Review: structure of amyloid fibril in diseases

doi:10.4236/jbise.2009.25050

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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/

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.

ABSTRACT

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-

filaments.

Keywords: Amyloid; Protofilaments; Aggregation;

Neurodegenerative

1. INTRODUCTION

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

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

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].

2. AMYLOIDOSIS DISEASES

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

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

3. THE ALZHEIMER’S DISEASE

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,

43,44].

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].

4. THE STRUCTURE OF AMYLOID IN

ALZHEIMER DISEASE

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

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

YEVHHQKLVFFAED23VGSNK28GAIIGLMVGGVV4

0I42, where subscripts indicate residue numbers.

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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 A42 [61]. (b) The in-

tramolecular anti-parallel -sheet at the C-terminus of A42 [112].

350 A. Ghahghaei et al. / J. Biomedical Science and Engineering 2 (2009) 345-358

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

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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

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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].

5. HUNTINGTON

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

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].

6. PARKINSON

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

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].

7. SPONGIFORM AND FAMILIAL

AMYLOIDOSIS DISEASES

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].

8. CONCLUSIONS

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

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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Appendix

APP Abbreviation: Amyloid precursor protein

AD Alzheimer’s disease

HD Huntington’s disease

PD Parkinson’s disease

LBs Lewy bodies

TSEs Transmissible spongiform encephalopathies