J. Biomedical Science and Engineering, 2009, 2, 345-358
doi: 10.4236/jbise.2009.25050 Published Online September 2009 (http://www.SciRP.org/journal/jbise/
Published Online September 2009 in SciRes. http://www.scirp.org/journal/jbise
Review: structure of amyloid fibril in diseases
Arezou Ghahghaei1*, Nasim Faridi1
1Department of Biology, Faculty of Science, University of Sistan and Baluchestan, Zahedan, Iran.
Email: Arezou@chem.usb.ac.ir
Received 8 May 2009; revised 13 July 2009; accepted 21 July 2009.
Tissue deposition of normally soluble proteins,
or their fragments, as insoluble amyloid fibrils
causes both acquired and hereditary systemic
amyloidoses, which is usually fatal. Amyloid is
associated with serious diseases such as Alz-
heimer’s disease, type 2 diabetes, Parkinson’s
Disease, Huntington’s Disease, cancer and the
transmissible spongiform encephalopathies. In-
formation concerning the structure and mecha-
nism of formation of fibrils in these diseases is
critical for understanding the process of pathol-
ogy of the amyloidoses and to the development
of more effective therapeutic agents that target
the underlying disease mechanisms. Structural
models have been made using information from
a wide variety of techniques, including electron
microscopy, X-ray diffraction, solid state NMR,
and Congo red and CD spectroscopy. Although
each type of amyloidosis is characterised by a
specific amyloid fibril protein, the deposits share
pathognomonic histochemical properties and the
structural morphology of all amyloid fibrils is
very similar. In fact, the structural similarity that
defines amyloid fibres exists principally at the
level of β-sheet folding of the polypeptides within
the protofilament, while the different types vary
in the supramolecular assembly of their proto-
Keywords: Amyloid; Protofilaments; Aggregation;
Amyloid fibril formation arises from the slow aggrega-
tion of intermediately folded peptide or protein mole-
cules. During this process the protein goes from its na-
tive soluble form to insoluble fibril, which is highly β-
sheet in character [1,2,3,4]. The aggregation of the amy-
loid β peptide has been examined in various solvents and
conditions and this has led to a model by which a con-
formational switching occurs from an α-helix or random
coil to a β-sheet structure early on the amyloid-forming
pathway [5], prior to a nucleation-dependent process
leading to the elongation of the fibril. Along this path-
way, small oligomeric intermediates and short fibrillar
structures (protofibrils) have been observed. In cross-
section, the fibril appears to be composed of several sub-
fibrils or protofilaments. Each of these protofilaments is
rich in stacked β-sheet structures in which hydrogen
bonded β-strands run perpendicular to the fibril’s axis,
and the backbone hydrogen bonds are parallel to it [6].
The basic process of amyloid formation is known to in-
volve the construction of fibrils from individual polypep-
tide monomer units held together by noncovalent interac-
tions generated by the formation of intermolecular β-sheets
or the correct stacking of intramolecular β-sheet structures
[7]. Small polypeptide fragments (as small as five or six
residues long) are able to form amyloid fibrils from fully
denatured conformations, larger sequences, typically be-
tween 80 and 150 residues, appear to require the popula-
tion of more compact or partially folded states to form
amyloids [7]. In a number of systems it has been demon-
strated that amyloid fibril formation by proteins in vitro is
preceded by the formation of metastable, nonfibrillar forms
often referred to as protofibrils. These species often have
the appearance of spherical beads 2–5 nm in diameter,
beaded chains, where the individual beads again have a
diameter of 2–5 nm, or annular structures, formed appar-
ently by the circularisation of the beaded chains.
Figure 1 suggests that the various fates awaiting a
polypeptide chain, once it has been synthesized in the
cell, will depend on the kinetics and thermodynamics of
the various equilibria between various possible states
[5,8]. In its monomeric state, the protein is believed to
fold from its highly disordered unfolded state (3)
through a partially structured intermediate state (2) to a
globular native state (1) [8,9]. The unfolded and partially
folded states can form aggregated species that are fre-
quently disordered. Highly ordered amyloid fibrils can
form through a mechanism of nucleation and elongation
[8,9,10] showed that the β-domain is the destabilized
region of the human lysozyme. This implies that the ag-
gregation process may be initiated by intermolecular
rather than intramolecular association [8,10,11].
346 A. Ghahghaei et al. / J. Biomedical Science and Engineering 2 (2009) 345-358
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Figure 1. A proposed mechanism for lysozyme amyloid
fibril formation [8]. An intermediate structural state form
of the protein (2) aggregates through the β domain (4) to
commence fibril formation. This intermediate (4) shows
a pattern for the development of the fibril.
Negative-stain electron microscopy (EM) showed that
the amyloid fibrils associated with the various diseases
mostly appear straight, and unbranched, and are 30-120
Å in diameter. They consist of two or more filamentous
subunits, which twist around each other with 11.5 nm,
24 β-strand periodicity resulting in a protofilament di-
ameter of 5-6 nm. The amyloid fibril, shown in Figure
2, is about 25-35 Å wide and consists of two or three
subunit strands helically arranged with a 35-50 Å diame-
ter repeat [12,13,14,15,16,17].
