Báo cáo khoa học: Protein-misfolding diseases and chaperone-based therapeutic approaches pdf

Molecular chaperones assist other proteins to achieve a functionally active 3D structure and thus prevent the formation of a misfolded or aggregated structure, essentially enhancing fold

Protein-misfolding diseases and chaperone-based

therapeutic approaches

Tapan K Chaudhuri and Subhankar Paul

Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, New Delhi, India

In order to be functionally active, a protein has to

acquire a unique 3D conformation via a complicated

folding pathway, which is described by the primary

amino acid sequence and the local cellular environment

[1] Protein folding is vital for a living organism

because it adds flesh to the gene skeleton A small

error in the folding process results in a misfolded

structure, which can sometimes be lethal [2] However,

within the cellular environment, which is highly

vis-cous, many proteins cannot fold properly by

them-selves and require the assistance of a special kind of

ubiquitous protein, the molecular chaperones [3] Molecular chaperones assist other proteins to achieve

a functionally active 3D structure and thus prevent the formation of a misfolded or aggregated structure, essentially enhancing folding efficiency by influencing the kinetics of the process and inhibiting events that lead to unproductive end points (e.g aggregation) Chaperones are located at various points in the cell and interact with nascent polypeptides during synthesis and translocation to different cellular compartments Chaperones are able to distinguish between the native

Keywords

chaperone-based therapeutic approaches;

chemical and pharmacological chaperones;

molecular chaperones; protein

conformational diseases; protein misfolding

and aggregation

Correspondence

T K Chaudhuri, Department of Biochemical

Engineering and Biotechnology, Indian

Institute of Technology Delhi, Hauz Khas,

New Delhi 110016, India

Fax: +91 11 2658 2282

Tel: +91 11 2659 1012

E-mail: tapan@dbeb.iitd.ac.in

(Received 3 January 2006, revised 10

Febru-ary 2006, accepted 14 FebruFebru-ary 2006)

doi:10.1111/j.1742-4658.2006.05181.x

A large number of neurodegenerative diseases in humans result from pro-tein misfolding and aggregation Propro-tein misfolding is believed to be the primary cause of Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Creutzfeldt–Jakob disease, cystic fibrosis, Gaucher’s disease and many other degenerative and neurodegenerative disorders Cellular mole-cular chaperones, which are ubiquitous, stress-induced proteins, and newly found chemical and pharmacological chaperones have been found to be effective in preventing misfolding of different disease-causing proteins, essentially reducing the severity of several neurodegenerative disorders and many other protein-misfolding diseases In this review, we discuss the prob-able mechanisms of several protein-misfolding diseases in humans, as well

as therapeutic approaches for countering them The role of molecular, chemical and pharmacological chaperones in suppressing the effect of pro-tein misfolding-induced consequences in humans is explained in detail Functional aspects of the different types of chaperones suggest their uses as potential therapeutic agents against different types of degenerative diseases, including neurodegenerative disorders

Abbreviations

AD, Alzheimer’s disease; ADH, antidiuretic hormone; AVP, arginine vasopressin; BSE, bovine spongiform encephalopathy; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane regulator; CJD, Creutzfeldt–Jacob disease; DMSO, dimethyl sulfoxide; ER, endoplasmic reticulum; FAP, familial amyloid polyneuropathy; GD, Gaucher’s disease; GSH-MEE, glutathione monoethyl ester; HbS, hemoglobin S; HD,

Huntington’s disease; HSP, heat shock protein; MCD, mad cow disease; MJD, Machado-Joseph disease; NAC, N-acetyl- L -cysteine; NDI, nephrogenic diabetes insipidus; NOV, N-octyl-h-valienamine; PCD, protein conformational disease; PD, Parkinson’s disease; PGD, polyglutamine disease; RP, retinitis pigmentosa; SCA, spinocerebeller ataxia; SSA, senile systemic amyloidosis; TMAO, trimethylamine-N-oxide; UPP, ubiquitin proteasome pathway.

and non-native states of targeted proteins, but how

they discriminate between correctly and incorrectly

folded proteins and how they selectively retain and

tar-get the latter for degradation is yet to be understood

Proteins that are not able to achieve the native state,

due either to an unwanted mutation in their amino acid

sequence or simply because of an error in the folding

process, are recognized as misfolded and subsequently

targeted to a degradation pathway This is referred to

as a protein ‘quality control’ (QC) system and is

com-posed of two components: molecular chaperones and

the ubiquitin proteasome system (UPS) [4] The QC

system plays a critical role in cell function and survival

A special class of chaperone, for example, calnexin,

forms part of the ‘quality control monitors’ that

recog-nize and target abnormally folded proteins for rapid

degradation [5] One class of QC chaperone associated

with the endoplasmic reticulum (ER), e.g calnexin and

calreticulin, BiP and ERp 57 [6], is able to recognize

misfolded proteins and help their retention in the ER,

allowing only correctly folded proteins to reach the

cytosol [5] One very strong and crucial aspect of QC in

the cell is the ubiquitin proteasome pathway (UPP)

