Structure

Review

Biology of Amyloid: Structure, Function,

and Regulation
Jason Greenwald1 and Roland Riek1,*
1ETH Zurich, Physical Chemistry, ETH Honggerberg, 8093 Zurich, Switzerland

*Correspondence: roland.riek@phys.chem.ethz.ch
DOI 10.1016/j.str.2010.08.009

Amyloids are highly ordered cross-b sheet protein aggregates associated with many diseases including Alz-
heimer’s disease, but also with biological functions such as hormone storage. The cross-b sheet entity
comprising an indefinitely repeating intermolecular b sheet motif is unique among protein folds. It grows
by recruitment of the corresponding amyloid protein, while its repetitiveness can translate what would be
a nonspecific activity as monomer into a potent one through cooperativity. Furthermore, the one-dimensional
crystal-like repeat in the amyloid provides a structural framework for polymorphisms. This review summa-
rizes the recent high-resolution structural studies of amyloid fibrils in light of their biological activities. We
discuss how the unique properties of amyloids gives rise to many activities and further speculate about
currently undocumented biological roles for the amyloid entity. In particular, we propose that amyloids could
have existed in a prebiotic world, and may have been the first functional protein fold in living cells.

Introduction one-dimensional nature of the order in the fibrils makes them

Amyloid fibrils have long been associated with dozens of
diseases including Alzheimer’s disease (AD), Parkinson’s
disease, and prion diseases (Chiti and Dobson, 2006). Recently,
however, it has become evident that there exist many amyloids,
termed ‘‘functional amyloids,’’ that have normal biological activ-
ities (Fowler et al., 2007). Amyloid is a term whose definition has
evolved along with our understanding of its underlying structure
(Sipe and Cohen, 2000). In this review, we adopt the modern
biophysical definition: an unbranched protein fiber whose
repeating substructure consists of b strands that run perpendic-
ular to the fiber axis, forming a cross-b sheet of indefinite length
(Figure 1). Thus, amyloids are composed of an ordered arrange-
ment of many (usually thousands) copies of a peptide or protein.
They are easily identified using electron microscopy (EM) as
long, nonbranched filaments with diameters of 6–12 nm (Sunde
and Blake, 1997) (Figure 1). The repeating cross-b sheet motif
gives rise to characteristic X-ray fiber diffraction patterns with
a meridional reflection at 4.7 Å corresponding to the inter-b
strand spacing and an equatorial reflection at 6–11 Å corre-
sponding to the distance between stacked b sheets. (Astbury
et al., 1935; Sunde et al., 1997) (Figure 1). Among protein folds,
this structural entity is unique and it gives rise to a plethora of
functions (both good and bad for the cell). It is the aim of this
review to summarize the recently established high-resolution
structural data on amyloids and to set this information in
perspective with their biological activities.
High Resolution Structures and Their Implications
By definition, all amyloid fibrils have a translational symmetry
element that lies parallel to the fibril axis, whereas electron
micrographs indicate that most have in addition a rotational
element (combined rotational/translational) yielding a helical or
screw symmetry. Thus, well-aligned fibrils can sometimes give
diffraction patterns that can be used to model (at low resolution)
the fibril structure. Despite their highly ordered nature, amyloids
are difficult to study by high-resolution structural methods. The
poor candidates for three-dimensional crystallization, and to
date, the only representative crystal structures are of amyloido-
genic peptides that are short enough to pack in a three-dimen-
sional lattice. These structures indicate that the most basic
cross-b structure is in fact a one-dimensional crystal with single
translational and rotational symmetry elements. Alternatively,
solid-state nuclear magnetic resonance (NMR) is a well-suited
high-resolution structure method for the study of amyloids, yet
to date, only a single high-resolution NMR structure of an
amyloid is available.
Amyloid Crystal Structures of Short Amyloidogenic
Peptides: The Cross-b Spine at Atomic Resolution
Researchers in the Eisenberg lab have taken a reductionist
approach to determine atomic resolution structures of amyloids.
By using short fibril-forming peptide segments of amyloid
proteins (e.g., Sup35, insulin, Ab, tau, and amylin), they were
able to grow three-dimensional microcrystals that likely repre-
sent the structure in the fibril form (i.e., in the one-dimensional
crystal-like form) (Ivanova et al., 2009; Nelson et al., 2005;
Sawaya et al., 2007; Wiltzius et al., 2009; Wiltzius et al., 2008).
Because all of the peptides formed both fibrils and microcrystals,
the researchers were able to demonstrate that the peptides
share a common structure in the two ordered states The
evidence for this includes that the microcrystals and fibrils
grow in the same conditions with some fibrils developing off of
the tips of crystals, that the long axis of the microcrystals is the
same as the fibril axis, that the microcrystals can seed the
growth of amyloid fibrils and that the calculated fiber diffraction
of the microcrystals and the diffraction from their fibril counter-
parts are very similar.
Using this approach, they have solved the structures of
25 microcrystals representing 22 unique peptides. The struc-
tures represent a survey of the types of interactions that can
support a cross-b core including the b strand and b sheet
symmetries that can exist in a cross-b fibril. Considering the
facts that several peptides form more than one distinct cross-b

1244 Structure 18, October 13, 2010 ª2010 Elsevier Ltd All rights reserved
Structure
Review
Figure 1. Underlying Structure of Amyloids
(A) Amyloid fibrils are composed of long filaments that are visible in negatively stained transmission electron micrographs.
(B) The schematic diagram of the cross-b sheets in a fibril, with the backbone hydrogen bonds represented by dashed lines, indicates the repetitive spacings that
give rise to (C) the typical fiber diffraction pattern with a meridional reflection at 4.7 Å (black dashed box) and an equatorial reflection at 6–11 Å (white dashed
box).
spine with distinct intersheet packing, and peptide fragments
with overlapping sequences from the same amyloid proteins
form distinct structures, the microcrystal-derived structures
are not likely to represent the conformations of the peptides
in the context of their biological amyloids. Furthermore, solid-
state NMR studies of both fibrils and microcrystals of the
peptide GNNQQNY show that the peptide conformation in the
crystals and fibrils differ from one another in subtle but distinct
ways (van der Wel et al., 2007). Nonetheless, the wealth of infor-
mation contained within these atomic resolution models is
invaluable for understanding the fundamentals of amyloid struc-
ture. For example, the collection of structures reveals two
classes of b sheet stacking interfaces, termed the ‘‘dry’’ and
‘‘wet’’ interfaces. The dry interface is devoid of water mole-
cules, consisting of complementary side chain interdigitation
that results in a high peptide packing density. Such a side chain
interdigitation is termed a ‘‘steric zipper’’ motif. The wet inter-
face is composed of hydrogen bonds between side chains,
some via water molecules, in a manner similar to intermolecular
contacts in protein crystals. This, along with the fact that some
peptides were found in multiple crystal forms with the same dry
but different wet interfaces, suggests that the stable structural
unit of the microcrystals, and hence of the fibrils, is a pair of
b sheets. The complete exclusion of water between the b sheets
maximizes the entropy gain for the formation of the interface
whereas the complementarity required to form such an inter-
face explains the sequence specificity of amyloid fibrils.
Although the amyloids of full-length proteins should have
more complex structures, the types of interactions that stabilize
them are likely to be the same as those observed in the short
peptides. As exemplified in Figure 2, these interactions (both
intra- and intersheet) can be classified as nonpolar (Van der
Waals and aromatic stacking) or polar (alternating charges
and hydrogen-bond ladders).
Structure 18, October 13, 2010 ª2010 Elsevier Ltd All rights reserved 1245
The Bigger Picture: Amyloid Fibrils of HET-s (218–289)
The only high-resolution model of a complete amyloid fibril
is the solid-state NMR structure of the prion-forming domain of
HET-s (Wasmer et al., 2008, 2009). HET-s is a functional prion
from the filamentous fungi Podospora anserina that is involved
in a self-nonself discrimination process (Coustou et al., 1997).
Like most prion proteins, HET-s is a multidomain protein. It has
a globular N-terminal domain and a flexible and disordered
C-terminal prion-forming domain (PFD) (Balguerie et al.,
2003). The PFD (comprising residues 218–289) can undergo
a large structural rearrangement to form a stable amyloid that
is itself the infectious entity, able to induce other HET-s mole-
cules to form the amyloid structure. The three-dimensional
structure of HET-s(218–289) fibrils was determined using 134
experimental inter- and intramolecular distance restraints
collected in PDSD (proton-driven 13C spin diffusion) experi-
ments (Wasmer et al., 2008). The fibril is a left-handed b-sole-
noid with each protein molecule forming two helical turns.
Because there are two helical windings per molecule, the
b strands alternate between inter- and intramolecular hydrogen
bonding along the fiber axis. In addition, there is an offset in the
winding of the solenoid so that the side chain packing
between the sheets is both inter- and intramolecular (Figure 3),
increasing the size and complexity of the intermolecular
interface (note: a similar intermolecular side chain arrangement
has been proposed for the Ab(1–42) fibrils associated with AD
[Luhrs et al., 2005]). In contrast to all of the small peptide struc-
tures in which none of the interstrand contacts are intramolec-
ular, the HET-s fibril b strands pack with 50% intramolecular
contacts (interstrand and intersheet). Furthermore, the pseudo-
repeat in the PFD amino acid sequence generates a pattern of
alternating charges along the fibril axis that supports the
correct in-register alignment of the b sheets (Figure 4). Thus,
it seems that the HET-s monomer contains structural elements
 Structure

