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

REVIEW ARTICLE
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:
(Received 3 January 2006, revised 10 Febru-
ary 2006, accepted 14 February 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. Protein 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.
FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS 1331
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 a
1
-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
Protein-misfolding diseases T. K. Chaudhuri and S. Paul
1332 FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS
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 b-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 sequence 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 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 induction 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.
Disease Proteins affected Mutations ⁄ mutated gene Ref.
CF CFTR DF508 [20]
a-Antitrypsin deficiency a-Antitrypsin D342K [21]
NDI Aquaporin-2 ⁄ V2asopressin
1
T126M, A147T, R187C
R187C ⁄ D62–64, L59P, L83Q,
Y128S, S16L, A294P, P322H, R337X
[22]
Fabry a-Galactosidase A R301Q, Q279E [23]
Cancer p-53 R175, G245, R248, R249,
R273 and R282
[24]
PD a-synuclein A53T, A30P [16]
AD
a-
Amyloid precursor protein AD 1, AD 2, AD 3, AD 4 Tau, preselinin 1 and 2,
a-macroglobulin
[25]
HD Huntingtin HD [25]

SCA Ataxin SCA [25]
T. K. Chaudhuri and S. Paul Protein-misfolding diseases
FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS 1333
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 protein 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
AB C
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.
Protein-misfolding diseases T. K. Chaudhuri and S. Paul
1334 FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS

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 PrP
C
are converted into b-sheet-dominant
PrP
Sc
, 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
(PrP
Sc
) drives the conversion of cellular PrP
C
into

disease-causing PrP
Sc
(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 PrP
Sc
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: OligomerizationI: Dimerization
Tetramer: Forming
aggregate
MonomerDimerMonomer
Fig. 2. Protein oligomerization. Misfolded monomers forming aggregate through intermolecular hydrogen bonding interaction leading to
b-sheet formation.

T. K. Chaudhuri and S. Paul Protein-misfolding diseases
FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS 1335
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

PrP
Sc
PrP
C
PrP
C
PrP
C
PrP
C
(i)
Newly converted
prions again infect
other normal
cellular prions
All the normal cellular
functional prion
molecules converted
into transmissible form
PrP
C
PrP
C

PrP
Sc
PrP
Sc
PrP
Sc
PrP
Sc
PrP
Sc
PrP
Sc
PrP
Sc
PrP
Sc
(ii) (iii)
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
mutant protein Ref.
Huntington Huntingtin 11–34 40–120 [45,75–78]
Spinal and bulbar
muscular atrophy
Androgen receptor 11–33 40–62 [79]
Spinocerebellar ataxia
Type 1 Ataxin 1 25–36 41–81 [80]
Type 2 Ataxin 2 15–24 35–59 [81]
Type 3 Ataxin 3 13–36 62–82 [82,83]
Type 6 Ataxin 6 4–16 21–27 [84]
Type 7 Ataxin 7 7–35 37–130 [85]
Dentatorubropallido-
Luysian atrophy
Atrophin 1 7–25 49–85 [86]
Protein-misfolding diseases T. K. Chaudhuri and S. Paul
1336 FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS
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
T. K. Chaudhuri and S. Paul Protein-misfolding diseases
FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS 1337
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 localized. 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.
Precursor protein Designation Diffusion Syndrome
Immunoglobulin light chain AL G, L Isolated or associated with myeloma
Immunoglobulin heavy chain AH G, L Isolated
Transthyretin ATTR G Familial amyloid neuropathy
Familial cardiac amyloid b-2-microglobulin Ab2M G Hemodialysis amyloidosis
Prostatic amyloid
Apolipoprotein A-I ApoA-I G, L Familial systemic amyloidosis
Apolipoprotein A-I ApoA-II G
Apolipoprotein A-IV ApoA-IV

Lysozyme Alys G Familial systemic amyloidosis
Atrial natriuretic factor AANF L
Insulin Ains L Iatrogenic
Cystatin Acys L Thyroid medullary cancer
Amylin
Insulinoma
IAPP L Diabetes type 2 islets of Langerhans,
Gelsolin AGel G Familial
Fibrinogen A a AFib – Nephropathy, hyperpathy
Protein-misfolding diseases T. K. Chaudhuri and S. Paul
1338 FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS
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 vitro reactivation 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].
Proteins Disease Cause Ref.
Hemoglobin Sickle cell anemia Aggregation [96]
CFTR protein Cystic fibrosis Trafficking [89]
Prion protein (PrP) Creutzfeld Jakob disease Aggregation [110]
S
F
Scrapie (Mad Cow Disease),
Familial insomnia
Huntingtin Huntington’s disease Aggregation [45,75–78]
b-amyloid protein Alzheimer’s disease Aggregation [46]

b-glucosidase Gaucher’s disease Trafficking [103,105]
a-Synuclein Parkinson’s disease Aggregation [51]
V2 vasopressin receptor Nephrogenic diabetes insipidus Trafficking [97,98]
Transthyretin Transthyretin amyloidoses Aggregation [67–74]
M Machado-Joseph atrophy
Rhodopsin Retinitis pigmentosa Trafficking [99]
aB
1B
-Antitrypsin aB
1B
-Antitrypsin Trafficking ⁄ aggregation [90]
a-Galactosidase Fabry Trafficking [101,102]
P53 Cancer Trafficking [92]
T. K. Chaudhuri and S. Paul Protein-misfolding diseases
FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS 1339
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
S
62
e
m
o
so
e
t
o
r
P
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
d
e
r
i
a

p
m
I
n
i
t
i
uq
i
b
u
Misfolded
protein
Misfolded
protein
impaired
proteasom
e
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.
Protein-misfolding diseases T. K. Chaudhuri and S. Paul
1340 FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS
system is ubiquitous from bacteria to mammals. The
Hsp40 family regulates the chaperone activity of the
Hsp family by upregulating their ATPase activity
[121]. A classic in vivo experiment was performed on
Drosophila melanogaster [8] in which human Hsp70
was overexpressed. Complete suppression of the polyQ
neurodegenerative disorder, which causes an external
eye defect, took place. Overexpression of Hsp70 sup-
pressed neurodegeneration and increased the lifespan
of the fruit fly by twofold. The same experiment was
later performed in mammals. In a mouse model,
Hsp70 was overexpressed and found to be effective for
type 1 spinocerebellar ataxin (SCA1) disease [103]. It
has also been suggested that the chaperones Hsp70
and Hsp40 may play a role in some human neurode-
gerarative disorders like PD and AD [122]. Hsp70
molecular chaperone was reported to mitigate dopam-
inergic neuron loss induced by a-synuclein protein in
in vivo studies in a Drosophila model. Overexpression
of Hsp70 also suppresses a-synuclein neurotoxicity.
Many wild-type proteins fold inefficiently in the ER,
even with the help of Hsp40, Hsp70 and Hsp90 which