X-ray fibre diffraction showed that all types of fibrils
have a common core structure [18,19,20,21]. They con-
sist of a helical array of β-sheets along the length of the
fibre. This indicates that the polypeptide chain in fibres
are hydrogen-bonded together along their entire length
which increases their stability [18,19,20,21]. Cross β-
sheets consist of two dominant reflections: a sharp and
intense meridional reflection at 4.8 Å, which corre-
sponds to the average distance between the hydrogen-
bonded β-strands that comprise β-sheets and a strong
and more diffuse reflection on the equator between 9-11
Å arising from the distance between stacked β-sheets,
[18,19,20,21]. The X-ray diffraction pattern of PI3-SH3
fibril is shown in Figure 3 [21]. The fibril contains two
reflections: the main reflection, which is intense, occurs
at 4.71 Å while the weaker reflections appear at 9.42.
Figure 2. Modeling of an amyloid fibril structure. (a) Overview of the fibril structure showing proto-
filaments extended in regular helical twists along the length of the fibre (b) Side view of inter-sheet spac-
ing between subprotofilaments of 9-11 Å perpendicular to the axis (c) Cross-section of amyloid fibril (d)
Slightly tilted side view of the fibril showing inter-strand spacing of 4.7 Å along the fibre axis [16].
A. Ghahghaei et al. / J. Biomedical Science and Engineering 2 (2009) 345-358 347
SciRes Copyright © 2009 JBiSE
Figure 3. X-ray diffraction pattern of PI3-SH3 fibrils.
The meridional (interstrand spacing) and equatorial (in-
tersheet spacing) reflections are indicated at 4.7 Å and
9.4 Å, respectively [21].
In some systems, small amounts of preformed amy-
loid fibrils, added to the sample to act as seeds, induce
the formation of amyloid fibrils more readily if protofi-
brils are present in the sample rather than non-
aggregated or smaller oligomeric species. This observa-
tion suggests that, in these cases at least, fibrils can form
preferentially from the assembly of protofibrils. Evi-
dence exists, however, that some oligomeric aggregates
assembling into chains and rings with “bead-on-string”
morphology may be off-pathway species that are not
amyloid-competent [22].
The soluble precursors of amyloid deposits do not
share any sequence homology or common fold. How-
ever, amyloid aggregates have common structural
features: 1) they show the same optical behavior (such
as birefringence) on binding certain dye molecules
like Congo red; 2) they present very similar mor-
phologies (long, unbranched and often twisted fibril-
lar structures a few nanometers in diameter); and 3)
they display the characteristic cross-β X-ray diffrac-
tion pattern which indicates that the “core” structure
is composed of β-sheets running perpendicular to the
fibril axis. [5,23].
Amyloid formation is associated with a number of
diseases e.g. Alzheimer’s, Parkinson’s, Huntington’s,
Creutzfeldt-Jakob, Spongiform and other neurodegen-
erative diseases in which the formation of β-sheet
aggregates known as amyloid plaques that, among
other features, are extremely resistant to protease ac-
tion, an indication of close packed clusters. All these
diseases are primarily associated with old age [3,
24,25]. It is a general observation that solubility and
protease resistance depend on actual secondary struc-
ture. A helical and or random coil content has been
shown to be coincident with high solubility and prote-
ase susceptibility whereas a β-sheet content has been
shown to determine poor solubility and resistance to
proteolysis [26,27]. The clinical classification of amy-
loidosis includes primary (de novo), secondary (a
complication of a previously existing disorder), famil-
ial, and isolated types [28,29]. Each disease is charac-
terized by a particular protein or polypeptide that ag-
gregates in an ordered manner to form insoluble amy-
loid fibrils [30]. These amyloid fibrils are deposited in
the tissues, where they are associated with the pathol-
ogy of the disease [31].
There are relatively few examples of nonpathological
functions. Given that many proteins have the ability to
form amyloids. Among the few known functions are the
amyloid curli, adhesive appendages of gram-negative
bacteria, and yeast regulatory amyloids that may mediate
epigenetic diversification. Recently, a natural amyloid
has been found to accelerate melanin assembly in
melanocytes. Amyloids have been proposed to have a
role in the establishment of memory, as well. The dis-
covery of new functions for amyloids would greatly in-
crease our knowledge of their roles in the normal func-
tioning of cells [32]. The role of amyloid β-peptide in
the pathogenesis of neurodegenerative disorders is not
completely elucidated, but its toxic effect is not neces-
sarily correlated with senile plaque deposition, since it
has been shown that the neurotoxic effect of amyloid β-
peptide is independent of plaque formation in transgenic
mouse models. It has been suggested that neurotoxic
effects can be induced by diffusible amyloid β-peptide
oligomers or by intraneuronal accumulation of amyloid
β-peptide [33].
