing extruded transcript to +30' is present.
Reaction
41.2.5.2 Phosphorylation (Ser5) of RNA pol II CTD
Authors
Description
Phosphorylation of serine 5 residue at the CTD of pol II largest subunit is an important step signaling the end of initiation and escape into
processive elongation processes. Cdk7 protein subunit of TFIIH phosphorylates RNA Pol II CTD serine 5 residues on its heptad repeats.
Reaction
41.2.5.3 RNA Polymerase II CTD (phosphorylated) binds to CE
Description
At the beginning of this reaction, 1 molecule of 'mRNA capping enzyme', and 1 molecule of 'Pol II transcription complex with (ser5)
phosphorylated CTD containing extruded transcript to +30' are present. At the end of this reaction, 1 molecule of 'RNA Pol II with phosphorylated
CTD: CE complex' is present.
Reaction
41.2.5.4 Activation of GT
Description
At the beginning of this reaction, 1 molecule of 'RNA Pol II with phosphorylated CTD: CE complex' is present. At the end of this reaction, 1
molecule of 'RNA Pol II with phosphorylated CTD: CE complex with activated GT' is present.

Reaction
41.2.5.5 SPT5 subunit of Pol II binds the RNA triphosphatase (RTP)
Authors
Description
The capping enzyme interacts with the Spt5 subunit of transcription elongation factor DSIF. This interaction may couple the capping reaction
with promoter escape or elongation, thereby acting as a Ã¢â‚¬Å"checkpointÃ¢â‚¬Â• to assure that capping has occurred before the polymerase
proceeds to make the rest of the transcript.
Reaction
41.2.6 RNA Polymerase II Transcription Elongation
Authors
Conaway, J, Conaway, R, 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
The mechanisms governing the process of elongation during eukaryotic mRNA synthesis are being unraveled by recent studies. These studies
have led to the expected discovery of a diverse collection of transcription factors that directly regulate the activities of RNA Polymerase II and
unexpected discovery of roles for many elongation factors in other basic processes like DNA repair, recombination, etc. The transcription
machinery and structural features of the major RNA polymerases are conserved across species. The genes active during elongation fall under
different classes like, housekeeping, cell-cycle regulated, development and differentiation specific genes etc. The list of genes involved in
elongation has been growing in recent times, and include: -TFIIS,DSIF, NELF, P-Tefb etc. that are involved in drug induced or
sequence-dependent arrest - TFIIF, ELL, elongin, elongator etc. that are involved in increasing the catalytic rate of elongation by altering the Km
and/or the Vmax of Pol II -FACT, Paf1 and other factors that are involved chromatin modification - DNA repair proteins, RNA processing and
export factors, the 19S proteasome and a host of other factors like Spt5-Spt5, Paf1, and NELF complexes, FCP1P etc. (Arndt and Kane, 2003).
Elongation also represents processive phase of transcription in which the activities of several mRNA processing factors are coupled to
transcription through their binding to RNA polymerase (Pol II). One of the key events that enables this interaction is the differential
phosphorylation of Pol II CTD. Phosphorylation pattern of CTD changes during transcription, most significantly at the beginning and during
elongation process. TFIIH-dependent Ser5 phosphorylation is observed primarily at promoter regions while P-Tefb mediated Ser2
phosphorylation is seen mainly in the coding regions, during elongation. Experimental evidence suggests a dynamic association of RNA
processing factors with differently modified forms of the polymerase during the transcription cycle. (Komarnitsky et al., 2000). [Komarnitsky et al
2000, Arndt & Kane 2003, Shilatifard et al 2003]
References
41.2.6.1 Formation of the Early Elongation Complex
Authors
Description
Transcription elongation by RNA polymerase II (RNAPII) is controlled by a number of trans-acting transcription elongation factors as well as by
cis-acting elements. Transcription elongation is a rate-limiting step for proper mRNA production in which the phosphorylation of Pol II CTD is a
crucial biochemical event. The role of CTD phosphorylation in transcription by Pol II is greatly impaired by protein kinase inhibitors such as
5,6-dichloro-1- ribofuranosylbenzimidazole (DRB), which block CTD phosphorylation and induce arrest of elongating Pol II. DRB-sensitive
activation Pol II CTD during elongation has enabled the isolation of two sets of factors -Negative Elongation Factors (NELF) and DRB sensitivity
inducing factor (DSIF). P-Tefb is a DRB-sensitive, cyclin-dependent CTD kinase composed of Cdk9 that carries out Serine-2 phosphorylation of
Pol II CTD during elongation.
The mechanism by which DSIF, NELF and P-TEFb act together in Pol II-regulated elongation is yet to be fully understood. Various biochemical
evidences point to a model in which DSIF and NELF negatively regulate elongation through interactions with polymerase containing a
hypophosphorylated CTD. Subsequent phosphorylation of the Pol II CTD by P-Tefb might promote elongation by inhibiting interactions of DSIF
and NELF with the elongation complex.
References
41.2.6.1.1 Hypophosphorylation of RNA Pol II CTD by FCP1P protein
Authors
Description
FCP1 dephosphorylates RNAP II in ternary elongation complexes as well as in solution and, therefore, is thought to function in the recycling of
RNAP II during the transcription cycle. Biochemical experiments suggest that human FCP1 targets CTDs that are phosphorylated at serine 2
(CTD-serine 2) and/or CTD-serine 5. It is also observed to stimulate elongation independent of its catalytic activity. Dephosphorylation of Ser2 -
phosphorylated Pol II results in hypophosphorylated form that disengages capping enzymes (CE).
References
Reaction
41.2.6.1.2 DSIF complex binds to RNA Pol II (hypophosphorylated)
Authors
Description
DSIF is a heterodimer consisting of hSPT4 (human homolog of yeast Spt4- p14) and hSPT5 (human homolog of yeast Spt5-p160). DSIF
association with Pol II may be enabled by Spt5 binding to Pol II creating a scaffold for NELF binding (Wada et al.,1998). Spt5 subunit of DSIF
can be phosphorylated by P-TEFb.
References
Dev, 12, 1998, 343-56.
Reaction
41.2.6.1.3 Formation of DSIF:NELF:early elongation complex
Authors
Description
NELF complex is a ~ 300 kDa multiprotein complex composed of 5 peptides (A - E): ~66,61,59,58 and 46 kDa. All these peptides are required
for NELF-mediated inhibition of Pol II elongation. NELF complex has been reported to bind to the pre-formed DSIF:RNA Pol II complex that may
act as a scaffold for its binding. NELF-A is suspected to be involved in Wolf-Hirschhorn syndrome.
Binding of DSIF:NELF to RNA Pol II CTD results in abortive termination of early elongation steps by the growing transcripts.
References
Reaction
41.2.6.2 Formation of RNA Pol II elongation complex
Authors
Description
TFIIS is a transcription factor involved in different phases of transcription, occurring in a major ubiquitous form and other tissue specific forms.
TFIIS stimulates RNA Pol II complex out of elongation arrest.
Other transcription factors like ELL, Elongin family members and TFIIF interact directly with elongating Pol II and increase its elongation rate.
These factors have been observed to act on naked DNA templates by suppressing transient pausing by the enzyme at all or most steps of
nucleotide addition. In Drosophila, ELL is found at a large number of transcriptionally active sites on polytene chromosomes. In general, ELL is
suspected to have more unidentified functions.
Elongin is a heterotrimeric protein complex that stimulates the overall rate of elongation. In addition, Elongin may act as an E3 Ubiquitin ligase.
Ubiquitylation of RNA Pol II occurs rapidly after genotoxic assault by UV light or chemicals, and results in degradation by proteasome. The FACT
complex appears to promote elongation by facilitating passage of polymerase through chromatin.
All these factors contribute to the formation of a processive elongation complex centered around the RNA Pol II complex positioned on the
DNA:RNA hybrid. This enables the RNA Pol II elongation complex to function as a platform that coordinates mRNA processing and export
(Reviewed by Shilatifard et al., 2003).
References
41.2.6.2.1 Hyperphosphorylation (Ser2) of RNA Pol II CTD by P-TEFb complex
Authors
Description
Cdk-9 is the kinase subunit of P-TEFb that phosphorylates Serine 2 on the heptapeptide repeats of Pol II CTD alleviating the negative action of
DSIF-NELF complex. This reaction is considered to be a rate limiting step for processive elongation. P-TEFb complex, that has a DRB-sensitive
cyclin-dependent kinase activity, is composed of ~43 kDa, Cdk9 kinase (PITALRE), and either Cyclin T1, Cyclin T2a, Cyclin T2b, or Cyclin K.
The exact mechanism by which P-TEFb removes the inhibition of elongation by DSIF-NELF is not yet known. P-TEFb is also capable of
phosphorylating Spt5 subunit of DSIF complex.
A P-TEFb complex (which contains only the Cyclin T1) is implicated in the efficient synthesis of human immunodeficiency virus-1 (HIV-1)
transcripts. Cyclin T1 subunit of the P-TEFb(Cyclin T1:Cdk9) complex interacts with HIV-1 encoded Tat protein that binds to the transactivation
response (TAR) element in the nascent HIV-1 transcript (reviewed in Price,2000).
The mechanism by which DSIF, NELF and P-TEFb or TAK/P-TEFb act together in Pol II-regulated elongation is yet to be fully understood.
Various biochemical evidences point to a model in which DSIF and NELF negatively regulate elongation through interactions with polymerase
containing a hypophosphorylated CTD. Subsequent phosphorylation of the Pol II CTD by P-TEFb might promote elongation by inhibiting
interactions of DSIF and NELF with the elongation complex.
References
Reaction
41.2.6.2.2 Recruitment of elongation factors to form elongation complex
Description
At the beginning of this reaction, 1 molecule of 'FACT complex', 1 molecule of 'Elongin Complex', 1 molecule of 'Early elongation complex with
hyperphosphorylated Pol II CTD', 1 molecule of 'TFIIH', 1 molecule of 'RNA polymerase II elongation factor ELL', and 1 molecule of 'TFIIS
protein' are present. At the end of this reaction, 1 molecule of 'Elongation complex' is present.
Reaction
41.2.6.3 Addition of nucleotides leads to transcript elongation
Authors
Description
High-resolution structures of free, catalytically active yeast Pol II and of an elongating form reveal that Pol II elongation complex includes
features like:
References
1863-76.
Reaction
41.2.6.4 Pol II elongation complex moves on the template as transcript elongates

Description
At the beginning of this reaction, 1 molecule of 'Processive elongation complex', and 1 molecule of 'NTP' are present. At the end of this reaction,
1 molecule of 'Elongation complex prior to separation', and 1 molecule of 'NTP' are present.
Reaction
41.2.6.5 Separation of elongating transcript from template
Description
At the beginning of this reaction, 1 molecule of 'Elongation complex prior to separation' is present. At the end of this reaction, 1 molecule of
'Elongation complex with separated and uncleaved transcript' is present.
Reaction
41.2.7 RNA Polymerase II Transcription Termination
Authors
Proudfoot, NJ, 2003-09-11.

Editors
Joshi-Tope, G, 0000-00-00.
Description
The detailed annotation of this section will be completed in the next release.
41.2.7.1 Cleavage of Growing Transcript in the Termination Region
Authors
Proudfoot, NJ, 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
This section includes the cleavage of both polyadenylated and non-polyadenylated transcripts.
In the former case polyadenylation has to precede transcript cleavage, while in the latter case there is no polyadenylation.
41.2.7.1.1 Cleavage of mRNA at the 3'-end
Editors
Joshi-Tope, G, 0000-00-00.
Description
polyadenylation.
References
Reaction
41.2.7.1.2 Cleavage of the 3'-end of Replication Dependent Histone Pre-mRNA
Authors
Editors
Description
References
Reaction
41.2.7.1.3 Cleavage of the 3'-end of the Histone Pre-mRNA
Authors
Editors

Description
References
Reaction
41.2.7.1.4 Cleavage of Intronless Pre-mRNA at 3'-end
Authors
Wahle, E, 2003-06-05.
Editors
Description
The polypeptide catalyzing the hydrolysis of the phosphodiester bond remains to be identified. Cleavage produces a 3'-OH on the upstream
fragment and a 5'-phosphate on the downstream fragment. At some unknown point after cleavage, the downstream fragment, CstF, CF I and CF
II are thought to be released, whereas CPSF and poly(A) polymerase remain to carry out polyadenylation.
References
Reaction
41.3 RNA Polymerase III Transcription
Authors
Hernandez, N, 2003-09-11.
Editors
Joshi-Tope, G, 0000-00-00.
Description
RNA polymerase III is one of three types of nuclear RNA polymerases present in eucaryotic cells. About 10% of the total transcription in dividing
cells can be attributed to its activity. It synthesizes an eclectic collection of catalytic or structural RNA molecules, some of which are involved in
protein synthesis, pre-mRNA splicing, tRNA processing, and the control of RNA polymerase II elongation, whereas some others have still
unknown functions. Like other RNA polymerases, RNA polymerase III cannot recognize its target promoters directly. Instead it is recruited to
specific promoter sequences through the help of transcription factors. There are three basic types of RNA polymerase III promoters, called types
1, 2, and 3(Geiduschek and Kassavetis, 1992). Although in vivo, RNA polymerase III may be recruited to these promoters as part of a large
complex (holo RNA polymerase III) containing the polymerase and its initiation factors (Wang et al., 1997), in vitro the reaction can be divided
into several steps. First, the promoter elements are recognized by DNA binding factors, which then recruit a factor known as TFIIIB. TFIIIB itself
then directly contacts RNA polymerase III. In human cells but not in S. cerevisiae, there are at least two versions of TFIIIB. One contains TBP,
Bdp1, and Brf1 (Brf1-TFIIIB), and the other TBP, Bdp1, and Brf2 (Brf2-TFIIIB) (Schramm et al., 2000; Teichmann et al., 2000).
References
G Kassavetis, "RNA polymerase III Transcription Complexes", Transcriptional Regulation, eds. McKnight, S.L., Yamamoto, K.R., 22, 1992,
247-280.
L Schramm, PS Pendergrast, Y Sun, N Hernandez, "Different human TFIIIB activities direct RNA polymerase III transcription from
TATA-containing and TATA-less promoters", Genes Dev, 14, 2000, 2650-63.
M Teichmann, Z Wang, RG Roeder, "A stable complex of a novel transcription factor IIB- related factor, human TFIIIB50, and associated
proteins mediate selective transcription by RNA polymerase III of genes with upstream promoter elements", Proc Natl Acad Sci U S A, 97, 2000,
14200-5.
Z Wang, T Luo, RG Roeder, "Identification of an autonomously initiating RNA polymerase III holoenzyme containing a novel factor that is
selectively inactivated during protein synthesis inhibition", Genes Dev, 11, 1997, 2371-82.
41.3.1 RNA Polymerase III Transcription Initiation
Authors
Editors
Joshi-Tope, G, 0000-00-00.
Description
There are three basic types of RNA polymerase III promoters. The three types of RNA polymerase III promoters are known as type 1, type 2,
and type 3 promoters. Type 1 promoters are found in the 5S genes and consist of a gene-internal element called the internal control region
(ICR), that is subdivided into A block, intermediate element, and C block (Bogenhagen, 1985; Sakonju et al., 1980). Type 2 promoters are found
in tRNA genes, Adenovirus 2 VAI gene, and other genes (Galli et al., 1981; Sharp et al., 1981). These promoters consists of two gene-internal
elements called the A and the B boxes. Type 3 promoters consist of a distal sequence element (DSE) that serves as an enhancer, a proximal
sequence element (PSE), and a TATA box (Baer et al., 1989; Lobo and Hernandez, 1989).
Some promoters combine elements from type 2 and 3 promoters. For example, the S. cerevisiae U6 promoter, also shown in the figure, contains
the TATA box typical of type 3 promoters and the A and B boxes typical of type 2 promoters. Moreover, in S. pombe, nearly all tRNA and 5S
genes contain a TATA box in addition to gene-internal elements, and the TATA box is required for transcription. Ã‚Â
References
SM Lobo, N Hernandez, "A 7 bp mutation converts a human RNA polymerase II snRNA promoter into an RNA polymerase III promoter", Cell,
58, 1989, 55-67.
DF Bogenhagen, S Sakonju, DD Brown, "A control region in the center of the 5S RNA gene directs specific initiation of transcription: II. The 3'
border of the region.", Cell, 19, 1980, 27-35.
S Sharp, D DeFranco, T Dingermann, P Farrell, D Soll, "Internal control regions for transcription of eukaryotic tRNA genes", Proc Natl Acad Sci
U S A, 78, 1981, 6657-61.
DD Brown, "A control region in the center of the 5S RNA gene directs specific initiation of transcription: I. The 5' border of the region.", Cell, 19,
1980, 13-25.
G Galli, H Hofstetter, ML Birnstiel, "Two conserved sequence blocks within eukaryotic tRNA genes are major promoter elements", Nature, 294,
1981, 626-31.
M Baer, TW Nilsen, C Costigan, S Altman, "Structure and transcription of a human gene for H1 RNA, the RNA component of human RNase P",
41.3.1.1 RNA Polymerase III Transcription Initiation From Type 1 Promoter
Authors
Editors
Joshi-Tope, G, 0000-00-00.
Description
The type 1 promoters recruit TFIIIA, the founding member of the C2H2 zinc finger family of DNA-binding proteins (Engelke et al., 1980; Sakonju
et al., 1981). The binding of TFIIIA then allows the binding of TFIIC (Lassar et al., 1983), a complex consisting of five subunits (which differs from
the six subunits in S. cerevisiae) in human cells and S. pombe. Once the DNA/TFIIIA/TFIIIC complex is formed, Brf1-TFIIIB joins the complex
and this in turn allows the recruitment of RNA polymerase III (Bieker et al., 1985; Setzer and Brown, 1985).
References
DR Engelke, SY Ng, BS Shastry, RG Roeder, "Specific interaction of a purified transcription factor with an internal control region of 5S RNA
genes", Cell, 19, 1980, 717-28.
DR Setzer, DD Brown, "Formation and stability of the 5 S RNA transcription complex", J Biol Chem, 260, 1985, 2483-92.
JJ Bieker, PL Martin, RG Roeder, "Formation of a rate-limiting intermediate in 5S RNA gene transcription", Cell, 40, 1985, 119-27.
S Sakonju, DD Brown, D Engelke, SY Ng, BS Shastry, RG Roeder, "The binding of a transcription factor to deletion mutants of a 5S ribosomal
RNA gene", Cell, 23, 1981, 665-9.
AB Lassar, PL Martin, RG Roeder, "Transcription of class III genes: formation of preinitiation complexes", Science, 222, 1983, 740-8.
41.3.1.1.1 Binding of TFIIIA To type 1 Promoter
Authors
Editors
Description
TFIIIA contains nine C2H2 zinc fingers (Arakawa et al., 1995). It binds to both the ICR region of the 5S RNA genes and to 5S RNA to form the
7S storage ribonucleoprotein particle (Pelham and Brown, 1980). Upon TFIIIA binding to the 5S gene, the TFIIIA zinc fingers are aligned over
the length of the ICR with the C-terminal zinc fingers in proximity to the 5 end, and the N-terminal zinc fingers in proximity to the 3 end, of the
ICR. Zinc fingers 1-3 contact the C block within the ICR and have been reported to contribute most of the binding energy of the full-length protein
(Clemens et al., 1992). However, TFIIIA fragments containing zinc fingers 4-9 bind to the A block and intermediate element within the ICR with
affinities close to those of the full-length protein. This and other observations suggest that simultaneous binding by all nine TFIIIA zinc fingers
requires energetically unfavorable distortions within the DNA, the protein, or both (Kehres et al., 1997).
References
HR Pelham, DD Brown, "A specific transcription factor that can bind either the 5S RNA gene or 5S RNA", Proc Natl Acad Sci U S A, 77, 1980,
4170-4.
DG Kehres, GS Subramanyan, VS Hung, GW Jr Rogers, DR Setzer, "Energetically unfavorable interactions among the zinc fingers of
transcription factor IIIA when bound to the 5 S rRNA gene", J Biol Chem, 272, 1997, 20152-61.
H Arakawa, H Nagase, N Hayashi, M Ogawa, M Nagata, E Takahashi, S Shin, Y Nakamura, "Molecular cloning, characterization, and
chromosomal mapping of a novel human gene (GTF3A) that is highly homologous to Xenopus transcription factor IIIA", Cytogenet Cell Genet,
70, 1995, 235-8.
KR Clemens, X Liao, V Wolf, PE Wright, JM Gottesfeld, "Definition of the binding sites of individual zinc fingers in the transcription factor IIIA-5S
RNA gene complex", Proc Natl Acad Sci U S A, 89, 1992, 10822-6.
Reaction
41.3.1.1.2 Binding of TFIIIC to TFIIIA:Type I Promoter complex
Authors
Editors
Description
Proteolytic and scanning electron microscopy studies indicate that S. cerevisiae TFIIIC consists of two globular domains separated by a flexible
linker, one of which, designated tau B, binds strongly to the B box, and the other, designated tau A, binds weakly to the A box, of type 2
promoters (Schultz et al., 1989). DNA footprinting and protein-protein interaction studies (Hsieh et al., 1999a; Hsieh et al., 1999b; Kovelman and
Roeder, 1992; Shen et al., 1996; Yoshinaga et al., 1989) support the models shown in the figure. The components of Brf1-TFIIIB (see TFIIIB
entries) are shown in grey, and TFIIIA is shown in blue. Sites of strong protein-DNA cross-linking are indicated by small ovals. Black and grey
rectangles show protein-protein contacts observed in human and S. cerevisiae TFIIIC subunits, respectively. The general arrangement of the
TFIIIC subunits on type 1 and 2 promoters is strikingly similar (Bartholomew et al., 1990; Braun et al., 1992a).
On type 1 promoters, S. cerevisiae TFIIIA cross-links strongly to the A box and more weakly over most of the gene, suggesting that it extends
over most of the gene (Braun et al., 1992a). Tfc3 is shifted downstream as compared to its position in the tRNA gene, with a main cross-link at
the 3 end of the C box and another one further downstream. The Tfc6 subunit cross-links at the end of the gene, like in type 2 genes. There is no
indication that the Tfc7 subunit contacts DNA in type 1 genes, but the Tfc1 subunit cross-links strongly upstream of the A box. The Tfc4 subunit
crosslinks to sites around and upstream of the start site of transcription (Braun et al., 1992a).
Numerous protein-protein contacts between various TFIIIC subunits have been described, which are symbolized by small rectangles in the
figure. The black rectangles indicate contacts identified with human TFIIIC subunits, the grey rectangles with S. cerevisiae TFIIIC subunits. Thus,
Tfc7 interacts directly with Tfc1 (Manaud et al., 1998). TTFIIIC90 interacts with TFIIIC220, TFIIIC110, and TFIIIC63 (Hsieh et al., 1999).
TFIIIC102 interacts with TFIIIC63 (Hsieh et al., 1999). Various TFIIIC subunits also interact directly with Brf1-TFIIIB subunits, as shown in the
figure. These protein-protein contacts are discussed below.
References
R Kovelman, RG Roeder, "Purification and characterization of two forms of human transcription factor IIIC", J Biol Chem, 267, 1992, 24446-56.
P Schultz, N Marzouki, C Marck, A Ruet, P Oudet, A Sentenac, "The two DNA-binding domains of yeast transcription factor tau as observed by
scanning transmission electron microscopy", EMBO J, 8, 1989, 3815-24.
YJ Hsieh, Z Wang, R Kovelman, RG Roeder, "Cloning and characterization of two evolutionarily conserved subunits (TFIIIC102 and TFIIIC63) of
human TFIIIC and their involvement in functional interactions with TFIIIB and RNA polymerase III", Mol Cell Biol, 19, 1999, 4944-52.
TK Kundu, RG Roeder, "The TFIIIC90 subunit of TFIIIC interacts with multiple components of the RNA polymerase III machinery and contains a
histone-specific acetyltransferase activity.", Mol Cell Biol, 19, 1999, 7697-704.
Reaction
41.3.1.1.3 Binding of TFIIIB to TFIIIC:TFIIIA:Type I Promoter complex