Studies suggest that disturbance in or impairment of

the UPP, which may be induced by the accumulation

of misfolded proteins in the ER or loss of function of

the enzymes involved in the ubiquitin conjugation and

deconjugation pathway, leads to altered UPS function,

which positively affects the accumulation of protein

aggregates in the cell [4] The formation of oligomers

and aggregates occurs in the cell when a critical

concen-tration of misfolded protein is reached Aggregated

proteins inside the cell often lead to the formation of

an amyloid-like structure, which eventually causes

dif-ferent types of degenerative disorders and ultimately

cell death [4]

In almost all protein-misfolding disorders, an error in

folding occurs because of either an undesirable

muta-tion in the polypeptide or, in a few cases, some

less-known reason The harmful effect of the misfolded

protein may be due to: (a) loss of function, as observed

in cystic fibrosis (CF) and a1-antitrypsin deficiency; or

(b) deleterious ‘gain of function’ as seen in many

neuro-degenerative diseases such as Alzheimer’s disease (AD),

Parkinson’s disease (PD) and Huntington’s disease

(HD), in which protein misfolding results in the

forma-tion of harmful amyloid [7] Protein aggregates are

sometimes converted to a fibrillar structure containing a

large number of intermolecular hydrogen bonds which

is highly insoluble These are commonly called amyloids

and their accumulation occasionally results in a

plaque-like structure [8] In some cases, the mutations are so

severe that they render the gene product biologically

inactive [cystic fibrosis transmembrane regulator (CFTR) protein] In other cases, however, the mutations are relatively minor and the resulting proteins show only

a partial loss of normal activity Despite having partial biological activity, these mutant proteins are not deliv-ered to their correct location, either inside the cell or in the extracellular space One example of disease invol-ving abnormal protein trafficking is a1-antitrypsin defi-ciency [9] In almost all cases of protein misfolding-mediated disorders, mutation in the gene (encoding the disease-causing protein) is very common However, the more frequent amyloid-related neurodegenerative dis-eases are characterized by the appearance of a toxic function caused by the misfolded proteins [10]

One or more of a chaperone’s activities result in the prevention⁄ suppression of a few devastating neurode-generative diseases Reduction in the intracellular level

of chaperones results in an increase in abnormally folded proteins inside the cell [5] Therefore, toxicity in different neurodegenerative disorders may result from

an imbalance between normal chaperone capacity and the production of misfolded protein species Increased chaperone expression can suppress the neurotoxicity caused by protein misfolding, suggesting that chaper-ones could be used as possible therapeutic agents [11] Natural, chemical or pharmacological chaperones have been shown to be promising agents for the control of many protein conformational disorders (PCD) These diseases include CF, AD, PD and HD, as well as sev-eral forms of prion diseases Here, we discuss the causes of protein misfolding, aggregation and amyloid formation in the cell, and the use of different chaperones as therapeutic agents against various protein-misfolding disorders

Protein misfolding and aggregation cause several diseases

Protein misfolding and its pathogenic consequences have become an important issue over the last two dec-ades According to the prion researcher Susan Lind-quist, ‘protein misfolding could be involved in up to half of all human diseases’ [12] Protein misfolding is also responsible for many p53-mediated cancers, which are also the result of incorrect protein folding Many cancers and other protein-misfolding disorders are caused by mutations in proteins (Table 1) that are key regulators of growth and differentiation Structural changes in a few proteins subsequently lead to aggre-gated masses, which occasionally result in neuro-toxicity and cell death Hooper [13] reported that aggregated⁄ misfolded proteins become neurotoxic (e.g prion protein in mad cow disease; MCD) because of

an inhibition of proteasome function Csermely [14]

