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
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: [email protected]
(Received 3 January 2006, revised 10 February 2006, accepted 14 February 2006)
doi:10.1111/j.1742-4658.2006.05181.x
A large number of neurodegenerative diseases in humans result from protein 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 molecular 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 probable 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 protein 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.
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 viscous, many proteins cannot fold properly by themselves 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
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, trimethylamineN-oxide; UPP, ubiquitin proteasome pathway.
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T. K. Chaudhuri and S. Paul
and non-native states of targeted proteins, but how
they discriminate between correctly and incorrectly
folded proteins and how they selectively retain and target 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 composed 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 recognize 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 concentration of misfolded protein is reached. Aggregated
proteins inside the cell often lead to the formation of
an amyloid-like structure, which eventually causes different 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 mutation in the polypeptide or, in a few cases, some lessknown 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 neurodegenerative diseases such as Alzheimer’s disease (AD),
Parkinson’s disease (PD) and Huntington’s disease
(HD), in which protein misfolding results in the formation 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 plaquelike structure [8]. In some cases, the mutations are so
severe that they render the gene product biologically
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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 delivered to their correct location, either inside the cell or in
the extracellular space. One example of disease involving abnormal protein trafficking is a1-antitrypsin deficiency [9]. In almost all cases of protein misfoldingmediated disorders, mutation in the gene (encoding the
disease-causing protein) is very common. However, the
more frequent amyloid-related neurodegenerative diseases 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 neurodegenerative 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 chaperones 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 several 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 decades. According to the prion researcher Susan Lindquist, ‘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 aggregated masses, which occasionally result in neurotoxicity and cell death. Hooper [13] reported that
aggregated ⁄ misfolded proteins become neurotoxic (e.g.
prion protein in mad cow disease; MCD) because of
FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS
T. K. Chaudhuri and S. Paul
Protein-misfolding diseases
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
a-Antitrypsin deficiency
NDI
CFTR
a-Antitrypsin
Aquaporin-2 ⁄ V2asopressin
[20]
[21]
[22]
Fabry
Cancer
1
a-Galactosidase A
p-53
DF508
D342K
T126M, A147T, R187C
R187C ⁄ D62–64, L59P, L83Q,
Y128S, S16L, A294P, P322H, R337X
R301Q, Q279E
R175, G245, R248, R249,
R273 and R282
A53T, A30P
AD 1, AD 2, AD 3, AD 4 Tau, preselinin 1 and 2,
a-macroglobulin
HD
SCA
PD
AD
aHD
SCA
a-synuclein
Amyloid precursor protein
Huntingtin
Ataxin
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 mutations 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 thermodynamically 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. Classic 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 hemoglobin.
A common feature of almost all protein conformational 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 bsheets 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 molecule 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 presence 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 conformational changes [26]. Based on the kinetic
[23]
[24]
[16]
[25]
[25]
[25]
modeling of protein aggregation, it has been proposed
that the critical event in PCD is the formation of protein oligomers that can then act as seeds to induce
protein misfolding [27–29]. In this model, misfolding
occurs as a consequence of aggregation (polymerization hypothesis) [26], which follows a crystallizationlike 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 misfolded protein, which may or may not form an aggregate. But in this hypothesis the critical question is
what factors are responsible for changes in conformation 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 oxidative 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 conformation 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 composed of b sheets, via intermolecular interactions. These
small oligomers form an ordered fibril-like structure
called amyloid via an intermolecular interaction [33,34].
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Protein-misfolding diseases
T. K. Chaudhuri and S. Paul
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 kinetically trapped intermediates in the protein folding or
assembly pathway [35,36]. Protein aggregation is facilitated by partial unfolding during thermal and oxidative stress and by alterations in the primary structure
caused by mutation, RNA modification or translational 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 diseases associated with protein misfolding, one or more
proteins are converted from the native structure to an
aggregated mass, which is commonly called an ‘amyloid’. 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 accumulation 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. intracellular bundles of neurofibrillary tangles in AD [40–43].
Although the initial process might be different in different diseases, a common trend is that during the formation of aggregates, a-helical domains disappear,
leading to an increase of b-sheet-dominated secondary
structure (Fig. 1) [44]. Recently, many other physiological disorders have been recognized as being caused
by the formation of protein aggregation, which subsequently 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 progressive are characterized by the selective and symmetrical loss of neurons in motor, sensory or cognitive
systems. The most common feature of all the neurodegenerative disorders is the occurrence of brain
lesions, formed by the intra- or extracellular accumulation 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 deposition 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 normal membrane precursor protein [46,47]. The main protein is a membrane protein called amyloid precursor
protein, which after being cleaved by b-secretase produces a b-amyloid precursor peptide fragment, this is
further cleaved by another protease b-secretase to produce 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 neurotoxicity has been generated by dimer and oligomer formation (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
A
α-helix
β-sheet
B
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.