Authors
Editors
Description
The recruitment of Brf1-TFIIIB to type 1 and 2 promoters has been intensively studied in S. cerevisiae (Joazeiro et al., 1996). The Tfc4 subunit of
TFIIIC, which protrudes upstream of the transcription start site (Bartholomew et al., 1990), can interact with the Brf1 subunit of Brf1-TFIIIB (Moir
et al., 1997). The Tfc4 subunit, which contains 11 copies of the tetratricopeptide repeat (TPR), appears to undergo conformational changes
during binding that promote association with ScBrf1 and accommodate variable placements of TFIIIB (Moir et al., 1997). As shown in the figure,
a number of protein-protein associations involving both S. cerevisiae and human TFIIIC and TFIIIB subunits have been described, which may
participate in the recruitment of TFIIIB to type 1 and 2 promoters. Thus, Tfc8 has been show to interact with Bdp1 and TBP, and the
corresponding human protein TFIIIC90 with Brf1 (Hsieh et al., 1999); Tfc4 with Brf1 and Bdp1, and the corresponding human protein TFIIIC102
with Brf1 and TBP (Hsieh et al., 1999); and the human protein TFIIIC63 with Brf1 and TBP (Hsieh et al., 1999).
References
B Bartholomew, CF Meares, ME Dahmus, "Photoaffinity labeling of RNA polymerase III transcription complexes by nascent RNA", J Biol Chem,
265, 1990, 3731-7.
RD Moir, KV Puglia, IM Willis, "Autoinhibition of TFIIIB70 binding by the tetratricopeptide repeat-containing subunit of TFIIIC", J Biol Chem, 277,
2002, 694-701.
CA Joazeiro, GA Kassavetis, EP Geiduschek, "Alternative outcomes in assembly of promoter complexes: the roles of TBP and a flexible linker in
placing TFIIIB on tRNA genes", Genes Dev, 10, 1996, 725-39.
Reaction
41.3.1.1.4 Recruitment of RNA polymerase III to TFIIIB:TFIIIC:TFIIIA:Type 1 Promoter Complex

Authors
Editors
Description
Cross-linking experiments performed in the yeast system have shown that within the transcription initiation complex, eight RNA polymerase III
subunits can be cross-linked to DNA (Bartholomew et al., 1993). The C34 subunit, which is known to be required specifically for transcription
initiation but not elongation (Wang and Roeder, 1997; Werner et al., 1993), maps the furthest upstream of the transcription start site, in close
proximity to Brf1-TFIIIB (Bartholomew et al., 1993). Indeed, this subunit interacts with Brf1 (Khoo et al., 1994; Werner et al., 1993). The figure
illustrates this and other protein-protein contacts involving RNA polymerase III subunits and either TFIIIC or Brf1-TFIIIB subunits. The contacts
identified with S. cerevisiae proteins are indicated by stippled arrows, those identified with human protein by solid arrows. Both the S. cerevisiae
RNA polymerase III subunits C53 and ABC10a interact with Tfc4 (Dumay et al., 1999; Flores et al., 1999), and both C17 and C34 interact with
Brf1. The human subunit RPC62 interacts with TIIIC63, and RPC39 with the TFIIIC subunits TFIIIC90 and TFIIIC63 (Hsieh et al., 1999a) and the
Brf1-TFIIIB subunits Brf1 and TBP (Wang and Roeder, 1997). The contacts between RNA polymerase III and TFIIIC subunits are not absolutely
required for transcription in vitro with the S. cerevisiae system, in which TFIIIC can be stripped from the DNA after assembly of TFIIIB without
compromising transcription (Kassavetis et al., 1990) or, indeed, where transcription can be performed in the absence of TFIIIC on TATA
box-containing promoters (Kassavetis et al., 1995; Rth et al., 1996). Nevertheless, they may contribute to the recruitment of RNA polymerase III
in vivo.
Reaction
41.3.1.1.5 RNA Polymerase III Promoter Opening at Type 1 Promoters
Authors
Geiduschek, E, 2004-03-29.
Editors
Description
Pol III initiation complexes open the promoter spontaneously. Indeed, this is the general case for DNA-dependent RNA polymerases. Only pol II,
with its requirement for TFIIH-directed and ATP-dependent promoter opening is exceptional. TFIIH introduces a layer of mechanism that is not in
the repertoire of any other transcriptase. Thus, it is pol III-mediated transcription that is, from a mechanistic perspective, most directly
comparable with archaeal and also bacterial transcription.
As promoter opening has been analyzed only in the S. cerevisiae this event is Inferred from the homologous pathway in yeast.
Source reaction
This reaction was inferred from the corresponding reaction "RNA Polymerase III Promoter Opening" in species Saccharomyces cerevisiae.
P Fruscoloni, M Zamboni, G Panetta, A De Paolis, GP Tocchini-Valentini, "Mutational analysis of the transcription start site of the yeast
tRNA(Leu3) gene.", Nucleic Acids Res, 23, 1995, 2914-8.
GA Kassavetis, GA Letts, EP Geiduschek, "The RNA polymerase III transcription initiation factor TFIIIB participates in two steps of promoter
opening.", EMBO J, 20, 2001, 2823-34.
S Hahn, S Roberts, "The zinc ribbon domains of the general transcription factors TFIIB and Brf: conserved functional surfaces but different roles
in transcription initiation.", Genes Dev, 14, 2000, 719-30.
GA Kassavetis, JA Blanco, TE Johnson, EP Geiduschek, "Formation of open and elongating transcription complexes by RNA polymerase III.", J
Mol Biol, 226, 1992, 47-58.
Reaction
Authors
Editors
Joshi-Tope, G, 0000-00-00.
Description
The type 2 promoters can recruit TFIIIC without the help of TFIIIA because TFIIIC binds directly to the A and B boxes. As for the type 1
promoters, this then allows the binding of Brf1-TFIIIB and RNA polymerase III. Importantly, in the yeast system, once Brf1-TFIIIB has been
recruited to type 1 or 2 promoters, TFIIIA and/or TFIIIC can be stripped from the DNA with high salt or heparin treatment. Brf1-TFIIIB remains
bound to the DNA and is sufficient to direct multiple rounds of transcription .
41.3.1.2.1 Binding of TFIIIC to Type 2 promoter
Authors
Editors
Description
Proteolytic and scanning electron microscopy studies indicate that S. cerevisiae TFIIIC consists of two globular domains separated by a flexible
linker, one of which, designated tau B, binds strongly to the B box, and the other, designated tau A, binds weakly to the A box, of type 2
promoters (Marzouki et al., 1986). DNA footprinting and protein-protein interaction studies (Hsieh et al., 1999; Hsieh et al., 1999; Kovelman and
Roeder, 1992; Shen et al., 1996; Yoshinaga et al., 1989) support the models shown in the figure. The components of Brf1-TFIIIB (see TFIIIB
entries) are shown in grey, and TFIIIA is shown in blue. Sites of strong protein-DNA cross-linking are indicated by small ovals. Black and grey
rectangles show protein-protein contacts observed in human and S. cerevisiae TFIIIC subunits, respectively. The general arrangement of the
TFIIIC subunits on type 1 and 2 promoters is strikingly similar (Bartholomew et al., 1990; Braun et al., 1992a).
On a type 2 promoter, the S. cerevisiae Tfc3 subunit cross-links primarily just upstream of the B box and Tfc6 cross-links at the end of the gene
(Bartholomew et al., 1990). Tfc1 and Tfc7 have strong cross-links within and near the 3 end of the A box, respectively (Bartholomew et al.,
1990). Tfc8 does not cross-link to DNA, and after partial protease digestion of TFIIIC, is found in the tB domain. In addition, however, Tfc8
displays genetic interactions with Tfc1, TBP, and ScBdp1, and it associates with TBP in vitro, suggesting that it is also present in the tA domain.
The Tfc4 subunit cross-links to sites around and upstream of the transcription start site (Bartholomew et al., 1990) and directly contacts both the
ScBrf1 and ScBdp1 subunits of TFIIIB.
Numerous protein-protein contacts between various TFIIIC subunits have been described, which are symbolized by small rectangles in the
figure. The black rectangles indicate contacts identified with human TFIIIC subunits, the grey rectangles with S. cerevisiae TFIIIC subunits. Thus,
Tfc7 interacts directly with Tfc1. TTFIIIC90 interacts with TFIIIC220, TFIIIC110, and TFIIIC63 (Hsieh et al., 1999). TFIIIC102 interacts with
TFIIIC63 (Hsieh et al., 1999). Various TFIIIC subunits also interact directly with Brf1-TFIIIB subunits, as shown in the figure.
References
N Marzouki, S Camier, A Ruet, A Moenne, A Sentenac, "Selective proteolysis defines two DNA binding domains in yeast transcription factor
tau", Nature, 323, 1986, 176-8.
R Kovelman, RG Roeder, "Purification and characterization of two forms of human transcription factor IIIC", J Biol Chem, 267, 1992, 24446-56.
SK Yoshinaga, ND L'Etoile, AJ Berk, "Purification and characterization of transcription factor IIIC2", J Biol Chem, 264, 1989, 10726-31.
265, 1990, 3731-7.
Y Shen, M Igo, P Yalamanchili, AJ Berk, A Dasgupta, "DNA binding domain and subunit interactions of transcription factor IIIC revealed by
dissection with poliovirus 3C protease", Mol Cell Biol, 16, 1996, 4163-71.
Reaction
41.3.1.2.2 Binding of TFIIIB to TFIIC: Type 2 Promoter Complex
Authors
Editors
Description
The recruitment of Brf1-TFIIIB to type 1 and 2 promoters has been intensively studied in S. cerevisiae (Joazeiro et al., 1996). The Tfc4 subunit of
TFIIIC, which protrudes upstream of the transcription start site (Bartholomew et al., 1990), can interact with the Brf1 subunit of Brf1-TFIIIB (Moir
et al., 1997). The Tfc4 subunit, which contains 11 copies of the tetratricopeptide repeat (TPR), appears to undergo conformational changes
during binding that promote association with ScBrf1 and accommodate variable placements of TFIIIB (Moir et al., 1997). As shown in the figure,
a number of protein-protein associations involving both S. cerevisiae and human TFIIIC and TFIIIB subunits have been described, which may
participate in the recruitment of TFIIIB to type 1 and 2 promoters. Thus, Tfc8 has been show to interact with Bdp1 and TBP, and the
corresponding human protein TFIIIC90 with Brf1 (Hsieh et al., 1999); Tfc4 with Brf1 and Bdp1, and the corresponding human protein TFIIIC102
with Brf1 and TBP (Hsieh et al., 1999); and the human protein TFIIIC63 with Brf1 and TBP (Hsieh et al., 1999).
References
265, 1990, 3731-7.
RD Moir, KV Puglia, IM Willis, "Autoinhibition of TFIIIB70 binding by the tetratricopeptide repeat-containing subunit of TFIIIC", J Biol Chem, 277,
2002, 694-701.
CA Joazeiro, GA Kassavetis, EP Geiduschek, "Alternative outcomes in assembly of promoter complexes: the roles of TBP and a flexible linker in
placing TFIIIB on tRNA genes", Genes Dev, 10, 1996, 725-39.
Reaction
41.3.1.2.3 Recruitment of RNA Polymerase III to the TFIIIB:TFIIIC: Type 2 Promoter Complex
Description
At the beginning of this reaction, 1 molecule of 'TFIIIB:TFIIIC:Type 2 Promoter Complex', and 1 molecule of 'RNA Polymerase III Holoenzyme'
are present. At the end of this reaction, 1 molecule of 'RNA Polymerase III:TFIIIB:TFIIIC:Type 2 Promoter Complex' is present.
Reaction
Authors
Editors
Description
Source reaction
opening.", EMBO J, 20, 2001, 2823-34.
Mol Biol, 226, 1992, 47-58.
Reaction
Authors
Editors
Joshi-Tope, G, 0000-00-00.
Description
The metazoan-specific type 3 promoters, which are exemplified by the human U6 promoter, recruit a complex variously called the snRNA
activating protein complex (SNAPc) (Sadowski et al., 1993), the PSE binding protein (PBP) (Waldschmidt et al., 1991), or the PSE transcription
factor (PTF) (Murphy et al., 1992). The complex contains five types of subunits and binds to the PSE. Type 3 promoters also recruit Brf2-TFIIIB
through a combination of protein-protein contacts with SNAPc and a direct association of the TBP component of Brf2-TFIIIB with the TATA box.
This then allows RNA polymerase III to join the complex.
The down stream element (DSE) of type 3 promoters, which enhances transcription from the core promoter, almost invariably contains an
octamer sequence and an SPH element (also called NONOCT element)(Cheung et al., 1993; Danzeiser et al., 1993; Kunkel et al., 1996;
Myslinski et al., 1992). The octamer sequence recruits the POU domain protein Oct-1 (Herr et al., 1988; Sturm et al., 1988), and the SPH
element recruits a zinc finger protein known as Staf or SPH binding factor (SBF), which has been cloned from humans (Myslinski et al., 1998;
Rincon et al., 1998).
References
CL Sadowski, RW Henry, SM Lobo, N Hernandez, "Targeting TBP to a non-TATA box cis-regulatory element: a TBP-containing complex
activates transcription from snRNA promoters through the PSE", Genes Dev, 7, 1993, 1535-48.
R Waldschmidt, I Wanandi, KH Seifart, "Identification of transcription factors required for the expression of mammalian U6 genes in vitro", EMBO
J, 10, 1991, 2595-603.
DA Danzeiser, O Urso, GR Kunkel, "Functional characterization of elements in a human U6 small nuclear RNA gene distal control region", Mol
Cell Biol, 13, 1993, 4670-8.
E Myslinski, A Krol, P Carbon, "ZNF76 and ZNF143 are two human homologs of the transcriptional activator Staf", J Biol Chem, 273, 1998,
21998-2006.
RA Sturm, G Das, W Herr, "The ubiquitous octamer-binding protein Oct-1 contains a POU domain with a homeo box subdomain", Genes Dev, 2,
1988, 1582-99.
JC Rincon, SK Engler, BW Hargrove, GR Kunkel, "Molecular cloning of a cDNA encoding human SPH-binding factor, a conserved protein that
binds to the enhancer-like region of the U6 small nuclear RNA gene promoter", Nucleic Acids Res, 26, 1998, 4846-52.
M Tanaka, U Grossniklaus, W Herr, N Hernandez, "Activation of the U2 snRNA promoter by the octamer motif defines a new class of RNA
polymerase II enhancer elements", Genes Dev, 2, 1988, 1764-78.
GR Kunkel, TC Cheung, JH Miyake, O Urso, KJ McNamara-Schroeder, WE Stumph, "Identification of a SPH element in the distal region of a
human U6 small nuclear RNA gene promoter and characterization of the SPH binding factor in HeLa cell extracts", Gene Expr, 6, 1996, 59-72.
S Murphy, JB Yoon, T Gerster, RG Roeder, "Oct-1 and Oct-2 potentiate functional interactions of a transcription factor with the proximal
sequence element of small nuclear RNA genes", Mol Cell Biol, 12, 1992, 3247-61.
41.3.1.3.1 Binding of SNAPc, Oct-1, and Staf to Type 3 Promoter
Authors
Editors
Description
SNAPc binds specifically to the PSE. This binding is mediated in part by an unusual Myb domain within SNAP190 (Mittal et al., 1999; Wong et
al., 1998). However, even though a SNAP190 segment consisting of just the Myb domain binds DNA, within the complex the Myb domain is not
sufficient for binding. The smallest characterized subassembly of SNAPc subunits that binds specifically to DNA consists of SNAP190 aa
84-505, SNAP43 aa 1-268, and SNAP50 (Ma and Hernandez, 2000). Consistent with the requirement for parts of SNAP190 and SNAP50 for
DNA binding, UV cross-linking experiments suggest that both SNAP190 (Yoon et al., 1995) and SNAP50 (Henry et al., 1996) are in close
contact with DNA.
The binding of SNAPc to the PSE is stabilized by a number of cooperative interactions with other members of the transcription initiation complex
including Oct-1, TBP, and Brf2.
The binding of SNAPc to the core promoter is stabilized by a direct protein-protein contact with the Oct-1 POU domain.
SNAPc does not bind very efficiently to the PSE on its own. It contains a damper of DNA binding that resides within the C-terminal two thirds of
SNAP190 and/or SNAP45, because a subcomplex of SNAPc (mini-SNAPc) lacking these sequences binds much more efficiently to DNA than
complete SNAPc (Mittal et al., 1999). The damper within SNAPc is deactivated, probably through a conformational change, by a direct
protein-protein contact with the Oct-1 POU domain. The transcription initiation complex is illustrated in Figure 6. The protein-protein contact
between the Oct-1 POU domain and SNAPc involves a glutamic acid at position 7 within the Oct-1 POUS domain and a lysine at position 900
within SNAP190, which are symbolized in Figure 6 by small triangles (Ford et al., 1998; Hovde et al., 2002; Mittal et al., 1999). The octamer
sequence within the DSE and the PSE are separated by more than 150 base pairs, but the direct protein-protein contact is rendered possible by
the presence of a positioned nucleosome between the DSE and the PSE, which, as shown in the figure, probably brings into close proximity the
Oct-1 POU domain and SNAPc (Stunkel et al., 1997; Zhao et al., 2001).
Reaction
41.3.1.3.2 Binding of TFIIIB to SNAPc:Oct-1:Staf:Type 3 Promoter Complex