suggested a ‘chaperone overload’ hypothesis, which

explains that with aging, there is an overburden of

accumulated misfolded protein that prevents molecular

chaperones from repairing phenotypically silent

muta-tions which might cause disease It has been shown

that the yield of correctly folded protein obtained from

in vitro refolding is low due to the formation of

ther-modynamically stable folding intermediates These

conformations are called ‘dead-end’ conformations and

are ‘off-pathway’ intermediates, they generally lead to

the formation of insoluble aggregates [15] that may

eventually causes different degenerative diseases

Clas-sic examples of these degenerative diseases are CF,

which is caused by the deletion of a single residue

phenylalanine in the CFTR protein, and sickle cell

anemia, which originated due to a mutation in

hemo-globin

A common feature of almost all protein

conforma-tional diseases is the formation of an aggregate caused

by destabilization of the a-helical structure and the

simultaneous formation of a sheet [16] These

b-sheets are formed between alternating peptide strands

Linkages between these strands result from hydrogen

bonding between their aligned pleated structures Such

b-linkages [17] with a pleated strand from one

mole-cule being inserted into a pleated sheet of the next lead

to hydrogen-bond formation between molecules [18]

The prerequisites for b-linkage formation are the

pres-ence of a donor peptide sequpres-ence that can adopt a

pleated structure and a b sheet that can act as an

acceptor for the extra strand [19]

It is not clear whether misfolding triggers protein

aggregation or protein oligomerization induces

con-formational changes [26] Based on the kinetic

modeling of protein aggregation, it has been proposed that the critical event in PCD is the formation of pro-tein oligomers that can then act as seeds to induce protein misfolding [27–29] In this model, misfolding occurs as a consequence of aggregation (polymeriza-tion hypothesis) [26], which follows a crystalliza(polymeriza-tion- crystallization-like process dependent on nucleus formation

The alternative model suggests that the underlying protein is stable in both the folded and misfolded forms in solution (conformational hypothesis) [30–32] This hypothesis proposes that spontaneous or induced conformational changes result in formation of the mis-folded protein, which may or may not form an aggre-gate But in this hypothesis the critical question is what factors are responsible for changes in conforma-tion without the inducconforma-tion of aggregates Studies have described several factors that play a crucial role, such

as mutation in the gene, which destabilizes the correct structure For example, mutation is common in all neurodegenerative disorders, which reduces the folding efficiency by changing the proper folding energetic Induced protein misfolding has been described as being responsible for all familial diseases In addition to mutation, other environmental stresses such as oxida-tive stress, alkalosis, acidosis, pH shift and osmotic shock are able to change the structure of a protein without involving aggregates

In a third hypothesis, the native protein conforma-tion is changed to an amyloidogenic intermediate, which is not stable in the cellular environment This intermediate has many exposed hydrophobic regions and therefore develops small oligomers, mainly com-posed of b sheets, via intermolecular interactions These small oligomers form an ordered fibril-like structure called amyloid via an intermolecular interaction [33,34]

Table 1 Mutation observed in different disease causing proteins CF, cystic fibrosis; NDI, nephrogenic diabetes insipidus; PD, Parkinson’s disease; AD, Alzheimer’s disease; HD, Huntington’s disease; SCA, spinocerebellar ataxia.

1

T126M, A147T, R187C R187C ⁄ D62–64, L59P, L83Q, Y128S, S16L, A294P, P322H, R337X

[22]

R273 and R282

[24]

AD

a-Amyloid precursor protein AD 1, AD 2, AD 3, AD 4 Tau, preselinin 1 and 2,

a-macroglobulin

[25]

Protein aggregation is an inevitable consequence of

a cellular existence and these aggregates are oligomeric

complexes of non-native conformers that arise from

intermolecular interactions among structured and

kin-etically trapped intermediates in the protein folding or

assembly pathway [35,36] Protein aggregation is

facili-tated by partial unfolding during thermal and

oxida-tive stress and by alterations in the primary structure

caused by mutation, RNA modification or

transla-tional misincorporation [36,37] Protein aggregates can

be either structured (e.g amyloid) or amorphous In

either case, they are insoluble and metabolically stable

in the physiological environment [38] For various

dis-eases associated with protein misfolding, one or more

proteins are converted from the native structure to an

aggregated mass, which is commonly called an

‘amy-loid’ The net accumulation of toxic protein aggregates

in the cell depends on the stability, compactness and

hydrophobic exposure of the aggregates, as well as on

the rate of protein synthesis in the cell [39] The

accu-mulation of toxic aggregates in the cell depends on

chaperone expression and protease networks [39]

Environmental stress may induce the synthesis of

higher levels of chaperones and proteases in the cell,

which can better remove toxic aggregates [39] Fibrillar

amyloids are commonly extracellular, but intracellular

fibrillar deposits are also seen in patients, e.g

intracel-lular bundles of neurofibrillary tangles in AD [40–43]