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T. K. Chaudhuri and S. Paul
Protein-misfolding diseases
I: Dimerization
Monomer
II: Oligomerization
Dimer
Monomer
Tetramer: Forming
aggregate
Fig. 2. Protein oligomerization. Misfolded monomers forming aggregate through intermolecular hydrogen bonding interaction leading to
b-sheet formation.
tau. NFTs are aggregations of the microtubular protein 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, progressive 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 progressive disorder and the pathology of PD involves the
degeneration of dopaminergic neurons in the substantia nigra and the deposition of intracytoplasmic inclusion 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 bodies in sporadic PD [53] and in dementia with Lewy
bodies [54]. Two missense mutations in the gene encoding 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 responsible for causing a disease in cattle, called bovine
spongiform encephalopathy (BSE, or ‘mad cow disease’), 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 wildtype protein PrPC are 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 sporadic 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 neuroprotective function and the defective prion can induce
normal as well as huntingtin protein to change conformation, 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 autosomal dominant ⁄ X-linked and appears in mid-life leading to severe neuronal dysfunction and neuronal cell
death [67]. In all of these diseases, the CAG trinucleotides, which code for phenylalanine in the coding
regions of genes, are thought to be translated into
polyglutamine (polyQ) tracts. As a result, the protein
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Protein-misfolding diseases
T. K. Chaudhuri and S. Paul
Normal cellular
prion protein are
infected by Scrapie
prion molecule
Newly converted
prions again infect
other normal
cellular prions
PrPSc
PrPSc
PrPSc
PrPSc
PrPC
PrPC
PrPC
PrPC
PrPC
(i)
All the normal cellular
functional prion
molecules converted
into transmissible form
PrPSc
PrPSc
PrPSc
PrPSc
PrPSc
PrPC
(ii)
(iii)
Fig. 3. Propagation of PrPSc takes place through the interaction of PrPSc with normal cellular protein PrPC. Binding between PrPSc and PrPC
induces conformational change in PrPC protein that results in the formation of PrPSc, which form aggregates through intermolecular association. (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 responsible proteins.
Disorder
Huntington
Spinal and bulbar
muscular atrophy
Spinocerebellar ataxia
Type 1
Type 2
Type 3
Type 6
Type 7
DentatorubropallidoLuysian atrophy
Protein
responsible
Normal No.
of repeats
No. repeats in
mutant protein
Ref.
Huntingtin
Androgen receptor
11–34
11–33
40–120
40–62
[45,75–78]
[79]
Ataxin 1
Ataxin 2
Ataxin 3
Ataxin 6
Ataxin 7
Atrophin 1
25–36
15–24
13–36
4–16
7–35
7–25
41–81
35–59
62–82
21–27
37–130
49–85
[80]
[81]
[82,83]
[84]
[85]
[86]
product, now containing an usually long string of glutamine 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 inherited 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
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analyzed with the truncated ataxin-3 with an expanded
polyglutamine stretch, in which polyglutamine-containing 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 transmembrane 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
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T. K. Chaudhuri and S. Paul
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 position 508 (DF508 CFTR) took place within a polypeptide 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 diagnosed 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 protein folding in the cell, allowing the accumulation of
an intermediate, which then polymerizes [90], impeding
its release and leading to plasma deficiency. The a1antitrypsin is a serpin – an inhibitor of proteolytic
enzymes with serine at the active site, which, on binding to its target proteinase(s), undergoes a conformational 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 selfassociation 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 example of a protein misfolding-mediated disorder. Inactivation 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, however, 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 removing 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
Protein-misfolding diseases
swapping an incorrect amino acid at one point in its
polypeptide chain. In these mutants, the normal function 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 intermolecular bonding among adjacent hemoglobin molecules 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-pleated 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 vasopressin. 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 mutations 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 identified, 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 saturation of normal protein processing, transport and
degradation. In transfected cells, rhodopsin with mutations in the intradiscal, transmembrane and cytoplasmic 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 lysosomes, resulting in an accumulation of glycosphingolipid globotriosylceramide (Gb3). The majority of
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T. K. Chaudhuri and S. Paul
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
Immunoglobulin heavy chain
Transthyretin
Familial cardiac amyloid b-2-microglobulin
Prostatic amyloid
Apolipoprotein A-I
Apolipoprotein A-I
Apolipoprotein A-IV
Lysozyme
Atrial natriuretic factor
Insulin
Cystatin
Amylin
Insulinoma
Gelsolin
Fibrinogen A a
AL
AH
ATTR
Ab2M
G, L
G, L
G
G
Isolated or associated with myeloma
Isolated
Familial amyloid neuropathy
Hemodialysis amyloidosis
ApoA-I
ApoA-II
ApoA-IV
Alys
AANF
Ains
Acys
IAPP
G, L
G
Familial systemic amyloidosis
G
L
L
L
L
Familial systemic amyloidosis
Iatrogenic
Thyroid medullary cancer
Diabetes type 2 islets of Langerhans,
AGel
AFib
G
–
Familial
Nephropathy, hyperpathy
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 misfolded, recognized by the ER’s protein quality control and
degraded before sorting into lysosomes. Fabry disease
is specific for those missense mutations that cause misfolding of a-Gal A.