Authors
Editors
Description
The snRNA activating protein complex (SNAPc) (Sadowski et al., 1993), the PSE binding protein (PBP) (Waldschmidt et al., 1991), or the PSE
transcription factor (PTF) (Murphy et al., 1992). The complex contains five types of subunits and binds to the PSE. Type 3 promoters also recruit
Brf2-TFIIIB through a combination of protein-protein contacts with SNAPc and a direct association of the TBP component of Brf2-TFIIIB with the
TATA box. This then allows RNA polymerase III to join the complex.
References
CL Sadowski, RW Henry, SM Lobo, N Hernandez, "Targeting TBP to a non-TATA box cis-regulatory element: a TBP-containing complex
activates transcription from snRNA promoters through the PSE", Genes Dev, 7, 1993, 1535-48.
R Waldschmidt, I Wanandi, KH Seifart, "Identification of transcription factors required for the expression of mammalian U6 genes in vitro", EMBO
J, 10, 1991, 2595-603.
S Murphy, JB Yoon, T Gerster, RG Roeder, "Oct-1 and Oct-2 potentiate functional interactions of a transcription factor with the proximal
sequence element of small nuclear RNA genes", Mol Cell Biol, 12, 1992, 3247-61.
Reaction
41.3.1.3.3 Recruitment of RNA Polymerase III to TFIIIB:SNAPc:Type 3 Promoter Complex
Authors
Editors

Description
The binding of SNAPc to the PSE is stabilized not only by cooperative interactions with the Oct-1 POU domain, but also by cooperative
interactions with TBP and Brf2 (Hinkley et al., 2003 ; Ma and Hernandez, 2002; Mittal and Hernandez, 1997). Moreover, Brf2, which cannot bind
to DNA on its own, recognizes and stabilizes TBP bound to the TATA box (Cabart and Murphy, 2001; Cabart and Murphy, 2002; Ma and
Hernandez, 2002). Thus, the U6 transcription initiation complex is stabilized by a complex network of protein-protein and protein-DNA
interactions. Nothing is known, however, about how the complex recruits RNA polymerase III.
References
CS Hinkley, HA Hirsch, L Gu, B LaMere, RW Henry, "The small nuclear RNA-activating protein 190 Myb DNA binding domain stimulates TATA
box-binding protein-TATA box recognition", J Biol Chem, 278, 2003, 18649-57.
P Cabart, S Murphy, "BRFU, a TFIIB-like factor, is directly recruited to the TATA-box of polymerase III small nuclear RNA gene promoters
through its interaction with TATA-binding protein", J Biol Chem, 276, 2001, 43056-64.
V Mittal, N Hernandez, "Role for the amino-terminal region of human TBP in U6 snRNA transcription", Science, 275, 1997, 1136-40.
B Ma, N Hernandez, "Redundant cooperative interactions for assembly of a human U6 transcription initiation complex", Mol Cell Biol, 22, 2002,
8067-78.
"Assembly of human small nuclear RNA gene-specific transcription factor IIIB complex de novo on and off promoter.", J Biol Chem, 277, 2002,
26831-8.
Reaction
Authors
Editors
Description
Source reaction
opening.", EMBO J, 20, 2001, 2823-34.
Mol Biol, 226, 1992, 47-58.
Reaction
41.3.2 RNA Polymerase III Chain Elongation
Authors
Kassavetis, GA, Geiduschek, EP, 2004-02-27.
Editors
Description
Pol III initiation complexes open the promoter spontaneously similar to the mechanism employed in archaeal and bacterial transcription.
41.3.2.1 Initiation of RNA Polymerase III Productive Transcription

Authors
Editors
Description
The transition from abortive to productive transcription may occur at bp +5. The primary transcripts of pol III-transcribed genes are short, ~90 to
120 nt for tRNA and 5s RNA genes (which constitute the great majority of products) and even the longest transcripts (e.g. the RNA of the signal
recognition particles) are only ~500 nt. This event is inferred from an event in Saccharomyces cerevisiae.
Source reaction
This reaction was inferred from the corresponding reaction "Beginning of RNA Polymerase III Productive Transcription" in species
Saccharomyces cerevisiae.
H Matsuzaki, GA Kassavetis, EP Geiduschek, "Analysis of RNA chain elongation and termination by Saccharomyces cerevisiae RNA
polymerase III.", J Mol Biol, 235, 1994, 1173-92.
C Bardeleben, GA Kassavetis, EP Geiduschek, "Encounters of Saccharomyces cerevisiae RNA polymerase III with its transcription factors
during RNA chain elongation.", J Mol Biol, 235, 1994, 1193-205.
DN Roberts, AJ Stewart, JT Huff, BR Cairns, "The RNA polymerase III transcriptome revealed by genome-wide localization and
activity-occupancy relationships.", Proc Natl Acad Sci U S A, 100, 2003, 14695-700.
SA Shaaban, EV Bobkova, DM Chudzik, BD Hall, "In vitro analysis of elongation and termination by mutant RNA polymerases with altered
termination behavior.", Mol Cell Biol, 16, 1996, 6468-76.
Reaction
41.3.2.2 RNA Polymerase III Productive Transcription