Although the initial process might be different in

dif-ferent diseases, a common trend is that during the

for-mation of aggregates, a-helical domains disappear,

leading to an increase of b-sheet-dominated secondary

structure (Fig 1) [44] Recently, many other

physiolo-gical disorders have been recognized as being caused

by the formation of protein aggregation, which

subse-quently forms a plaque-like structure containing a

large number of amyloid fibrils, these are polymerized

to cross b-sheet structures with the b-strands arranged

perpendicular to the long axis of the fiber

Toxic amyloid formation causes many human neurodegenerative disorders

Neurodegenerative disorders that are chronic and pro-gressive are characterized by the selective and symmet-rical loss of neurons in motor, sensory or cognitive systems The most common feature of all the neuro-degenerative disorders is the occurrence of brain lesions, formed by the intra- or extracellular accumula-tion of misfolded, aggregated or ubiquitinated proteins [4] Proteins associated with some neurodegenerative diseases like AD, PD and HD, are tau⁄ b-amyloid (Ab), a-synuclein and huntingtin, respectively [8] For

AD, PD and CJD a few cases are familial or inherited but the remainder are sporadic in nature

AD is a progressive degenerative disease of the brain

in the elderly which clouds memory and causes impaired behavior [45] The neuropathological features

of this devastating disease are the extracellular depos-ition of Ab and neurofibrilary tangles (NFT) in the brain A central process of AD is the cleavage of a 42 amino acid b-amyloid peptide from an otherwise nor-mal membrane precursor protein [46,47] The main pro-tein is a membrane propro-tein called amyloid precursor protein, which after being cleaved by b-secretase produ-ces a b-amyloid precursor peptide fragment, this is further cleaved by another protease b-secretase to pro-duce Ab-42 instead of Ab-40, which is amyloidogenic

It is thought that cellular degradation of Ab-42 is the normal fate of this peptide fragment when produced in small amounts under normal conditions, however, in some lesser known conditions it forms extracellular aggregates and subsequently generates amyloid plaques Studies have reported that impairment of the UPS may

be involved in this disorder [16] An increase in neuro-toxicity has been generated by dimer and oligomer for-mation (Fig 2) of the Ab fragment [48]

According to many scientists, AD should be first defined by the presence of NFTs caused by the protein

α-helix α-helix

β-sheet

β-sheet α-helix

Fig 1 During amyloid formation most of the a-helical structures in the polypeptide chain of a native protein are converted into b-pleated sheets (A) Native polypeptide chain composed of mainly a-helical secondary structure (B) Misfolding causes conversion of a-helical structure to b-pleated sheets and (C) final misfolded structure of polypeptide chain contains mostly b-pleated sheets.

tau NFTs are aggregations of the microtubular

pro-tein tau, which are found to be hyperphosphorylated

in the neuronal cells of AD patients Although, tau

polymer formation is a hallmark of other degenerative

disorders, such as corticobasal degeneration,

progres-sive supranuclear palsy and pick disease [49], all differ

from AD in that they lack Ab plaque deposition [50]

In contrast to AD, it is believed that in PD, protein

accumulates in the intracellular space [51] PD is the

second most common, late-onset neurodegenerative

disorder, and is characterized by muscular rigidity,

postural instability and resting tremor It is a slow

pro-gressive disorder and the pathology of PD involves the

degeneration of dopaminergic neurons in the

substan-tia nigra and the deposition of intracytoplasmic

inclu-sion bodies called Lewy bodies in brain cells The

exact mechanism by which these cells are lost is not

known Heritable forms of PD are caused by gene

mutations To date, three genes encoding a-synuclein,

parkin and ubiquitin C-terminal hydrolase L1 protein

have been shown to be associated with familial forms

of PD [52] All three proteins are present in Lewy

bod-ies in sporadic PD [53] and in dementia with Lewy

bodies [54] Two missense mutations in the gene

enco-ding a-synuclein are linked to dominantly inherited

PD, thereby directly implicating a-synuclein in the

pathogenesis of the disease Recent studies suggest that

the intracellular accumulation of a-synuclein [55] leads

to mitochondrial dysfunction [56], oxidative stress

[57,58] and caspase degradation [59] accentuated by

mutations associated with familial parkinsonism

[60,61]