Review

Figure 2. Steric Zippers, Hydrogen Bonding, and van der Waals Packing in Atomic Resolution Amyloid Crystal Structures
The interactions found in cross-b fibrils are exemplified by the structures of two peptides (A) NNQNTF (Protein Data Bank code: 3FVA) and (B) NVGSNTY (Protein
Data Bank code: 3FTL). The left panels are the views looking down the axes of the microcrystal ‘‘fibrils’’ with the neighboring dry interfaces depicted as a solvent-
accessible surface (gray) to show the tight interdigitation of the side chains. The intersheet hydrogen bonds are shown as yellow dashed lines (only a unique set
are shown, not symmetry equivalent bonds). The coloring scheme is white for main-chain carbon, yellow for side chain carbon, blue for nitrogen, and red for
oxygen. The position of the two-fold screw axes that relate the individual molecules in the fibril are indicated with black hurricane symbols. The right panels
are the view from the side of the fibrils showing only the b sheet corresponding to the colored strand from the left panel. The intrasheet hydrogen-bonds are shown
(black for main-chain only bonds and yellow for any side chain interactions) and the coloring of the main-chain carbons of individual strands alternates blue and
white. The front face of the sheet in (A) has two Asn ladders and a Phe ladder. The peptide in (B) is actually comprised of two short b strands with a kink between
them. The kink allows the Asn in the ladder to make a hydrogen bond to the main-chain oxygen of residue three (Gly) within the same molecule. In (A) the spacing
between the repetitive structures is indicated (the left panel has only the average spacing between main-chain atoms because the intersheet distances are not
related by crystallographic translations along unit cells).

that facilitate amyloid fibril nucleation (pseudo-repeats), growth
(winding offset) (Ritter et al., 2005), and ‘‘in register’’ intermo-
lecular alignment (alternate charged side chains). Within this
context, it is worth mentioning that, as a functional amyloid,
HET-s has evolved to fold into a cross-b motif and therefore
may be more complex than the disease-related amyloids for
which the cross-b structure is an unfortunate energy minimum
on the folding landscape. The b-solenoid is in fact a large
structural family of soluble proteins (Kajava and Steven,
2006), and the complexity of the HET-s PFD amyloid structure
is on par with other protein folds. It is ironic though, that with its
mere two helical turns per molecule, the HET-s PFD is the
shortest b-solenoid in sequence but a structure that can form
fibrils longer than 100 mm. Unlike in the peptide microcrystal
structures, there is a pattern of order/disorder visible on all
sides of the fibril (Figure 4) that arises from the flexible non-
b stranded loops of the molecule and that is distributed around
the fibril by a twist in the HET-s fiber model of about 14 per
monomer.
Despite its complexity, close inspection reveals that the HET-s
fibril consists of the same structural features that are found in the
microcrystals: a hydrophobic core, steric zipper-like interactions
for the intersheet packing, and Asn ladders and p-p stacking
running along the length of the fiber (Figures 3 and 4). However,
solid-state NMR and distance restraint-based refinements in
general cannot generate models with the accuracy required to
assign the conformation of every side chain. For example, as
shown in Figure 3, the side chains of the Asn residues within
the double Asn ladder of HET-s are less well defined than those
in the peptide structures (Figure 2). This ‘‘disorder’’ in the HET-s
model is unlikely to be a true representation of the structure
because the conformation of the Asn is dictated by the large
entropy gain achieved in satisfying a hydrogen-bond for every
Asn in the ladder without immobilizing any water molecules.
In a long fibril with hundreds or thousands of repeats, this
entropic effect can lead to a very large restraint on side chain
dynamics. Thus, much of the conformational variability in the
HET-s PFD structure is a result of the model refinement method.
Additionally, the twist rate of 14 per monomer is poorly
described by the NMR restraints and thus has an inherent
uncertainty. It is therefore very useful to have the atomic
resolution peptide crystal structures as a complement to the

1246 Structure 18, October 13, 2010 ª2010 Elsevier Ltd All rights reserved
Structure
Review
solid-state NMR-derived structure of the HET-s PFD, and
a synthesis of both NMR and crystallographic data can be
used to generate much more information about amyloids.
The Amyloid as a Universal Structure of Polypeptides
The reductionist approach of the Eisenberg team has shown
that the cross-b fold can be as simple as a single four-residue
b strand (the asymmetric unit from some of the microcrystals)
that assembles in a repetitive manner (Nelson et al., 2005). On
the other hand, the HET-s amyloid fold is as complex as that of
globular proteins. The predictive power of several algorithms for
the cross-b aggregation propensity of polypeptide sequences
(Fernandez-Escamilla et al., 2004; Tartaglia et al., 2008; Trovato
et al., 2006) suggests that the cross-b state is less complex than
most tertiary structures, for which the only predictive capabilities
are based on sequence similarity to known structures. Thus the
complexity of the cross-b fold can fall somewhere between
a secondary and a tertiary structural feature. The simplicity of
the most basic repeating subunit of the amyloid (e.g., a b strand)
might suggest that any polypeptide that can form a b strand
could eventually form an amyloid. In fact, many proteins aggre-
gate into amyloids or amyloid-like states when the delicate
balance between folding and aggregation is disturbed: overex-
pression in Escherichia coli can cause many proteins to form
inclusion bodies that have an amyloid-like substructure (Ventura
and Villaverde, 2006; Wang et al., 2008); some proteins can
aggregate into amyloid-like structures when heat denatured;
and some proteins that have stable soluble folds can be pushed
into an amyloid state in nonbiological conditions (Chiti et al.,
1999; Guijarro et al., 1998). However, amyloid aggregation is
highly amino acid sequence specific as demonstrated by the
‘‘steric zipper’’ side chain interactions observed in the micro-
crystals discussed above (Nelson and Eisenberg, 2006a; Nelson
et al., 2005; Sawaya et al., 2007). The essential involvement of
side chain interactions in the aggregation process is evident
from the observed sequence-specific nature of amyloid aggre-
gation (Margittai and Langen, 2006; Tjernberg et al., 2002; Zanuy
and Nussinov, 2003) and from the predictive power of the algo-
rithms for aggregation propensities. Nonetheless, an exhaustive
screening of nonphysiological conditions such as high protein
concentration, extreme pH, nonaqueous solvents (Chiti et al.,
Structure 18, October 13, 2010 ª2010 Elsevier Ltd All rights reserved 1247
Figure 3. The b-Solenoid Motif in HET-s
The left view is a ribbon representation of the
HET-s PFD fibril with the peptide chains alter-
nating blue and white and a ball on the N-termini
(residue 225). The four b strands are numbered
and with ‘‘a’’ and ‘‘b’’ designations for the codirec-
tional strands that are broken by a short kink and
‘‘–’’ and ‘‘+’’ designations for the strands from
the molecule below and above the central white
one. In both views, it is clear the side chain interac-
tions between the two sheets occur between
strands 1 and 4 of the same molecule and between
strands 2 and 3 of neighboring subunits. The right
view is 90 rotated from the left and is showing
only the Ca trace of the cross-b core and the
four Asn side chains in the interior of the core
that form a double Asn ladder. The absence of
the visible hydrogen-bond network in the Asn
ladder reflects the limitations of the technique
and refinement method rather than the actual
conformation of the Asn side chains (see main
text).
1999; Guijarro et al., 1998; Marcon et al., 2005; Polverino de Lau-
reto et al., 2003) can alter the influence of the side chains in the
aggregation process, perhaps driving any protein into an
amyloid conformation.
Structural Polymorphism of Amyloids
Although the current atomic resolution structural data on func-
tional amyloids is limited, it appears that under physiological
conditions, the amyloid fibrils of highly evolved structures like
HET-s are isomorphic structural entities, whereas ‘‘accidental’’
amyloids like the disease-associated amyloids are often found
in multiple structural states, or polymorphs. One can argue that
because the disease-related amyloids result from a misfolding
of the polypeptide chain, their structures do not always represent
the deepest or only accessible local minimum in the folding
energy landscape (or in this case, the aggregation energy land-
scape). Furthermore, this landscape may also be sensitive to
the environment such that changes in the conditions of aggrega-
tion will lead to different local minima, each representing
a different polymorph (Wang et al., 2010b). This energetic
description explains why polymorphism exists, but currently
much more can be said about what are the structural origins of
polymorphism.
EM images can differentiate polymorphic fibrils based on the
degree of twisting, the number of filaments per fibril, and the
diameter or mass per length of the fibrils (Fandrich et al.,
2009). Such structural heterogeneity can also be observed in
solid-state NMR spectra, either as multiple NMR signals per
atom or as very broad resonance lines, indicating that the origin
of the macroscopically observed polymorphisms in distinct
structures is at the atomic level. Indeed, the various steric zipper
packings observed in the amyloid peptide crystal structures
(Figure 4) represent two types of polymorphisms, packing and
segmental. Packing polymorphs (Figure 4A) have the same resi-
dues involved in the cross-b core but are packed differently (i.e.,
parallel versus anti-parallel strands) whereas segmental poly-
morphs differ in which stretches of residues are involved in the
cross-b core.
In addition to packing and segmental polymorphs, another
source of multiple energetically similar minima could be the
repetitive nature of the aggregate state. In a soluble protein,