Authors
Editors
Description
The principal cleavage products are dinucleotides, and they are produced in large stoichiometric excess over complete transcripts. Overall
productive RNA chain elongation proceeds quite rapidly. This event is inferred from an event in Saccharomyces cerevisiae.
Source reaction
This reaction was inferred from the corresponding reaction "Beginning of RNA Polymerase III Productive Transcription" in species
C Bardeleben, GA Kassavetis, EP Geiduschek, "Encounters of Saccharomyces cerevisiae RNA polymerase III with its transcription factors
during RNA chain elongation.", J Mol Biol, 235, 1994, 1193-205.
DN Roberts, AJ Stewart, JT Huff, BR Cairns, "The RNA polymerase III transcriptome revealed by genome-wide localization and
activity-occupancy relationships.", Proc Natl Acad Sci U S A, 100, 2003, 14695-700.
SA Shaaban, EV Bobkova, DM Chudzik, BD Hall, "In vitro analysis of elongation and termination by mutant RNA polymerases with altered
termination behavior.", Mol Cell Biol, 16, 1996, 6468-76.
Reaction
41.3.3 RNA Polymerase III Transcription Termination
Authors
Kassavetis, GA, Geiduschek, EP, 2004-02-27.
Editors
Joshi-Tope, G, 0000-00-00.
41.3.3.1 RNA Polymerase III Transcriptional Pause at Terminator Sequence
Authors
Editors
Description
RNA Polymerase III terminates transcription at extremely simple sites, consistent with its role in producing small transcripts. These sites are
essentially "Tn" (in the non-transcribed strand).
References
Source reaction
This reaction was inferred from the corresponding reaction "RNA Polymerase III Transcriptional Pause at Terminator Sequence" in species
Y Huang, RJ Maraia, "Comparison of the RNA polymerase III transcription machinery in Schizosaccharomyces pombe, Saccharomyces
cerevisiae and human.", Nucleic Acids Res, 29, 2001, 2675-90.
DD Brown, "Nucleotide sequences in Xenopus 5S DNA required for transcription termination.", Cell, 24, 1981, 261-70.
S Gunnery, Y Ma, MB Mathews, "Termination sequence requirements vary among genes transcribed by RNA polymerase III.", J Mol Biol, 286,
1999, 745-57.
S ChÃ©din, M Riva, A Sentenac, C Carles, "The RNA cleavage activity of RNA polymerase III is mediated by an essential TFIIS-like subunit and
is important for transcription termination.", Genes Dev, 12, 1999, 3857-71.
M Hamada, AL Sakulich, SB Koduru, RJ Maraia, "Transcription termination by RNA polymerase III in fission yeast. A genetic and biochemically
tractable model system.", J Biol Chem, 275, 2000, 29076-81.
NR Cozzarelli, SP Gerrard, M Schlissel, DD Brown, "Purified RNA polymerase III accurately and efficiently terminates transcription of 5S RNA
genes.", Cell, 34, 1983, 829-35.
DS Allison, BD Hall, "Effects of alterations in the 3' flanking sequence on in vivo and in vitro expression of the yeast SUP4-o tRNATyr gene.",
EMBO J, 4, 1985, 2657-64.
A Mazabraud, D Scherly, F MÃ¼ller, D Rungger, SG Clarkson, "Structure and transcription termination of a lysine tRNA gene from Xenopus
laevis.", J Mol Biol, 195, 1987, 835-45.
Reaction
41.3.3.2 RNA Polymerase III Termination and release of transcribed mRNA
Authors
Editors
Description
Efficient transcript production requires efficient release of RNA polymerase at the terminator; slow release at the terminator of a short
transcription unit quickly becomes rate limiting for transcription at steady state. Although pol III autonomously recognizes sequence terminators,
proteins that help to rapidly detach pol III from the terminator can affect the productivity of transcription if they eliminate termination as the
rate-limiting step.
La, NF1 family proteins, PC4 and topoisomerase I have been proposed as accessory pol III transcription factors that facilitate multi-cycle
transcription by hspol III, and are hence described as positive regulators of termination.
References
L Bai, RG Roeder, "Nuclear factor 1 (NF1) affects accurate termination and multiple-round transcription by human RNA polymerase III.", EMBO
J, 19, 2001, 6823-32.
CJ Yoo, SL Wolin, "La proteins from Drosophila melanogaster and Saccharomyces cerevisiae: a yeast homolog of the La autoantigen is
dispensable for growth.", Mol Cell Biol, 14, 1994, 5412-24.
RG Roeder, "DNA topoisomerase I and PC4 can interact with human TFIIIC to promote both accurate termination and transcription reinitiation by
RNA polymerase III.", Mol Cell, 1, 1998, 749-57.
Source reaction
This reaction was inferred from the corresponding reaction "RNA Polymerase III Termination and release of transcribed mRNA" in species
G Dieci, A Sentenac, "Detours and shortcuts to transcription reinitiation.", Trends Biochem Sci, 28, 2003, 202-9.
RJ Maraia, "Transcription termination factor La is also an initiation factor for RNA polymerase III.", Proc Natl Acad Sci U S A, 93, 1996, 3383-7.
N Hernandez, "Recruitment of RNA polymerase III to its target promoters.", Genes Dev, 16, 2002, 2593-620.
L Bai, RG Roeder, "Nuclear factor 1 (NF1) affects accurate termination and multiple-round transcription by human RNA polymerase III.", EMBO
J, 19, 2001, 6823-32.
EP Geiduschek, GA Kassavetis, "The RNA polymerase III transcription apparatus.", J Mol Biol, 310, 2001, 1-26.
CJ Yoo, SL Wolin, "La proteins from Drosophila melanogaster and Saccharomyces cerevisiae: a yeast homolog of the La autoantigen is
dispensable for growth.", Mol Cell Biol, 14, 1994, 5412-24.
N Lin-Marq, SG Clarkson, "Efficient synthesis, termination and release of RNA polymerase III transcripts in Xenopus extracts depleted of La
protein", EMBO J, 17, 1998, 2033-41.
RG Roeder, "DNA topoisomerase I and PC4 can interact with human TFIIIC to promote both accurate termination and transcription reinitiation by
RNA polymerase III.", Mol Cell, 1, 1998, 749-57.
G Dieci, A Sentenac, "Facilitated recycling pathway for RNA polymerase III.", Cell, 84, 1996, 245-52.
H Fan, AL Sakulich, JL Goodier, X Zhang, J Qin, RJ Maraia, "Phosphorylation of the human La antigen on serine 366 can regulate recycling of
RNA polymerase III transcription complexes.", Cell, 88, 1997, 707-15.
RJ Maraia, RV Intine, "Recognition of nascent RNA by the human La antigen: conserved and divergent features of structure and function.", Mol
Cell Biol, 21, 2001, 367-79.
Reaction
41.4 Transcription from mitochondrial promoters
Authors
Gustafsson, C, 2005-04-25.
Editors
Matthews, L, 0000-00-00.
Reviewers
Cantatore, P, 0000-00-00.
Description
Thirteen of the ~80 different proteins present in the respiratory chain of human mitochondria are encoded by the mitochondrial genome
(mtDNA). The circular mtDNA, which is present in 1000 to 10000 copies in the human cell, also encodes for 2 ribosomal RNAs, and 22 transfer
RNAs. The double-stranded mitochondrial genome lacks introns and the longer non-coding region contains the control elements for transcription
and replication of mtDNA (Shadel and Clayton, 1997). The two mtDNA strands are referred to as the heavy (H-strand) and the light (L-strand)
due to their differing G+T content. In human cells, each strand contains one single promoter for transcriptional initiation, the light-strand promoter
(LSP) or the heavy-strand promoter (HSP). Transcription from the mitochondrial promoters produce polycistronic precursor RNA encompassing
all the genetic information encoded in each of the specific strands. The primary transcripts are processed to produce the individual tRNA and
mRNA molecules (Clayton, 1991; Ojala et al., 1981). There is likely a second initiation site for heavy strand transcription, which produces RNAs
spanning the rDNA region. The resulting transcript including the genes for the two mitochondrial rRNAs and ends at the boundary between the
16 S rRNA and the tRNALeu(UUR) genes (Montoya et al., 1982; Montoya et al.,1983; Christianson and Clayton 1986). The existence of such a
separate transcription unit may explain why the steady-state levels of rRNAs are much higher than the steady state levels of mRNAs.
References
J Montoya, T Christianson, D Levens, M Rabinowitz, G Attardi, "Identification of initiation sites for heavy-strand and light-strand transcription in
human mitochondrial DNA", Proc Natl Acad Sci U S A, 79, 1982, 7195-9.
TW Christianson, "In vitro transcription of human mitochondrial DNA: accurate termination requires a region of DNA sequence that can function
bidirectionally", Proc Natl Acad Sci U S A, 83, 1986, 6277-81.
J Montoya, GL Gaines, G Attardi, "The pattern of transcription of the human mitochondrial rRNA genes reveals two overlapping transcription
units", Cell, 34, 1983, 151-9.
"Replication and transcription of vertebrate mitochondrial DNA", Annu Rev Cell Biol, 7, 1991, 453-78.
GS Shadel, "Mitochondrial DNA maintenance in vertebrates", Annu Rev Biochem, 66, 1997, 409-35.
D Ojala, J Montoya, G Attardi, "tRNA punctuation model of RNA processing in human mitochondria", Nature, 290, 1981, 470-4.
41.4.1 Mitochondrial transcription initiation
Authors
Editors
Matthews, L, 0000-00-00.
Reviewers
Description
Human mtDNA is transcribed by a dedicated mitochondrial RNA polymerase (POLRMT), which displays significant sequence similarity to the
monomeric RNA polymerases found in bacteriophages. In contrast to the phage T7 RNA polymerase, POLRMT cannot interact with promoter
DNA and initiate transcription on its own, but requires the presence of the mitochondrial transcription factor A (TFAM), and either transcription
factor B1 (TFB1M) or B2 (TFB2M). The 4 proteins of the basal mitochondrial transcription machinery have been purified in recombinant form and
used to reconstitute transcription in vitro with a promoter containing DNA fragment (Falkenberg et al., 2002). Although both TFB1M and TFB2M
can support in vitro transcription with POLRMT, TFB2M is at least two orders of magnitude more active than TFB1M and the physiological role of
TFB1M in mitochondrial transcription has not yet been completely defined. The TFB1M and TFB2M display primary sequence similarity to a
family of rRNA methyltransferases, which dimethylates two adjacent adenosine bases near the 3Ã¢â‚¬â„¢ end of the small subunit rRNA during
ribosome biogenesis (Falkenberg et al., 2002; McCulloch et al., 2002). Human TFB1M is, in fact, a dual function protein, which not only support
mitochondrial transcription in vitro, but also acts as a rRNA methyltransferase (Seidel-Rogol et al., 2003). The methyltransferase activity is not
required for transcription, since point mutations in conserved methyltransferase motifs of TFB1M revealed that it stimulates transcription in vitro
independently of S-adenosylmethionine binding and rRNA methyltransferase activity.
References
V McCulloch, BL Seidel-Rogol, GS Shadel, "A human mitochondrial transcription factor is related to RNA adenine methyltransferases and binds
S-adenosylmethionine", Mol Cell Biol, 22, 2002, 1116-25.
P Fernandez-Silva, JA Enriquez, J Montoya, "Replication and transcription of mammalian mitochondrial DNA", Exp Physiol, 88, 2003, 41-56.
"Transcription and replication of mitochondrial DNA", Hum Reprod, 15, 2000, 11-7.
BL Seidel-Rogol, V McCulloch, GS Shadel, "Human mitochondrial transcription factor B1 methylates ribosomal RNA at a conserved stem-loop",
Nat Genet, 33, 2003, 23-4.
M Falkenberg, M Gaspari, A Rantanen, A Trifunovic, NG Larsson, CM Gustafsson, "Mitochondrial transcription factors B1 and B2 activate
transcription of human mtDNA", Nat Genet, 31, 2002, 289-94.
41.4.1.1 TFAM binds to mitochondrial promoters
Authors
Editors
Matthews, L, 0000-00-00.
Reviewers
Description
Studies of human LSP have revealed that a minimal DNA fragment corresponding to position -28 to +16 relative to the transcription initiation site
is able to support transcription initiation in a mitochondrial extract (Chang and Clayton, 1984). TFAM interacts directly with nucleotides between
positions -35 and -17 (Fisher et al., 1987), and the exact distance between the TFAM-binding site and the transcription start site is essential for
promoter activity (Dairaghi et al., 1995).
References
RP Fisher, JN Topper, "Promoter selection in human mitochondria involves binding of a transcription factor to orientation-independent upstream
regulatory elements", Cell, 50, 1987, 247-58.
DJ Dairaghi, GS Shadel, "Human mitochondrial transcription factor A and promoter spacing integrity are required for transcription initiation",
DD Chang, "Precise identification of individual promoters for transcription of each strand of human mitochondrial DNA", Cell, 36, 1984, 635-43.
Reaction
41.4.1.2 Association of TFAM:mt promoter complex with POLRMT:TFB2M
Description
At the beginning of this reaction, 1 molecule of 'POLRMT:TFB2M complex', and 1 molecule of 'TFAM:mitochondrial promoter complex' are
present. At the end of this reaction, 1 molecule of 'POLRMT:TFB2M:TFAM:mitochondrial promoter complex' is present.
References
M Gaspari, M Falkenberg, NG Larsson, CM Gustafsson, "The mitochondrial RNA polymerase contributes critically to promoter specificity in
mammalian cells", EMBO J, 23, 2004, 4606-14.
M Falkenberg, M Gaspari, A Rantanen, A Trifunovic, NG Larsson, CM Gustafsson, "Mitochondrial transcription factors B1 and B2 activate
transcription of human mtDNA", Nat Genet, 31, 2002, 289-94.
Reaction
41.4.2 POLRMT chain elongation
Authors
Editors
Matthews, L, 0000-00-00.
Reviewers
Description
The details of the mechanism of POLRMT chain elongation have not yet been determined.
41.4.3 Mitochondrial transcription termination
Authors
Editors
Matthews, L, 0000-00-00.
Reviewers
Description
Transcription of the heavy (H)-strand of mitochondrial DNA (mtDNA) involves two overlapping transcription units (Montoyaet al.,1982; Montoya
et al., 1983). The first unit starts right upstream of the tRNAPhe gene and spans the tRNAPhe, rRNA 12S, rRNA 16S and tRNAVal genes
(initiation site IH1). The other starts about 100 bp further downstream (initiation site IH2), at the boundary between tRNAPhe and rRNA12S
genes, and produces a single polycistronic RNA that encompasses almost the entire length of the H-strand. The ribosomal transcription unit is
transcribed at a much higher rate compared to the other transcription unit and control of its expression is exerted both at the level of initiation
and termination (Gelfand and Attardi, 1981; Attardi et al., 1990). A central role in the control of termination has been attributed to the
mitochondrial transcription termination factor (mTERF), a 39-kDa protein that binds to a 28-base pair region of mtDNA located within the
tRNALeu(UUR) gene, at a position immediately downstream of the rRNA 16S gene (Fernandez-Silva et al.,1997; Kruse et al., 1989).
References
B Kruse, N Narasimhan, G Attardi, "Termination of transcription in human mitochondria: identification and purification of a DNA binding protein
factor that promotes termination", Cell, 58, 1989, 391-7.
J Montoya, T Christianson, D Levens, M Rabinowitz, G Attardi, "Identification of initiation sites for heavy-strand and light-strand transcription in
human mitochondrial DNA", Proc Natl Acad Sci U S A, 79, 1982, 7195-9.
TW Christianson, "In vitro transcription of human mitochondrial DNA: accurate termination requires a region of DNA sequence that can function
bidirectionally", Proc Natl Acad Sci U S A, 83, 1986, 6277-81.
P Fernandez-Silva, F Martinez-Azorin, V Micol, G Attardi, "The human mitochondrial transcription termination factor (mTERF) is a multizipper
protein but binds to DNA as a monomer, with evidence pointing to intramolecular leucine zipper interactions", EMBO J, 16, 1997, 1066-79.
J Montoya, GL Gaines, G Attardi, "The pattern of transcription of the human mitochondrial rRNA genes reveals two overlapping transcription
units", Cell, 34, 1983, 151-9.
G Attardi, A Chomyn, MP King, B Kruse, PL Polosa, NN Murdter, "Regulation of mitochondrial gene expression in mammalian cells", Biochem
Soc Trans, 18, 1990, 509-13.
R Gelfand, G Attardi, "Synthesis and turnover of mitochondrial ribonucleic acid in HeLa cells: the mature ribosomal and messenger ribonucleic
acid species are metabolically unstable", Mol Cell Biol, 1, 1981, 497-511.
41.4.3.1 Association of mTERF with the termination sequence
Authors
Editors
Matthews, L, 0000-00-00.
Reviewers
Description
The 39-kDa mitochondrial transcription termination factor (mTERF), binds to a 28-base pair region of mtDNA (3237-3249) located within the
tRNALeu(UUR) gene, at a position immediately downstream of the rRNA 16S gene (Fernandez-Silva et al., 1997; Kruse et al., 1989; Daga et al.,
1993). mTERF binding to the termination sequence block transcription bidirectionally in a partially purified human mitochondrial system (Shang
and Clayton, 1994). A polar termination activity has been also observed for the sea urchin homologue of mTERF, referred to as mtDBP with
respect of phage RNA polymerases (Fernandez Silva et al. 2001).
References
B Kruse, N Narasimhan, G Attardi, "Termination of transcription in human mitochondria: identification and purification of a DNA binding protein
factor that promotes termination", Cell, 58, 1989, 391-7.
P Fernandez-Silva, PL Polosa, M Roberti, B Di Ponzio, MN Gadaleta, J Montoya, P Cantatore, "Sea urchin mtDBP is a two-faced transcription
termination factor with a biased polarity depending on the RNA polymerase", Nucleic Acids Res, 29, 2001, 4736-43.
A Daga, V Micol, D Hess, R Aebersold, G Attardi, "Molecular characterization of the transcription termination factor from human mitochondria", J
Biol Chem, 268, 1993, 8123-30.
P Fernandez-Silva, F Martinez-Azorin, V Micol, G Attardi, "The human mitochondrial transcription termination factor (mTERF) is a multizipper
protein but binds to DNA as a monomer, with evidence pointing to intramolecular leucine zipper interactions", EMBO J, 16, 1997, 1066-79.
J Shang, DA Clayton, "Human mitochondrial transcription termination exhibits RNA polymerase independence and biased bipolarity in vitro", J
Biol Chem, 269, 1994, 29112-20.
Reaction
42 Translation
Authors
Merrick, WC, Bedwell, D, Gebauer, F, Anand, M, Balar, BA, Gross, S, Ortiz, PA, Ozturk, S, Pittman, YR, Ulloque, R, Hentze, MW, Kinzy, TG,
2005-03-29.
Editors
Tello-Ruiz, MK, Gillespie, ME, Gopinathrao, G, Matthews, L, 0000-00-00.
Reviewers
Hershey, JW, Sonenberg, N, Hinnebusch, AG, 0000-00-00.
Description
Protein synthesis is accomplished through the process of translation of an mRNA sequence into a polypeptide chain. This process can be
divided into three distinct stages: initiation, elongation and termination. During the initiation phase, the two subunits of the ribosome are brought
together to the translation start site on the mRNA where the polypeptide chain is to begin. Extension of the polypeptide chain occurs when a
specific aminoacyl-tRNA, as determined by the template mRNA, binds an elongating ribosome. The protein chain is released from the ribosome
when any one of three stop codons in the relevant reading frame on the mRNA is reached. Individual reactions at each one of these stages are
catalyzed by a number of initiation, elongation and release factors, respectively.
42.1 Eukaryotic Translation Initiation
Description
Initiation of translation in the majority of eukaryotic cellular mRNAs depends on the 5'-cap (m7GpppN) and involves ribosomal scanning of the 5'
untranslated region (5'-UTR) for an initiating AUG start codon. Therefore, this mechanism is often called cap-dependent translation initiation.
Proximity to the cap, as well as the nucleotides surrounding an AUG codon, influence the efficiency of the start site recognition during the
scanning process. However, if the recognition site is poor enough, scanning ribosomal subunits will ignore and skip potential starting AUGs, a
phenomenon called leaky scanning. Leaky scanning allows a single mRNA to encode several proteins that differ in their amino-termini. Several
eukaryotic cell and viral mRNAs initiate translation by an alternative mechanism that involves internal initiation rather than ribosomal scanning.
These mRNAs contain complex nucleotide sequences, called internal ribosomal entry sites, where ribosomes bind in a cap-independent manner
and start translation at the closest downstream AUG codon.
42.1.1 Cap-dependent Translation Initiation
Description
Translation initiation is a complex process in which the Met-tRNAi initiator, 40S, and 60S ribosomal subunits are assembled by eukaryotic
initiation factors (eIFs) into an 80S ribosome at the start codon of an mRNA. The basic mechanism for this process can be described as a series
of five steps: 1) formation of a pool of free 40S subunits, 2) formation of the ternary complex (Met-tRNAi/eIF2/GTP), and subsequently, the 43S
complex (comprising the 40S subunit, Met-tRNAi/eIF2/GTP, eIF3 and eIF1A), 3) activation of the mRNA upon binding of the cap-binding
complex eIF4F, and factors eIF4A, eIF4B and eIF4H, with subsequent binding to the 43S complex, 4) ribosomal scanning and start codon
recognition, and 5) GTP hydrolysis and joining of the 60S ribosomal subunit.
42.1.1.1 Formation of a pool of free 40S subunits
Description
The 80S ribosome dissociates into free 40S (small) and 60S (large) ribosomal subunits. Each ribosomal subunit is constituted by several
individual ribosomal proteins and rRNA.
42.1.1.1.1 Release of 40S and 60S subunits from the 80S ribosome
Description
80S monosomes dissociate into 40S and 60S ribosomal subunits. eIF1A promotes this dissociation.
References
JW Hershey, WC Merrick, "Pathway and mechanism of initiation of protein synthesis.", Translational Control of Gene Expression. (Sonenberg,
Hershey and Mathews, eds.), 2000, 33-88.
Reaction
42.1.1.1.2 eIF3 and eIF1A bind to the 40S subunit
Description
eIF3 and eIF1A bind to the 40S ribosomal subunit.
References
3078-87.
R Majumdar, A Bandyopadhyay, U Maitra, "Mammalian translation initiation factor eIF1 functions with eIF1A and eIF3 in the formation of a stable
40 S preinitiation complex.", J Biol Chem, 278, 2003, 6580-7.
H Goumans, A Thomas, A Verhoeven, HO Voorma, R Benne, "The role of eIF-4C in protein synthesis initiation complex formation.", Biochim
Biophys Acta, 608, 1980, 39-46.
Reaction
42.1.1.2 Formation of the ternary complex, and subsequently, the 43S complex
Description
Binding of the methionyl-tRNA initiator to the active eIF2:GTP complex results in the formation of the ternary complex. Subsequently, this
Met-tRNAi:eIF2:GTP (ternary) complex binds to the complex formed by the 40S subunit, eIF3 and eIF1A, to form the 43S complex.
References
3078-87.
1976, 9076-82.
(eds)., 1990, 521-526.
42.1.1.2.1 De novo formation of eIF2:GTP
Description
Activation of eIF2 through direct binding of GTP.