facilitate the refolding or retrotranslocation of misfold-
ed proteins back into the cytoplasm, where they are
degraded by the proteasome [7].
The role of chemical and pharmaco-
logical chaperones in rescuing protein
conformational defects
Chemical chaperones
Another strategy to prevent misfolding or correct a
mutant protein’s lethal conformation is to influence
the protein folding environment inside the cell. In
order to test this idea, DF508 CFTR protein was tested
for its ability to fold at 37° C and < 30 °C. It was
observed that at the higher temperature part of the
newly synthesized protein was misfolded and degraded,
whereas at the lower temperature a portion formed the
native structure. This helped in the discovering of
some chemical compounds that stabilize proteins
against thermal denaturation and might help to correct
folding defects. These compounds were collectively
called chemical chaperones [123]. Recent studies sug-
gest that chemical chaperones are effective in inhibiting
the formation of misfolded structure and subsequent
amyloid formation [124]. They are low molecular mass
compounds known to stabilize protein conformation
against thermal and chemical denaturation [9]. Chem-
ical chaperones have been shown to reverse the
intracellular retention of several different misfolded
proteins such as CFTR [21,22], a-antitrypsin [125],
aquaporin-2 [22], vasopressin V2 receptor [95], a-galac-
tosidase A [99], p53 and P-glycoprotein [126].

Recently, some of these compounds have been shown
in cell culture models to correct folding and trafficking
defects in DF508 CFTR in CF [20], the prion protein
PrP [127], and temperature-sensitive mutants of the
tumor suppressor protein p53, the viral oncogene pro-
tein pp60 and the ubiquitin-activating enzyme E1
[128]. Glycerol is an example of a chemical chaperone
and enhances protein stability by decreasing the sol-
vent-accessible surface area of the protein [129,130]. It
thus increases the rate of in vitro protein folding [131]
and enhances the rate of oligomeric assembly [132].
Although chemical chaperones have not been tested in
human organs, they have been studied in mouse cells
Step1 Step3Step2
ATP
ClpB
DnaK/DnaJ/
GrpE
GroEL/
GroES
ATPATP
NativePartially
folded chain
Matured
aggregate
Loose aggregate
Fig. 5. Protein disaggregation process in E.coli by chaperones [121]. Step1: ClpB chaperone preproceses the aggregate and produces a
loose structure having more hydrophobic surfaces (HS) exposed to the solvent. Step2: Dnak binds to those newly exposed HS along with
co-chaperones DnaJ and GrpE, disaggregates the loose aggregates and refolds them partially. Step3: GroEL and GroES assist final refolding
from monomeric partially folded form to native state.

T. K. Chaudhuri and S. Paul Protein-misfolding diseases
FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS 1341
in vitro and the response was satisfactory. Hou Lin
and colleagues [104] have shown that N-octyl-h-valien-
amine (NOV) can be used as a carbohydrate mimic
that would act as an inhibitor of b-glucosidase and
could be used in the suppression of GD, in which a
defect in b-glucosidase (b-Glu) leads to a misfolded
conformation. Sawkar and colleagues [133] reported
that a subset of N-alkylated deoxynorjirimycin and
simpler six-member ring N-heterocycles increase the
activity of b-glucosidase within human cells, this is the
best compound discovered to date. This may be a con-
venient alternative to intravenous enzyme-replacement
therapy. However, relevant transgenic animal models
to test this approach are not yet available.
The failure to secrete a1-AT into the circulation
allows neutrophil elastase to degrade lung parenchyma,
causing emphysema. Burrows and colleagues [125] have
shown that osmolytes such as 4-phenylbutyric acid and
glycerol increase the secretion efficiency of a1-AT in
cell lines and transgenic animals. Although these com-
pounds do not bind to a1-AT specifically, they increase
the secretion efficiency of variants of a1-AT that are
normally retained in the ER. Glycerol and 4-phenylbu-
tyric acid accomplish a1-AT hypersecretion ability
without influencing the secretion efficiency of other
proteins or decreasing proteasome activity.
Chemical chaperones have also been tried as thera-
peutic agents in prion disease. A variety of compounds

including anthracyclines, porphyrins and diazo dyes
block prion replication when administered with PrP
Sc
,
the aggregated infectious form of the prion protein, in
animal models [134,135]. Unfortunately, this is not a
clinically relevant model for therapeutic intervention,
because subclinical disease exists for months in mice
and years in humans. However, quinacrine has been
clinically approved, and clinical trials are being carried
out to test the usefulness of this molecule in patients
with CJD. Recently, Vogtherr et al. [136] used NMR
spectroscopy and chemical shift titration to show that
quinacrine binds specifically to PrP
C
the normally
folded cellular isoform of the prion protein. On bind-
ing with PrP
C
, quinacrine stabilizes the conformation
of the protein and hence the conversion of PrP
C
to
PrP
Sc
can be prevented, this in turn suppresses the pro-
gression of CJD.
In AD, no effective therapy using a chaperone sys-
tem has been found. Inhibition of Ab-fibril formation
might be a reasonable therapeutic strategy because

familial mutations that lead to an increase in Ab con-
centration or to its aggregation increase neuropatholo-
gy [137–139]. Peptidomimetics, based on the peptide
LVFFA from Ab, modified at the N- or C-terminus,
and the all-d (right-handed) version and several retro-
inverso peptidomimetics, block both Ab seeding and
growth. Unfortunately, the pharmacological profile of
these compounds is far from ideal. Nonpeptidic, aro-
matic-rich Ab aggregation inhibitors have also been
reported by many pharmaceutical and biotechnology
companies. But their lack of specificity is a problem.
In CF, the DF508 mutation can be rescued by treat-
ing cells with chemical chaperones like glycerol,
dimethyl sulfoxide (DMSO), trimethylamine-N-oxide
(TMAO) and deuterated water [140,141]. Mutation in
DF508 position leads to the appearance of small num-
ber of chloride channels in the plasma membrane.
These can be recovered by incubating cells at a higher
temperature. Glycerol, DMSO and TMAO mimic the
same act and thus rescue the mutation. The therapeu-
tic effect of chemical chaperones has been studied on
MJD, in which organic solvent DMSO, cellular osmo-
lytes glycerol and TMAO were used. Using an in vitro
cell culture system, the same effect has been observed
when chemical chaperones were used. These reagents
include the organic solvent DMSO and cellular osmo-
lytes glycerol and TMAO, these are called chemical
chaperones because of their influence on protein con-
formation [127]. Application of these three chemical
chaperones has been shown to be useful in suppressing