The function of the majority of proteins is attribut-
able to its unique three dimensional structure [34].
Failure to fold to into its correct structure limits of
abrogates the protein’s ability to perform the biologi-
cal roles for which it was produced [35]. Not only
does the aggregation of folding intermediates strips the
cell of an important resource, the presence of the ag-
gregate themselves can have dire consequence and
results in a wide range of disease such as described in
the Table 1.
Conformational diseases such as those in Table 1 are
associated with proteins that do not attain or maintain
their native structure resulting in aggregation and in-
solubilization [36,37]. The insoluble deposits in this dis-
ease form either plaques or fibrillar tangles within tis-
sues and their accumulation ultimately result in cell
damage or death [35,38].
348 A. Ghahghaei et al. / J. Biomedical Science and Engineering 2 (2009) 345-358
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Table 1. Representative human disease associated with defective protein folding [113,114,115].
Disease Protein Phenotype
Alzheimer’s disease β-Amyloid Aggregation
Tay-Sachs disease β-Hexasominidase Improper trafficking
Cancer PS3 Misfolding/improper trafficking
Spongiform Prion Protein Aggregation
Creutz feldt-jakob diseases Prion protein Aggregation
Maifan syndrom Fibrillin Misfolding
Parkinson diseases α-Synuclein Aggregation
Gaucher’s disease β-Glucosidase Improper trafficking
Scurvy Collagen Misfolding
Huntington’s diseases Huntington Aggregation
Familial amyloiosis Transthyretin Aggregation
Alzheimer’s disease (AD) is one of the most common
neurodegenerative disorders associated with aging, and
is characterized by fibrillar deposits of amyloid β (Aβ)
peptides in the brain parenchyma and cortical blood ves-
sels [5,37,39]. Patients in Alzheimer’s disease often have
psychiatric manifestations of disease, such as psychosis
(e.g., delusions and hallucinations) and disruptive behav-
iors (e.g., psychomotor agitation and physical aggres-
sion), especially in the later stages of the disease (1).
One of the characteristics of brains afflicted with AD is
the presence of extracellular structural elements referred
to as amyloid plaques. Plaques are, in part, composed of
masses of filaments, which are in turn composed of the
insoluble form of the Aβ peptide [5,39,40]. These Aβ
aggregates may cause neuronal injury directly by acting
on synapses, or indirectly by activating microglia and
astrocytes and therefore pharmacological interventions
have been developed to target the sequential events
originating from Aβ synthesis [40,41]. Usually neuritic
plaques composed of amyloid β-protein (Aβ) are an
early and invariant neuropathological feature of Alz-
heimer's disease (AD), [41]. Genetic and neuropatho
logical studies suggest that the processing of amyloid
precursor protein (APP) to yield amyloid β-protein (Aβ),
and its subsequent aggregation, play important roles in
the pathogenesis of Alzheimer’s disease (AD) as the
numbers of A plaques and A burden increased over
time in the brain with AD and A deposition precedes
clinical symptom of AD. The other pathological feature
of this disease is intraneural neurofibrillary tangles [42,
The Aβ family of peptides is derived by the enzymatic
breakdown of the amyloid precursor protein (APP), a
563-770 residue membrane protein expressed in neu-
ronal and non-neuronal tissue [45]. Patients with Down
syndrome develop AD because of an extra copy of the
amyloid precursor protein (APP) gene on chromosome
21 [46]. Amyloid precursor protein (APP) is a ubiquitous
membrane glycoprotein encoded by a single gene on
chromosome 21 and formed as a cleavage byproduct by
three proteases, β-, γ- nd α-secretase [47]. It’s cleavege
via the α-secretase or the β-secretase pathway, often re-
ferred to as the amyloidogenic pathway. When APP is
cleaved by α-secretase, it produces a large amino-
terminal fragment APPα destined for secretion and a
smaller carboxyl-terminal fragment. Further processing
of the carboxyl-terminal fragment by γ-secretase pro-
duces a 22- to 24-residue fragment termed P3, which
may or may not be amyloidogenic. Alternatively, when
APP is cleaved by β-secretase it produces a soluble
amino-terminal fragment, APPβ, and a carboxyl-terminal
fragment containing the Aβ peptide. Cleavage of the
carboxyl-terminal fragment by γ-secretase results in the
formation of multiple Aβ variants of 40-43 amino acids,
which are prone to aggregate. The most abundant forms
are 40 and 42 amino acids in length, Aβ 40 and Aβ 42.
Both forms are capable of assembling into 60-100 Å
diameter β-sheet fibrils that exhibit the characteristic
cross-β X-ray fiber diffraction pattern, and yield a red-
green birefringence when stained with Congo red [45].
The ratio of Aβ 42 to Aβ 40 is about 1:10. Aβ 42 plays a
critical role in the pathogenesis of AD since its aggrega-
tive ability and neurotoxicity are much greater than those
of Aβ 40. Aβ 42 oligomers initially formed as a seed
accelerate the aggregation of Aβ 40 to form the amyloid
plaques that eventually lead to the neurodegeneration
(amyloid cascade hypothesis) [47].