References
1976, 9076-82.
Reaction
42.1.1.2.2 Met-tRNAi binds to eIF2:GTP to form the ternary complex
Description
The ternary complex forms upon binding of the initiator methionyl-tRNA to the active eIF2:GTP complex.
References
1976, 9076-82.
Reaction
42.1.1.2.3 Formation of the 43S pre-initiation complex
Description
The ternary complex (Met-tRNAi:eIF2:GTP) binds to the complex formed by the 40S subunit, eIF3 and eIF1A, to form the 43S complex. eIF1A
promotes binding of the ternary complex to the 40S subunit within 43S. The initiator methionyl-tRNA from the ternary complex is positioned at
the ribosomal P site.
References
3078-87.
(eds)., 1990, 521-526.
Reaction
42.1.1.3 Activation of the mRNA upon binding of the cap-binding complex and eIFs, and subsequent binding to 43S
Description
The cap-binding complex is constituted by the initiation factors eIF4A, eIF4G and eIF4E. First, eIF4E must be released from the inactive
eIF4E:4E-BP complex. Then eIF4A interacts with eIF4G, and eIF4E binds to the amino-terminal domain of eIF4G, resulting in the formation of
the cap-binding complex eIF4F. eIF4A together with eIF4B or eIF4H is thought to unwind RNA secondary structures near the 5'-end of the
mRNA. The translation initiation complex is formed when the 43S complex binds the cap-bound mRNA.
References
3078-87.
854-9.
258, 1983, 5804-10.
42.1.1.3.1 Release of eIF4E from the inactive eIF4E:4E-BP complex
Description
eIF4E gets released from the inactive eIF4E:4EBP complex.
References
A Pause, GJ Belsham, AC Gingras, O DonzÃ©, TA Lin, JC Lawrence, N Sonenberg, "Insulin-dependent stimulation of protein synthesis by
phosphorylation of a regulator of 5'-cap function.", Nature, 371, 1994, 762-7.
Reaction
42.1.1.3.2 Formation of the cap-binding eIF4F complex
Description
eIF4A interacts with eIF4G, and eIF4E interacts with the amino-terminal domain of eIF4G to form the cap-binding complex eIF4F.
References
J Yoder-Hill, A Pause, N Sonenberg, WC Merrick, "The p46 subunit of eukaryotic initiation factor (eIF)-4F exchanges with eIF-4A.", J Biol Chem,
268, 1993, 5566-73.
Reaction
42.1.1.3.3 eIF4F binds to mRNP
Description
The factor eIF4E within the eIF4F (cap-binding) complex directly binds the 5'-cap on eukaryotic mRNAs. Note that the mRNA is in complex with
cytoplasmic proteins constituting an mRNP complex.
References
N Sonenberg, KM Rupprecht, SM Hecht, AJ Shatkin, "Eukaryotic mRNA cap binding protein: purification by affinity chromatography on
sepharose-coupled m7GDP.", Proc Natl Acad Sci U S A, 76, 1980, 4345-9.
258, 1983, 5804-10.
Reaction
42.1.1.3.4 Cap-bound mRNA is activated by helicases
Description
The DEAD-box RNA helicase eIF4A, together with the RNA-binding proteins eIF4B or eIF4H, is thought to unwind RNA secondary structures
near the 5'-end of the mRNA and in the presence of ATP.
References
258, 1983, 5804-10.
Reaction
42.1.1.3.5 Translation initiation complex formation
Description
References
3078-87.
854-9.
258, 1983, 5804-10.
42.1.1.3.5.1 Formation of translation initiation complexes containing mRNA that does not circularize
Description
References
3078-87.
854-9.
258, 1983, 5804-10.
Reaction
42.1.1.3.5.2 Formation of translation initiation complexes yielding circularized Ceruloplasmin mRNA in a 'closed-loop' conformation
Authors
Matthews, L, 2004-12-13.
Editors
Matthews, L, 0000-00-00.
Description
The precise order of events leading to the circularization of poly (A) mRNA during translation initiation is unknown. Here the association of PABP
with the poly (A) mRNA and the association of PABP with eIF4F are represented as occuring simultaneously after formation of the initiation
complex. However, it is also possible that these interactions occur during the formation of the translation initiation complex. The binding of eIF4F
to the cap and binding of PABP to the poly (A) tail, for example, may occur at the same time. In fact, the eIF4G-PABP interaction helps eIF4F to
bind tighter to the cap (Borman et al. 2000.) In addition, eIF4B and eIF4H bind more transiently to the mRNA and may not be part of an initial
complex in which PABP has not yet touched eIF4G.
References
H Imataka, A Gradi, N Sonenberg, "A newly identified N-terminal amino acid sequence of human eIF4G binds poly(A)-binding protein and
functions in poly(A)-dependent translation", EMBO J, 17, 1998, 7480-9.
AM Borman, YM Michel, KM Kean, "Biochemical characterisation of cap-poly(A) synergy in rabbit reticulocyte lysates: the eIF4G-PABP
interaction increases the functional affinity of eIF4E for the capped mRNA 5'-end", Nucleic Acids Res, 28, 2000, 4068-75.
Source reaction
This reaction was inferred from the corresponding reaction "Translation initiation complex formation" in species Homo sapiens.
3078-87.
854-9.
258, 1983, 5804-10.
Reaction
42.1.1.4 Ribosomal scanning and start codon recognition
Description
The 80S ribosome bound to the mRNA moves along the mRNA molecule from its initial site to the initiation codon and forms a 48S complex, in
which the initiation codon is base paired to the anticodon of the Met-tRNAi. Proper recognition of the AUG initiation codon depends on base
pairing with the anticodon of the Met-tRNAi and requires eIF1, eIF1A, eIF2 and eIF5.
References
3078-87.
854-9.
42.1.1.4.1 Ribosomal scanning
Description
The mRNA-bound ribosomal complex moves along the 5'-untranslated region (5'-UTR) of the mRNA from its initial site to the initiation codon to
form a 48S complex, in which the initiation codon (AUG) is base paired to the anticodon of the Met-tRNAi. It is not known whether eIF4A (or
another ATPase, such as DED1) facilitates scanning by melting mRNA secondary structures or by actively propelling the ribosome.
References
3078-87.
RY Chuang, PL Weaver, Z Liu, TH Chang, "Requirement of the DEAD-Box protein ded1p for messenger RNA translation.", Science, 275, 1997,
1468-71.
I Iost, M Dreyfus, P Linder, "Ded1p, a DEAD-box protein required for translation initiation in Saccharomyces cerevisiae, is an RNA helicase.", J
Biol Chem, 274, 1999, 17677-83.
TV Pestova, IN Shatsky, CU Hellen, "Functional dissection of eukaryotic initiation factor 4F: the 4A subunit and the central domain of the 4G
subunit are sufficient to mediate internal entry of 43S preinitiation complexes.", Mol Cell Biol, 16, 1997, 6870-8.
Reaction
42.1.1.4.2 Start codon recognition
Description
The AUG initiation codon in the mRNA is recognized by base pairing with the anticodon of the Met-tRNAi. This reaction requires eIF1, eIF1A,
eIF2 and eIF5.
References
854-9.
Reaction
42.1.1.5 GTP hydrolysis and joining of the 60S ribosomal subunit
Description
Hydrolysis of eIF2-GTP occurs after the Met-tRNAi has recognized the AUG. This reaction is catalyzed by eIF5 (or eIF5B) and is thought to
cause dissociation of all other initiation factors and allow joining of the large 60S ribosomal subunit. The 60S subunit joins - a reaction catalyzed
by eIF5 or eIF5B - resulting in a translation-competent 80S ribosome. Following 60S subunit joining, eIF5B hydrolyzes its GTP and is released
from the 80S ribosome, which is now ready to start elongating the polypeptide chain.
References
Biol, 116, 1978, 727-53.
3078-87.
Biol Chem, 250, 1975, 5556-62.
Biol Chem, 266, 1991, 14039-45.
2000, 332-5.
42.1.1.5.1 eIF2:GTP is hydrolyzed, eIFs are released

Description
Once the Met-tRNAi has recognized the AUG, eIF2-bound GTP is hydrolyzed. The reaction is catalyzed by eIF5 (or eIF5B) and is thought to
cause dissociation of all other initiation factors and allow joining of the large 60S ribosomal subunit. Release of the initiation factors from 40S
leaves the Met-tRNAi in the ribosomal P-site base-paired to the start codon on the mRNA.
References
Biol, 116, 1978, 727-53.
3078-87.
Biol Chem, 250, 1975, 5556-62.
Biol Chem, 266, 1991, 14039-45.
2000, 332-5.
Reaction
42.1.1.5.2 eIF5B:GTP is hydrolyzed and released
Description
Once the 60S subunit joins the translation initiation complex, eIF5B hydrolyzes its GTP and is released from the now 80S monosome. The fully
assembled 80s ribosome is now ready to start elongation of the polypeptide chain.
References
Biol Chem, 250, 1975, 5556-62.
2000, 332-5.
Reaction
42.1.1.5.3 The 60S subunit joins the translation initiation complex
Description
Joining of the 60S subunit to form the 80S ribosome is catalyzed by the presence of GTP-bound eIF5B.
References
Biol, 116, 1978, 727-53.
Biol Chem, 250, 1975, 5556-62.
2000, 332-5.
Reaction
42.1.1.6 Recycling of eIF2:GDP
Description
The active eIF2:GTP complex may be formed by direct binding of GTP to free eIF2 or by GDP-GTP exchange on the eIF2:GDP:eIF2B complex.
The eIF2:GDP complex binds eIF2B forming an eIF2:GDP:eIF2B intermediate complex. eIF2B is a guanine nucleotide releasing factor required
to cause GDP release so that a new GTP molecule can bind and activate eIF2. Phosphorylated eIF2:GDP sequesters all eIF2B as an inactive
complex, and thus, reuse of eIF2 is inhibited as a consequence of the tight bond it forms with eIF2B, which prevents nucleotide exchange.
Therefore, in the absence of free eIF2B, excess eIF2 remains in its inactive GDP-bound form and protein synthesis slows dramatically.
References
MJ Clemens, VM Pain, ST Wong, EC Henshaw, "Phosphorylation inhibits guanine nucleotide exchange on eukaryotic initiation factor 2.",
Nature, 296, 1982, 93-5.
42.1.1.6.1 Formation of eIF2:GDP:eIF2B intermediate
Description
Inactive eIF2:GDP binds eIF2B to form an eIF2:GDP:eIF2B intermediate.
References
Reaction
42.1.1.6.2 eIF2 activation
Description
eIF2B is a guanine nucleotide releasing factor that is required to cause GDP release so that a new GTP molecule can bind and activate eIF2, so
that it can be reused.
References
Reaction
42.1.2 Cap-independent Translation Initiation
Description
Initiation on several viral and cellular mRNAs is cap-independent and is mediated by binding of the ribosome to internal ribosome entry site
(IRES) elements. These elements are often found in characteristically long structured regions on the 5'-UTR of an mRNA that may or may not
have regulatory upstream open reading frames (uORFs). Both of these features on the 5'-end of the mRNA hinder ribosomal scanning, and thus
promote a cap-independent translation initiation mechanism. IRESs act as specific translational enhancers that allow translation initiation to
occur in response to specific stimuli and under the control of different trans-acting factors, as for example when cap-dependent protein synthesis
is shut off during viral infection. Such regulatory elements have been identified in the mRNAs of growth factors, protooncogenes, angiogenesis
factors, and apoptosis regulators, which are translated under a variety of stress conditions, including hypoxia, serum deprivation, irradiation and
apoptosis. Thus, cap-independent translational control might have evolved to regulate cellular responses in acute but transient stress conditions
that would otherwise lead to cell death, while the same mechanism is of major importance for viral mRNAs to bypass the shutting-off of host
protein synthesis after infection. Encephalomyocarditis virus (EMCV) and hepatitis C virus exemplify two distinct mechanisms of IRES-mediated
initiation. In contrast to cap-dependent initiation, the eIF4A and eIF4G subunits of eIF4F bind immediately upstream of the EMCV initiation codon
and promote binding of a 43S complex. Accordingly, EMCV initiation does not involve scanning and does not require eIF1, eIF1A, and the eIF4E
subunit of eIF4F. Nonetheless, initiation on some EMCV-like IRESs requires additional non-canonical initiation factors, which alter IRES
conformation and promote binding of eIF4A/eIF4G. Initiation on the hepatitis C virus IRES is simpler: a 43S complex containing only eIF2 and
eIF3 binds directly to the initiation codon as a result of specific interaction of the IRES and the 40S subunit.
References
M Holcik, N Sonenberg, RG Korneluk, "Internal ribosome initiation of translation and the control of cell death.", Trends Genet, 16, 2000, 469-73.
42.2 Eukaryotic Translation Elongation
Authors
Description
The translation elongation cycle adds one amino acid at a time to a growing polypeptide according to the sequence of codons found in the
mRNA. The next available codon on the mRNA is exposed in the aminoacyl-tRNA (aa-tRNA) binding site (A site) on the 30S subunit.
A: Ternary complexes of aa -tRNA:eEF1A:GTP enter the ribosome and enable the anticodon of the tRNA to make a codon/anticodon interaction
with the A-site codon of the mRNA. B: Upon cognate recognition, the eEF1A:GTP is brought into the GTPase activating center of the ribosome,
GTP is hydrolyzed and eEF1A:GDP leaves the ribosome. C: The peptidyl transferase center of ribosome catalyses the formation of a peptide
bond between the incoming amino acid and the peptide found in the peptidyl-tRNA binding site (P site). D: In the pre-translocation state of the
ribosome, the eEF2:GTP enters the ribosome, physically translocating the peptidyl-tRNA out of the A site to P site and leaves the ribosome
eEF2:GDP. This action of eEF2:GTP accounts for the precise movement of the mRNA by 3 nucleotides.Consequently, deacylated tRNA is
shifted to the E site. A ribosome associated ATPase activity is proposed to stimulate the release of deacylated tRNA from the E site subsequent
to translocation (Elskaya et al., 1991). In this post-translocation state, the ribosome is now ready to receive a new ternary complex.
This process is illustrated below with: an amino acyl-tRNA with an amino acid, a peptidyl-tRNA with a growing peptide, a deacylated tRNA with
an -OH, and a ribosome with A,P and E sites to accommodate these three forms of tRNA.
References
WC Merrick, J Nyborg, "The Protein Biosynthesis Elongation Cycle", Translational Control of Gene Expression, 2000, 89-127.
AV El'skaya, GV Ovcharenko, SS Palchevskii, ZM Petrushenko, FJ Triana-Alonso, KH Nierhaus, "Three tRNA binding sites in rabbit liver
ribosomes and role of the intrinsic ATPase in 80S ribosomes from higher eukaryotes", Biochemistry, 36, 1997, 10492-7.
LD Kapp, JR Lorsch, "The molecular mechanics of eukaryotic translation", Annu Rev Biochem, 73, 2004, 657-704.
42.2.1 eEF1A complexes with GTP