The prion protein, which is thought to be

respon-sible for causing a disease in cattle, called bovine

spongiform encephalopathy (BSE, or ‘mad cow

dis-ease’), and a disease in humans, called variant

Creutz-feldt–Jakob disease (vCJD) [62] is thought to undergo

a conformational change in which a helices of the wild-type protein PrPCare converted into b-sheet-dominant PrPSc, resulting in misfolding and aggregation [63,64] CJD is inherited as an autosomal dominant disorder and the most common human prion disease, the spor-adic form, accounts for  85% of cases;  10–15% of cases are familial Sporadic CJD results from the endogenous generation of prions In general, familial CJD has an earlier age-of-onset and a longer clinical course than sporadic CJD Fatal familial insomnia is the strangest phenotype of familial prion diseases The symptoms are dominated by progressive insomnia, autonomic dysfunction and dementia In the case of infectious prion disease, the infectious scrapie protein (PrPSc) drives the conversion of cellular PrPC into disease-causing PrPSc (Fig 3) [63] The normal prion protein is protease sensitive, soluble, and has a high a-helix content, but its normal function is unknown The disease-causing prion protein (the transmissible isoform) is protease resistant and insoluble, forms amyloid fibrils, and has a high b-sheet content Studies have reported that prion protein PrPSc has a neuro-protective function and the defective prion can induce normal as well as huntingtin protein to change confor-mation, which later form aggregates [63,65,66]

In some human disorders, protein misfolding takes place due to repetition of glutamine in the polypeptide chain, which is called polyglutamine disease (PGD) This disorder is progressive, inherited, either auto-somal dominant⁄ X-linked and appears in mid-life lead-ing to severe neuronal dysfunction and neuronal cell death [67] In all of these diseases, the CAG trinucleo-tides, which code for phenylalanine in the coding regions of genes, are thought to be translated into polyglutamine (polyQ) tracts As a result, the protein

II: Oligomerization I: Dimerization

Tetramer: Forming aggregate Monomer

Dimer Monomer

Fig 2 Protein oligomerization Misfolded monomers forming aggregate through intermolecular hydrogen bonding interaction leading to b-sheet formation.

product, now containing an usually long string of

glu-tamine residues, appears to misfold and form large

detergent-insoluble aggregates within the nucleus or

cytoplasm, thereby leading to the eventual demise of

the effected neuron [5] To date eight different

inher-ited neurodegenerative diseases (Table 2) have been

found to be due to expansion of glutamine repeats in

the affected proteins HD is the most frequent of

them

Machado–Joseph disease⁄ spinocerebellar ataxia-3

(MJD⁄ SCA-3) is another inherited neurodegenerative

disorder caused by expansion of the polyglutamine

stretch in the MJD gene-encoded protein ataxin-3 The

truncated form of mutated ataxin-3 causes aggregation

and cell death in vitro and in vivo In vitro cellular

models and transgenic animals have been created and

analyzed with the truncated ataxin-3 with an expanded polyglutamine stretch, in which polyglutamine-contain-ing aggregates and cell death were invariably observed [68–74]

Protein misfolding and loss of function leads to several lethal diseases

CF is characterized by thick mucous secretions in the lung and intestines [8] Amino acid sequence analysis

of CFTR protein has shown that the protein resides within membranes, contains 12 potential transmem-brane domains, two nucleotide-binding domains, and

a highly charged hydrophilic region, which has been shown to act as a regulatory domain [5] Although many mutations in the CFTR sequence have been

Normal cellular

prion protein are

infected by Scrapie

prion molecule

(i)

Newly converted prions again infect other normal cellular prions

All the normal cellular functional prion molecules converted into transmissible form

Fig 3 Propagation of PrP Sc takes place through the interaction of PrP Sc with normal cellular protein PrP C Binding between PrP Sc and PrP C

induces conformational change in PrP C protein that results in the formation of PrP Sc , which form aggregates through intermolecular associ-ation (i) Transmissible isoform of one prion protein molecule infects other normal cellular prion molecules (ii) Infection causes induction in conformation of normal prions that converts them to transmissible prion molecules, which again start infecting other normal prion molecules (iii) All the cellular normal prions are transformed into disease causing scrapie prion proteins.

Table 2 Neurodegenerative diseases caused by repetition of CAG codon which encodes glutamine in the polypeptide chain of the respon-sible proteins.