the polyglutamine diseases and preventing cell death.
Because DMSO was found to be an antioxidant [142],
two others, N-acetyl-l-cysteine (NAC) and glutathione
monoethyl ester (GSH-MEE) [143], were used to check
whether the antioxidant property of DMSO has any
effect on the reduction in aggregate formation. How-
ever, NAC and GSH-MEE did not prevent protein
aggregation, and so DMSO acts on the polypeptide
chain in a similar way to glycerol and TMAO [124].
Pharmacological chaperones
The use of chemical chaperones in reversing mutant
protein folding are well documented, but their use
requires high concentrations, at least micromolar level.
Although, small molecular mass molecules, including
osmolytes like glycerol, have been used to reverse
misfolding in several disease-causing proteins, their
lack of specificity means that they are a far from prac-
tical therapeutic approach in humans.
Because stability of the protein molecule can be
achieved by binding to substrates molecules, Loo &
Clarke examined the idea of using different substrates
of P-glycoprotein in cells expressing mutant P-glyco-
protein, which causes retention in the ER and subse-
quent degradation of the protein. Their work proved
effective from a therapeutic point of view because
synthesis of this mutant protein in the presence of sub-
Protein-misfolding diseases T. K. Chaudhuri and S. Paul
1342 FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS
strate molecules resulted in mature and active P-glyco-
protein, and based on the same type of approach,

other similar molecules were found which are collec-
tively named as pharmacological chaperones. Their use
is needed only at the micro molar level in the cell to
prevent misfolding of mutant proteins.
Pharmacological chaperones have proved very
effective in rescuing a few receptor proteins from pro-
teasomal degradation. Pharmacological ligands act by
binding to specific conformations of receptor proteins
and stabilizing them. Selective VB
2B
receptor antago-
nists, which are retained in the ER and are responsible
for NDI, were assessed to reveal whether they facilitate
the folding of mutant VB
2B
receptor protein. Biosyn-
thesis of mutant VB
2B
receptors was monitored in the
presence of the selective V2 receptor nonpeptide antag-
onist SR121463A. Morello et al. [144] proposed a
model for the mode of action of pharmacological
chaperones. Small nonpeptide VB
2B
receptor antago-
nists permeate the cell and bind to unstable folding
intermediates of the mutant receptors; this would sta-
bilize a conformation of the receptor that allows its
release from the ER quality control system. The stabil-
ized receptor proteins would then be targeted to the

cell surface where they bind AVP and promote signal
transduction upon dissociation from the antagonist.
These antagonists are VB
2B
specific and have the same
function as a chaperone, hence they are referred to as
pharmacological chaperones.
Loo & Clarke functionally characterizedartificialmuta-
tions of the multidrug resistance 1 gene ( ABCB1), which
codes for P-glycoprotein 1, an energy-dependent trans-
porter at the plasma membrane that interacts with a
wide variety of cytotoxic agents [126]. Morello et al.
[144] proved that selective V2 receptor antagonists
(SR121463A, VPA985) can permeate the cell surface
and facilitate the folding of mutant V2 receptors which
are retained in the ER and cause NDI.
Different molecular, chemical and pharmacological
chaperones, which have been already studied experi-
mentally and reported to reverse the mutational effect
of the protein conformation and suppress the pheno-
type are shown in Tables 5 and Table 6.
Conclusions
From the discussion on the mechanisms of different
protein misfolding disorders, it is clear that a nascent
polypeptide chain can become misfolded due to a spe-
cific gene mutation, which takes place in almost all
familial neurodegenerative diseases, or a matured
native protein can also achieve a misfolded conforma-
tion inside the cell, an example is the cause of prion
disease. The fates of these misfolded proteins in var-

ious disorders are different, in one class of diseases
misfolded proteins interact further with each other
through intermolecular interaction and form structured
aggregates thus gaining toxicity. Neurodegenerative
disorders are good examples of this specific pathway.
It might be that the proteasome pathway is not effi-
cient enough to degrade these misfolded proteins prior
to aggregation because of impairment of the UPS. In
another case, misfolded proteins are directed to the
UPP with the help of many other chaperones in addi-
tion to ubiquitins, and are consequently degraded by
the action of proteasome. Hence these proteins cannot
be secreted from the ER, but are degraded and
their disappearance from the specific site inside the
cell where they function causes disease. Good
examples of this are CF and a-antitrypsin deficiency
disorders.
Whatever the reason for a protein not achieving its
functional form, it is the conformational defect that
leads to disease. Therapy should therefore aim to inhi-
bit and ⁄ or reverse conformational changes in the
protein molecules responsible. In most PCDs the mis-
folded protein is rich in b sheet, and therapy
should involve designing a peptide to prevent and
reverse b-sheet formation. It might be possible to cor-
rect these diseases by persuading the misfolded proteins
Table 5. Mutational effect in human proteins corrected by molecular, chemical and pharmacological chaperones [5]. HD, Huntington’s dis-
ease; MJD, Machado-Joseph disease; AD, Alzheimer’s disease; PD, Parkinson’s disease; CF, cystic fibrosis; NDI, nephrogenic diabetes
insipidus; CJD, Creuzfeld-Jacob diease; GD, Gaucher’s disease.
Type of chaperone Chaperone name Disease

Molecular Hsp70, Hsp104, Hsp40 HD, MJD, AD, PD
Chemical DMSO, glycerol, TMAO AD, PD, CF, NDI, atherosclerosis, CJD, HD
Pharmacological SR121463A, VPA985,
1-deoxy-galactonojirimycin
CP31398, CP257042,
amyloidoses, capsaicin,
cycloporin, Vinblatin, verapamil
GD, NDI, Transthyretin
congenital hypogonadotropic
hypogonadism
T. K. Chaudhuri and S. Paul Protein-misfolding diseases
FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS 1343
to fold correctly. There is no doubt that chaperones
play a critical role in controlling protein misfolding and
thus reducing the threat of associated neurodegenera-
tive diseases. Many questions remain regarding their
mode of action in suppressing and correcting the mis-
folding of disease-causing proteins. In MJD and SCA1
disease Hsp70 and Hsp40 have been shown to be highly
effective in suppressing the degeneration of polyQ-
mediated disorders and increasing the lifespan of fruit
flies and mice. However, for other major neurodegener-
ative diseases like AD and PD the result of the same
experiment is not known. It has been shown that a
combination of heat shock proteins Hsp70 and Hsp40
is most effective in inhibiting huntingtin aggregation
in vitro and in a mammalian cell culture model system.
In some cases, the actual relationship between the dis-
ease and its phenotype is not known [10]. However,
experimental data suggest that correction of protein