small structural differences such as loop or side chain confor-
mations usually interchange on a fast time scale and comprise
a part of the large ensemble of structures that exists for one
particular protein. However, in highly ordered states such as
protein crystals, many side chains that are known to be dynamic
in solution are found in only a single well-ordered state. Further-
more, it is common to find the side chain conformations to be
distinct between two crystals of the same protein. Thus, the
one-dimensional crystal-like nature of the amyloid entity will
also place restrictions on the ensemble of conformations that
can exist within one fibril/crystal. An example of such side chain
conformers is presented in Figure 4A, in which the observed
crystal structure conformation is shown on the left and the Glu
side chain rotated to its most favored conformation while main-
taining its hydrogen bond network on the right. Although the two
amyloids differ only by the side chain orientation of Glu, their
surface features are markedly different. It is not possible to
have a mixture of Glu side chain conformations within one fiber
because the two are sterically mutually exclusive. This type of
polymorphism is subtler than the other two and we refer to it
as side chain polymorphism.
Because amyloid fibers are often bundles of protofilaments
(the most basic unit of the cross-b entity), polymorphism is
often observed as different supramolecular assemblies of the
1248 Structure 18, October 13, 2010 ª2010 Elsevier Ltd All rights reserved
Structure
Review
Figure 4. Polymorphism in Amyloids
(A) The effect of packing polymorphism (left panel)
and side chain polymorphism (right panel) on the
surface features of amyloids. The left panel
compares the packing polymorphic structures of
the peptide NVGSNTY (PDB 3FTK top and 3FTL
bottom) with three orthogonal views. The right
panel shows a single view of the peptide LVEALYL
(PDB 3HYD) in the true crystal structure conforma-
tion on the left and with the Glu side chain rotated
to its most favored conformation while maintaining
its hydrogen bond network on the right. The
solvent-accessible surfaces are colored by atom
with yellow for carbon, blue for nitrogen and red
for oxygen.
(B) The HET-s PFD surface showing a complete
360 twist of the fibril (broken into two panels).
The amino acid residues are colored yellow for
nonpolar (F,W,Y,P,V,A,I,L,C,M,G) green for polar
(S,T,N,Q,H) and blue (R,K) and red (D,E) for
charged. The alternating surface charges on the
amyloid core are more visible in the top view
whereas the long loop connecting strands 2 and
3 is the largely yellow and green stripe of less
ordered features visible in the bottom view.
protofilaments. It has been postulated
that the tight binding between protofila-
ments of a fibril is based on weak
and nonspecific interactions that are
translated into potent interaction by the
repeat-induced cooperativity. Such
different supramolecular structures may
be the result of underlying packing,
segmental, or side chain polymorphisms
in the protofilments (Figure 4A) or may
themselves be an independent form of
polymorphism, termed ‘‘assembly poly-
morphism’’ (Paravastu et al., 2008). Regardless of their molec-
ular origins, all four classes of polymorphism (i.e., segmental,
packing, side chain, and assembly polymorphism) can be prop-
agated by the self-templating nature of amyloids that ensures
the persistence of distinct amyloid fibrils, each consisting of
a unique conformation or polymorph. However, due to the
stochastic nature of nucleation and the fact that nucleation is
often a rate-limiting step in the aggregation process, the Boltz-
mann distribution of peptide conformations is not necessarily
reflected in mixtures of polymorphic amyloid aggregates. For
example, any polymorph, once it is established, may replicate
itself more quickly than a more stable polymorph that nucleates
only slowly.
The Amyloid Compared to Other Protein Aggregates
Although the amyloid-like conformation is a common feature of
many protein aggregates, there are other mechanisms by which
proteins can assemble into ordered aggregates without invoking
the cross-b motif. This includes domain-swapping, end-to-end
stacking (Bennett et al., 2006; Eisenberg et al., 2006; Nelson
and Eisenberg, 2006a, 2006b), and silk-type b sheet polymeriza-
tion. The functional aggregation of proteins in an end-to-end or
lateral manner is most well studied in the cytoskeletal complexes
of the actins and tubulins. Their correct function requires that
there be a tight control over the assembly and disassembly of
Structure
Review
their ordered aggregates (i.e., actin filaments and microtubules).
In the case of actin, the filaments retain the ability to make
branch points that are critical for the rigidity of the cytoskeleton
by incorporating specialized branch-inducing complexes into
the filament. Because it does not involve a gross rearrangement
of the protein fold, the end-to-end type of aggregation is revers-
ible and does not limit much the speed at which the assembly
and disassembly occur. The regulation of the assembly process
is similar for both actin and the tubulins in which nucleating
factors stimulate the initiation of aggregation whereas NTP
(nucleoside triphosphate) binding and hydrolysis regulates the
assembly/disassembly. Another example of end-to-end aggre-
gation is the assembly of hemoglobin S (HbS), the glutamate to
valine point mutant of hemoglobin A, into end-to-end and lateral
fibers. The fibers of HbS distort the red blood cells into the
abnormal rigid shape that gives the name to the resulting
disease: sickle cell anemia. The polymerization of HbS is
affected by several factors including oxygen saturation, hemo-
globin concentration, and hemoglobin composition. This latter
effect spares HbS/HbA individuals from severe anemia, and
because there is no biological control over the assembly/disas-
sembly, HbS/HbS individuals are afflicted to various degrees.
In contrast to the end-to-end/lateral aggregates are those of
the arthropod silks whose ordered assembly is rapid and
obtained on demand from specialized glands. The enormous
variety of silks represents many uncharacterized mechanisms
for polypeptide assembly, yielding structures that range from
primarily a-helical content to parallel b sheets and even to
cross-b amyloid-like structures. However, the more well-studied
spider fibroins present an interesting mechanism that involves
dynamic control like the end-to-end aggregates, a large and irre-
versible structural rearrangement like the amyloid aggregates
and a specialized production machinery like the keratin/collagen
aggregates. The fibroins have multiple repeating domains of
several types with highly conserved terminal nonrepeating (NR)
globular domains. Protein constructs that contain just two types
of repeats (Gly/Ala rich and Gly/Pro/Gln rich) can form a b-rich
aggregate whereas the addition of the C-terminal NR domain
to the construct produces a protein that in vitro displays many
of the aggregation properties of silk. The different types of
repeating domains give rise to the many properties of silk,
most importantly its tensile strength and flexibility. On silk fiber
assembly, the Gly/Ala rich regions (thought to be helical in solu-
tion) form a b sheet rich structure in which, unlike amyloids, the
strands run parallel to the fiber axis, whereas the Gly/Pro/Gln
segments adopt an uncharacterized structure that gives the
elasticity to the silk. A recent study has determined the structure
of the C-terminal nonrepeating domain from the dragline silk of
Araneus diadematus, and showed that it is essential for
controlled aggregation and ordered fiber assembly (Hagn
et al., 2010). The current data support the hypothesis that it is
the terminal NR domains that allow for the storage of the aggre-
gation prone repeats at a very high concentration in the spinning
gland (%50% w/v). The conversion from the soluble proteins
(thought to be in either a liquid crystal or micellar suspension)
into the ordered insoluble fibers is stimulated by a change in
the salt type (from Cl to PO43 ), a decrease in pH, dehydration,
and by the shear forces as the proteins pass through the narrow
spinning duct. Thus it appears that both a prior ordering and
Structure 18, October 13, 2010 ª2010 Elsevier Ltd All rights reserved 1249
a mechanical force are required to achieve the ordered assembly
of fibroins into a dragline silk fiber.
Yet another type of protein aggregation occurs in the
assembly of the structural proteins like collagen and the keratins.
These structural proteins comprise a variety of folds that we will
not discuss here except to say that what separates them from
other ordered aggregates is the fact that they do not spontane-
ously form ordered aggregates, rather they require a large
degree of specialized cellular machinery and processing to
achieve their final state. Finally, a group of aggregates that are
classified solely on their macroscopic appearance are the amor-
phous aggregates, a type that is often obtained by heat precip-
itation or concentration precipitation of proteins. Although the
classical macroscopic amorphous aggregate is on the meso-
scopic scale different from the fibrous aggregate, a recent study
from our lab indicates that amorphous aggregates may also
contain a defined cross-b-structure, however without the long
range (>mm) order found in fibrils (Wang et al., 2010b). This
finding suggests that the manner in which a polypeptide back-
bone achieves a kinetically accessible local energy minimum
might require that there is some local order or repeated structure
throughout the aggregate. Thus, there seems to be a caveat
about using the terms ‘‘ordered’’ versus ‘‘amorphous’’ protein
aggregates and it is perhaps more correct to describe aggre-
gates based on an order/disorder continuum or an extent of
long-range ordering (i.e., coherence length).
In summary, a comparison of the various types of aggregates
described above reveals two significant features that set the
amyloid apart from other ordered aggregates. First, the cross-b
spine of the amyloid can be formed from peptides as short as
four residues. Second, and partly due to the first, the repeat
distance is short; in the most basic amyloid it is the interstrand
distance of 4.7 Å. The close spacing of identical side chains
can generate specificity where none would exist without
a repeating structure. For example, a single amphipathic b strand
would have no affinity for a membrane, but a fibril made up of this
peptide could bind tightly to lipids. The same is true of a basic
peptide interacting with DNA (see below). In addition to support-
ing cooperative binding, the amyloid structure allows very short
peptides to assemble into complexes with regular secondary
structure, thus mimicking some features of larger folded proteins.
Hence, amyloids have unique structural properties that can result
in unique activities, as we shall see in the following section.
Structure-Activity Relationship
The two-state (soluble/insoluble) nature of amyloids and the
structural repetitiveness of the aggregates add a level of
complexity that most soluble proteins cannot access. Hence,
amyloids can possess a variety of biophysical and biological
properties, supporting a diversity of functions that rivals that of
soluble proteins. The growing list of known amyloid related activ-
ities (Table 1) shows that the formation of amyloids may result
both in a loss of function of the polypeptide, as described for
the yeast prions (Osherovich and Weissman, 2002; True and
Lindquist, 2000), or in a gain of function, as in the case of the
HET-s prion (Maddelein et al., 2002). Pmel17 amyloids serve
as a template for ligand binding (Fowler et al., 2006), whereas
other amyloids induce a specific toxic response as shown for
the HET-s prion system (Maddelein et al., 2002). The structural
 Structure