The formation of insoluble Aβ deposits in the brain is a
pathological hallmark of AD. If the hypothesis that the
neurotoxicity of Aβ is mediated by amyloid fibril forma-
tion is correct, inhibition of Aβ fibril formation might
A. Ghahghaei et al. / J. Biomedical Science and Engineering 2 (2009) 345-358 349
SciRes Copyright © 2009
slow progression or prevent the disease. However, more
recent studies have shown that fibrils are not the only
neurotoxic structures and that Aβ also assembles into
soluble forms like small oligomers and protofibrils,
which could be responsible for neurotoxicity [48]. Aβ
(1-40) and Aβ (1-42) differ structurally by the absence or
presence of two C-terminal amino acids [49]. The amino
acid sequence of the Aβ 1–42 peptide is D1AEFRHDSG9
0I42, where subscripts indicate residue numbers.
The solid state NMR data are consistent with an Aβ1–
40 monomer secondary structure composed of a struc-
turally disordered N-terminal region followed by two β-
strand segments connected by a “loop” or “bend” seg-
ment. Structural model resulted by NMR data support a
parallel β-sheet structure in fibrils formed by both the 40
and 42-residue β amyloid peptides (Aβ 1–40,42). Paral-
lel β-sheets have been found by solid state NMR origi-
nally in fibrils formed by residues 10–35 of β-amyloid
(Aβ 10–35) [23,31,50]. However, antiparallel β-sheets
have also been found by solid state NMR in fibrils
formed by shorter β-amyloid fragment. In agreement
with the NMR experiment MD analysis of various struc-
tural models of short β-amyloid fragments, including
parallel and antiparallel β-sheet structures suggested a
strand-loop-strand structure with parallel β-sheets for
Aβ10–35 fibrils [51]. Recent solid state NMR and elec-
tron microscopy experiments have shown that Aβ 1–40
can form at least two distinct amyloid fibril structures,
with distinct and self-propagating morphologies and
molecular-level structural features, dependent on subtle
variations in fibril growth conditions. Although all Aβ1–
40 fibril structures studied to date contain parallel β-
sheets, distinct structures differ in the specific details of
side-chain-side-chain contacts and in mass-per length
(MPL) values [52].
Thin sections of amyloid fibrils formed from fragments
of amyloid β (Aβ), the amyloid fibril protein of Alz-
heimer's disease, show cross-sections containing five or
six protofilaments [23,53,54,55,56]. The protofilaments
were over 40Å in diameter and appeared as “beaded”
structures with a 200 Å periodicity [53,54,57,58,59].
Electron microscopy studies have shown that amyloid
fibrils consist of three to six protofilaments. It is possible
that the exposed hydrophobic residues could be involved
in helical packing among the protofilaments. It is known,
for instance, that side-chain packing plays an important
role in stabilizing helical coiled-coil bundles. While the
center of pleated β-sheet structure is hydrophobic, the
ends of the sheets are somewhat hydrophilic. Ionic inter-
actions and/or hydrogen bond interactions as additional
forces stabilizing the cross-β structure have been pro-
posed [60].
Oligomerization of Aβ42 induced by the intermolecu-
lar β-sheet at positions 15–21 and 24–32 would confine
this radical species in the oligomer, making it possible to
damage the cells continuously. However, this oligomer
would not lead to the fibrils since the C-terminal β-sheet
is intramolecular (Figure 4(b)). As shown in Figure 4(a)
and (b), the C-terminal residues in Aβ42 play a critical
role in its aggregative and neurotoxicity [61] and the
intermolecular β-sheet in the C-terminal region of Aβ42
seems to be preferable to long fibrils [47,62]. Aβ (1-42)
did not adopt a unique fold, but rather a mixture of rap-
idly interconverting conformations that were classified
into three distinct families. The secondary structure
analysis revealed that these conformations were domi-
nated by loops and turns but that some helical structure
formed in the C-terminal hydrophobic tail. Experimental
studies of full-length Aβ monomers in water (organic
solvent mixtures) showed that the monomer structure
consists of two α-helical regions connected through an
exible turn- or bend-like kink. The model of one helical
turn of the twisted pleated β-sheet is composed of
48 monomers of Aβ. Each monomer contains an antipar-
allel β-sheet. Overall, there are 96 β-strands. Four
strands form a unit and 24 units stack together along the
fibril axis. If each unit were twisted by 15° relative to its
immediate neighbors (above and below) in the same way,
24 units would make a complete helical turn, with the
Figure 4. (a) A representation of the hydrophobic residue at the C-terminus of A42 [61]. (b) The in-
tramolecular anti-parallel -sheet at the C-terminus of A42 [112].