misfolding constitutes a viable therapeutic strategy for
diseases caused by protein misfolding. Most of these
genetic disorders are progressive, and treatment is
therefore difficult. However, for some diseases, a grow-
ing number of treatment options such as drugs, antioxi-
dants, cell transplantation, surgery, rehabilitation
procedures and preimplantation diagnosis are available
[52]. In most cases, they have proved to be risk worthy
and having little adverse effect, whereas overexpressed
molecular chaperone-induced therapy has been shown
to be highly effective in fruit flies and even mammals
like the mouse [103,120]. Chemical chaperones like
DMSO and TMAO have been studied in vitro, and
showed reduced cytotoxicity and cell death, which has
been reported to be a good therapeutic strategy [124].
Chaperone treatment in humans and its benefits are yet
to be reported. In order to have chaperone treatment it
is worth knowing the exact mechanism of aggregate
formation in the underlying protein. In future, an
understanding of the causes of protein aggregation and
the genetic and environmental susceptibility of a speci-
fic individual may provide a better opportunity for
effective therapeutic intervention.
In a few cases, drugs may boost chaperone activa-
tion ⁄ upregulation, which would then prevent protein
misfolding. In 2001, Wanker and colleagues (Annual
Conference of the Genetics Society of Australia, July
2004) at the Max Planck Institute found that geldana-
mycin, an antibiotic, activated a heat shock response
and inhibited huntingtin aggregation in a cell culture

model of HD. This was promising but aggregates,
although not normal, are not a primary problem in
HD. However, Nancy Bonini [12] has shown that this
antibiotic not only prevented protein aggregation in a
fruit fly model of neurodegeneration but also stopped
the degeneration. Geldanamycin may be a treatment
for HD. This unpublished result was also discussed
at the Annual Conference of the Genetics Society of
Australia (July 2004).
It may be possible that in the near future molecular,
chemical and pharmacological chaperones might
change the mode of treatment and open a new door in
clinical research into the neurodegenerative diseases.
References
1 Anfinsen CB (1973) Principles that govern the folding
of protein chains. Science 181, 223–230.
2 Ellis RJ & Pinheiro TJ (2002) Danger – misfolding pro-
teins. Nature 416, 483–484.
3 Kinjo AR & Takada S (2003) Competition between
protein folding and aggregation with molecular chaper-
ones in crowded solutions: insight from mesoscopic
simulations. Biophys J 85, 3521–3531.
Table 6. Mutations rescued by chemical and pharmacological chaperones. CF, cystic fibrosis; NDI, nephrogenic diabetes insipidus.
Disease Protein
Mutations
recovered Agents used Ref.
CF CFTR DF508 Glycerol, DMSO, TMAO [20]
a-Antitrypsin deficiency a-Antitrypsin D342K Glycerol [21]
NDI Aquaporin-2 T126M,
A147t, r187c

Glycerol, DMSO, TMAO [22]
NDI Aquaporin-2 ⁄
V2 vasopressin
T126M, A147T,
R187C,R187C ⁄
SR121463A, VPA985 [146]
D62–64,l59p, l83q,
Y128S, S16L,
A294P,
P322H, R337X
Fabry a-Galactosidase A R301Q, Q279E 1-Deoxy galactonojirimycin [23]
Cancer p53 L173A, L175S,
V249S, M273H
CP31398, CP257042 [24]
Protein-misfolding diseases T. K. Chaudhuri and S. Paul
1344 FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS
4 Berke SJS & Paulson HL (2003) Protein aggregation
and the ubiquitin proteasome pathway: gaining the
UPPer hand on neurodegeneration. Curr Opin Genet
Dev 13, 253–261.
5 Welch WJ (2003) Role of quality control pathways in
human diseases involving protein misfolding. Semin
Cell Dev Biol 15, 31–38.
6 Swanton E, High S & Woodman P (2003) Role of cal-
nexin in the glycan-independent quality control of
proteolipid protein. EMBO J 22, 2948–2958.
7 Cohen FE & Kelly JW (2003) Therapeutic approaches
to protein misfolding diseases. Nature 426, 905–909.
8 Muchowski PJ (2002) Protein misfolding, amyloid for-
mation, and neurodegeration: a critical role for molecu-

lar chaperones? Neuron 35, 9–12.
9 Welch WJ & Brown CR (1996) Influence of molecular
and chemical chaperones on protein folding. Cell Stress
Chaperone 1, 109–115.
10 Forman MS, Lee VM & Trojanowski JQ (2003)
‘Unfolding’ pathways in neurodegenerative disease.
Trends Neurosci 26, 407–408.
11 Barrel JM, Broadley SA, Schaffar G & Hartl FU
(2004) Roles of molecular chaperones in protein mis-
folding diseases. Semin Cell Dev Biol 15, 17–29.
12 Bradbury J (2003) Chaperones: keeping a close eye on
protein folding. Lancet 361 (9364), 1194–1195.
13 Hooper NM (2001) Could inhibition of the proteasome
cause mad cow disease? Trends Biotechnol 21, 144–145.
14 Csermely P (2001) Chaperone overload is a possible
contributor to ‘civilization diseases’. Trends Genet 17,
701–704.
15 Kopito RR (2000) Aggresomes, inclusion bodies and
protein aggregation. Trends Cell Biol 10, 524–530.
16 Dobson CM (1999) Protein misfolding, evolution and
disease. Trends Biochem Sci 24, 329–332.
17 Darby NJ & Creighton TE (1993) Protein Structure.
Oxford: IRL Press.
18 Bennet MJ, Schlunegger MP & Eisenberg D (1995) 3D
domain swapping: a mechanism for oligomer assembly.
Protein Sci 4 , 2455–2468.
19 Perutz MF (1997) Mutations make enzyme polymerize.
Nature 385, 773–775.
20 Brown CR, Hong-Brown LQ, Biwersi J, Verkman AS
& Welch WJ (1996) Chemical chaperones correct the

mutant phenotype of the DF508 cystic fibrosis trans-
membrane conductance regulator protein. Cell Stress
Chaperone 1, 1117–1125.
21 Sato S, Ward CL, Krouse ME, Wine JJ & Kopito RR
(1996) Glycerol reverses the misfolding phenotype of
the most common cystic fibrosis mutation. J Biol Chem
271, 635–638.
22 Tamarappoo BK & Verkman AS (1999) Defective
aquaporin-2 trafficking in nephrogenic diabetes insipi-
dus and correction by chemical chaperones. J Clin
Invest 101, 2257–2267.
23 Fan JQ, Ishii S, Asano N & Suzuki Y (1999) Acceler-
ated transport and maturation of lysosomal a-galac-
tosidase A in Fabry lymphoblasts by exzyme inhibitor.
Nat Med 5, 112–115.
24 Nikolova PV, Wong KB, DeDecker B, Henckel J &
Fersht AR (2000) Mechanism of rescue of common
p53 cancer mutations by second-site suppressor muta-
tions. EMBO J 19, 370–378.
25 Martin JB (1999) Molecular basis of the neurodegener-
ative disorders. N Engl J Med 340, 1970–1980.
26 Soto C (2001) Protein misfolding and disease; protein
refolding and therapy. FEBS Lett 498, 204–207.
27 Jarrett JT, Lansbury PT Jr (1993) Seeding ‘one-dimen-
sional crystallization’ of amyloid: a pathogenic mechan-
ism in Alzheimer’s disease and scrapie? Cell 73, 1055–
1058.
28 Harper JD, Lansbury PT Jr (1997) Models of amyloid
seeding in Alzheimer’s disease and scrapie: mechanistic
truths and physiological consequences of the time-