Review

Table 1. Functional Amyloids

Amyloid Function Reference
Curli Component of extracellular matrix involved in adhesion, aggregation, Chapman et al., 2002
invasion and biofilm formation
Microcin E492 Bacteriocin, membrane pore-forming peptide, amyloid form is Bieler et al., 2005
inactive
Chaplins Assisting aerial hyphae formation in Streptomycetes Claessen et al., 2003
Harpins Secreted by plant pathogenic bacteria, destabilize plant membranes, Oh et al., 2007
induce cell death
Sup35a Translation termination, prion form is inactive True and Lindquist, 2000
Ure2pa Regulatory function Baxa et al., 2005
in the nitrogen
catabolite repression pathway, prion form inactive
Rnq1pa Enhances the inducibility of other prions Derkatch et al., 2000
Swi1pa Chromatin remodeling factor, prion form inactive Du et al., 2008
a
Mot3 Transcriptional regulator of cell wall remodeling genes, prion form is Alberti et al., 2009
inactive
Hydrophobins Surface attachment and aerial hyphae formation Wösten and de Vocht, 2000
HET-s Heterokaryon incompatibility (see text) Coustou et al., 1997
Pmel17 Templates the synthesis of melanin Fowler et al., 2006
Neuronal CPEB Cytoplasmic polyadenylation element-binding protein regulates Si et al., 2003a
mRNA translation (see text)
Peptide hormones Sorting, storage, and release of diverse hormones (see text) Maji et al., 2009
a
Although the functional nature of these prions is still debated (Nakayashiki et al., 2005), they are included as functional because under certain stresses
they may confer some selective advantage, and there are dozens of other potential prions in yeast that have been conserved throughout fungal evolu-
tion (Alberti et al., 2009).

repetitiveness of the amyloid fiber provides an ideal template for
making it into a transmissible or infectious fold. In fact, most
prions (infectious proteins) have been found in their infectious
form to be amyloids whereas the amyloid structure itself appears
to be responsible for prion strain diversity (see below) (Toyama
et al., 2007). Although the in vitro formation of most amyloids
can be nucleated or ‘‘seeded’’ by the amyloid itself from a soluble
pool of the amyloidogenic peptide or protein, not all amyloids
have been shown to be transmissible and thus not all amyloids
can be considered prions (Riek, 2006). Thus the simplicity of
a cross-b-structure with its one-dimensional crystal-like nature
can encompass a wide range of activities. As we will highlight
in the following specific examples, their activity can be depen-
dent on the repetitive nature of the amyloid but also on its two-
state nature and the compactness of the fold.
Peptide Hormone Amyloids in the Formation
of Secretory Granules
Our recent finding that the amyloid structure is involved in the
formation of secretory granules in mammalian cells (Maji et al.,
2009) hints at the biological ubiquity of functional amyloids.
Eukaryotic cells have specialized pathways for both the constitu-
tive and regulated transport of secretory proteins/peptides to
the extracellular space (Kelly, 1985, 1987). During constitutive
secretion, a newly synthesized peptide hormone is transported
to the cell surface whereupon it is immediately released and
the rate of peptide secretion is limited by the rate of peptide
synthesis (Arvan and Castle, 1998; Lacy, 1975; Palade, 1975;
Tooze, 1998). In contrast, many secretory cells have an addi-
tional secretion pathway through which they can store peptide
hormones for extended periods of time. By packaging the secre-
tory peptides in highly concentrated membrane-enclosed secre-
tory granules (Arvan and Castle, 1998; Dannies, 2001; Kelly,
1985) the cells can store a large quantity of the hormone until
a signal triggers its release, at which point they can secrete
hormones much faster than their synthesis rates would permit.
During the maturation process, the prohormones are proteo-
lytically processed into their active hormone entity followed by
hormone aggregation into hormone-specific granules that are
membrane-enclosed. The granules are generally composed of
a single peptide species or, when coaggregated, the distinct
peptides exist exclusively in a specific integer ratio. Until
recently, there was no explanation for how peptide hormones
self-segregated, but the amyloid nature of peptide hormones in
secretory granules (Maji et al., 2009) explains much of what
were mysteries in the processes of granule formation, selection,
storage, and hormone release. The nature of amyloids provides
a series of controls over secretory granules that are semi-auton-
omous, requiring minimal peripheral cell machinery.
1. The timing and location of amyloid aggregation in the Golgi
can be controlled by the processing of the prohormone.
Because the amyloid aggregation of a small peptide is
sensitive to the sequence and chemical modifications of
the peptide, there are multiple ways in which processing
could initiate aggregation. In addition to controlling aggre-
gation through modification of the hormone, there is the
possibility of controlling aggregation through pH or the
presence of helper molecules such as glycosaminogly-
cans (GAGs) that can stabilize the amyloid conformation
(Maji et al., 2009).