Disorder

Protein responsible

Normal No.

of repeats

No repeats in

Spinal and bulbar

muscular atrophy

Spinocerebellar ataxia

Dentatorubropallido-Luysian atrophy

identified, one in particular has been noted in over 705

patients examined, in this mutation deletion of three

nucleotides coding for a phenylalanine residue at

posi-tion 508 (DF508 CFTR) took place within a

polypep-tide of 1480 amino acids [87] The DF508 allele of

CFTR has been confirmed as a trafficking mutation

that blocks maturation of the protein in the ER and

targets it for premature proteolysis [88] The clinical

importance of this mutation becomes evident when

considering that it accounts for 70% of patients

diag-nosed with CF [89]

The most common and severe form of a1-antitrypsin

deficiency is caused by the Z mutation, a single base

substitution (Gul342-Lys) in the a1-antitrypsin gene

Misfolding of proteins during synthesis can initiate an

ordered polymerization, which leads to aggregation of

the protein within the cell This slows the rate of

pro-tein folding in the cell, allowing the accumulation of

an intermediate, which then polymerizes [90], impeding

its release and leading to plasma deficiency The

a1-antitrypsin is a serpin – an inhibitor of proteolytic

enzymes with serine at the active site, which, on

bind-ing to its target proteinase(s), undergoes a

conforma-tional change It is known that serpin polymerization

involves the interaction of one serpin molecule with

the b-sheet of another molecule of the same type;

extensive knowledge of this mechanism may help in

the development of b-strand blockers to prevent

self-association of these proteins [91]

The tumor suppressor protein p53, which is a

sequence-specific transcription factor whose function is

to maintain genome integrity, presents a classic

exam-ple of a protein misfolding-mediated disorder

Inacti-vation of p53 by mutation is a key molecular event,

and is detected in > 50% of all human cancers [24]

The p53 tumor suppressor is one of our defenses

against uncontrolled cell growth which leads to tumor

proliferation Under normal conditions there is a low

level of p53 tumor suppressor protein in the cell,

how-ever, when DNA damage is sensed, p53 levels rise and

initiate protective measures p53 protein binds to many

regulatory sites in the genome and begins production

of proteins that halt cell division until the damage is

repaired If the damage is too severe, p53 initiates the

process of programmed cell death, or apoptosis, which

directs the cell to commit suicide, permanently

remov-ing the damage The human p53 suppressor gene is

mutated with high frequency in cancers [91] Most of

these are missense mutations, affecting residues that

are critical for maintaining the structural fold of this

highly conserved DNA-binding protein, changing the

information in the DNA at one position and causing

the cell to produce p53 protein with an error through

swapping an incorrect amino acid at one point in its polypeptide chain In these mutants, the normal func-tion of p53 is lost and the protein is unable to prevent multiplication in the damaged cell [92–94]

Sickle cell anemia is a genetic disorder in which the amino acid valine at the sixth position of the b-globin chain is replaced by glutamine Galkin and Vekilov [95] have reported that this mutation promotes inter-molecular bonding among adjacent hemoglobin mole-cules and results in stable long polymer fiber formation Mutant hemoglobin S (HbS) also leads to a stable fiber-like structure while HbS is in deoxy state This polymerization changes the shape and rigidity of red blood cells and triggers abnormality Lot of b-plea-ted sheet accumulates as ‘amyloid plaques’

Nephrogenic diabetes insipidus (NDI) is a disorder known to be caused by misfolding of one hormonal protein, antidiuretic hormone, also known as vasopres-sin NDI is characterized by an inability of the kidneys

to remove water from the urinea and by resistance of the kidneys to the action of arginine vasopressin [96] Wildin et al [97] reported that a mutation in the AVPR2 gene, which encodes arginine vasopressin, is most common in NDI More than 70 different muta-tions have been identified; the majority are missense and nonsense mutations Furthermore, 18 frameshift mutations due to nucleotide deletions or insertions (up

to 35 bp) and four large deletions have been reported Retinitis pigmentosa (RP) is the most common cause

of inherited blindness with over 25 genetic loci identi-fied, it is characterized by night-blindness and loss of peripheral vision, followed by loss of central vision Mutations in the gene encoding rhodopsin have been identified [98] and more than 100 mutations have now been described that account for 15% of all inherited human retinal degenerations The failure of rhodopsin

to translocate to the outer segment per se does not appear to be enough to cause RP; rather, it would appear that misfolded rhodopsin acquires a ‘gain of function’ that leads to cell death The nature of this gain of function is unclear, but may be related to sat-uration of normal protein processing, transport and degradation In transfected cells, rhodopsin with muta-tions in the intradiscal, transmembrane and cytoplas-mic domains fails to translocate to the plasma membrane, and accumulates in the ER and Golgi Hence these mutant proteins fail to translocate because

of misfolding and this causes the disorder [99]