dependent solubility of amyloid proteins. Annu Rev
Biochem 66, 385–407.
29 Lomakin A, Teplow DB, Kirschner DA & Benedek
GB (1997) Kinetic theory of fibrillogenesis of amyloid
beta-protein. Proc Natl Acad Sci USA 94, 7942–
7947.
30 Thomas PJ, Qu BH & Pedersen PL (1995) Defective
protein folding as a basis of human disease. Trends
Biochem Sci 20, 456–459.
31 Cohen FE & Prusiner SB (1998) Pathologic confor-
mations of prion proteins. Annu Rev Biochem 67,
793–819.
32 Prusiner SB (1998) Prions. Proc Natl Acad Sci USA 95,
13363–13383.
33 Serpell SC, Sunde M, Fraser PE, Luther PK, Morris
EP, Sangren O, Lundgren E & Black CC (1995) Exami-
nation of the structure of the transthyretin amyloid
fibril by image reconstruction from electron micro-
graphs. J Mol Biol 254, 113–118.
34 Kelly JW (1996) Alternative conformations of amy-
loidogenic proteins govern their behavior. Curr Opin
Struct Biol 6 , 11–17.
35 London J, Skrzynia C & Goldberg ME (1974)
Renaturation of Escherichia coli tryptophanase after
exposure to 8 m urea. Evidence for the existence of
nucleation centers. Eur J Biochem 47, 409–415.
36 Wetzel R (1994) Mutations and off-pathway aggrega-
tion of proteins. Trends Biotechnol 12, 193–198.
37 Fink A. (1998) Protein aggregation: folding aggre-
gates, inclusion bodies and amyloids. Fold Design 3,

R9–R23.
38 Troulinaki K & Tavernarakis N (2003) Neurodegenera-
tive conditions associated with ageing: a molecular
interplay? Mech Ageing Dev 126, 23–33.
39 Ben-Zvi AP & Goloubinoff P (2001) Mechanism of
disaggregation and refolding of stable protein
T. K. Chaudhuri and S. Paul Protein-misfolding diseases
FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS 1345
aggregates by molecular chaperones. J Struc Biol 135,
84–93.
40 Temussi PA, Masino L & Pastore A (2003) From Alz-
heimer to Huntington: why is a structural understand-
ing so difficult? EMBO J 22, 355–361.
41 Hardy J & Selkoe DJ (2002) The amyloid hypothesis of
Alzheimer’s disease: progress and problems on the road
to therapeutics. Science 297, 353–356.
42 Mochizuki A, Tamaoka A, Shimohata A, Komatsuzaki
Y & Shoji S (2000) A 42-positive non-pyramidal neu-
rons around amyloid plaques in Alzheimer’s disease.
Lancet 355, 42–43.
43 Kienlen-Campard P, Miolet S, Tasiaux B & Octave JN
(2002) Intracellular amyloid-1–42, but not extracellular
soluble amyloid-peptides, induces neuronal apoptosis.
J Biol Chem 277, 15666–15670.
44 Ursini F, Davies KJ, Maiorino M, Parasassi T & Seva-
nian A (2002) Atherosclerosis: another protein misfold-
ing disease? Trends Mol Med 8, 370–374.
45 Martin JB (1996) Pathogenesis of neurodegenerative
disorders: the role of dynamic mutations. Neuroreport
20, i–vii.

46 Games D, Adams D, Alessandrini R et al. (1995)
Alzheimer-type neuropathology in transgenic mice
overexpressing V717F b-amyloid precursor protein.
Nature 373, 523–527.
47 Oltersdorf T, Fritz LC, Schenk DB et al. (1989) The
secreted form of the Alzheimer’s amyloid precursor
protein with the Kunitz domain is protease nexin-II.
Nature 341, 144–147.
48 Higuchi M (2005) Understanding molecular mechanism
of proteolysis in Alzheimer’s disease: progress towards
therapeutic intervention. Biochim Biophys Acta 1751,
60–67.
49 Guillozet-Bongaarts AL, Garcia-Sierra F, Reynolds
MR, Horowitz PM, Fu Y, Wang T, Cahill MM,
Bigio EE, Berry RW & Binder LI (2005) Tau
truncation during neurofibrillary tangle evolution
in Alzheimer’s disease. Neurobiol Aging 26, 1015–
1022.
50 Berry RW, Quinn B, Johnson N, Cochran EJ, Ghoshal
N, Binder LI (2001) Pathological glial tau accumula-
tions in neurodegenerative disease: review and case
report. Neurochem Int 39, 469–479.
51 Forloni G, Terreni L, Bertani I et al. (2002) Protein
misfolding in Alzheimer’s and Parkinson’s disease:
genetics and molecular mechanisms. Neurobiol Aging
23, 957–976.
52 Shastry BS (2003) Neurodegenerative disorders of pro-
tein aggregation. Neurochem Int 43, 1–7.
53 Mouradian MM (2002) Recent advances in the genetic
and pathogenesis of Parkinson’s disease. Neurology 58,

179–185.
54 Schlossmacher MG, Frosch MP, Gai WP et al. (2002)
Parkin localizes to the Lewy bodies of Parkinson
disease and dementia with Lewy bodies. Am J Patholol
160, 1655–1667.
55 El-Agnaf O & Irvine GB (2000) Review: formation and
properties of amyloid-like fibrils derived from alpha-
synuclein and related proteins. J Struct Biol 130, 300–
309.
56 Hsu LJ, Sagara Y, Arroyo A, Pockenstein E, Sisk A,
Mallory M, Wong J, Takenouchi T, Hashimoto M &
Masliah E (2000) a-Synuclein promotes mitochondrial
deficiencies and oxidative stress. Am J Pathol 157, 401–
410.
57 Osterova-Golts N, Petruceli L, Hardy J, Lee JM, Farer
M & Wolozin B (2000) The A53T alpha-synuclein
mutation increases iron-dependent aggregation and
toxicity. J Neurosci 20, 6048–6054.
58 Souza JM, Giasson BI, Chen Q, Lee VMY & Ischiro-
poulos H (2000) Dityrosine cross-linking promotes for-
mation of stable a-synuclein polymers. Implication of
nitrative and oxidative stress in the pathogenesis of
neurodegenerative synucleinopatheis. J Biol Chem 275,
18344–18349.
59 Da Costa CA, Ancolio K & Checler F (2000) Wild
type but not Parkinson’s disease-related Ala-53 fi Thr
mutant alpha synuclein protects neuronal cells
from apoptotic stimuli. J Biol Chem 275, 24065–
24069.
60 Forloni G, Bertani I, Calella AM, Thaler F & Inve-