350 A. Ghahghaei et al. / J. Biomedical Science and Engineering 2 (2009) 345-358
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helical axis parallel to the fibril axis. Given that the angle
between the two immediate strands from two different
monomers was set at 15°, the helical twist formed by the
24 units in the model was actually nearly a complete turn
(~350°). Four strands in each unit layer are in an identi-
cal orientation, and those between two adjacent (above,
below) unit layers are in an antiparallel arrangement
(Figure 5(a) and (b)). In other words, the unit layers
stack in an antiparallel fashion. The distances between
the two adjacent strands in different unit layers and that
between two strands within a unit layer are ~4.7 and
~9.7 Å, respectively (Figure 5(c) and (d)). These dis-
tances are consistent with results from x-ray fibril dif-
fraction studies on amyloid fibrils. NMR experiments on
Aβ (10-35) monomer structure in an aqueous solution
show a collapsed structure with loops, strands, and turns
without any significant amount of α-helical or β-strand
content. These studies suggest that Aβ monomer struc-
ture is very sensitive to external conditions, such as tem-
perature, pH, and solvent. An Aβ (1-40) monomer struc-
ture considered with little α-helix or β-strand at low
temperatures. As the temperature was increased to
physiological, substantial β-sheet content developed. A
folded Aβ (1-42) monomer, but not an Aβ (1-40) mono-
mer, possesses a turn at G37-G38 stabilized by a hydro-
phobic interaction between V36 and V39 [63]. Prelimi-
nary cryo-electron microscopy of the amyloid fibrils
formed from Aβ (11-25) and from Aβ (1-42) revealed
that although they resembled one another closely in di-
ameter and morphology, Aβ (11-25) appeared to form
more consistently homogeneous, straight, uniform fibrils
with clearly definededges (Figure 6(a). The Aβ (1-42)
fibril showed less contrast, poorly defined edges and did
not appear as straight or rigid (Figure 6(b). Close in-
spection of Aβ (1-42) fibril images did not reveal any
additional features (Figure 6(b). The Aβ (1-42) fibrils
showed the spacing between the bands measured 4.7-4.8
Å, which corresponds to the hydrogen bonding distance
between β-strands. These results appear to directly re-
veal the β-sheet structure a single amyloid fibril [5,56].
A model has been suggested for Aβ amyloid protofila-
ments in which, Aβ (12-42) folds into a β-hairpin and
associates into four β-sheets, which twist around a cen-
tral axis. In these models the β-strands are in register be-
tween the sheets. Aβ (11-25) fibrils were more ordered,
uniform structures. Aβ (1-42) fibrils appeared to have less
defined edges and this meant that selecting fibril images
was more difficult. It is possible that this is because Aβ
(1-42) fibril structure is complicated by loops and disor-
dered protein around the periphery of the fibrils whereas
Aβ (11-25) fibrils constitute the core β-sheet structure.
The β-strands clearly run perpendicular to the fibre axis.
The strong, obvious striations visible across the fibre in
these projections indicate that the β-sheets within the
Figure 5. (a) A four-strand antiparallel β-sheet unit from the dimer interface. (b) A dimer of Aβ 12-42
in an antiparallel β-sheet conformation. The distance between any two adjacent strands is ~4.7 Å. (c)
The dimer is translated three times in a direction perpendicular to the plane defined by the C atoms
of the dimer, each by ~9.7 Å. An 8-mer results. (d) A perfectly stacked 16-mer. It was constructed by
stacking (parallel) two 8-mers together in the direction of the fibril axis [61].
A. Ghahghaei et al. / J. Biomedical Science and Engineering 2 (2009) 345-358 351
SciRes Copyright © 2009
Figure 6. Low dose cryo electron micrograph of (a) A(11 to 25) amyloid fibrils and (b) A(1 to 42)
amyloid fibrils. The fibrils formed from both peptides are clearly long, straight and predominantly sin-
gle. (b) A magnified view of A(1-42) fibril. It is clear from these images that the A(11 to 25) fibrils
are straighter and more clearly defined than those of A(1 to 42) [5].
fibril are likely to be in exact register with one another,
where a β-strand of one sheet is in register with the
β-strand of the adjacent sheet, so that the strands rein-
force one another in the image. Amyloid fibrils com-
posed of Aβ peptide are composed of continuous β-sheet
structure where the β-strands run perpendicular to the
fibre axis.
Electron microscopy has been used as a tool to exam-
ine the structure and morphology of these aggregates
from ex vivo materials, but predominantly from synthetic
amyloid fibrils assembled from proteins or peptides in
vitro. Electron microscopy has shown that the fibrils are
straight, unbranching, and are of a similar diameter (60-
100 A) irrespective of the precursor protein. Image proc-
essing has enhanced electron micrographs to show that
amyloid fibrils appear to be composed of protofilaments
wound around one another. In combination with other
techniques, including X-ray fiber diffraction and solid
state NMR, electron microscopy has revealed that the
internal structure of the amyloid fibril is a ladder of β-
sheet structure arranged in a cross-β conformation [64].