1250 Structure 18, October 13, 2010 ª2010 Elsevier Ltd All rights reserved
Structure
Review
2. The formation of amyloid fibrils is highly sequence-specific
so that once initiated, the amyloid aggregation of the
hormone is self-selective, yielding granule cores
composed of specific hormones only. Specific coaggre-
gation of some hormones is possible as has been demon-
strated for ACTH and b-endorphin, two hormones that are
processed from a single prohormone. Because some
amyloid proteins are able to cross-seed (Giasson et al.,
2003; Han et al., 1995), it should be expected that some
hormones will form mixed fibrils, although still in a specific
manner.
3. After initiation of aggregation and sorting of the peptides
into specific secretory granule cores, the amyloid struc-
ture provides the most dense packing possible (Nelson
et al., 2005), both excluding nonaggregation-prone consti-
tutively secreted proteins and providing an extremely
stable storage system.
4. As part of the granule maturation process, the hormone
amyloids are surrounded by membrane as they separate
from the Golgi. There are many examples to suggest that
membrane binding is an inherent property of amyloid
aggregates (Gellermann et al., 2005; Sparr et al., 2004)
and so formation of a membrane around the hormone
aggregate may be spontaneous.
5. Finally, on receiving the appropriate signal, the granules
are secreted and the amyloid structure of the hormone
aggregates enables a controlled release of monomeric,
functional hormone (Maji et al., 2008). Each peptide
hormone will have its own dissociation rate and this can
again be controlled by factors such as pH, ion concentra-
tion, and extracellular chaperones.
The Structure-Activity Relationship in the HET-s
Amyloid Prion
The structure of the prion-forming domain (PFD) from HET-s
revealed that the infectious state of this functional amyloid is
a b-solenoid and thus a member of a large structural class of
soluble proteins (Maddelein et al., 2002; Ritter et al., 2005;
Wasmer et al., 2008). The complexity of its structure reflects its
evolutionarily optimized role as an amyloid. The b-solenoid
structure is not a chance misfolding of the PFD, but rather
a very stable conformation that can spontaneously occur at
moderate protein concentration. The presence of a pseudo-
repeat in the sequence allows a single PFD to adopt two turns
of the b-solenoid, forming the nucleus of the amyloid fold. As
well, it gives rise to alternating charges along the fibril axis that
support the correct in-register alignment of the b sheets
(Figure 4). The pairing of these repeats guarantees that there is
a unique low-energy fold without detectable polymorphism in
physiological HET-s fibrils (Wasmer et al., 2008). With its hydro-
phobic core and hydrophilic solvent-exposed side chains, the
HET-s PFD fibril has many of the hallmarks of soluble proteins.
In fact, unlike many amyloidogenic proteins, there is no toxicity
associated with its overexpression in bacterial or fungal hosts
or even in rats (unpublished data).
It had been previously shown that the introduction of the
b sheet-breaking residue proline within the predicted b strands
resulted in a loss of infectivity in the fungus, whereas proline
mutations in the loop positions had no effect (Ritter et al.,
Structure 18, October 13, 2010 ª2010 Elsevier Ltd All rights reserved 1251
2005). Therefore, the high-resolution structure was able to
confirm that the amyloid core is the infectious entity. However,
of its many functions, only the infectivity of the HET-s prion is
completely contained within its PFD. The heterokaryon incom-
patibility function requires a full-length protein that includes the
N-terminal globular domain (termed the HeLo domain based
on its conservation with other fungal proteins) and the PFD
(Balguerie et al., 2004). Furthermore, the HeLo domain has
a prion regulatory function that also requires a full-length protein
with the proper spacing between the domains (Balguerie et al.,
2003; Greenwald et al., 2010) (see also below). In the latter
report, we conclude that the formation of the amyloid structure
in the PFD causes a structural rearrangement of the HeLo
domain that leads to the other activities of the HET-s prion. In
this manner, the combination of a globular fold and an amyloid
could create an immeasurable functional landscape and one
that has certainly been explored by nature and that is awaiting
discovery in the laboratory. The evolutionarily optimized fibril
structure of HET-s has many features that are likely to be found
in other highly evolved functional amyloids (Barnhart and
Chapman, 2006; Fowler et al., 2006, 2007). In contrast, the
disease-associated amyloids discussed in the following exam-
ples have functions that are detrimental to the organism and
structures that are not as easily characterized due to their high
degree of polymorphism arising from their metastable nature.
Distinct Aggregates, Distinct Activities
Our recent structural studies on HypF-N aggregates shows that
even the rapid, forced aggregation of a protein can lead to
several distinct amyloid-like states whose conformations
depend on the conditions in which they were formed (Wang
et al., 2010b). Furthermore, the activities of the aggregates in
several biochemical and biological assays varied with the
conformation of the aggregates. HypF-N is the N-terminal
domain of the prokaryotic hydrogenase maturation factor, which
has been shown to form toxic oligomers on way to forming
amyloid fibrils. In addition to these amyloid fibrils, we studied
the bacterial inclusion bodies, heat-precipitated aggregates,
concentration-induced aggregates, and trichloroacetic acid-
precipitated aggregates of HypF-N. Despite the wide range of
conditions used to induce protein aggregation, all five types of
HypF-N aggregates contain the cross-b sheet motif. However,
each of the aggregates is structurally distinct, having different
segments of the protein involved in protected secondary struc-
tures. We compared the affinity of each aggregate to adenosine
triphosphate (ATP), thioflavin T, oligonucleotides, and micelles,
as well as the ability of each to interfere with cell viability.
Although the highest activities were found in the fibrils, the other
aggregates had significant activities that were not intercorre-
lated (e.g., strong ATP binding was not correlated with cell
viability). We believe that the key to their varying properties is
that each aggregate is built around a cross-b sheet motif
composed of different amino acid segments. The repetitive
nature of the cross-b sheet motif supports cooperative interac-
tions that can generate unique and potent activities. This one
example of the landscape of aggregate structure-activity rela-
tionships highlights the existence of a multifactorial structure-
activity function for amyloids and begins to shed light on the
complexities of polymorphs (see above) and prion strains (see
below).