mizzi R (2000) Alpha-synuclein and Parkinson’s dis-
ease: selective neurodegenerative effect of alpha-
synuclein fragment on dopaminergic neurons in vitro
and in vivo. Ann Neurol 47, 632–640.
61 Kanda S, Bishop J, Eglitis MA, Yang Y & Mouradian
MM (2000) Enhanced vulnerability to oxidative stress
by a-synuclein mutations and C-terminal truncation.
Neuroscience 97, 279–284.
62 Prusiner SB (2001) Neurodegenerative diseases and
prions. N Engl J Med 344, 1516–1526.
63 Cohen FE, Pan KM, Huang Z, Baldwin M, Fletterick
RJ & Prusiner SB (1994) Structural clues to prion repli-
cation. Science 264, 530–531.
64 Goldfarb LG, Brown P, Haltia M et al. (1992) Creutz-
feldt–Jakob disease cosegregates with the codon 178-
asn PRNP mutation in families of European origin.
Ann Neurol 31, 274–281.
65 Zanata SM, Lopes MH, Mercadante AF et al. (2002)
Stress-inducible protein 1 is a cell surface ligand for
cellular prion that triggers neuroprotection. EMBO J
21, 3307–3316.
66 Chiarini LB, Freitas ARO, Zanata SM, Brentani RR,
Martins VR & Linden R (2002) Cellular prion protein
transduces neuroprotective signals. EMBO J 21, 3317–
3326.
67 Jana NR & Nukina N (2003) Recent advances in
understanding the pathogenesis of polyglutamine
diseases: involvement of molecular chaperones and
Protein-misfolding diseases T. K. Chaudhuri and S. Paul
1346 FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS

ubiquitin proteasome pathway. J Chem Neuroanat 26,
95–101.
68 Ikeda H, Yamaguchi M, Sugai S, Aze Y, Narumiya S
& Kakizuka A (1999) Expanded polyglutamine in the
Machado–Joseph disease protein induces cell death
in vitro and in vivo. Nature Genet 13, 196–202.
69 Paulson HL, Perez MK, Trotteir Y, Trojanowski JQ,
Subramony SH, Das SS, Vig P, Mandei JL, Fishbeck
KH & Pittman RN (1997) Intranuclear inclusions of
expanded polyglutamine protein in spinocerebellar
ataxia type 3. Neuron 19, 333–344.
70 Jackson GR, Salecker I, Dong X, Yao X, Arnheim N,
Faber PW, MacDonald ME & Zipursky SI (1998)
Polyglutamine-expanded human huntingtin transgenes
induce degradation of Drosophila photoreceptor neu-
rons. Neuron 21, 633–642.
71 Warrick JM, Paulson HL, Gray-Board GL, Bui QT,
Fishbeck KH, Pittman RN & Bonini NM (1998)
Expanded polyglutamine protein forms nuclear inclu-
sions and causes neural degeneration in Drosophila.
Cell 93, 939–949.
72 Evert BO, Wullner U, Schulz JB, Weller M, Groscurth
P, Trottier Y, Brice A & Klockgether T (1999) High
level expression of expanded full-length ataxin-3 in vitro
causes cell death and formation of intranuclear inclu-
sions in neuronal cells. Hum Mol Genet 8, 1169–1176.
73 Yoshizawa T, Yamagishi Y, Koseki N, Goto J, Yosh-
ida H, Shibasaki F, Shoji S & Kanazawa I (2000) Cell
cycle arrest enhances the in vitro cellular toxicity of the
truncated Machado–Joseph disease gene product with

an expanded polyglutamine stretch. Hum Mol Genet 9,
69–78.
74 Yoshizawa T, Yoshida H & Shoji S (2001) Differential
susceptibility of cultured cell lines to aggregate forma-
tion and cell death produced by the truncated
Machado–Joseph disease gene product with an expan-
ded polyglutamine stretch. Brain Res Bull 56, 349–352.
75 Gusella JF, Persichetti F & MacDonald ME (1997)
The genetic defect causing Huntington’s disease: repea-
ted in order contexts? Mol Med 3, 238–246.
76 MacDonald ME, Barnes G, Srinidhi J et al. (1993)
Gametic but not somatic instability of CAG repeat
length in Huntington’s disease. J Med Genet 30, 982–
986.
77 Meyers RH, MacDonald ME, Koroshetz WJ et al.
(1993) De novo expansion of a CAG repeat in a Japan-
ese patient with sporadic Huntington’s disease. Nat
Genet 5, 168–173.
78 Du
¨
rr A, Dode
´
C, Hahn V, Peˆ cheux C, Pillon B, Fein-
gold J, Kaplan J-C, Agid Y & Brice A (1995) Diagno-
sis of ‘sporadic’ Huntington’s disease. J Neurol Sci 129,
51–55.
79 Del Favero J, Krols L, Michalik A et al. (1998)
Molecular genetic analysis of autosomal dominant cere-
beller ataxia with retinal degeneration (ADCA type II)
caused by CAG triplet repeat expansion. Hum Mol

Genet 7, 177–186.
80 Banfi S, Servadio A, Chung MY, Kwiatkowski TJ Jr,
McCall AE, Duvick LA, Shen Y, Roth EJ, Orr HT &
Zoghbi HY (1994) Identification and characterization
of the gene causing type 1 spinocerebeller ataxia. Nat
Genet 7, 513–520.
81 Sanpie K, Takano H, Igarashi S et al. (1996) Identifica-
tion of the spinocerebeller ataxia type 2 gene using a
direct identification of repeat expansion and cloning
technique, DIRECT. Nat Genet 14, 277–284.
82 Schols L, Amoiridis G, Buttner T, Przuntek H, Epplen
J & Riess O (1997) Autosomal dominant cerebeller
ataxia: phenotypic differences in genetically defined
subtypes. Ann Neurol 42, 924–932.
83 Ikeda H, Yamaguchi M, Sugai S, Aze Y, Arumiya S &
Kakizuka A (1996) Expanded polyglutamine in the
Machado–Joseph disease protein induces cell death
in vitro and in vivo. Nat Genet 13, 196–202.
84 Zhuchenko O, Bailey J, Bonnen P, Ashizawa T, Stock-
ton DW, Amos C, Dobyns WB, Subramony SH,
Zoghbi HY & Lee CC (1997) Autosomal dominant cer-
ebeller ataxia (SCA6) associated with small polygluta-
mine expansions in the aB
1aB
-voltage-dependent
calcium channel. Nat Genet 15, 62–69.
85 David G, Dorr A, Steaming G et al. (1998) Molecular
and clinical correlations in autosomal dominant cere-
beller ataxia with progressive macular dystrophy
(SCA7). Hum Mol Genet 7, 165–170.