The nonfibrillar oligomeric species are pathogenic, al-
though reports differ as to whether soluble dimers or
higher molecular weight protofibrils are toxic. The
highly amyloidogenic Aβ 42 forms soluble oligomers
extremely rapidly and Aβ 42 could form the pathogenic
oligomeric species and/or amyloid fibrils and deposit in
senile plaques faster than Aβ 40 [23,31,50,65].
Numerous evidence suggests that β-amyloid found
abundantly in the brains of Alzheimer disease patients, is
toxic in neuronal cell cultures through a mechanism in-
volving free radicals. Therefore, anti-amyloid strategies
are currently being investigated to lower the production
of A. Vitamin E prevents the oxidative damage induced
by β-amyloid in cell culture and delays memory deficits
[66], Another approach is using inhibitor investigation of
anti-amyloid strategies to lower the production of A.
Using of chemical agents such as Congo red, rifampicin
and benzofurans can prevent A oligomerization and the
formation of neurotoxic protofibrils but are not appro-
priate in vivo due to their toxicity [67,68].
Huntington’s disease (HD) is an autosomal-dominant
neurodegenerative disorder caused by a CAG triplet re-
peat expansion coding for a poly-glutamine (polyQ) se-
quence in the N-terminal region of the Huntington (htt)
protein [69]. The first symptoms of Huntington’s disease
usually occur in the third to fifth decade [70]. As the
disease progresses, a variety of motor, emotional/ behav-
ioral, and cognitive symptoms are experienced, includ-
ing unsteadiness, trouble holding onto things, trouble
walking, changes in sleeping patterns, delusions and
hallucinations, intellectual decline, and memory loss
Which Psychiatric symptoms are among the most com-
mon features [71,72].
CAG repeats resulting in long polyglutamine tracts
have been implicated in the pathogenesis of at least eight
neurodegenerative diseases including Huntington and
several spinocerebellar ataxias [73]. Normally, Hunting-
ton is a cytoplasmic protein expressed at high levels in
the striatal neurons vulnerable to degeneration in HD
352 A. Ghahghaei et al. / J. Biomedical Science and Engineering 2 (2009) 345-358
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and at low or undetectable levels in the neurons resistant
to degeneration. In HD brain, N-terminal fragments of
mutant huntington were reported to accumulate and form
inclusions in the nucleus and identified as a characteris-
tic neuronal intracellular inclusion (NII) [74]. This inclu-
sions contained β-pleated sheet stabilized by hydrogen
bonds. In several cases, NI is spherical aggregates,
sometimes assembled by insoluble amyloid-like fibrils.
Aggregates outside the nucleus have also been observed,
but it has been shown recently that nuclear localization
is required for toxicity [75]. The poly-Q disorders are
also associated with the formation of neuronal nuclear
inclusions (NI) whose average size is about 5–7 μm, and
formed by aggregates of the protein with the expanded
polyglutamine sequence [75,76,77].
The hypothesis that amyloid structures are formed in
Huntington’s disease (HD) is supported by evidence that
polyglutamine forms amyloid like protein aggregates in
vitro, which stain with Congo Red (a histological stain
for amyloid) and exhibit green birefringence under po-
larized light, [78,79]. Ultrastructural studies of brains of
HD transgenic mice revealed neuronal intranuclear in-
clusion that contained aggregated huntingtin protein with
granular or fibrillar morphology. Neuronal intranuclear
inclusion with similar structural features also were de-
tected in postmortem brains of HD and spinocerebellar
ataxia type 3 patients as well as in stable and transiently
transfected cell lines. Furthermore, recombinant proteins
with an expanded poly(Q) stretch (51–122 glutamines)
were found to form insoluble high molecular weight
protein aggregates in vitro [80].
Increasing PG length correlates with the number of
inclusions, which contributes to earlier onset of illness.
In vivo inclusions occur primarily in subpopulations of
neurons that are affected in the illnesses. In vitro expres-
sion of PG tracts has been shown to lead to aggregation
of proteins by either cross linking or by polar-zipper
hydrogen bonding [81,82]. Consistent with this, it was
found that the rate of aggregate formation in vitro di-
rectly correlates with repeat length: the longer the
poly(Q) tract, the faster the aggregation rate. Similarly,
the protein concentration required for aggregate forma-
tion decreased with an increase of the poly(Q) repeat
length. In vitro aggregation of N-terminal, poly(Q)-
containing huntingtin peptides is self initiated and, like a
crystallization or Aβ formation, follows a nucleation-
dependent pathway. [83].
Thus the formation of amyloidlike huntingtin aggre-
gates in vitro not only depends on poly(Q) repeat length
but also critically depends on protein concentration and
time [79]. Among these, formation of ordered huntingtin
aggregates is highly poly(Q) repeat length-dependent,
which correlated the age of onset and the severity of HD.
The majority of adult-onset cases have expansions rang-
ing from 41 to 55 units, whereas expansions of 70 and
above invariably cause the juvenile form of the disease.
Interestingly, pathological effects occur in patients only
when the length of poly-Q exceeds a rather sharp thresh-
old of 35±40 glutamines. Thus length of the poly-Q tract
correlates directly with the age of onset and with the
severity of the symptoms in the diseases [79,83].