The Infectious Nature of Amyloids
An amyloid can in principle grow by recruiting the corresponding
soluble amyloid protein. Hence, under certain circumstances,
amyloid replication can occur in an organism once infected by
the corresponding amyloid (seeds). The question of what makes
an amyloid infectious has been addressed in a recent mathemat-
ical model that was experimentally validated in vivo (Tanaka et al.,
2004). The infectivity of an amyloid depends on the concentration
of soluble and aggregated amyloid protein, the growth rate of the
amyloid, and the rate of breakage of the amyloid fiber into seeds.
According to this theoretical framework, manipulation of these
variables could transform any amyloid into a prion (Riek, 2006).
For example, an increase of soluble amyloid protein in the host
might be sufficient to transform an amyloid into a prion (Meyer-
Luehmann et al., 2006). In contrast, the formation of long fibrils
decreases the seed to total amyloid protein ratio, thus reducing
the prion titer (Silveira et al., 2005). Based on the nucleated poly-
merization model of amyloids it is theoretically possible that all
amyloids (under the correct circumstances) can be infectious.
The normally noninfections Ab amyloid-associated AD becomes
infectious in a mouse that overexpresses the precursor to Ab
(Kane et al., 2000; Meyer-Luehmann et al., 2006) as well as in non-
transgenic primates (Ridley et al., 2006). Other amyloids that
appear to have an infectious component to them are the amyloid
entity of foie gras (Westermark and Westermark, 2009) and the
tau amyloid associated with AD. For further details, we refer the
reader to an excellent review by Aguzzi and Calella (2009) that
concentrates on this topic.
The Toxic Nature of an Amyloid
The long list of diseases associated with amyloid formation
suggests that something is inherently toxic about amyloids.
However, this observation is countered by the growing list of
functional amyloids (Table 1). Thus, toxicity, like other protein
activities, is rather something to be associated with a particular
conformation of a peptide, such that one polymorph will have
different activities from another polymorph. But what are the
structural features of an amyloid that can lead to toxicity? The
lack of toxicity of the functional HET-s fibrils might be due to their
hydrophilic surface that would suppress nonspecific hydro-
phobic interactions with other proteins or membranes. Alterna-
tively, the folding/aggregation pathway of functional amyloids
in general may be more cooperative, lacking oligomeric or proto-
fibril intermediates of the kind that have been suggested to be
the toxic species in AD (Hardy and Selkoe, 2002; Klein et al.,
2004; Lazo et al., 2005). Although b sheet-rich (Chimon et al.,
2007), these conformational intermediates have a structural
motif that is distinct from fibrils as evidenced by the establish-
ment of both oligomer-specific and fibril-specific antibodies
(Kayed et al., 2003). Consequently, it has been suggested that
the disease-associated amyloid fibrils actually have a beneficial
function in that they sequester the toxic oligomers into nontoxic
mature amyloid fibrils (Hardy and Selkoe, 2002). Finally, as
discussed next, the fibrillogenesis of functional amyloids may
be highly regulated, which represses any potentially toxic effects
such as mislocalization or hyperaggregation.
Control Of Aggregation
Considering the often delicate balance between properly folded
and misfolded proteins as well as the widespread occurrence of
1252 Structure 18, October 13, 2010 ª2010 Elsevier Ltd All rights reserved
Structure
Review
toxic protein aggregates in biology, it is not surprising that
complex regulatory systems have evolved to maintain the
balance of functional proteins necessary for life. These systems,
collectively referred to as protein homeostasis, or ‘‘proteostasis’’
(Powers et al., 2009) comprise a vast network that ranges from
synthesis and folding (i.e., ribosome, chaperones, aggregases,
disaggregases) to degradation (i.e., proteases, autophagy, lyso-
somal targeting). Outside of this proteostasis network, functional
aggregates have their own controls that can involve external
regulatory elements (i.e., enzymatic activation) or self-contained,
autoregulatory elements.
Proteostasis and the Role of Chaperones in Aggregation
Transgenic Caenorhabiditis elegans that express the 42-residue
peptide of Ab in their body wall muscle cells suffer an age-related
paralysis that is accompanied by Ab amyloid deposits. In this
C. elegans model of Ab toxicity, two opposing protective path-
ways have been identified in the proteostasis network (Cohen
et al., 2006). Both pathways are activated by inhibition of the
IGF1-R (insulin/insulin growth factor 1-like receptor) signaling
pathway, which negatively regulates the heat shock transcription
factor HSF1. The first pathway, enabled by upregulation of HSF1
activity, leads to the induction of a disaggregase activity that
decreases the amount of Ab amyloid aggregates. This protective
disaggregase activity is detectable in an in vitro assay in which
homogenized worms are mixed with Ab fibrils with continuous
monitoring of fibril content by Thioflavin T fluorescence.
A second pathway, also downstream of IGF1-R signaling and
dependent on the FOXO transcription factor activity, acts by
enhancing the formation of large aggregates and thereby
protecting the cell from smaller, putatively more toxic oligomers.
The study of the yeast prion Sup35, whose amyloid state is
propagated in [PSI+] variants, has greatly expanded our under-
standing of the role of chaperones in the aggregation and disag-
gregation of proteins (Serio and Lindquist, 2001). Many chaper-
ones bind to aggregation-prone segments of proteins and
protect them from unfavorable interactions, thereby assisting
in protein folding and preventing unwanted aggregation. This
description can lead to a simplistic view that predicts that chap-
erones would negatively regulate the amyloid based prions.
However, several examples from the yeast literature paint
a more nuanced picture of chaperone activity. The chaperone
Hsp104 is a protein-remodeling factor whose disaggregase
activity, which can reduce the size of Sup35 fibrils, ironically is
required for [PSI+] propagation. Whereas the deletion of
Hsp104 leads to [PSI+] curing, so does its overexpression
(Chernoff et al., 1995). Thus, the proper balance of disaggregase
activity is required to maintain the correct size and numbers of
fibril seeds for prion propagation as the larger Sup35 aggregates
formed in the absence of Hsp104 have lost their infectivity.
Hsp104 is not acting alone and members of the Hsp70 family
of chaperones are also involved in [PSI+] maintenance and
curing. Despite their highly conserved sequence and domain
organization, the Hsp70 proteins can also have opposing effects
on the Sup35 aggregate load and on the [PSI+] prion state. Two
subclasses of cytosolic Hsp70 proteins in yeast are Ssa (Ssa1)
and Ssb (Ssb1 and Ssb2). Ssb behaves as expected for a chap-
erone by inhibiting the spontaneous formation of [PSI+] in [psi ]
cells and by increasing the curing efficiency of overexpressed
Hsp104. Ssa has the exact opposite effect: inhibiting [PSI+]
Structure
Review
curing by overexpressed Hsp104 and increasing the sponta-
neous formation of [PSI+]. Studies with Ssa/Ssb chimeras
show that the peptide-binding domain determines the [PSI+]
activity (Allen et al., 2005). It is proposed that Ssa stabilizes an
unfolded/prion-competent form of Sup35 whereas Ssb either
stimulates refolding into nonprion conformation or targets mis-
folded proteins for degradation. It is also shown that in a [PSI+]
culture that overproduces Sup35, the proportion of [psi ] cells
increases with the overexpression of Ssa, demonstrating that
Ssa can also have antagonistic effects on [PSI+] propagation.
This latter effect, like the Hsp104 deletion, is likely to be due to
an increase in the size of the Sup35 aggregates and decrease
in the number of seeds. Thus, the proteostasis network of chap-
erones in yeast demonstrates the importance of the balance of
protein expression and folding for the fate of amyloidogenic
proteins. Furthermore, these results highlight the fact that the
infectivity of an amyloid is dependent on the number and size
of the aggregates in the cell.
Enzymatic Control of Aggregation
Perhaps the most intricate enzymatic control of aggregation is in
the coagulation cascade. Here the control of a nonamyloid
aggregation is achieved by a series of proteolytic activations
leading to the cleavage of fibrinogen by thrombin to yield fibrin,
the protein that forms the fibrous aggregate that strengthens
a clot. Plasminogen binds to the fibrin molecules in a clot and
is activated by tissue plasminogen activator, which also binds
to fibrin and cleaves plasminogen to form plasmin. Plasmin,
proceeds to digest fibrin, thus dissolving the clot. Although fibrin
is not an amyloid, its regulation is analogous to the enzymatic
control of peptide hormone aggregation. The proteolytic pro-
cessing of the nonamyloidogenic prohormone by the corre-
sponding prohormone convertase, which like thrombin is synthe-
sized as an inactive zymogen, is required to form the mature
hormone. Because only the mature hormones are found in the
secretory granules, it follows that the prohormone convertases
are required to initiate the amyloid aggregation of peptide
hormones. The subsequent disaggregation is not enzymatically
controlled, but rather by a change in the environment of the
amyloid aggregate, through the dilution of the monomeric
peptides as they are released from the aggregate, and possibly
by extracellular chaperones. A disease-related example of enzy-
matic control of aggregation is the processing of the amyloid
precursor protein (APP) by sequential cleavage reactions to yield
Ab. Because the ‘‘control’’ of Ab formation does not have a known
biological function, it may be better classified as an aberrant
degradation. However, there are striking similarities between
the processing of APP (the precursor to Ab) and the functional
amyloid Pmel17 (Hurbain et al., 2008). Pmel17 aggregation has
many levels of control in that the protein must first pass through
multivesicular endosomal compartments before it can be
proteolytically processed by a proprotein convertase. The pro-
cessing is required for amyloidogenesis and the vesicular traf-
ficking is required for the processing (Berson et al., 2003; Hurbain
et al., 2008). Other similarities include the fact that like APP,
Pmel17 is a membrane glycoprotein and that their amyloidogenic
fragments are without glycosylation. Although Ab still has no
known function, its presence in the body and the above similar-
ities with Pmel17 suggest that it may have a function (Kimura and
Schubert, 1993), which even might involve the amyloid entity.
Structure 18, October 13, 2010 ª2010 Elsevier Ltd All rights reserved 1253
Self-Regulating Aggregates
In contrast to the aggregation controls described above, there
exist both functional and nonfunctional aggregates whose
aggregation control is encoded within their protein sequence
or structure. Nearly all globular proteins contain these sorts of
controls because their stable three-dimensional structure itself
prevents the aggregation of their aggregation-prone regions.
Furthermore, a multiproteome analysis has found that many
highly aggregation prone stretches of amino acids in proteins
are flanked by ‘‘gatekeeper’’ residues of lysine, arginine, or
proline (Rousseau et al., 2006). These gatekeeper residues
inhibit the formation of aggregates in these aggregation-prone
stretches. An analogous use of gatekeeper residues is also
found in the functional amyloid, curli, where they act as a regu-
lator of aggregation and toxicity (Wang et al., 2010c). The curli
gatekeepers are aspartate and glycine, but they probably act
similarly to the arginine, lysine, and proline gatekeepers. Curli
fibers are composed of two subunits, CsgA and CsgB. In vivo,
the polymerization of the major curli subunit protein, CsgA, is
dependent on CsgB-mediated nucleation. The amyloid core of
CsgA features five imperfect repeats (R1–R5) and the internal
three repeats were shown to have nonconsensus (gatekeeper)
residues. When the gatekeeper residues were mutated to the
consensus residues, there was a CsgB-independent growth of
curli fibers in vivo. These fibers were mislocalized and were
associated with cytotoxicity. Thus the curli functional amyloid
aggregation is spatially controlled through a multicomponent
nucleation with gatekeeper residue control. In addition to gate-
keeper residues, other controls can be encoded in the amyloid
itself. The aggregation propensity of a peptide can be altered
by pH or salt (e.g., spider silks) and as mentioned for the peptide
hormones, can require a cofactor such as a charged polymer
(heparin). An example of pH control has recently been reported
for the amyloid core of Pmel17 (McGlinchey et al., 2009) that
in vitro, only forms amyloids under mildly acidic conditions like
those found in the melanosome lumen.
Another type of self-regulation is found in the functional aggre-
gates of spider silk (see above) and the HET-s prion. These multi-
domain proteins have, in addition to their natively disordered
aggregation domain, a globular domain (i.e., HeLo in HET-s)
whose functions include an aggregation regulatory role. In the
HET-s system, aggregation is used as an identity marker
that is detected through amyloid-based intermolecular interac-
tions: soluble HET-S recognizes amyloid HET-s. Thus the aggre-
gation control in the HET-s/S system is a little more complex in
that the two ‘‘isoforms’’ (95% identical) have very different
aggregation propensities. HET-S does not aggregate in vivo
whereas HET-s spontaneously forms aggregates in vivo and
their in vitro behavior is consistent with the one in vivo. The prion
forming domains (PFDs) of HET-s and HET-S, when expressed in
isolation, both aggregate 100 times faster than HET-s indi-
cating that the globular HeLo domains of both proteins have
some inhibitory effect on aggregation. However, in HET-S the
effect is much stronger and Het-S can actually inhibit the aggre-
gation of HET-s in trans (Balguerie et al., 2003). Our structural
study of the HeLo domains of HET-S and HET-s led us to the
conclusion that the thermodynamic stability of the HeLo domain
is what regulates aggregation of the PFD (Greenwald et al.,
2010). The cross-b folding of the PFD puts a structural strain