86 Riordan JR (1993) The cystic fibrosis transmembrane
conductance regulator. Annu Rev Physiol 55, 609–630.
87 Kopito RR (1999) Biosynthesis and degradation of the
CFTR. Physiol Rev 79, S167–S173.
88 Kerem B, Rommens JM, Buchanan JA, Markiewicz D,
Cox TK, Chakravarti A, Buchwald M & Tsui LC
(1989) Identification of the cystic fibrosis gene: genetic
analysis. Science 245, 1073–1080.
89 James EL, Whisstock JC, Gore MG & Bottomley SP
(1999) Probing the unfolding pathway of alpha-1-anti-
trypsin. J Biol Chem 274, 9482–9488.
90 Carrell RW, Lomas DA, Sidhar S & Foreman R (1996)
Alpha-1-antitrypsin deficiency. A conformational
disease. Chest 110, 243S–247S.
91 Foster BA, Coffey HA, Morin MJ & Rastinejad F
(1999) Pharmacological rescue of mutant p53 confor-
mation and function. Science 286, 2507–2510.
92 Cho Y, Gorina S, Jeffrey PD & Pavletich NP (1994)
Crystal structure of a p53 tumor suppressor–DNA
complex: understanding tumorigenic mutations. Science
265, 346–355.
93 Soussi T & May P (1996) Structural aspects of the p53
protein in relation to gene evolution: a second look.
J Mol Biol 260, 623–637.
94 Walker DR, Bond JP, Tarone RE, Harris CC,
Makalowski W, Boguski MS & Greenblatt MS (1999)
T. K. Chaudhuri and S. Paul Protein-misfolding diseases
FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS 1347
Evolutionary conservation and somatic mutation hot-
spot maps of p53: correlation with p53 protein struc-

tural and functional features. Oncogene 19, 211–218.
95 Galkin O & Vekilov PG (2004) Mechanisms of homo-
geneous nucleation of polymers of sickle cell anemia
hemoglobin in the deoxy state. J Mol Biol 336, 43–59.
96 Inaba S, Hatakeyama H, Taniguchi N & Miyamori I
(2001) The property of a novel V2 receptor mutant in a
patient with nephrogenic diabetes insipidus. J Clin
Endocrin Metab 86, 1381–1385.
97 Wildin RS, Antush MJ, Bennett RL, Schoof JM &
Scott CR (1994) Heterogeneous AVPR2 gene muta-
tions in congenital nephrogenic diabetes insipidus.
Am J Hum Genet 55, 266–277.
98 Dryja TP, McGee T, Reichel E, Hahn L, Cowley G,
Yandell DW, Sandberg MA & Berson EL (1990) A
point mutation of the rhodopsin gene in one form of
retinitis pigmentosa. Nature 343, 364–366.
99 Chapple JP, Grayson C, Hardcastle AJ, Saliba RS,
Van der Spuy J & Cheetham ME (2001) Unfolding ret-
inal dystrophis: a role for molecular chaperones?
Trends Mol Med 7, 414–421.
100 Eng CM, Resnick-Silverman LA, Niehaus DJ, Astrin
KH & Desnick RJ (1993) Nature and frequency of
mutations in the a-galactosidase A gene that cause
Fabry disease. Am J Hum Genet 53, 1186–1197.
101 Ishii S, Nakao S, Minamikawa-Tachino R, Desnick RJ
& Fan JQ (2002) Alternative splicing in the a-galactosi-
dase A gene: increased exon inclusion results in the Fa-
bry cardiac phenotype. Am J Hum Genet 70, 994–1002.
102 Anthony H, Futerman JL, Horowitz SM, Silman I &
Zimran A (2004) New directions in the treatment of

Gaucher disease. Trends Pharmacol Sci 25, 147–151.
103 Michelin K, Wajner A, Goulart Lda S, Fachel AA,
Pereira ML, de Mello AS, Souza FT, Pires RF, Giugli-
ani R & Coelho JC (2004) Biochemical study on b-glu-
cosidase in individuals with Gaucher’s disease and
normal subjects. Clin Chim Acta 343, 145–153.
104 Lin H, Sugimoto Y, Ohsaki Y et al. (2004) N-Octyl-
h-valienamine up-regulates activity of F2131 mutant
h-glucosidase in cultured cells: a potential chemical
chaperone therapy for Gaucher disease. Biochim
Biophys Acta: Mol Basis Dis 1–10.
105 Blake C & Serpell L (1996) Synchrotron X-ray studies
suggest that the core of the transthyretin amyloid is a
continuous b-sheet helix. Structure 4, 989–998.
106 Booth DR, Sunde M, Bellotti V et al. (1997) Instability,
unfolding and aggregation of human lysozyme variants
underlying amyloid fibrillogenesis. Nature 385, 787–793.
107 Tan SY & Pepys MB (1994) Amyloidosis. Histopathol-
ogy 25, 403–414.
108 Turnell WG & Finch JT (1992) Binding of the dye
Congo red to the amyloid protein pig insulin reveals a
novel homology amongst amyloid-forming peptide
sequences. J Mol Biol 227, 1205–1223.
109a Gilles G (2005) Amyloidoises. Orphanet Encyclopedia
(Guillevin L, ed.). Service de Medicine, Paris.
109 Hartl UF & Hayer-Hartl M (2002) Molecular chaper-
ones in the cytosol: from nascent chain to folded pro-
tein. Science 295, 1852–1858.
110 Mogk A, Tomoyasu T, Goloubinoff P, Rudiger S,
Roder D, Langen H & Bukau B (1999) Identification

of thermolabile Escherichia coli proteins: prevention
and reservation of aggregation by DnaK and ClpB.
EMBO J 18, 6934–6949.
111 Seonga IS, Oha JY, Leeab JW, Tanakab K & Chunga
CH (2000) The HslU ATPase acts as a molecular
chaperone in prevention of aggregation of SulA, an
inhibitor of cell division in Escherichia coli. FEBS Lett
477, 224–229.
112 Zahn R, Perrett S & Fersht AR (1996) Conformational
states bound by the molecular chaperones GroEL and
secB: a hidden unfolding (annealing) activity. J Mol
Biol 261, 43–61.
113 Skowyra D, Georgopoulos C & Zylicz M (1990)
The E. coli dnaK gene product, the hsp70 homolog,
can reactivate heat inactivated RNA polymerase in
an ATP hydrolysis-dependent manner. Cell 62,
939–944.
114 Schrimer EC, Glover JR, Singer MA & Lindquist S
(1996) HSP100 ⁄ Clp proteins: a common mechanism
explains diverse functions. Trends Biochem Sci 21, 289–
296.
115 Sanchez Y, Parsell DA, Taulien J, Vogel JL, Craig EA
& Lindquist S (1993) Genetic evidence for a functional
relationship between Hsp104 and hsp70. J Bacteriol
175, 6484–6491.
116 Chernoff YO, Lindquist SL, Ono B, Inge-Vechtomov
SG & Liebman SW (1995) Role of the chaperone pro-
tein Hsp104 in propagation of the yeast prion-like fac-
tor [psi+]. Science 268, 880–884.
117 Patino MM, Liu JJ, Glover JR & Lindquist S (1996)