Discovery of the gene underlying HD can be used as a
genetic therapy for a logical step towards finding a cure.
Animal models that closely mimic the neurobiological
and clinical symptoms of the disease can be used to test
experimental treatments for HD across different stages
of the disease [71,84].
Parkinson’s disease (PD) is an age-related neurodegen-
erative disorder. In 80% cases PD is most common
Parkinsonism which clinically is a syndrome character-
ized by tremor at rest, rigidity, slowness or absence of
voluntary movement, postural, instability, and freezing.
PD is a progressive disease, which affects both women
and men. It has an effect on 1% of people beyond 65
years of age, with a higher prevalence in men [85-88].
The postmortem PD substantia nigra is characterized by
sporadic intraneuronal cytoplasmic inclusions known as
Lewy bodies (LB). The presence of Lewy bodies are
associated with neurodegenerative disorders such as
sporadic and familial Parkinson’s disease (PD), dementia
with LBs and the LB variant of Alzheimer’s disease. The
principal component of LBs is the protein α-synuclein,
but they also contain various amounts of other proteins,
including the molecular chaperones, αB-crystallin, clus-
terin, torsin A, Hsp27 and Hsp70 [89,90,91,92,93].
α-Synuclein protein and a fragment of it, called NAC.
α-Synuclein is a presynaptic protein, which was origi-
nally identified as the precursor protein for the non-β-
amyloid component (NAC) of Alzheimer's disease (AD)
senile plaques. NAC is a 35 amino acid peptide compris-
ing amino acids 61-95 of the α-syn sequence and has
been identified as the second major constituent in the
plaques of AD brains [94,95,96]. The formation of NAC
peptides in a crossed β-pleated sheet conformation was
assessed by thioflavine-S staining. Only the aged sam-
ples of NAC(1-35) and NAC(1-18) were thioflavine-S
positive, indicative of the presence of amyloid-like fila-
ments [97,98].
The involvement of α-synuclein in neurodegenerative
diseases was first suspected after the isolation of a α-
synuclein fragment (NAC) from amyloid plaques in
Alzheimer’s disease (AD). Later, two different α-synu-
clein mutations were shown to be associated with auto-
somal-dominant Parkinson’s disease (PD), but only in a
small number of families. However, the discovery that
α-synuclein is a major component of Lewy bodies and
Lewy neurites, the pathological hallmarks of PD, con-
firmed its role in PD pathogenesis [99].
A. Ghahghaei et al. / J. Biomedical Science and Engineering 2 (2009) 345-358 353
SciRes Copyright © 2009 JBiSE
Two autosomal dominant mutations in the α-synuclein
gene were linked to familial early onset PD: A53T and
A30P. The mutations, A30P and A53T, do not affect the
conformational behavior of the monomeric protein;
wild-type α-synuclein (WT), A53T, and A30P are all
‘‘natively unfolded’’ at low concentration. At higher
concentrations, WT, A53T, and A30P all form amyloid
fibrils of similar morphology by what appears to be a
nucleation-dependent mechanism [97]. All three proteins
also produce nonfibrillar oligomers that may be assem-
bly intermediates, analogous to the Aβ protofibril. Fibril-
lization of α-synuclein is clearly accelerated by the A53T
mutation but the effect of the A30P mutation on fibril
formation has not been determined, although A30P ac-
celerates the formation of nonfibrillar oligomers and the
disappearance of unsedimentable protein from solution
[100,101]. Circular dichroism spectroscopy indicated
that α-synuclein undergoes a conformational change
from random coil to β-sheet structure during assembly.
X-ray diffraction and electron diffraction of the α-
synuclein assemblies showed a cross-β conformation
characteristicof amyloid [90].
In vitro multiple factors have been shown to acceler-
ate α-synuclein aggregation. In aqueous solution, α-
synuclein is natively unfolded within extended structure
composed of random coil without a hydrophobic core. In
vivo, it binds to rat brain vesicles via the first four 11-
mer N-terminal repeats. In vitro, it binds to monolayer
phospholipid membranes, acquiring an α-helical secon-
dary structure probably formed by the seven N-terminal
11-residue repeats containing the conserved core se-
quence Lys-Thr-Lys-Glu-Gly-Val [102]. Therapeutic stra-
tegies aimed to prevent this aggregation are therefore
envisaged. Although little has been learned about its
normal function, α-synuclein appears to interact with a
variety of proteins and membrane phospholipids, and
may therefore participate in a number of signaling path-
ways. In particular, it may play a role in regulating cell
differentiation, synaptic plasticity, cell survival, and
dopaminergic neurotransmission. Thus, pathological
mechanisms based on disrupted normal function are also
possible [102,103].