on the HeLo domain that for HET-S, but not HET-s, leads to
a structural rearrangement that prevents further amyloid growth.
In this manner, HET-S can inhibit its own aggregation as well as
that of the nearly identical HET-s. It is this mechanism that allows
the PFD of HET-S to act as an ‘‘amyloid-sensor’’ that sets off the
cell-death reaction when it encounters the prion form of HET-s.
Although the downstream processes that lead to cell-death are
currently not known, we hypothesize that the structurally altered
HeLo domain of HET-S is directly involved in a toxic species.
Hypothesis
There are many hypotheses that concern the amyloid entity. The
two most often reviewed ones—summarized only briefly here
while referring to published reviews—are the amyloid cascade
hypothesis of Ab and a-synuclein associated with AD and
Parkinson’s disease (Hardy and Selkoe, 2002), and the protein-
only hypothesis first proposed by Alper et al. (1967) concerning
the origin of prions and prion strains (Prusiner, 1998). Other
hypotheses of interest are the involvement of amyloids in the
origin of life (Carny and Gazit, 2005) and in the evolution of
protein folding.
Prions: The Protein-Only Hypothesis
The ‘‘protein-only hypothesis’’ (Alper et al., 1967; Griffith, 1967;
Prusiner, 1982) states that prion diseases such as scrapie in
sheep, bovine spongiform encephalopathy (BSE), and Creutz-
feldt-Jakob disease in human are distinct from infectious
diseases caused by bacteria, viruses, or viroids, in that the origin
of the disease is related to conformational alterations of a ubiqui-
tous protein and that nucleic acids are not essential for the prop-
agation of the infectious agent. Thus, prions are infectious
proteins (Prusiner, 1982). In the prion diseases, the prions prop-
agate by converting the normal form of the prion protein (PrPC)
into an altered infectious b sheet-rich, protease resistant confor-
mation (PrPres) (Prusiner, 1998), which is proposed to be an
amyloid entity. The amyloid serves as an infectious seed that
recruits soluble host PrPC for growth. The growing amyloid
may break into pieces, producing more infectious seeds.
Prions have also been identified in lower eukaryotes, namely
yeast and the filamentous fungus Podospora anserina (Coustou
et al., 1997; Serio and Lindquist, 2001; Wickner et al., 2001). The
[URE3] and [PSI] non-Mendelian genetic elements of yeast were
shown to correspond to the prion state of the Ure2p and Sup35p
proteins, respectively. Ure2p and Sup35p are soluble proteins in
the normal conformation but aggregate in vivo on conversion to
the prion state (Edskes et al., 1999; Patino et al., 1996). In vitro,
purified Sup35p and Ure2p proteins undergo self-seeded poly-
merization into amyloid-like fibrils (Glover et al., 1997; King
et al., 1997). It has been shown that the HET-s prion and the
yeast prions that are expressed in E. coli are infectious once
they have been converted into amyloid fibrils (Maddelein et al.,
2002; Sparrer et al., 2000). Hence, for these prions the protein-
only hypothesis has been many times proven. However, the de
novo generation of the infectious prion-agent starting from
noninfectious recombinant PrPC has only recently been
achieved. The landmark study from the lab of Deleault et al.
(2007) showed that PrPC purified from cell lines can be infectious
on the formation of protease-resistant PrPres using protein-mis-
folding cyclic amplification (PMCA). Legname et al. (2004)
showed for the first time that mouse PrP produced recombi-
1254 Structure 18, October 13, 2010 ª2010 Elsevier Ltd All rights reserved
Structure
Review
nantly in E. coli can be polymerized into amyloid fibrils that are
infectious. However, the infectivity titer is very low and only
observed on inoculation intracerebrally into transgenic mice
that have a many-fold overexpression of the murine PrP frag-
ment PrP(89-231). Brain extracts from these infected mice con-
tained the protease resistant PrPres, and neuropathological
findings suggest that a novel prion strain was created. Very
recently, much more compelling evidence has been presented
in which cofactors were used to convert recombinant PrP into
highly infectious PrPres via PMCA in the presence of RNA and
membrane-mimetics (Wang et al., 2010a). These experiments
together with similar results from Castilla et al. (2005) suggest
that the protein-only hypothesis is now also proven for mamma-
lian prion disease. There is, however, one caveat concerning all
of the above experiments that support the protein-only hypoth-
esis: although they demonstrate that the presence of PrPC
(Bueler et al., 1993), PrPres, and PrPres growth are necessary
for infectivity, it is still possible that there is an endogenous
viral-like DNA/RNA that gets expressed through the presence
of PrPres and uses PrPres as a container for infectivity.
Prion Strains
In mammals, more than a dozen prion strains are known (Weiss-
mann, 2005). Prion strains are distinguished by several factors
including the incubation time, the age of disease onset, the ability
to cross a species barrier, and the brain area of deposited prions.
Because according to the protein-only hypothesis (see above)
the prion protein in its aggregated conformation is both the toxic
and the infectious culprit of prion diseases, the strain-specific
properties must be attributed to altered conformations of prion
protein amyloids. Although it is perhaps difficult to imagine
how all of the prion strain-specific properties are due to altered
conformations of PrPres, there is experimental evidence to
support this hypothesis: the mammalian prion strains can be
differentiated by PrP glycosylation patterns (Asante et al.,
2002), conformation-sensitive antibodies to PrP (Cali et al.,
2009), amyloid conformation-specific fluorescent dyes (Sigurd-
son et al., 2007), and PrP protease-resistance. Furthermore,
conformationally distinct amyloid aggregates of the yeast prion
Sup35 can be propagated in vitro that on infection, lead to
several distinct prion strains (King and Diaz-Avalos, 2004;
Tanaka et al., 2004). Specifically, two polymorphs of Sup35
fibrils that lead to different strains have been shown to have
distinct cross-b cores (Toyama et al., 2007).
The fact that there are more than a dozen documented
prion strains can be explained by the multitude of possible poly-
morphisms. The combination of packing and segmental
polymorphisms with side chain polymorphisms, assembly poly-
morphisms (Figure 4), and possible chemical modifications (such
as distinct glycosylation patterns) may generate many amyloid
states with distinct activities. As exemplified in Figure 4, just
a single rotation of a side chain can change the surface charge
distribution of the amyloid and hence its potential to bind
charged molecules such as membranes, RNA, and DNA.
Polymorphs may differ in their stability of the amyloid fiber, which
can affect the shearing rate, thus leading to more or fewer infec-
tious seeds and thus a difference in infectivity or disease onset
rate. The strain specific species barrier may be explained by
the conformation of the amyloid that either is able to incorporate
the new host PrP that differs in the amino acid sequence (low
Structure
Review
species barrier) or is only poorly able to seed the new host PrP
(high species barrier). The often-observed adaptation of the
prion strain to the new host on serial transmission can be
explained by a new side chain or packing polymorphism that is
established and that enables the host PrP to replicate more
efficiently. It is more difficult to directly connect PrP polymor-
phism to the strain-specific brain lesion profiles, but because
cell-specific properties such as pH, membrane composition,
glycosylation, and available chaperone systems will likely vary
in different brain regions, they may influence the amyloid replica-
tion. The structure-dependent activities of the prion amyloid may
build the basis for the brain region-dependent toxicity. In
summary, with the multiple types of polymorphisms that can
form in an environment-dependent manner (Wang et al.,
2010b), the presence of hundreds of subtly different amyloid
entities that have distinct activities yet that replicate themselves
with great accuracy seems to be possible.
The Amyloid Cascade Hypothesis in AD
AD is the most common fatal neurodegenerative disorder, and
the devastating neuropathology of AD is tightly linked to the
cortical deposition of fibrillar Ab plaques (Glenner and Wong,
1984). The major component of these deposits is Ab(1-42), which
is generated from the amyloid precursor protein (APP) by the
proteolytic activities of b- and g-secretase (Kang et al., 1987;
Masters et al., 1985). Elevated physiological levels of Ab(1-42)
lead to early onset AD in Down Syndrome patients (Goldgaber
et al., 1987; Tanzi et al., 1987), and in several hereditary forms
of AD that have either been linked to mutations in the APP
gene, or in the presenilin genes, which code for the g-secretase
(Suzuki et al., 1994). Mouse models that are transgenic for
human hereditary APP variants and that overexpress human
g-secretase rapidly develop AD-like neurological disorders
(Games et al., 1995). These in vivo findings and the dramatically
increased in vitro aggregation propensity of Ab(1-42) compared
to Ab(1-40) (Riek et al., 2001) suggest fibril formation by Ab(1-42)
as the process primarily responsible for the fatal neuropatholog-
ical manifestation of AD.
However, because of the weak correlation between plaque
load and AD pathology the amyloid hypothesis has been chal-
lenged. More recent findings indicate that Ab dimers (McDonald
et al., 2010; Shankar et al., 2008), small diffusible Ab oligomers,
and a membrane-bound Ab oligomer are highly toxic and are
associated with memory dysfunction in the early stage of AD
(Cohen et al., 2006; Hardy and Selkoe, 2002; Lambert et al.,
1998; Lesne et al., 2006). Although the above toxic oligomers
are enriched in b sheet content, they are in a distinct conforma-
tion from amyloid fibrils (Kayed et al., 2003). In vitro, Ab has been
described in many states including a membrane-bound mono-
mer, a dimer, small oligomers, worm-like fibrils, ring-like oligo-
mers, protofibrils, and dozens of polymorphic mature fibrils,
the latter observable even within the same preparation (Mein-
hardt et al., 2009). It has also been shown that different solid-
state NMR spectra can be obtained depending on how the Ab
fibrils were prepared (Balbach et al., 2002; Petkova et al.,
2005). These different NMR spectra were correlated with the
EM-observable macroscopic fibril morphologies, indicating
that the polymorphisms have their roots at the atomic level
(see above). Each of these various structural entities appear to
be toxic, but which of them is the most toxic culprit in AD and
Structure 18, October 13, 2010 ª2010 Elsevier Ltd All rights reserved 1255
what is the cause of toxicity is under debate (Finder and Glock-
shuber, 2007; Selkoe, 2004). However, for an aggregation
disease there may be many different amyloid structures present
in a disease sufferer and each of these structural entities can
have distinct activities. Hence, Ab aggregates may be toxic to
cells and the host on many levels. The plurality of potential
aggregates, each having several toxic mechanisms, may
thereby account in addition to genetic factors for the docu-
mented complex multi facet nature of amyloid diseases. It is
evident that many important scientific challenges lie ahead in
the amyloid disease field.
Prebiotic Biology and the Origin of Protein Folds
The many activities of protein aggregates support the hypothesis
that under the harsh conditions of the prebiotic earth some
3.9 billion years ago, the first self-propagating biomolecules
were peptide aggregates composed of the cross-b sheet entity
(Maury, 2009). The synthesis of amino acids under primitive earth
conditions has been demonstrated in the lab (Miller, 1953) and
the subsequent formation of peptides can be achieved by one
of several prebiotic syntheses: (1) by carbonyl sulfide, a volcanic
gas (Leman et al., 2004); or (2) by copper in high salt and high
temperature, conditions that mimic a shallow sun-baked lagoon
(Schwendinger and Rode, 1992). In a prebiotic world, those
peptides that were able to form amyloids would have been
protected from hydrolysis (Toledano et al., 2006) or other chem-
ical modification while exposed to intense ultraviolet irradiation,
large temperature fluctuations, and high salt concentration. The
preferential assembly of certain sequences over others could
have been an early evolutionary pressure that selected for the
fittest amino acid sequences (Williams et al., 2009), whereas
some of these peptide amyloids may have replicated themselves
in a template-assisted peptide synthesis (Takahashi and Mihara,
2004). Next, the amyloid entities may have recruited membranes
by their intrinsic activity to bind other repetitive structures (Wang
et al., 2010b). There are numerous selective advantages of
a membrane enclosure for such a self-replicating system, as it
would control the concentration of both the peptides and amino
acids that are critical for peptide aggregation and template-as-
sisted peptide synthesis, as well as protect the system further
from the harsh environmental conditions. At some time in their
evolutionary history, amyloids may also have recruited DNA or
RNA molecules, again via their activity to bind other quasi-repet-
itive structures, thereby stabilizing the nucleic acids while
optimizing its own replication (Deleault et al., 2003). This may
have set the stage for an RNA-protein world with the required
complexity to evolve into life as we know it (Carny and Gazit,
2005).
Even if life’s origins are not tied to a self-replicating amyloid, it
is still likely that the first protein folds in a living cell would have
been amyloids. There are many arguments to support the idea
that protein aggregation has played a key role in the early evolu-
tion of proteins (Chernoff, 2001; Maury, 2009; True and Lind-
quist, 2000) the simplest being the improbability that folded
soluble proteins existed early in the evolution of polypeptide
sequences. It is probable that primitive polypeptides were
comprised of only a subset of today’s amino acids, and a model
of the codon usage in the Last Universal Ancestor predicts that in
this subset (Ala, Val, Ile, Asn, Ser, Thr, His, Asp) amyloidogenic
amino acids are well represented. The observation that protein