Support for the prion hypothesis for inheritance of a
phenotypic trait in yeast. Science 273 , 622–626.
118 Goloubinoff P, Mogk A, Ben Zvi AP, Tomoyasu T &
Bukau B (1999) Sequential mechanism of solubilization
and refolding of stable protein protein aggregates by a
bichaperone network. Proc Natl Acad Sci USA 96,
13732–13737.
119 Tomoyasu T, Mogk A, Langen H, Goloubinoff P &
Bukau B (2001) Genetic dissection of the roles of
chaperones and proteases in protein folding and
degradation in the E. coli cytosol. Mol Biol 40, 397–
413.
120 Sherman MY & Goldberg AL (2001) Cellular defenses
against unfolded proteins. A cell biologist thinks about
neurodegenerative diseases. Neuron 29, 15–32.
121 Kobayashi Y & Sobue G (2001) Protective effect of
chaperones on polyglutamine diseases. Brain Res Bull
56, 165–168.
Protein-misfolding diseases T. K. Chaudhuri and S. Paul
1348 FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS
122 Auluck PK, Chan HY, Trojanowski JQ, Lee VM &
Bonini NM (2002) Chaperone suppression of a-synuc-
lein toxicity in a Drosophila model for Parkinson’s dis-
ease. Science 295 , 865–868.
123 Denning GM, Anderson MP, Amara JF, Marshall J,
Smith AE & Welsh MJ (1992) Processing of mutant
cystic fibrosis transmembrane conductance regulator is
temperature-sensitive. Nature 358, 1797–1799.
124 Yoshida H, Yoshizawa T, Shibasaki F, Shoji S &
Kanazawa I (2002) Chemical chaperones reduce aggre-

gate formation and cell death caused by the truncated
Machado–Joseph disease gene product with an expan-
ded polyglutamine stretch. Neurobiol Dis 10, 88–99.
125 Burrows JAJ, Willis LK & Perlmutter H (2000) Chemi-
cal chaperones mediate increased secretion of mutant
a1-antitrypsin (a1-AT) Z: a potential pharmacological
strategy for prevention of liver injury and emphysema
in a1-AT deficiency. Proc Natl Acad Sci USA 97,
1796–1801.
126 Loo TW & Clarke DM (1997) Correction of defective
protein kinesis of human P-glycoprotein mutants by
substrates and modulars. J Biol Chem 272, 709–712.
127 Tatzelt J, Pruisener SB & Welch WJ (1996) Chemical
chaperones interfere with the formation of scrapie
prion protein. EMBO J 15, 6363–6373.
128 Brown CR, Hong-Brown LQ & Welch WJ (1997)
Correcting temperature-sensitive protein folding
defects. J Clin Invest 99, 1432–1444.
129 Gekko K & Timasheff SN (1981) Mechanisms of pro-
tein stabilization by glycerol: preferential hydration in
glycerol–water mixtures. Biochemistry 20, 4667–4676.
130 Reibekas AA & Massey V (1997) Glycerol-assisted
restorative adjustment of flavoenzyme conformation
perturbed by site-directed mutagenesis. J Biol Chem
272, 22248–22252.
131 Sawano H, Koumoto Y, Ohta K, Sasaki Y, Segawa S
& Tachibana H (1992) Efficient in vitro folding of the
three-disulfide derivatives of hen lysozyme in the pre-
sence of glycerol. FEBS Lett 303, 11–14.
132 Shelanski ML, Gaskin F & Cantor CR (1973) Micro-

tubule assembly in the absence of added nucleotides.
Proc Natl Acad Sci USA 70, 765–768.
133 Sawkar AR, Cheng WC, Beutler E, Wong CH, Balch
WE & Kelly JW (2002) Chemical chaperones increase
the cellular activity of N370S b-glucosidase: a therapeu-
tic strategy for Gaucher disease. Proc Natl Acad Sci
USA 99, 15428–15433.
134 Korth C, May BCH, Cohen FE & Pruisner SB (2001)
Acridine and phenothiazine derivatives as pharma-
cotherapeutics for prion disease. Proc Natl Acad Sci
USA 98, 9836–9841.
135 May BC, Fafarman AT, Hong SB, Rogers M, Deady
LW, Prusiner SB & Cohen FE (2003) Potent inhibition
of scrapie prion replication in cultured cells by bis-acri-
dines. Proc Natl Acad Sci USA 100, 3416–3421.
136 Vogtherr M, Grimme S, Elshorst B, Jacobs DM, Fiebig
K, Griesinger C & Zahn R (2003) Antimalarial drug
quinacrine binds C-terminal helix of cellular prion pro-
tein. J Med Chem 46, 3563–3564.
137 Findies MA (2002) Peptide inhibitors of beta amyloid
aggregation. Curr Top Med Chem 2, 417–423.
138 Yang DS, Serpell LC, Yip CM, McLaurin J, Chrishti
MA, Horne P, Boudreau L, Kisilevsky R, Westaway D
& Fraser PE (2001) Assembly of Alzheimer’s amyloid-b
fibrils and approaches for therapeutic intervention.
Amyloid 8, 10–19.
139 Howlett DS et al. (2002) Assembly of Alzheimer’s amy-
loid aggregation. Curr Top Med Chem 2, 417–423.
140 Powell K & Zeitlin PL (2002) Therapeutic approaches
to repair defects in DF508 CFTR folding and cellular

targeting. Adv Drug Deliv Rev 54, 1395–1408.
141 Howard M & Welch WJ (2002) Manipulating the fold-
ing pathway of DF508 CFTR using chemical chaper-
ones. Methods Mol Med 70 , 267–275.
142 Yu ZW & Quinn PJ (1995) Dimethyl sulphoxide: a
review of its applications in cell biology. Biosci Rep 14,
259–258.
143 Wedi B, Straede J, Wieland B & Kapp A (1999) Eosi-
nophil apoptosis is mediated by stimulators of cellular
oxidative metabolisms and inhibited by antioxidants:
involvement of a thiosensitive redox regulation. Blood
94, 2365–2373.
144 Morello JP, Petaja-Repo UE, Bichet DG & Bouvier M
(2000) Pharmacological chaperones: a new twist on
receptor folding. Trends Pharmacol Sci 21, 466–469.
T. K. Chaudhuri and S. Paul Protein-misfolding diseases
FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS 1349