Authors
Description
The cycle of elongation starts with an empty ribosomal A-site and the peptidyl-tRNA in the P-site. eEF1A is activated by GTP binding and allows
for the subsequent binding of aminoacyl-tRNA (aa-tRNA).This process is illustrated below with a GTP molecule in white and eEF1A protein in
yellow.
Source reaction
This reaction was inferred from the corresponding reaction "reEF1A complexes with GTP" in species Oryctolagus cuniculus.
Reaction
42.2.2 eEF1A:GTP:aminoacyl tRNA ternary complex formation.
Authors
Description
The binding of eEF1A:GTP to aminoacyl tRNA (aa-tRNA) results in the formation of a ternary complex (eEF1A:GTP:aa-tRNA). Human eEF1A
and rabbit eEF1A are 100% identical, and prokaryotic homologue of eEF1A (EF-Tu) shows 59% identity in the GTP-binding domain.This
process is illustrated below with: a GTP molecule in white and eEF1A protein in yellow.
References
WC Merrick, TE Dever, TG Kinzy, SC Conroy, J Cavallius, CL Owens, "Characterization of protein synthesis factors from rabbit reticulocytes",
Source reaction
This reaction was inferred from the corresponding reaction "reEF1A:GTP:Aminoacyl-tRNA ternary complex formation" in species Oryctolagus
cuniculus.
Reaction
42.2.3 Peptide chain elongation
Authors
Description
The mechanism of a peptide bond requires the movement of three protons. First the deprotonation of the ammonium ion generates a reactive
amine, allowing a nucleophilic attack on the carbonyl group. This is followed by the loss of a proton from the reaction intermediate, only to be
taken up by the oxygen on the leaving group (from the end of the amino acid chain bound to the tRNA in the P-site). The peptide bond formation
results in the net loss of one water molecule, leaving a deacylated-tRNA in the P-site, and a nascent polypeptide chain one amino acid larger in
the A-site.
For the purpose of illustration, the figures used in the section show one amino acid being added to a peptidyl-tRNA with a growing peptide chain.
References
42.2.3.1 Aminoacyl-tRNA binds to the ribosome at the A-site
Authors
Description
resulting conformational change releases the aa-tRNA to the A-site, and GDP bound form eEF1A is released from the ribosome.
Insight into the mechanics of this system has been obtained from earlier works with rabbit reticulocytes and the E.coli system.
This process is illustrated below with: an amino acyl-tRNA with an amino acid, a peptidyl-tRNA with a growing peptide and a ribosome with A,P
References
MV Rodnina, T Pape, R Fricke, W Wintermeyer, "Elongation factor Tu, a GTPase triggered by codon recognition on the ribosome: mechanism
and GTP consumption", Biochem Cell Biol, 73, 1995, 1221-7.
Source reaction
This reaction was inferred from the corresponding reaction "Aminoacyl-tRNA binds to the ribosome at the A-site" in species Oryctolagus
cuniculus.
Reaction
42.2.3.2 Hydrolysis of eEF1A:GTP
Authors
Description
resulting conformational change releases the aa-tRNA to the A-site, and GDP bound form of eEF1A is released from the ribosome.
This process is illustrated below with: an amino acyl-tRNA with an amino acid,a peptidyl-tRNA with a growing peptide and a ribosome with A,P
References
Source reaction
This reaction was inferred from the corresponding reaction "Hydrolysis of reEF1A:GTP" in species Oryctolagus cuniculus.
Reaction
42.2.3.3 Peptide transfer from P-site tRNA to the A-site tRNA
Description
The A- and P-sites of the ribosome positions the aa-tRNA and peptidyl-tRNA such that a nucleophilic attack can occur between the amine group
of the A-site aa-tRNA and the carbonyl group of the growing peptide chain on the P-site tRNA, resulting in the formation of a peptide bond. The
carboxyl end of the peptide chain is uncoupled from the tRNA molecule in the P-site and forms a new peptide bond with the amino acid that is in
the A-site.
This process is illustrated below with: a peptidyl-tRNA with a growing peptide,a deacylated tRNA with an -OH and a ribosome with A,P and E
sites to accommodate these three forms of tRNA.
References
Source reaction
This reaction was inferred from the corresponding reaction "Peptide transfer from P-site tRNA to the A-site tRNA" in species Escherichia coli.
A, 66, 1970, 1164-9.
Reaction
42.2.3.4 Translocation of ribosome by 3 bases in the 3' direction
Authors
Description
Following peptide bond formation, GTP-bound eEF2 catalyzes the translocation of the deacylated tRNA in the P-site and the peptidyl-tRNA in
the A-site (the pre-translocation state) into the E- and P- sites (the post-translocation state), respectively. Thus, the mRNA advances by three
bases to expose the next codon in the A-site. After translocation, GDP-bound eEF2 leaves the ribosome to allow another round of elongation.
eEF2 is reactivated by the release of GDP and binds GTP for subsequent rounds.
This process is illustrated below with a peptidyl-tRNA with a growing peptide, a deacylated tRNA with an -OH and a ribosome with A,P and E
sites to accommodate these three forms of tRNA is also shown.
References
Biochemistry, 29, 1990, 10734-44.
Source reaction
This reaction was inferred from the corresponding reaction "Translocation of ribosome by 3 bases in the 3' direction" in species Escherichia coli.
Biochemistry, 29, 1990, 10734-44.
K Holschuh, D Riesner, HG Gassen, "Steps of mRNA translocation in protein biosynthesis", Nature, 293, 1981, 675-7.
A, 66, 1970, 1164-9.
Reaction
42.2.4 Regeneration of eEF1A:GTP by eEF1B activity
Authors
Description
The eEF1B complex binds to eEF1A and regulates its activity by catalyzing the release of GDP. Subsequently, GTP is able to bind eEF1A
allowing the formation of the ternary complex (eEF1A-GTP-aa-tRNA).In metazoans eEF1 protein family is composed of four subunits: eEF1A
and eEF1B alpha, beta, and gamma (formerly EF-1alpha, EF-1beta, EF-1delta, and EF-1gamma, respectively). Both eEF1B alpha and eEF1B
beta function as nucleotide exchange proteins. eEF1B gamma associates with eEF1B alpha and stimulates its exchange activity.
This process is illustrated below with a GTP molecule in white and eEF1A protein in yellow.The three subunits of eEF1B are also shown.
References
JM Perez, G Siegal, J Kriek, K Hard, J Dijk, GW Canters, W Moller, "The solution structure of the guanine nucleotide exchange domain of
human elongation factor 1beta reveals a striking resemblance to that of EF-Ts from Escherichia coli", Structure Fold Des, 7, 1999, 217-26.
Damme HT van, R Amons, R Karssies, CJ Timmers, GM Janssen, W Moller, "Elongation factor 1 beta of artemia: localization of functional sites
and homology to elongation factor 1 delta", Biochim Biophys Acta, 1050, 1990, 241-7.
GM Janssen, W Moller, "Elongation factor 1 beta gamma from Artemia. Purification and properties of its subunits.", Eur J Biochem, 171, 1988,
119-29.
Reaction
42.3 Eukaryotic Translation Termination
Authors
Bedwell, DM, 2004-11-09.
Editors
Description
The arrival of any of the three stop codons (UAA, UAG and UGA) into the ribosomal A-site triggers the binding of a release factor (RF) to the
ribosome and subsequent polypeptide chain release. In eukaryotes, the RF is composed of two proteins, eRF1 and eRF3. eRF1 is responsible
for the hydrolysis of the peptidyl-tRNA, while eRF3 provides a GTP-dependent function. The ribosome releases the mRNA and dissociates into
its two complex subunits, which can reassemble on another molecule to begin a new round of protein synthesis. It should be noted that at
present, there is no factor identified in eukaryotes that would be the functional equivalent of the bacterial ribosome release (or recycling) factor,
RRF, that catalyzes dissociation of the ribosome from the mRNA following release of the polypeptide
References
24, 2004, 7769-78.
S Ichikawa, A Kaji, "Molecular cloning and expression of ribosome releasing factor", J Biol Chem, 264, 1989, 20054-9.
42.3.1 GTP bound eRF3:eRF1 complex binds the peptidyl tRNA:mRNA:80S Ribosome complex
Authors
Description
References
L Frolova, Goff X Le, HH Rasmussen, S Cheperegin, G Drugeon, M Kress, I Arman, AL Haenni, JE Celis, M Philippe, "A highly conserved
eukaryotic protein family possessing properties of polypeptide chain release factor", Nature, 372, 1994, 701-3.
S Hoshino, M Imai, M Mizutani, Y Kikuchi, F Hanaoka, M Ui, T Katada, "Molecular cloning of a novel member of the eukaryotic polypeptide
chain-releasing factors (eRF). Its identification as eRF3 interacting with eRF1.", J Biol Chem, 273, 1998, 22254-9.
Source reaction
This reaction was inferred from the corresponding reaction "GTP bound eRF3:eRF1 complex binds the peptidyl-tRNA:mRNA:Ribosome
24, 2004, 7769-78.
Reaction
42.3.2 GTP Hydrolysis by eRF3 bound to the eRF1:mRNA:polypeptide:80S Ribosome complex
Authors
Description
References
16677-80.
Source reaction
This reaction was inferred from the corresponding reaction "GTP Hydrolysis by eRF3 bound to the eRF1:mRNA:polypeptide:80S Ribosome
24, 2004, 7769-78.
Reaction
42.3.3 Polypeptide release from the eRF3-GDP:eRF1:mRNA:80S Ribosome complex
Authors
Description
References
16677-80.
Source reaction
This reaction was inferred from the corresponding reaction "Polypeptide release from the eRF3-GDP:eRF1:mRNA:80S Ribosome complex" in
species Saccharomyces cerevisiae.
24, 2004, 7769-78.
Reaction
43 mRNA Processing
Authors
Carmichael, GG, Hammarskjold, ML, Hastings, M, Krainer, AR, Marzluff, WF, Wahle, E, Zhang, Z, 2003-06-05.
Editors
Reviewers
Nilsen, TW, Krainer, AR, 0000-00-00.
Description
Processing of mRNA encompasses the capping of mRNA, processing of mRNAs with and without introns, and also modification of mRNA, as in
mRNA editing.
43.1 mRNA Capping
Description
The 5'-ends of all eukaryotic pre-mRNAs studied thus far are converted to cap structures. The cap is thought to influence splicing of the first
intron, and is bound by 'cap-binding' proteins, CBP80 and CBP20, in the nucleus. The cap is important for translation initiation, and it also
interacts with the poly(A)terminus, via proteins, resulting in circularization of the mRNA to facilitate multiple rounds of translation. The cap is also
important for mRNA stability, protecting it from 5' to 3' nucleases, and is required for mRNA export to the cytoplasm.
The capping reaction usually occurs very rapidly on nascent transcripts; after the synthesis of only a few nucleotides by RNA polymerase II. The
capping reaction involves the conversion of the 5'-end of the nascent transcript from a triphosphate to a diphosphate by a RNA
5'-triphosphatase, followed by the addition of a guanosine monophosphate by the mRNA guanylyltransferase, to form a 5'-5'-triphosphate
linkage. This cap is then methylated by 2'-O-methyltransferases.
References
AJ Shatkin, JL Manley, "The ends of the affair: capping and polyadenylation.", Nat Struct Biol, 7, 2000, 838-42.
K Mizumoto, Y Kaziro, "Messenger RNA capping enzymes from eukaryotic cells.", Prog Nucleic Acid Res Mol Biol, 34, 1988, 1-28.
D Bentley, "Coupling RNA polymerase II transcription with pre-mRNA processing.", Curr Opin Cell Biol, 11, 1999, 347-51.
43.1.1 Capping complex formation
Authors
Editors
Description
The capping enzyme binds the 5'-end of the nascent transcript soon after it is synthesized on the DNA template, and results in the formation of
the capping complex along with the C-terminal domain of RNA polymerase II, and Spt5.
Reaction
43.1.2 Hydrolysis of the 5'-end of the nascent transcript by the capping enzyme
Authors
Description
After the capping complex is formed, the RNA triphosphatase activity of the capping enzyme hydrolyzes the 5'-end phosphate group of the
nascent mRNA transcript to a diphosphate.
The RNA triphosphatase (RTP) domain of mammalian capping enzyme is a member of a superfamily of phosphatases that include the protein
tyrosine phosphatases, some lipid phosphatases, and several nucleic acid phosphatases. This family uses a conserved nucleophilic cysteine
residue to attack the target phosphate. A transient phospho-cysteinyl enzyme intermediate is then hydrolyzed to regenerate the enzyme active
site. It should be noted that while higher eukaryotic capping enzymes use PTP-like triphosphatase domains, the yeast triphosphatases are a
completely different class of enzymes. The yeast RTPs are metal-dependent phosphatases. RNA 5'-triphosphatase (RTP) catalyzed first
reaction can be represented as:pppN(pN)n + GTP -> ppN(pN)n + Pi; (n=20-25)
References
Reaction
43.1.3 Formation of the CE:GMP intermediate complex
Authors
Description
A highly conserved lysine within the guanylyltransferase (GT) site of the mRNA capping enzyme attacks the alpha-phosphate of GTP. An
enzyme-GMP covalent intermediate is formed.
References
Reaction
43.1.4 Transfer of GMP from the capping enzyme GT site to 5'-end of mRNA
Authors
Description
The diphosphate 5'-end of the mRNA is joined to the GMP, releasing it from the enzyme. At this time, it is unclear how the RNA diphosphate end
is transferred from the active site of the triphosphatase to the guanylyltransferase site. The covalent enzyme-GMP complex can form in the
absence of RNA.
Guanylyltransferase (GT) catalyzed second reaction can be represented as:ppN(pN)n + GTP -> GpppN(pN)n + PPi
References
Reaction
43.1.5 Dissociation of transcript with 5'-GMP from GT
Authors
Description
GMP capped mRNA transcript dissociates from GT for further modification.
Reaction
43.1.6 Methylation of GMP-cap by RNA Methyltransferase
Authors
Description
In the final step of the capping reaction, the methyltransferase takes a methyl group from S-adenosyl-methionine to the N7 position of the cap
guanine. N7G-methyltransferase (MT) mediated reaction can be represented as:
GpppN(pN)n + S-adenosylmethionine (Adomet) ->m7GpppN(pN)n + S-adenosylhomocysteine (Adohcy).
References
T Tsukamoto, Y Shibagaki, Y Niikura, K Mizumoto, "Cloning and characterization of three human cDNAs encoding mRNA
(guanine-7-)-methyltransferase, an mRNA cap methylase.", Biochem Biophys Res Commun, 251, 1998, 27-34.
Reaction
43.1.7 Recognition and binding of the mRNA cap by the cap-binding complex
Authors
Editors
Description
The cap binding complex binds to the methylated GMP cap on the nascent mRNA transcript.
Reaction
43.2 Processing of Capped pre-mRNA
Authors
Joshi-Tope, G, 2004-06-23.
Description
This section describes the processing of pre-mRNAs with and without introns, after they are capped. Although uncapped pre-mRNAs can be
spliced in vitro, all pre-mRNAs are believed to be capped in vivo.
43.2.1 Processing of Capped Intron-Containing Pre-mRNA
Authors
Carmichael, GG, Hammarskjold, ML, Hastings, M, Krainer, AR, Marzluff, WF, Wahle, E, Zhang, Z, 2003-06-05.
Editors
Reviewers
Nilsen, TW, 0000-00-00.
Description
Any covalent change in a primary (nascent) mRNA transcript is mRNA Processing. For successful gene expression, the primary mRNA
transcript needs to be converted to a mature mRNA prior to its translation into polypeptide. Eucaryotic mRNAs undergo a series of complex
processing reactions; these begin on nascent transcripts as soon as a few ribonucleotides have been synthesized during transcription by RNA
Polymerase II, through the export of the mature mRNA to the cytoplasm, and culminate with mRNA turnover in the cytoplasm.
43.2.1.1 Internal Methylation of mRNA
Editors
Joshi-Tope, G, 0000-00-00.
Description
In addition to the methylation of the 5'-cap, there is methylation of internal nucleotides in the mRNA. This methylation can occur in translated and
untranslated regions. One to three methyl groups have been seen per mRNA molecule, but methylation is non-stoichiometric. The most frequent
methylation observed is at the N6 position of adenosine. The function of mRNA internal methylation, if any, is unknown.
References
JA Bokar, ME Shambaugh, D Polayes, AG Matera, FM Rottman, "Purification and cDNA cloning of the AdoMet-binding subunit of the human
mRNA (N6-adenosine)-methyltransferase.", RNA, 3, 1998, 1233-47.
Reaction
43.2.1.2 Formation of pre-mRNPs
Description
After the nascent pre-mRNA undergoes the initial capping and methylation reactions, it gets associated with numerous factors, including the
various heterogeneous nuclear ribonucleoproteins (hnRNPS), the nuclear Cap-Binding Complex, and many splicing factors that make the
pre-mRNA a substrate for splicing, 3'-end processing, and in some cases editing.
Reaction
43.2.1.3 mRNA Splicing
Description
The process in which excision of introns from the primary transcript of messenger RNA (mRNA) is followed by ligation of the two exon termini
exposed by removal of each intron, is called mRNA splicing. Most of the mRNA is spliced by the major pathway, involving the U1, U2, U4, U5
and U6 snRNPs. A minor fraction, about 1 %, of the mRNAs are spliced via the U12 dependent pathway.
References
Z Zhou, LJ Licklider, SP Gygi, R Reed, "Comprehensive proteomic analysis of the human spliceosome.", Nature, 419, 2002, 182-5.
43.2.1.3.1 mRNA Splicing - Major Pathway
Description
The splicing of pre-mRNA occurs within a large, very dynamic complex, designated the 'spliceosome'. The 50-60S spliceosomes are estimated
to be 40-60 nm in diameter, and have molecular weights in the range of 3-5 million kDa. Small nuclear RNAs (snRNAs) U1, U2, U4, U5, and U6,
are some of the best characterized components of spliceosomes, and are known to play key roles not only in spliceosomal assembly, but also in
the two catalytic steps of the splicing reaction. Over 150 proteins have been detected in spliceosomes, and only a subset of these has been
characterized. The characterization, and the determination of the functions of the protein components of the spliceosome, is still work in
progress.
During spliceosome assembly, the snRNAs and the spliceosomal proteins assemble on the pre-mRNA in a stepwise pathway. First the E
complex forms, followed by complexes A and B; the C complex forms next and contains the products of the first step of the splicing reaction.
Complexes called i and D form as a consequence of the second step of the splicing reaction, which contain the excised intron and the spliced
exons, respectively.
43.2.1.3.1.1 Formation of the Spliceosomal E complex
Description
Pre-mRNA transcripts become rapidly associated with many RNA-binding proteins, including hnRNP proteins, cap-binding proteins, SR proteins,
etc; in the test tube this binding does not require splice sites or ATP. The E complex, or early complex, is the first detectable functional
intermediate in spliceosome assembly in vitro. It is an ATP-independent complex. When a functional 5' splice site is present, it is bound by the
U1 snRNP. The splicing factor U2AF (65 and 35 kDa subunits) binds to the polypyrimidine tract (Y)n and the AG dinucleotide at the 3' splice site,
respectively. SF1/mBBP binds to the branch site. Binding of many of these factors is cooperative; e.g., SR proteins and U2AF apparently interact
with each other, facilitating their binding to the pre-mRNA. In the presence of ATP, the E complex is converted to the first ATP-dependent
spliceosomal complex, namely the A complex.
References
Reaction
43.2.1.3.1.2 Formation of the Spliceosomal A Complex
Description
The A complex is the first ATP-dependent complex in spliceosome assembly. U2AF recruits the U2 snRNP to bind to the branch site in the E
complex in an ATP-dependent fashion, to form the A complex. The U2 snRNA base-pairs with the branch site, causing the branch-site
adenosine to bulge out, which later positions it for nucleophilic attack at the 5' splice site. The A complex serves as a substrate for formation of
the B complex.
References
Reaction
43.2.1.3.1.3 Formation of the Spliceosomal B Complex
Authors
Krainer, AR, 2003-06-05.

Editors
Joshi-Tope, G, 0000-00-00.
Description
The formation of the B complex is ATP-dependent, and both the 5' and 3' splice sites are essential for B complex assembly. The U4 and U6
snRNPS are extensively base-paired, and this U4:U6 complex associates with the U5 snRNP to form a tri-snRNP particle. This tri-snRNP
particle then binds to the spliceosomal A complex, to form the spliceosomal B complex.
References
Reaction
43.2.1.3.1.4 Formation of an intermediate Spliceosomal C complex
Authors
Krainer, AR, 2003-06-05.
Editors
Joshi-Tope, G, 0000-00-00.
Description
The spliceosomal C complex is a very short-lived intermediate; the splicing intermediates are rapidly converted to splicing products. Also, the
spliced products are released very rapidly, and no complex containing both the splicing products has been isolated. Conversion of the
spliceosomal B complex to the spliceosomal C complex requires ATP. The extensive base-pairing between the U4 and U6 snRNAs is disrupted
during the formation of the C complex, which is thought to require helicase-type activity associated with the DEAD box factors.
References
Reaction
43.2.1.3.1.5 Formation of the active Spliceosomal C complex
Description
The active C complex is formed due to a conformational change in the intermediate C complex. After formation of the active C complex, the
splicing reactions occur very rapidly.
References
Reaction
43.2.1.3.1.6 Lariat Formation and 5'-Splice Site Cleavage