GD is an inherited lipid-storage disorder. It is
caused by mutation in the gene encoding acid b-glucosidase (GlcCerase) [102], an enzyme that participates
in the degradation of glycosphingolipids [103]. Symptoms may have neurological discrepancy or may be
non-neurological [104]. Deficiency of this enzyme causes 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 encoding 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 category 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 insoluble macrostructures [105,106], these macrostructures
are deposited in the tissues and cause disease (Table 3)
1338
[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 symptoms and biochemical type of amyloid protein
involved. Many amyloidoses are multisystemic, generalized 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 pathway where they are processed by a different chaperone
system, UPS, and subsequently reach their destination
by gain or loss of function leading to several degenerative 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
FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS
T. K. Chaudhuri and S. Paul
Protein-misfolding diseases
Table 4. Proteins involved in different human diseases caused by misfolding, aggregation and trafficking [5,26].
Proteins
Disease
Cause
Ref.
Hemoglobin
CFTR protein
Prion protein (PrP)
S
F
Huntingtin
b-amyloid protein
b-glucosidase
a-Synuclein
V2 vasopressin receptor
Transthyretin
M
Rhodopsin
aB1B-Antitrypsin
a-Galactosidase
P53
Sickle cell anemia
Cystic fibrosis
Creutzfeld Jakob disease
Scrapie (Mad Cow Disease),
Familial insomnia
Huntington’s disease
Alzheimer’s disease
Gaucher’s disease
Parkinson’s disease
Nephrogenic diabetes insipidus
Transthyretin amyloidoses
Machado-Joseph atrophy
Retinitis pigmentosa
aB1B-Antitrypsin
Fabry
Cancer
Aggregation
Trafficking
Aggregation
[96]
[89]
[110]
Aggregation
Aggregation
Trafficking
Aggregation
Trafficking
Aggregation
[45,75–78]
[46]
[103,105]
[51]
[97,98]
[67–74]
Trafficking
Trafficking ⁄ aggregation
Trafficking
Trafficking
[99]
[90]
[101,102]
[92]
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 distinct 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 molecular 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 solubilization. Stable aggregates are resistant to most ATPase
chaperone systems when functioning individually, for
example GroELS, Hsp90, ClpB, and low concentrations 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 chaperones 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 solubilizing prion-like aggregates in Saccharomyces cerevisiae
[116,117]. Many other positive responses have been
reported on cellular chaperone-mediated disaggregation in vivo. A classic experiment was performed
by Goloubinoff et al., who proved the phenomena of
in vitro reactivation and disaggregation of stable aggregates 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 disappear 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
FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS
1339
Protein-misfolding diseases
DNA
T. K. Chaudhuri and S. Paul
Ribosome
RNA
CHIP
Ubiquitin
conjugation
(A)
Native
Porotein
Misfolded
protein
(B)
ATP
Ubiquitin
E1
E3
E2
(C)
Impaired
ubiquiti n
Misfolded
protein
(D)
(L)
(M)
Ubiquitinated
protein
Aggregate/Fibrillar
amyloid
(E)
(F)
(K)
Partially
folded
protein
26S
P ro te o s o m e
(J)
(I)
impaired
proteasome
(O)
(N)
Hsp60
Ubiquitin
(H)
Hsp104
Hsp90
Hsp40
Gain of
toxicity
Hsp70
E1
E2
E3
Misfolded
protein
(G)
Degraded
protein
Amyloidoses
(Familial amyloid
neuropathy)
Cause several neurodegenerative
diseases and lead cell demise like
Alzheimer disease, Parkinson disease
Loss of protein function cause
several diseases like cystic
fibrosis
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 protein 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 proteasome 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 monomeric protein. (N) Aggregates or fibrillar amyloid may further interact each other to form plaque like structure and accumulates in the different cellular space and becomes toxic and this toxicity formation cause amyloidosis class of disorders. (O) Non-toxic matured amyloid cause
Amyloidoses type disorders.
