Post Translational Modifications

of Proteins
 Introduction
• It is the chemical modification of protein after
its translation.
• Key role in functional Proteomics.
• They regulate activity, localization and
interaction with other cellular molecules such
as proteins, nucleic acids, lipids and cofactors.
 Types of Post Translational
Modifications of Proteins
• Phosphorylation
• Glycosylation
• Ubiquitination
• S-Nitrosylation
• Methylation
• N-Acetylation
• Lipidation
• Proteolysis
 Phosphorylation
• Addition of phosphate group to a protein.
• Principally on serine, threonine or tyrosine
residues.
• Also known as Phospho regulation.
• Critical role in cell cycle, growth, apoptosis
and signal transduction pathways.

Protein kinases
ATP + protein ———————> phosphoprotein + ADP
Phosphorylation
Example
 Glycosylation
• The covalent attachment of oligosaccharides
• Addition of glycosyl group or carbohydrate
group to a protein.
• Principally on Asparagine, hydroxylysine,
serine or threonine.
• Significant effect on protein folding,
conformation, distribution, stability and
activity.
Example
 Classes of Glycans
• N-Linked glycans
– attached to nitrogen of Asparagine or arginine side
chains.
• O-Linked glycans
– attached to hydroxy oxygen of serine,threonine
• Phospho glycans
– linked through the phosphate of serine.
• C-Linked glycans
– Rare form, Sugar is added to a carbon on tryptophan
side chain.
 Ubiquitination
• Ubiquitin is a small regulatory protein that can
be attached to the proteins and label them for
destruction.
• Effects in cell cycle regulation, control of
proliferation and differentiation, programmed
cell death (apoptosis), DNA repair, immune
and inflammatory processes and organelle
biogenesis.
Ubiquitin cycle
 S-Nitrosylation

• Nitrosyl (NO) group is added to the protein.

• NO a chemical messanger that reacts with free
cysteine residues to form S-nitrothiols.
• Used by cells to stabilize proteins, regulate
gene expression.
 Alkylation/Methylation
• Addition of methyl group to a protein.
• Usually at lysine or arginine residues.
• Binds on nitrogen and oxygen of proteins
• Methyl donor is S-adenosylmethionine (SAM)
• Enzyme for this is methyltransferase
• Methylation of lysine residues in histones in
DNA is important regulator of chromatin
structure
 Example

Where SAM (S-adenosyl methionine) is converted into SAH(S-adenosyl homocysteine)

 N-Acetylation
• Addition of acetyl group to the nitrogen.
• Histones are acetylated on lysine residues in
the N-terminal tail as a part of gene
regulation.
• Involved in regulation of transcription factors,
effector proteins, molecular chaperons and
cytoskeletal proteins.
• Methionine aminopeptidase (MAP) is an
enzyme responsible for N-terminal acetylation
Example
Where,
HDACs = Histone deactyllase ,
KATs = N-acetyltransferase.
 Lipidation

• Lipidation attachment of a lipid group, such as

a fatty acid, covalently to a protein.
• In general, lipidation helps in cellular
localization and targeting signals, membrane
tethering and as mediator of protein-protein
interactions.
 Types of lipidation

• C-terminal glycosyl phosphatidylinositol (GPI)

anchor
• N-terminal myristoylation
• S-palmitoylation
• S-prenylation
 C-terminal glycosyl
phosphatidylinositol (GPI) anchor

• GPI anchors tether cell surface proteins to the

plasma membrane
• GPI-anchored proteins are often localized to
cholesterol- and sphingolipid-rich lipids, which
act as signaling platforms on the plasma
membrane.
 N-myristoylation

• It is the attachment of myristoyl group a 14-

carbon saturated fatty acid (C14) to a protein.
• It is facilitated by N-myristoyltransferase (NMT)
and uses myristoyl-CoA as the substrate.
 S-palmitoylation

• It is addition of C16 palmitoyl group from

palmitoyl-CoA
• Palmitoyl acyl transferases (PATs) enzyme
favors this step.
• Reversed by thioesterases
 S-prenylation

• Addition of a farnesyl (C15) or geranylgeranyl

(C20) group to proteins.
• Enzyme involved in this reaction is farnesyl
transferase (FT) or geranylgeranyl transferases
(GGT I and II).
 Disulfide Bonding

• Disulfide bonds are covalent bonds formed

between two cysteine residues (R-S-S-R).
• These bonds contribute to the correct folding of
proteins as other elements of secondary
structure
Disulfide Bonding
 Proteolysis

• Cleavage of peptide bonds by proteases.

• Examples of Proteases- Serine Proteases,
Cysteine Proteases, Aspartic acid Proteases.
• Involved in Antigen processing, Apoptosis, Cell
signalling
 Identification of modifications

• Mass spectrometry
• HPLC analysis
• Incorporation of radioactive groups by addition to
growing cells
– e.g., 75Se-labeling and chromatographic
isolation of proteins
• Antibody cross-reactivity
– e.g., antibody against phosphotyrosine
• Polyacrylamide gel electrophoresis (PAGE)
 Identification of modifications

use 2D gel electrophoresis to

detect modified proteins in
whole-cell (or partly purified)
lysates

O-GlcNAc is an abundant modification of nucleocytoplasmic proteins.

Nucleo-cytoplasmic proteins from HeLa cells were immunopurified with an
O-GlcNAc-specific antibody and stringently washed, and the O-GlcNAc-
containing proteins were specifically eluted with free GlcNAc. The resulting
proteins were separated on two-dimensional gels and visualized by silver
staining. pI, isoelectric point; MW, molecular weight.
From Wells et al. (2001) Science 291, 2376-8.
small-molecule modifications can affect not only the activity, but also the structure of
proteins, much as ligands such as ATP can affect the activity and structure of proteins
 • Jensen, O., N (2004) Modification-specific proteomics:
Characterization of post-translational modifications by mass
spectrometry. Current Openings in bio-chemistry. 8, 33-41
• Mann, M and Jensen, O., N (2003) Proteomic analysis of post-
translational modifications. Nature Biotechnology. 21, 255-261.
• Matsubayashi, Y (2012) Recent advances in research on small
post-translationally modified peptide signals in plants. Genes to
Cells 17, 1-10.
• Ralp, A. Bradshaw and Albert, E. Stewart (1994) Analysis of
protein modifications: Recent advances in detection,
References characterization and mapping. 5(1), 85-93.
• Walsh C. (2006) Posttranslational modification of proteins :
Expanding nature's inventory. Englewood, Colo.: Roberts and
Co. Publishers. xxi, 490 p. p.
• Gaston B. M. et al. (2003) S-nitrosylation signaling in cell
biology. Mol Interv. 3, 253-63.
• Jaffrey S. R. and Snyder S. H. (2001) The biotin switch method
for the detection of S-nitrosylated proteins. Sci STKE. 2001, pl1.
• Han P. and Chen C. (2008) Detergent-free biotin switch
combined with liquid chromatography/tandem mass
spectrometry in the analysis of S-nitrosylated proteins. Rapid
Commun Mass Spectrom. 22, 1137-45.
THANK YOU