Authors
Krainer, AR, 2003-06-05.
Description
In the first catalytic step of mRNA splicing, the 2' OH group of the bulged A at the branch site performs a nucleophilic attack on the 5' splice site
phosphodiester bond, resulting in cleavage of the bond between the 5' exon and the 5' end of the intron, and formation of a new bond between
the 5' end of the intron and the branch site A. This results in a lariat-shaped intermediate, with the intron still attached to the 3' exon. The branch
site A has a 2'-5' phosphodiester bond with the G at the beginning of the intron, in addition to the usual 5'-3' and 3'-5'phosphodiester bonds.
References
RA Padgett, MM Konarska, PJ Grabowski, SF Hardy, PA Sharp, "Lariat RNA's as intermediates and products in the splicing of messenger RNA
precursors.", Science, 225, 1984, 898-903.
B Ruskin, AR Krainer, T Maniatis, MR Green, "Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro.", Cell, 38,
1984, 317-31.
Reaction
43.2.1.3.1.7 Formation of Exon Junction Complex
Description
At the beginning of this reaction, 1 molecule of 'Magoh-Y14 complex', and 1 molecule of 'Spliceosomal active C complex with lariat containing,
5'-end cleaved pre-mRNP:CBC complex' are present. At the end of this reaction, 1 molecule of 'Exon Junction Complex' is present.
References
H Le Hir, D Gatfield, E Izaurralde, MJ Moore, "The exon-exon junction complex provides a binding platform for factors involved in mRNA export
and nonsense-mediated mRNA decay", EMBO J, 20, 2001, 4987-97.
H Le Hir, E Izaurralde, LE Maquat, MJ Moore, "The spliceosome deposits multiple proteins 20-24 nucleotides upstream of mRNA exon-exon
junctions", EMBO J, 19, 2000, 6860-9.
VL Reichert, Hir H Le, MS Jurica, MJ Moore, "5' exon interactions within the human spliceosome establish a framework for exon junction
complex structure and assembly", Genes Dev, 16, 2002, 2778-91.
Reaction
43.2.1.3.1.8 Cleavage at the 3'-Splice Site and Exon Ligation
Description
The second step of the splicing reaction results in cleavage of the transcript at the 3'splice site, and results in ligation of the two exons and
excision of the intron.
Reaction
43.2.1.3.2 mRNA Splicing - Minor Pathway
Editors
Joshi-Tope, G, 0000-00-00.
Description
The splicing of a subset of pre-mRNA introns occurs by a second pathway, designated the AT-AC or U12-dependent splicing pathway. AT-AC
introns have highly conserved, non-canonical splice sites that are removed by the AT-AC spliceosome, which contains distinct snRNAs (U11,
U12, U4atac, U6atac) that are structurally and functionally analogous to the major spliceosome. U5 snRNA as well as many of the protein factors
appear to be conserved between the two spliceosomes.
References
Q Wu, AR Krainer, "AT-AC pre-mRNA splicing mechanisms and conservation of minor introns in voltage-gated ion channel genes.", Mol Cell
Biol, 19, 1999, 3225-36.
WY Tarn, JA Steitz, "Pre-mRNA splicing: the discovery of a new spliceosome doubles the challenge.", Trends Biochem Sci, 22, 1997, 132-7.
43.2.1.3.2.1 Formation of AT-AC A complex
Authors
Joshi-Tope, G, 2003-10-27.
Description
U12-type AT-AC introns are distinguished from the major U2-type introns by the consensus sequences of their highly conserved splicing signals.
U12 introns have the 5' ss consensus sequence (G/A)TATCCTTT, the branchpoint sequence TTTCCTTAACT and the 3' ss (C/T)AG. Initial
recognition of AT-AC introns involves interaction of U12 snRNP with the branch-point sequence and U11 with the 5' ss. Unlike the major splicing
pathway, U11 and U12 are in a complex and interact with the pre-mRNA simultaneously, binding in an ATP-dependent manner as a di-snRNP
complex and likely bridging the 5' ss and 3' ss region.
Twenty proteins have been identified in the U11/U12 di-snRNP complex including the snRNP Sm proteins BÃ¢â‚¬â„¢, B, D3, D2, D1, E, F, and
G which are identical to the major splicing pathway Sm proteins. A U2 snRNP core protein complex, SF3b is also found in the U11/U12
di-snRNP, including p14, a protein that interacts with the branchpoint adenosine.
SR proteins are required for formation of A complex in AT-AC splicing. The same SR proteins involved in splicing of the major introns are also
active in splicing of AT-AC introns, though, as in the major pathway, there is substrate specificity.
Reaction
43.2.1.3.2.2 Formation of AT-AC B Complex
Authors
Joshi-Tope, G, 2003-10-27.
Description
The U4atac/U6atac enters the spliceosome and U6atac snRNA forms base pairing interactions with the 5' ss and also forms base pairing
interactions with U12 and U4atac is partially displaced. U5 snRNP, the only snRNP common to both the major and minor splicing pathways, also
joins the spliceosome to form the B complex and interacts with nucleotides within the 3' end of the exon flanking the 5' ss.
Reaction
43.2.1.3.2.3 Formation of AT-AC C complex
Description
At the beginning of this reaction, 1 molecule of 'ATAC B Complex' is present. At the end of this reaction, 1 molecule of 'U4 ATAC snRNP', and 1
molecule of 'ATAC C Complex' are present.

Reaction
43.2.1.3.2.4 ATAC spliceosome mediated Lariat formation,5' splice site cleavage
Description
At the beginning of this reaction, 1 molecule of 'ATAC C Complex' is present. At the end of this reaction, 1 molecule of 'ATAC C Complex with
lariat containing 5'-end cleaved mRNA' is present.

Reaction
43.2.1.3.2.5 ATAC spliceosome mediated 3' splice site cleavage, exon ligation
Description
At the beginning of this reaction, 1 molecule of 'ATAC C Complex with lariat containing 5'-end cleaved mRNA' is present. At the end of this
reaction, 1 molecule of 'U6 ATAC snRNP', 1 molecule of 'post exon ligation complex', 1 molecule of 'U12 snRNP', 1 molecule of 'U11 snRNP',
and 1 molecule of 'U5 snRNP' are present.

Reaction
43.2.1.4 mRNA 3'-end processing
Editors
Joshi-Tope, G, 0000-00-00.
Description
The 3' ends of eukaryotic mRNAs are generated by posttranscriptional processing of an extended primary transcript. For almost all RNAs, 3'-end
processing consists of two steps: (i) the mRNA is first cleaved at a particular phosphodiester bond downstream of the coding sequence, (ii) the
upstream fragment then receives a poly(A) tail of approximately 250 adenylate residues, whereas the downstream fragment is degraded. The
two partial reactions are coupled so that reaction intermediates are usually undetectable. While 3' processing can be studied as an isolated
event in vitro, it appears to be connected to transcription, splicing, and transcription termination in vivo.
The only known exception to the rule of cleavage followed by polyadenylation are the major histone mRNAs, which are cleaved but not
polyadenylated.
References
43.2.1.4.1 Cleavage of mRNA at the 3'-end
Editors
Joshi-Tope, G, 0000-00-00.
Description
polyadenylation.
References
Reaction
43.2.1.4.2 mRNA polyadenylation
Editors
Joshi-Tope, G, 0000-00-00.
Description
The upstream fragment generated by 3' cleavage of the pre-mRNA receives a poly(A) tail of approximately 250 AMP residues in a reaction
depending on the AAUAAA sequence 10 to 30 nucleotides upstream of the 3' end. Polyadenylation is carried out by three proteins: Poly(A)
polymerase carries the catalytic activity. The enzyme has no specificity for any particular RNA sequence, and it also has a very low affinity for
the RNA.
Under physiological conditions, the activity of poly(A) polymerase thus depends on two auxiliary factors, both of which bind to specific RNA
sequences and recruit the enzyme by a direct contact. One of these proteins is the heterotetrameric CPSF, which binds the AAUAAA sequence
and is also essential for 3' cleavage. The second is the nuclear poly(A) binding protein (PABPN1), which binds the growing poly(A) tails once this
has reached a length of about ten nucleotides. Stimulation of poly(A) polymerase by both proteins is synergistic and results in processive
elongation of the RNA, i.e. the polymerase adds AMP residues without dissociating from the RNA. The processive reaction is terminated when
the tail has reached a length of about 250 nucleotides.
References
Reaction
43.2.1.5 Transport of Mature Transcript to Cytoplasm
Authors
Joshi-Tope, G, 2003-09-02.
Description
Transport of mRNA through the Nuclear Pore Complex (NPC) is a dynamic process involving distinct machinery and receptor subsets. The
separation of the two compartments and the regulation of this transport provide spatial and temporal control over mRNA expression and
ultimately control over translation. It should be noted that mRNA export does not rely on a specific motif in the mRNA molecule, but rather
transport appears to be coupled to processing and regulation. The specific proteins that are bound to the mRNA determine when it will be
transported to the cytoplasm. This limitation insures that transport overwhelmingly favors transport of fully processed mRNA molecules.
43.2.1.5.1 Transport of Mature mRNA derived from an Intron-Containing Transcript
Description
processing. This coupling is achieved by the nuclear pore complex, which recognizes and transports only completed mRNAs.
43.2.1.5.1.1 Recruitment of TAP to the EJC
Authors
Joshi-Tope, G, 2005-02-17.
Editors
Joshi-Tope, G, 0000-00-00.
Description
Aly/Ref recruits TAP to the Exon Junction Complex. This makes the mRNP complex ready for export to the cytoplasm.
Reaction
43.2.1.5.1.2 Docking of the TAP:EJC Complex with the NPC
Description
At the beginning of this reaction, 1 molecule of 'TAP:3'-polyadenylated, capped mRNA complex' is present. At the end of this reaction, 1
molecule of 'SRp55', 1 molecule of 'U2AF 65 kDa subunit', 1 molecule of 'SR9 / SRp30', 1 molecule of 'hTra2', 1 molecule of 'hPrp16', 1
molecule of 'SR 11/ p54', 1 molecule of 'hPrp22', 1 molecule of 'SRp40', 1 molecule of 'hPrp17', 1 molecule of 'SF2/ASF/SFRS1', 1 molecule of
'hSLU7', 1 molecule of 'Export Receptor bound mature mRNA Complex', 1 molecule of 'U2AF 35 kDa subunit', 1 molecule of 'SR2 / SC35', 1
molecule of 'hPrp43', 1 molecule of 'SRp20', 1 molecule of 'SR7/ 9G8 protein', 1 molecule of 'hPrp18', and 1 molecule of 'SR4 / SRp75' are
present.
Reaction
43.2.1.5.1.3 Transport of the export-competent complex through the NPC
Description
In this reaction, 1 molecule of 'Export Receptor bound mature mRNA Complex' is translocated from nucleoplasm to cytosol.
Reaction
43.2.1.5.1.4 Release from the NPC and Disassembly of the mRNP

Description
At the beginning of this reaction, 1 molecule of 'eIF4E', and 1 molecule of 'Export Receptor bound mature mRNA Complex' are present. At the
end of this reaction, 1 molecule of 'THOC4(Aly/Ref)', 1 molecule of 'Mature mRNP Complex', 1 molecule of 'SRm160', and 1 molecule of 'TAP'
are present.
Reaction
43.2.1.5.2 Transport of Mature mRNAs Derived from Intronless Transcripts
Authors
Editors
Description
Transport of mature mRNAs derived from intronless transcripts require some of the same protein complexes as mRNAs derived from intron
containing complexes, including TAP and Aly/Ref. However a number of the splicing related factors are lacking from the intronless derived
mRNAs, as they required no splicing.
43.2.1.5.2.1 Transport of the SLBP Dependant Mature mRNA
Description
Transport of U7 snRNP and stem-loop binding protein (SLBP) processed mRNA.

43.2.1.5.2.1.1 Docking of Mature Replication Dependent Histone mRNA with the NPC
Authors
Editors
Description
stemloop and SLBP enhance the rate of transport of histone mRNAs in Xenopus oocytes, but are not essential for transport
References
Reaction
43.2.1.5.2.1.2 Transport of the Mature IntronlessTranscript Derived Histone mRNA:SLBP:TAP:Aly/Ref complex through the NPC
Authors
Editors
Description
Once the transport complex is fully assembled the mature mRNA can be translocated from the nucleoplasm to the cytoplasm. The assembled
complex starts at the nucleoplasmic basket, travels through the pore, and ends it journey at the cytoplasmic face of the nuclear pore complex.
Reaction
43.2.1.5.2.1.3 Release of the Mature intronless transcript derived Histone mRNA:SLBP:eIF4E Complex
Authors
Description
Reaction
43.2.1.5.2.2 Transport of the SLBP independent Mature mRNA
Description
Transport of the SLBP independent Mature mRNA through the nuclear pore.
43.2.1.5.2.2.1 Docking of Mature Histone mRNA complex:TAP at the NPC
Authors
Editors
Description
mature transcript docks at the NPC, in the course of transport CBC will be lost from the mRNA cap, and remain in the nucleous.
References
Reaction
43.2.1.5.2.2.2 Transport of the Mature Intronless Transcript Derived Histone mRNA:TAP:Aly/Ref Complex through the NPC
Authors
Description
The mature SLBP independent intronless histone mRNA is transported through the nucler pore to the cytoplasmic side.
Reaction
43.2.1.5.2.2.3 Release of the SLBP independent Histone mRNA from the NPC
Authors

Description
Reaction
43.2.1.5.2.3 Transport of Mature mRNA Derived from an Intronless Transcript
Description
processing.
43.2.1.5.2.3.1 Docking of the Mature intronless derived transcript derived mRNA, TAP and Aly/Ref at the NPC
Authors
Description
The polyadenylated, capped transcript and TAP dock at the nucleoplasmic side of the NPC. The Cap Binding Complex (CBC) and CPSF
complexes are released back into the nucleoplasm.
Reaction
43.2.1.5.2.3.2 Transport of the Mature intronless transcript derived mRNA:TAP:Aly/Ref Complex through the NPC
Authors
Description
The nucleoplasmic 3' polyadenylated, capped intronless mRNA and TAP are transported through the NPC to the cyotplasmic side of the pore.
Reaction
43.2.1.5.2.3.3 Release of the Mature intronless derived mRNA, TAP, and Aly/Ref from the NPC
Authors
Description
The cytoplasmic 3' polyadenylated, capped intronless mRNA and TAP are released from the NPC into the cytosol. Cytosolic TAP will be
recycled to the nucleous, while the 3' polyadenylated, capped intronless mRNA is bound by eIF4E and destined for translation.
Reaction
43.2.2 Processing of Capped Intronless Pre-mRNA
Authors
Editors
Joshi-Tope, G, 0000-00-00.
Description
There are two classes of intronless pre-mRNAs (mRNAs expressed from genes that lack introns). The first class encodes the replication
dependent histone mRNAs. These mRNAs have unique 3Ã¢â‚¬â„¢ ends that do not have a polyA tail. The replication dependent histone
mRNAs in all metazoans, as well as Chlamydomonas and Volvox fall into this class.
The second class of mRNAs end in polyA tails, which are formed by a mechanism similar to that which poly-adenylate pre-mRNAs containing
introns. In the intronless genes there is a different method of replacing the 3Ã¢â‚¬â„¢ splice site that activates polyadenylation.
References
WF Marzluff, P Gongidi, KR Woods, J Jin, LJ Maltais, "The human and mouse replication-dependent histone genes.", Genomics, 80, 2002,
487-98.
R Sanchez, WF Marzluff, "The stem-loop binding protein is required for efficient translation of histone mRNA in vivo and in vitro", Mol Cell Biol,
22, 2002, 7093-104.
Z Dominski, WF Marzluff, "Formation of the 3' end of histone mRNA.", Gene, 239, 1999, 1-14.
JG Gall, "Cajal bodies: the first 100 years.", Annu Rev Cell Dev Biol, 16, 2001, 273-300.
MR Frey, AG Matera, "Coiled bodies contain U7 small nuclear RNA and associate with specific DNA sequences in interphase human cells.",
43.2.2.1 SLBP Dependent Processing of Replication-Dependent Histone Pre-mRNAs
Authors
Editors

Description
There are two well-documented trans-acting factors required for histone pre-mRNA processing. These are:
1 Stem-loop binding protein (SLBP), also termed hairpin binding protein (HBP). This 32 kDa protein is likely the first protein that binds to the
histone pre-mRNA as it is being transcribed.
A third protein joins the U7 snRNP, ZFP100, a large zinc finger protein. ZFP100 interacts with SLBP bound to the histone pre-mRNA and with
Lsm11 and likely plays a critical role in recruiting U7 snRNP to the histone pre-mRNA.
It should be noted that there must be other trans-acting factors, including the factor that catalyzes the cleavage reaction. The cleavage occurs in
the presence of EDTA as does the cleavage reaction in polyadenylation, it is likely that this reaction is catalyzed by a protein. There may well be
additional proteins associated with U7 snRNP, and since under some conditions in vitro processing occurs in the absence of SLBP, it is possible
that all of the other factors required for processing are associated with the active form of U7 snRNP.
References
43.2.2.1.1 Binding of SLBP to Replication-Dependent Histone Pre-mRNA
Authors
Editors
Description
The 32 kDa stem-loop binding protein (SLBP), also termed hairpin binding protein (HBP) is likely the first protein that binds to the histone
pre-mRNA as it is being transcribed.
References
Z Dominski, LX Zheng, R Sanchez, WF Marzluff, "Stem-loop binding protein facilitates 3'-end formation by stabilizing U7 snRNP binding to
histone pre-mRNA.", Mol Cell Biol, 19, 1999, 3561-70.
ZF Wang, ML Whitfield, TC Ingledue, Z Dominski, WF Marzluff, "The protein that binds the 3' end of histone mRNA: a novel RNA-binding protein
required for histone pre-mRNA processing.", Genes Dev, 10, 1997, 3028-40.
F Martin, A Schaller, S Eglite, D SchÃ¼mperli, B MÃ¼ller, "The gene for histone RNA hairpin binding protein is located on human chromosome
4 and encodes a novel type of RNA binding protein.", EMBO J, 16, 1997, 769-78.
Reaction
43.2.2.1.2 Recruitment of U7 snRNP:ZFP100 complex to the SLBP Bound Pre-mRNA
Authors
Description
same as ones found in the spliceosomal snRNPs and there are 2, Lsm10 and Lsm11 that are unique to U7 snRNP. A third protein joins the U7
snRNP, ZFP100, a large zinc finger protein. ZFP100 interacts with SLBP bound to the histone pre-mRNA and with Lsm11 and likely plays a
critical role in recruiting U7 snRNP to the histone pre-mRNA.
References
Reaction
43.2.2.1.3 Cleavage of the 3'-end of Replication Dependent Histone Pre-mRNA
Authors
Editors
Description
References
Reaction
43.2.2.2 SLBP independent Processing of Histone Pre-mRNAs
Authors
Editors
Description
This class of mRNAs is expressed from genes that lack introns yet the transcripts end in polyA tails. These tails are formed by a mechanism
similar to that for pre-mRNAs containing introns. It is believed that there is a cis-element that replaces the 3Ã¢â‚¬â„¢ splice site that normally
serves to activate polyadenylation of intron containing pre-mRNAs.
43.2.2.2.1 Recruitment of U7 snRNP:ZFP100 complex to the Histone Pre-mRNA
Authors
Editors

Description
References
Reaction
43.2.2.2.2 Cleavage of the 3'-end of the Histone Pre-mRNA
Authors
Editors
Description
References
Reaction
43.2.2.3 Processing of Intronless Pre-mRNAs
Authors
Wahle, E, 2003-06-05.
Editors
Description
The 3' ends of eukaryotic mRNAs are generated by posttranscriptional processing of an extended primary transcript. For almost all RNAs, 3'
processing consists of two steps: The mRNA is first cleaved at a particular phosphodiester bond downstream of the coding sequence. The
upstream fragment then receives a poly(A) tail of approximately 250 adenylate residues whereas the downstream fragment is degraded. The two
partial reactions are coupled so that reaction intermediates are usually undetectable. While 3' processing can be studied as an isolated event in
vitro, it appears to be connected to transcription, splicing and transcription termination in vivo.
References
43.2.2.3.1 Recognition of AAUAAA sequence by CPSF
Authors
Wahle, E, 2003-06-05.
Editors

Description
Poly(A) polymerase has no specificity for any particular RNA sequence, as well as a very low affinity for the RNA. Under physiological
conditions, the activity of poly(A) polymerase depends on two auxiliary factors, both of which bind to specific RNA sequences and recruit the
poly(A) polymerase by a direct contact. One of these proteins is the heterotetrameric CPSF, which binds the AAUAAA sequence and is also
essential for 3' cleavage.
References
Reaction
43.2.2.3.2 Recruitment of CstF to the CPSF Bound Pre-mRNA
Authors
Wahle, E, 2003-06-05.
Editors
Description
Endonucleolytic cleavage separates the pre-mRNA into an upstream fragment destined to become the mature mRNA and a downstream
fragment that is rapidly degraded. Polyadenylation and cleavage occur concurrently, with complexes co-assembling, cleavage depends on two
signals in the RNA, a highly conserved hexanucleotide, AAUAAA, 10 to 30 nucleotides upstream of the cleavage site, and a poorly conserved
GU- or U-rich downstream element. Additional sequences, often upstream of AAUAAA, can enhance the efficiency of the reaction. Cleavage
occurs most often after a CA dinucleotide. A single gene can have more than one 3' processing site.
required for assembly. Cleavage depends on a number of protein factor. CPSF, a heterotetramer, binds specifically to the AAUAAA sequence.
The heterotrimer CstF binds the GU- or U-rich downstream element.
References
Reaction
43.2.2.3.3 Binding of Cleavage factors and Poly(A)Polymerase to the CstF:CPSF:Pre-mRNA Complex
Authors
Wahle, E, 2003-06-05.
Editors
Description
CF I, which appears to be composed of two subunits, one of several related larger polypeptides and a common smaller one, also binds RNA, but
with unknown specificity. RNA recognition by these proteins is cooperative. Cleavage also requires CF II, composed of at least two subunits, and
poly(A) polymerase, the enzyme synthesizing the poly(A) tail in the second step of the reaction.
References
Reaction
43.2.2.3.4 Cleavage and polyadenylation of Intronless Pre-mRNA
Authors
Wahle, E, 2003-06-05.
Editors
Description
The nuclear poly(A) binding protein (PABPN1), which binds the growing poly(A) tails once it has reached a length of about ten nucleotides.
Stimulation of poly(A) polymerase by both CPSF and PABPN1 is synergistic and results in processive elongation of the RNA (the polymerase
adds AMP residues without dissociating from the RNA). The processive reaction is terminated when the tail has reached a length of about 250
nucleotides.
References
Reaction
43.3 mRNA Editing
Authors
Carmichael, GG, 2003-08-22.