Another protein conformational disorder is Fabry disease, which is a lysosomal storage disorder, caused

by a deficiency of galactosidase A activity in lyso-somes, resulting in an accumulation of glycosphingo-lipid globotriosylceramide (Gb3) The majority of

cardiac Fabry patients have missense mutations in the

a-Gal A gene (GLA), although alternative splicing

mutations and small deletions have also been observed

[100,101] Such mutant enzymes appear to be

misfold-ed, recognized by the ER’s protein quality control and

degraded before sorting into lysosomes Fabry disease

is specific for those missense mutations that cause

mis-folding of a-Gal A

GD is an inherited lipid-storage disorder It is

caused by mutation in the gene encoding acid

b-glu-cosidase (GlcCerase) [102], an enzyme that participates

in the degradation of glycosphingolipids [103]

Symp-toms may have neurological discrepancy or may be

non-neurological [104] Deficiency of this enzyme

cau-ses accumulation of glucocerebrosides in macrophage

lysosome In very few cases, GD is caused by mutation

in the saposin C domain of the gene prosaposin, which

controls the optimum activity of GlcCerase by

enco-ding a protein saposin C [102]

Amyloidoses

In all the above cases either misfolded proteins form

fibrillar aggregates which become toxic and lead to cell

death (all neurodegenrative diseases) or, in other

cate-gory of disease, misfolded proteins are directed to the

proteasome pathway for degradation (proteolysis), and

protein deficiency causes the disease In a third case,

even if the fibrils themselves are not toxic, the ready

autolinkage of proteins and polypeptides by b-strand

bonding involves risks of further linkage to give

insol-uble macrostructures [105,106], these macrostructures

are deposited in the tissues and cause disease (Table 3)

[107] Different amyloidosis may be heterogeneous in nature but all have common properties in that they all bind the dye Congo red that intercalates between their

b strands [108]

Amyloidosis is classified according to clinical symp-toms and biochemical type of amyloid protein involved Many amyloidoses are multisystemic, gener-alized or diffuse but a few are also locgener-alized They mainly affect kidneys, heart, gastrointestinal tract, liver, skin, peripheral nerve and eyes It is a slowly progressive disease that can lead to morbidity and death Amyloid deposits are extracellular and not metabolized or cleared by the body, thus the deposits eventually impair the function of the organ where they accumulate

Table 4 shows the causes of different disorders by specific disease-causing proteins and Fig 4 shows the possible fate of misfolded proteins through the path-way where they are processed by a different chaperone system, UPS, and subsequently reach their destination

by gain or loss of function leading to several degener-ative disorders

Molecular chaperones can prevent protein misfolding and aggregation

Large multidomain proteins have been found to form a misfolded structure and aggregated mass during

in vitro refolding [109] The cellular environment is crowded with proteins and other macromolecules, and

so the chance of a newly synthesized unfolded protein forming aggregates is greater in vivo than in vitro Cellular molecular chaperones are proteins that change

Table 3 Classification of amyloidoses and name of precursor proteins and nomenclature [109a] Amyloidoses that affect central nervous system are not considered here G, generalized; L, localized.

Prostatic amyloid

Amylin

Insulinoma

this equation by selectively recognizing and binding to

the exposed hydrophobic surfaces of a non-native

protein via non-covalent interactions, thus inhibiting

irreversible aggregation of those proteins in vivo [5]

and in vitro

Molecular chaperones are composed of several

dis-tinct classes of sequence-conserved proteins, most of

which are stress inducible like heat shock proteins

(Hsp) Major classes of these Hsp are Hsp100 (in

E coli, ClpA⁄ B ⁄ X, HslU), Hsp90 (in E coli, HtpG),

Hsp70 (in E coli, DnaK), Hsp60 (in E coli, GroEL)

and the small Hsps (in E coli, IbpA⁄ B) These

mole-cular chaperones have important damage-control

functions during and following stress Under in vitro

conditions, many chaperones, such as E coli IbpB,

DnaK, DnaJ, GroEL, HtpG and SecB, and proteases

such as DegP, HslU and Ion can bind chemically

unfolded polypeptides and prevent aggregation [21,

110–112] They are also involved in aggregate

solubili-zation Stable aggregates are resistant to most ATPase

chaperone systems when functioning individually, for

example GroELS, Hsp90, ClpB, and low

concentra-tions of DnaK Skowyra et al [113] observed that the

DnaK chaperone system might reactivate some forms

of protein aggregate It has been observed that

Hsp100, which includes Ipb, ClpA, HslU and ClpX in

E coli, has disaggregation activity [114] ClpA and

ClpX have been shown to destabilize some native

protein structures, allowing them through the central

cavity into the ClpP for proteolysis [114]