sequences have evolved away from aggregation-prone amino
acid sequence stretches (Monsellier and Chiti, 2007) and that
organism complexity anticorrelates with proteomic b-aggrega-
tion propensity (Tartaglia et al., 2005) are further clues that point
to an amyloid as the mother of protein folds. Once peptides were
aggregating reproducibly into an amyloid entity with functionality
in the biologically useful range (Wang et al., 2010b), soluble
molten globule-like states (Pervushin et al., 2007) and later
well-folded soluble protein folds may have evolved from these
protein aggregates.
The Amyloid Entity for Long-Term Memory
Although the mechanism of long-term memory is not known, it is
believed that long-term memory must at some level require
stable changes in the molecular components of the neuronal
synapse, perhaps mediated by local regulation of protein
synthesis (Darnell, 2003). The cytoplasmic polyadenylation
element binding protein (CPEB) is thereby considered to be
a leading candidate for synaptic translational regulation that
acts by activating dormant mRNAs. In three very exciting
reports, Si et al. (2003a) showed that a neuronal isoform of
CPEB in Aplysia (ApCPEB) is necessary for long-term synaptic
changes, and that ApCPEB undergoes a conformational switch
into an amyloid state that appears to be the active form of the
protein (Si et al., 2003b). This aggregated state is stably trans-
missible in yeast and is necessary for long-term memory in
Aplysia (Si et al., 2010), and thus may provide a basis for
a long-lasting and permissive mark for long-term changes in
the synapse. The N-terminal region of ApCPEB, unique to the
neuronal isoform, is responsible for the amyloid-like properties
of the molecule. Like yeast prions that propagate as an amyloid,
it has a Gln and Asn content of 48% and lacks predictable
secondary structure. Because the mouse homolog has only
18% Gln and Asn content in the N-terminal segment, it is not
clear yet whether the corresponding process may also be
present in mammals. However, the concept of an amyloid entity
as the key to long-term memory, although perhaps surprising, is
interesting. The requirement of a lasting change to the synapse
after a single signaling event could be fulfilled by an amyloid,
a structural entity whose soluble-to-aggregate transition can
be tightly regulated (see above). Because the formation of the
ApCPEB amyloid results in a gain of function that leads to new
protein expression, the temporal regulation of the amyloid (i.e.,
increase or decrease in levels) may also allow for ‘‘strength-
ening’’ the memory during repetitive stimulations of the synapse.
While on the topic of functional amyloids in the brain, we would
like to go one step further to propose an intriguing hypothesis,
although one that lies outside the range of the current scientific
testability. An important unanswered question is whether
a computational machine (a Turing machine) that is governed
by classical physics can also be a source of intelligence or
even consciousness. Penrose (1994) argues that the brain can
go beyond what can be achieved by algorithms, and unlike
a computer, can carry out a noncomputable type of functioning.
He proposes that consciousness and free will can only be
explained by a new kind of physics that, for example, may
involve the translation of quantum mechanical states into
classical physics states (i.e., collapse of the wave function).
Penrose (1994) suggests that such a situation could be taking
place in microtubules, the rigid repetitive polymers of the tubu-
1256 Structure 18, October 13, 2010 ª2010 Elsevier Ltd All rights reserved
Structure
Review
lins. Because the protein within microtubules switch between
two conformational states, it is speculated that this conforma-
tional switch generates a quantum coherent state in neurons
that somehow (e.g., through quantum gravity) could affect and
influence the classical physics of neurons (Penrose, 1994). One
of the strongest counter arguments to this idea is one of feasi-
bility due to the short coherence times that have been calculated
(Tegmark, 2000), although coherence times of electron spin pairs
of several ms in proteins at biological relevant temperatures are
documented (Ritz et al., 2009). We propose amyloids as an
alternative to microtubules as a supramolecular structure that
could support quantum coherence-based biology. Several
reports have shown that the cross-b sheet motif of amyloids
has properties such as electron spin diffusion, electric conduc-
tance, and autofluorescence (Del Mercato et al., 2007; Margittai
and Langen, 2008) that may support quantum coherence
phenomena.
Conclusion
In 1935, the pioneering biophysicist Astbury published an X-ray
diffraction pattern from poached, stretched egg white that dis-
played the reflections typical for the cross b sheet entity (Astbury
et al., 1935). The authors concluded that when proteins/peptides
aggregate, they go into their energetically most favorable confor-
mational state, i.e., the cross-b sheet motif. In other words, when
a protein aggregates it generally forms an amyloid-like entity
comprising a specific structure. Protein aggregation can thus
be viewed as a primitive folding process that results in a defined
set of aggregated conformations. This makes the amyloid entity
a prime candidate both for the origin of life and for the first folds
of the known protein universe. Furthermore, it is consistent with
the fact that there are many functional amyloids that are involved
in diverse mechanisms such as protection, storage, infectivity,
and perhaps long-term memory. Although 75 years have passed
since the amyloid was proposed as a common structural motif,
the structural and functional characterization of the amyloid
world is just in the starting phase of discovery with much yet to
be revealed.
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