Editors
Description
After transcription, some RNA molecules are altered to contain bases not encoded in the genome. Most often this involves the editing or
modification of one base to another, but in some organisms can involve the insertion or deletion of a base. Such editing events alter the coding
properties of mRNA.
RNA editing can be generally defined as the co- or post transcriptional modification of the primary sequence of RNA from that encoded in the
genome through nucleotide deletion, insertion, or base modification mechanisms.
There are two pathways of RNA editing: the substitution/conversion pathway and the insertion/deletion pathway. The insertion/deletion editing
occurs in protozoans like Trypanosoma, Leishmania; in slime molds like Physarum spp., and in some viral categories like paramyxoviruses,
Ebola virus etc. To date, the substitution/conversion pathway has been observed in human along with other mammals, Drosophila, and some
plants. The RNA editing processes are known to create diversity in proteins involved in various pathways like lipid transport, metabolism etc. and
may act as potential targets for therapeutic intervention (Smith et al., 1997).
The reaction mechanisms of cytidine and adenosine deaminases is represented below. In both these reactions, NH3 is presumed to be
released:
References
K Stuart, AK Panigrahi, "RNA editing: complexity and complications.", Mol Microbiol, 45, 2002, 591-6.
JM Gott, RB Emeson, "Functions and mechanisms of RNA editing.", Annu Rev Genet, 34, 2001, 499-531.
43.3.1 mRNA Editing: C to U Conversion
Description
The best characterized case of C to U editing is in the intestinal apolipoprotein B transcript, where the editing event creates a premature
translation stop codon and consequently leads to a shorter form of the protein. In the liver, C to U editing is important in the expression of
specific isoforms of the apolipoprotein B enzyme. ApoB mRNA editing is a posttranscriptional, nuclear process that can be initiated after splicing,
at the time of polyadenylation and is completed by the time pre-mRNA matures fully (reviewed by Blanc and Davidson, 2003).
This editing event is a simple hydrolytic cytidine deamination to uridine, and is carried out by the Apobec-1 enzyme, along with the Apobec-1
complementing factor, ACF. The editing of apo-B mRNA involves the site-specific deamination of (C6666 to U), which converts codon 2153 from
a glutamine codon, CAA, to a premature stop codon, UAA. As ACF is distributed in a variety of tissues, and these genes contain multiple family
members, it is possible that editing events in additional targets will be found.
The cis-acting regulatory elements for C to U editing include: 22 nt editing site within ApoB mRNA, 5Ã¢â‚¬â„¢ tripartite motif with an enhancer
element adjacent to the target cytidine, a spacer element and mooring sequence both 3Ã¢â‚¬â„¢ to the cytidine (reviewed by Smith et al., 1997).
The editing complex can be represented as:
References
V Blanc, NO Davidson, "C-to-U RNA editing: mechanisms leading to genetic diversity.", J Biol Chem, 278, 2003, 1395-8.
JE Wedekind, GS Dance, MP Sowden, HC Smith, "Messenger RNA editing in mammals: new members of the APOBEC family seeking roles in
the family business.", Trends Genet, 19, 2003, 207-16.
A Chester, J Scott, S Anant, N Navaratnam, "RNA editing: cytidine to uridine conversion in apolipoprotein B mRNA.", Biochim Biophys Acta,
1494, 2000, 1-13.
JM Gott, RB Emeson, "Functions and mechanisms of RNA editing.", Annu Rev Genet, 34, 2001, 499-531.
43.3.1.1 Formation of the Editosome
Authors
Description
The editosome for C to U editing in mammals consist of a member of cytidine deaminase family of enzymes, apoB mRNA editig catalytic
polypeptide 1 (APOBEC-1) and a complementing specificity factor (ACF) in addition to the target mRNA.
43.3.1.1.1 Formation of stem-loop structure in ApoB mRNA
Authors
Description
It is predicted that the target RNA could form secondary structures including stem-loop confirmation prior to the formation of editosome.
Reaction
43.3.1.1.2 Binding of ACF to stem-looped RNA
Authors
Description
ACF protein is cytosolic in origin and is translocated to nucleus where it binds to the target RNA. The order of events in the formation of
editosomes, namely, binding of ACF and APOBEC-1 are not will elucidated in human cells.
Reaction
43.3.1.1.3 Binding of APOBEC-1 to form editosome
Authors
Description
APOBEC-1 binds to ACF:stem-looped mRNA complex forming the editosome.
Reaction
43.3.1.2 C4 deamination of cytidine
Authors
Description
Hydrolytic deamination of cytidine leads to uridine. Ammonia is presumed to be released during this reaction.
References
C Hadjiagapiou, F Giannoni, T Funahashi, SF Skarosi, NO Davidson, "Molecular cloning of a human small intestinal apolipoprotein B mRNA
editing protein.", Nucleic Acids Res, 22, 1994, 1874-9.
JO Henderson, V Blanc, NO Davidson, "Isolation, characterization and developmental regulation of the human apobec-1 complementation factor
(ACF) gene.", Biochim Biophys Acta, 1522, 2001, 22-30.
Reaction
43.3.2 mRNA Editing: A to I Conversion
Authors
Carmichael, GG, 2003-08-22.
Description
In humans the deamination of adenosines to inosines is the most common editing event. It is particularly prevalent in the brain, where it leads to
amino acid changes that affect the conductance of several ion channels. Inosines are recognized by the translation machinery as if they were
guanosines.
ADARs (Adenosine Deaminases Acting on RNA) modify pre-mRNA, acting as single peptides and recognize structural determinants in the RNA.
To date 3 members of this deaminase family are known: ADAR 1, ADAR 2, and ADAR 3 that share a common modular domain structure. ADAR
1 and 2 contain a catalytic deaminase domain, a double-stranded RNA binding domain and exhibit RNA editing activity. ADAR1 activity is found
in various mammalian tissues with the highest concentration in brain.
An increasing number of mammalian genes have been found to undergo deamination by ADARs. Deamination by editing in pre-mRNAs
encoding subunits of ionotropic glutamate receptors (GluRs) is another well studied example. An editing event at the Q/R site of the GluR2
(GluRB) subunit of AMPA Ã¢â‚¬"receptors converts a Gln codon CAG to an Arg codon CIG rendering the heteromeric receptor impermeable to
Ca 2+ ions. Another example is the editing of 5-HT2C subtype serotonin receptor mRNA resulting in receptor isoforms with reduced G-protein
coupling efficiency (reviewed by Gerber and Keller, 2001).
In mice, the editosomes with ADAR proteins require some cis-acting elements like an intronic 'editing-site complementary sequence (ECS)'.
Although evolutionarily conserved, the actual role of ECS is not yet elucidated in humans. The editing complex can be generally represented as:
References
T Melcher, S Maas, A Herb, R Sprengel, M Higuchi, PH Seeburg, "RED2, a brain-specific member of the RNA-specific adenosine deaminase
family.", J Biol Chem, 271, 1997, 31795-8.
V Blanc, NO Davidson, "C-to-U RNA editing: mechanisms leading to genetic diversity.", J Biol Chem, 278, 2003, 1395-8.
BL Bass, "RNA editing by adenosine deaminases that act on RNA.", Annu Rev Biochem, 71, 2002, 817-46.
AP Gerber, W Keller, "RNA editing by base deamination: more enzymes, more targets, new mysteries.", Trends Biochem Sci, 26, 2001, 376-84.
AG Polson, PF Crain, SC Pomerantz, JA McCloskey, BL Bass, "The mechanism of adenosine to inosine conversion by the double-stranded
RNA unwinding/modifying activity: a high-performance liquid chromatography-mass spectrometry analysis.", Biochemistry, 30, 1992, 11507-14.
43.3.2.1 Formation of dsRNA structure by looping
Authors
Description
Site-specific A to I conversion requires dsRNA structures present around the editing site. These structures can be formed by exonic sequences
and neighboring intronic sequences. An editing-site complementary sequence (ECS) has been identified in mice. Although minimal requirements
for A to I editing have been identified, detailed mechanisms describing the individual steps are not yet well studied in humans.
Reaction
43.3.2.2 Formation of editosomes by ADAR proteins
Authors
Description
It is still unclear how ADAR 1 and ADAR 2 proteins form the editosomes with the target RNA. Other components of these editosomes for A to I
editing are unknown.
43.3.2.2.1 Binding of ADAR1 homodimer to dsRNA duplex

Description
At the beginning of this reaction, 1 molecule of 'dsRNA duplex', and 1 molecule of 'ADAR1 homodimer ' are present. At the end of this reaction, 1
molecule of 'Editosome (ADAR1) complex' is present.
Reaction
43.3.2.2.2 Binding of ADAR2 homodimer to dsRNA duplex
Description
At the beginning of this reaction, 1 molecule of 'dsRNA duplex', and 1 molecule of 'ADAR2 homodimer' are present. At the end of this reaction, 1
molecule of 'Editosome (ADAR2) complex' is present.
Reaction
43.3.2.3 C6 deamination of adenosine
Authors
Description
Hydrolytic deamination of adenosine leads to inosine. Ammonia is presumed to be released during this reaction.
References
JB Patterson, CE Samuel, "Expression and regulation by interferon of a double-stranded-RNA-specific adenosine deaminase from human cells:
evidence for two forms of the deaminase.", Mol Cell Biol, 15, 1995, 5376-88.
F Lai, CX Chen, KC Carter, K Nishikura, "Editing of glutamate receptor B subunit ion channel RNAs by four alternatively spliced DRADA2
double-stranded RNA adenosine deaminases.", Mol Cell Biol, 17, 1997, 2413-24.
43.3.2.3.1 Deamination at C6 position of adenosine in Editosome (ADAR1)
Description
At the beginning of this reaction, 1 molecule of 'Editosome (ADAR1) complex', and 1 molecule of 'H2O' are present. At the end of this reaction, 1
molecule of 'A to I edited RNA:Editosome (ADAR1) complex', and 1 molecule of 'NH3' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'double-stranded RNA adenosine deaminase activity' of 'ADAR1 homodimer '.
Reaction
43.3.2.3.2 Deamination at C6 position of adenosine in Editosome (ADAR2)
Description
At the beginning of this reaction, 1 molecule of 'H2O', and 1 molecule of 'Editosome (ADAR2) complex' are present. At the end of this reaction, 1
molecule of 'NH3', and 1 molecule of 'A to I edited RNA:Editosome (ADAR2) complex' are present.
This reaction takes place in the 'nucleus' and is mediated by the 'double-stranded RNA adenosine deaminase activity' of 'ADAR2 homodimer'.
Reaction

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The Reactome Book

A textbook of biological pathways

MP Morrow; J Tschopp; L Van Aelst; M Bukrinsky; JA Borowiec; EH

1.2.3.6 Interaction of BIM with BCL-xl 110

1.4.1.8 Caspase-mediated cleavage of PKC theta 165

2.2.1.1 Formation of the active cofactor, UDP-glucuronate 294

3.4.2.3 BoNT Light Chain Type E cleaves SNAP-25 353

5.3.3.1.5 Proteasome mediated degradation of Cyclin D1 504

6.1.1.2.1.3 TDG glycosylase mediated recognition and binding of an ethenocytosine 611

7.3 Switching of origins to a post-replicative state 752

9.1.1 Gap junction assembly 798

10.2.4.1.1.1.2 DSIF complex binds to RNA Pol II (hypophosphorylated) 861

10.4.1.1.1 Formation of a pool of free 40S subunits 916

11.1.2.2.3 Rev multimer-bound HIV-1 mRNA associates with Crm1 1058

11.2.6.2.3.4 Formation of a Nef:ARF1:CD4 complex 1111

12.1.2.3.3.3.1.8 Exocytosis of pyrophosphate 1147

12.4.12 JAM-C homodimerises 1236

13.1.3.2 Uncoating of the Influenza Virion 1295

13.2.1.1.2 Binding of NS1 to poly(A)-binding protein II (PABII) 1355

15.1.4 Digestion of triacylglycerols by extracellular CEL (bile salt-dependent lipase) 1403

15.6.1.1.4 3-Oxopalmitoyl-CoA+CoA-SH<=>myristoyl-CoA 1459

15.8.2.1.2.9 5Beta-cholestan-7alpha,12alpha,24(S)-triol-3-one is reduced to 5beta-cholestan-3alpha,7alpha,12alpha,24(S)-tetrol 1568

15.8.3.3.2 Pregn-5-ene-3,20-dione isomerizes to progesterone 1627

16 Membrane Trafficking 1684

17.8.4 alpha-aminoadipate + alpha-ketoglutarate => alpha-ketoadipate + glutamate 1780

18.5.2 D-galactose 1-phosphate + UDP-glucose <=> D-glucose 1-phosphate + UDP-galactose 1907

21.1.3.1 Riboflavin is phosphorylated to FMN 1960

22.2.1 De novo synthesis of IMP 2003

22.2.3.2.10 inosine 5'-monophosphate (IMP) + H2O => inosine + orthophosphate 2046

22.3.3.10 2'-deoxycytidine 5'-monophosphate + H2O => 2'-deoxycytidine + orthophosphate 2089

22.5.2.4 2'-deoxyinosine 5'-monophosphate (dIMP) + H2O => 2'-deoxyinosine + orthophosphate 2129

24.1.3 S-acetyldihydrolipoamide + CoA => acetyl-CoA + dihydrolipoamide 2276

29.2 EGFR binds EGF ligand 2381

30.1.2.1.2 Autocatalytic phosphorylation of FGFR2b 2442

31.1.1.4.1 Ligand binds to TLR7 or TLR8 2488

31.1.1.7.2.2.4 First phosphorylation of IRAK1 by IRAK4 bound to activated TLR 2534

31.3.1 Phosphorylation of CD3 and TCR zeta chains 2589

31.4.21 Platelet-derived TREM-1 ligand binds to TREM-1 2648

32.3.2.2.1.2.1 PDE3B signalling 2702

33.2.2.1.2 NRAGE sequesters CHE1 in the cytoplasm 2753

33.3.2.2 By PACAP type 1 receptor 2809

33.4.1.3.1.6 (Frs2)Rap1-GTP binds to and activates B-Raf 2869

33.4.3.11 AKT phosphorylates targets in the nucleus 2925

34.7.2 NICD2 trafficks to the nucleus 2973

39.1.4 Neurotransmitter uptake and Metabolism In Glial Cells 3035

41.2.1.5 Formation of the closed pre-initiation complex 3098

42.1.1 Cap-dependent Translation Initiation 3157

Alnemri, E, Hengartner, M, Tschopp, J, Tsujimoto, Y, Hardwick, JM, 2004-01-16.

Gopinathrao, G, Matthews, L, Gillespie, ME, Joshi-Tope, G, 0000-00-00.

Hengartner, M, Ranganathan, S, Vaux, D, 0000--0-0-.

1.1 Extrinsic Pathway for Apoptosis

Gillespie, ME, 2004-08-10.

Gillespie, ME, 0000-00-00.

MO Hengartner, "The biochemistry of apoptosis.", Nature, 407, 2000, 770-6.

1.1.1 Death Receptor Signalling

Gillespie, ME, 2004-08-10.

Gillespie, ME, 0000-00-00.

1.1.1.1 FasL/ CD95L signaling

Gillespie, ME, 2004-08-18.

Gillespie, ME, 0000-00-00.

MO Hengartner, "The biochemistry of apoptosis.", Nature, 407, 2000, 770-6.

1.1.1.1.1 FASL binds FAS Receptor

This reaction takes place in the 'cell'.

1.1.1.1.2 Trimerization of the FASL:FAS receptor complex