The transmissible spongiform encephalopathies (TSEs)
or prion diseases form a group of fatal neurodegenera-
tive diseases including bovine spongiform encephalopa-
thy, Creutzfeldt-Jakob Disease (CJD) and Gerstmann-
Stra¨ussler-Scheinker syndrome (GSS). TSEs, or prion
diseases, are mammalian neurodegenerative disorders
characterized by a posttranslational conversion and brain
accumulation of an insoluble, protease-resistant isoform
(PrPSc) of the host-encoded cellular prion protein (PrPC)
[104]. The diseases are rare, but outbreaks of acquired
forms of CJD, such as variant CJD and iatrogenic CJD
with cadaveric growth hormone or dura grafts, have
prompted the development of therapeutic interventions
and new diagnostic methods [105]. In humans, prion
diseases result from infectious modes of transmission,
Gerstmann-Sträussler-Scheinker Syndrome, Fatal Famil-
ial Insomnia; and modes of transmission. The clinical
symptoms associated with each of the human prion dis-
ease forms vary dramatically [106,107].
Cellular prion protein, PrPC, is a predominantly α-
helical glycoprotein that remains attached to the outer
membrane of the cells through a glycophosphatidyl
inositol linkage. A -sheet-rich conformational isoform
of PrPC, termed PrPSC, has been considered to be the
infectious agent of the fatal neurodegenerative diseases
transmissible spongiform encephalopathies (TSE) and its
hereditary forms of spongiform encephalopathies (SE).
[104]. PrPSC exists as oligomers and amyloid polymers
(fibres) and, unlike PrPC, is resistant to digestion by pro-
teinase K (PK), which is considered as an indicator of
formation of PrPSC. However, PrPC may undergo dis-
ease-associated structural modifications that do not lead
to a protease-resistant molecule, indicating that prion
disease can occur in the absence of PrPSC; further prote-
ase-resistant prion protein forms without any infectivity
can be generated. [108].
Familial amyloidosis may occur in patients with fa-
milial Mediterranean fever or it may arise from
transthyretin mutations. Approximately 30 such muta-
tions have been described [28]. Transthyretin, previously
called prealbumin because it migrates ahead of albumin
in standard electrophoretic separations, is a serum carrier
of thyroid hormones and vitamin A. The mutant
transthyretin is deposited as extracellular twisted β-
pleated sheet fibrils in peripheral somatic and autonomic
nerves and visceral organs; it causes autonomic and pe-
ripheral somatic disorders, and the disease is ultimately
fatal [28]. Clinically, patients are susceptible to neuro-
pathic, cardiopathic, or nephropathic complications. Type
I familial hereditary generalized amyloidosis is inherited
as an autosomal dominant gene mutation with a single
amino acid substitution of methionine for valine at posi-
tion 30 in transthyretin [28].
The topics discussed in this review provide a great deal
of evidence for the proteins that have an intrinsic capac-
ity of aggregating and forming structures such as amy-
loidfibrils. Aggregates are most commonly formed from
the interaction of partially folded intermediates contain-
ing significant native-like structure. These interactions
involve extended chain or -sheet-like conformations.
Thus, both ordered and disordered aggregates show in-
creased structure relative to the native conformation (in
the case of all-proteins, the increased structure is dis-
354 A. Ghahghaei et al. / J. Biomedical Science and Engineering 2 (2009) 345-358
SciRes Copyright © 2009 JBiSE
tinct from that of the native protein).
Misfolded and aggregated species are likely to owe
their toxicity to the exposure on their surfaces of regions
of proteins that are buried in the interior of the structures
of the correctly folded native states. The exposure of
large patches of hydrophobic groups is likely to be par-
ticularly significant as such patches favour the interac-
tion of the misfolded species with cell membranes
[109,110,111]. Interactions of this type are likely to lead
to the impairment of the function and integrity of the
membranes involved, giving rise to a loss of regulation
of the intracellular ion balance and redox status and
eventually to cell death. The data reported so far strongly
suggest that the conversion of normally soluble proteins
into amyloid fibrils and the toxicity of small aggregates
appearing during the early stages of the formation of the
latter are common or generic features of polypeptide
chains. Moreover, the molecular basis of this toxicity
also appears to display common features between the
different systems that have so far been studied.
Amyloidosis comprises a group of diseases in which
protein tissue deposits have common morphologic struc-
tural, and staining properties but variable protein compo-
sition [28,37]. In this review we highlighted the rele-
vance of amyloid fibril formation of different protein to
diseases. The fibrillar morphology, diagnostic staining
characteristics and underlying β-sheet structure of the
proteins deposits led to them being classified as amyloid
fibrils, and the diseases are now regarded as a protein
misfolding disease. There are still many outstanding and
critical questions regarding protein aggregation. Among
these are questions about the detailed mechanism of the
aggregation process, factors determining the kinetics of
aggregation, the structural nature of the intermolecular
interactions, and how aggregation may be effectively
and efficiently prevented, especially in vivo.
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APP Abbreviation: Amyloid precursor protein
AD Alzheimer’s disease
HD Huntington’s disease
PD Parkinson’s disease
LBs Lewy bodies
TSEs Transmissible spongiform encephalopathies