Schrimer et al have shown that Hsp70 and Hsp100

function in combination to reactivate many protein

aggregates [114] They also showed that Hsp104

cooperates with Hsp70 and Hsp40 in a slow and

inefficient disaggregation, which is generally limited to small aggregates of luciferase and a-galactosidase Their findings have been supported by evidence that both chaperones collaborate in the cellular acquisition

of thermotolerance [115] It has been reported that the yeast non-Mendelian factor [psi+], which is analogous

to mammalian prions, is propagated at when there are intermediate amounts of the chaperone protein Hsp104 and overproduction or inactivation of Hsp104 caused loss of [psi+] [116] These results suggest that chaper-ones are crucial in prion disease progression and that a certain level of chaperone expression can rid cells of prions without affecting their viability Control of the expression level of Hsp104 may provide a therapy against prion disease In addition, Hsp104, along with Hsp70, has been shown to be responsible for solubiliz-ing prion-like aggregates in Saccharomyces cerevisiae [116,117] Many other positive responses have been reported on cellular chaperone-mediated disaggrega-tion in vivo A classic experiment was performed

by Goloubinoff et al., who proved the phenomena of

in vitroreactivation and disaggregation of stable aggre-gates of malate dehydrogenase by ClpB together with DnaK, DnaJ and GrpE (KJE), and further explained the mechanism of the whole disaggregation process (Fig 5) [118]

Mogk, Tomoyasu and colleagues [110,119] showed that, in E coli, stable protein aggregates rapidly disap-pear from the insoluble fraction following chaperone action during a short recovery period Under normal conditions, chaperones repair the conformational defects of some mutated proteins, thus reducing their phenotypic effects and dampening genome cleansing (elimination of damaged genes from the gene pool of a

Table 4 Proteins involved in different human diseases caused by misfolding, aggregation and trafficking [5,26].

S

F

Scrapie (Mad Cow Disease), Familial insomnia

population, which normally takes place via natural

selection) Sherman & Goldberg [120] first reported

that Hsp70 and Hsp40 molecular chaperones prevent

aggregation of polyglutamine-containing proteins It has been reported that Hsp70 and Hsp40 chaperone family members act together The chaperone complex

(B)

(D) (L)

(H)

(M)

(J)

Loss of protein function cause several diseases like cystic fibrosis

(E) (F)

(I) (K)

Gain of toxicity

Cause several neurodegenerative diseases and lead cell demise like Alzheimer disease, Parkinson disease

Degraded protein

Native

Porotein

Ubiquitin

Aggregate/Fibrillar amyloid

(N)

(G)

Hsp60 Ubiquitin Hsp104 Hsp90 Hsp40 Hsp70

DNA

RNA

Ribosome

(A)

(C)

E1

E2 E3

ATP

Ubiquitin conjugation

E1 E2

E3

Ubiquitinated protein

Partially

folded

protein

CHIP

Misfolded protein

Misfolded protein

impaired proteasome

Misfolded protein

Amyloidoses (Familial amyloid neuropathy) (O)

Fig 4 The fate of cellular misfolded protein is shown (A) Nascent polypeptide chain is converted into folded protein (B) Polypetide chain reaches misfolded structure (C) Native protein molecule is converted into misfolded structure due to specific mutation or cellular stress (D)

In the first step Hsp 40 ⁄ 70 ⁄ 90 facilitate to direct them to the proteasomal pathway and the second step is ubiquitination of misfolded pro-tein assisted by E1 (ubiquitin activating enzyme), E2 (ubiquitin conjugating enzyme) & E3 (ubiquitin ligase) (E) Due to the damage of ubiquitin enzymes, misfolded protein is directed to the aggregation pathway (F) Misfolded protein enters into the proteasome system with the help

of ubiquitin complex (G) Proteasome’s action degrades misfolded protein into small peptides and ubiquitin is regenerated (H) Impaired pro-teasome system couldn’t degrade misfolded protein (I, J) The misfolded protein forms aggregate (K) Cellular Hsp104 disaggregates the compact aggregates and develop partially folded monomer with the assistance of Hsp70 (L) Partially folded protein is converted into native protein by the action of Hsp60 chaperones (M) Hsp104 and Hsp70 chaperones can directly convert compact aggregate into native mono-meric protein (N) Aggregates or fibrillar amyloid may further interact each other to form plaque like structure and accumulates in the differ-ent cellular space and becomes toxic and this toxicity formation cause amyloidosis class of disorders (O) Non-toxic matured amyloid cause Amyloidoses type disorders.