population, which normally takes place via natural
selection). Sherman & Goldberg [120] first reported
that Hsp70 and Hsp40 molecular chaperones prevent
1340
aggregation of polyglutamine-containing proteins. It
has been reported that Hsp70 and Hsp40 chaperone
family members act together. The chaperone complex
FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS
T. K. Chaudhuri and S. Paul
Protein-misfolding diseases
Step1
Step2
Step3
ATP
ATP
ATP
ClpB
Matured
aggregate
DnaK/DnaJ/
GrpE
Loose aggregate
GroEL/
GroES
Partially
folded chain
Native
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.
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 suppressed 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 neurodegerarative disorders like PD and AD [122]. Hsp70
molecular chaperone was reported to mitigate dopaminergic 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 misfolded proteins back into the cytoplasm, where they are
degraded by the proteasome [7].
The role of chemical and pharmacological 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 suggest 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]. Chemical 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-galactosidase 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 protein pp60 and the ubiquitin-activating enzyme E1
[128]. Glycerol is an example of a chemical chaperone
and enhances protein stability by decreasing the solvent-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
FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS
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Protein-misfolding diseases
T. K. Chaudhuri and S. Paul
in vitro and the response was satisfactory. Hou Lin
and colleagues [104] have shown that N-octyl-h-valienamine (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 convenient 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 compounds 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-phenylbutyric 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 therapeutic agents in prion disease. A variety of compounds
including anthracyclines, porphyrins and diazo dyes
block prion replication when administered with PrPSc,
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 PrPC the normally
folded cellular isoform of the prion protein. On binding with PrPC, quinacrine stabilizes the conformation
of the protein and hence the conversion of PrPC to
PrPSc can be prevented, this in turn suppresses the progression of CJD.
In AD, no effective therapy using a chaperone system has been found. Inhibition of Ab-fibril formation
might be a reasonable therapeutic strategy because
familial mutations that lead to an increase in Ab concentration or to its aggregation increase neuropathology [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 retro1342
inverso peptidomimetics, block both Ab seeding and
growth. Unfortunately, the pharmacological profile of
these compounds is far from ideal. Nonpeptidic, aromatic-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 treating 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 number 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 therapeutic effect of chemical chaperones has been studied on
MJD, in which organic solvent DMSO, cellular osmolytes 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 osmolytes glycerol and TMAO, these are called chemical
chaperones because of their influence on protein conformation [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. However, 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 practical 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-glycoprotein, which causes retention in the ER and subsequent 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-
FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS
T. K. Chaudhuri and S. Paul
Protein-misfolding diseases
Table 5. Mutational effect in human proteins corrected by molecular, chemical and pharmacological chaperones [5]. HD, Huntington’s disease; 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
Chemical
Pharmacological
Hsp70, Hsp104, Hsp40
DMSO, glycerol, TMAO
SR121463A, VPA985,
1-deoxy-galactonojirimycin
CP31398, CP257042,
amyloidoses, capsaicin,
cycloporin, Vinblatin, verapamil
HD, MJD, AD, PD
AD, PD, CF, NDI, atherosclerosis, CJD, HD
GD, NDI, Transthyretin
congenital hypogonadotropic
hypogonadism
strate molecules resulted in mature and active P-glycoprotein, and based on the same type of approach,
other similar molecules were found which are collectively 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 proteasomal degradation. Pharmacological ligands act by
binding to specific conformations of receptor proteins
and stabilizing them. Selective VB2B receptor antagonists, which are retained in the ER and are responsible
for NDI, were assessed to reveal whether they facilitate
the folding of mutant VB2B receptor protein. Biosynthesis of mutant VB2B receptors was monitored in the
presence of the selective V2 receptor nonpeptide antagonist SR121463A. Morello et al. [144] proposed a
model for the mode of action of pharmacological
chaperones. Small nonpeptide VB2B receptor antagonists permeate the cell and bind to unstable folding
intermediates of the mutant receptors; this would stabilize a conformation of the receptor that allows its
release from the ER quality control system. The stabilized 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 VB2B specific and have the same
function as a chaperone, hence they are referred to as
pharmacological chaperones.
Loo & Clarke functionally characterized artificial mutations of the multidrug resistance 1 gene (ABCB1), which
codes for P-glycoprotein 1, an energy-dependent transporter 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 phenotype 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 specific gene mutation, which takes place in almost all
familial neurodegenerative diseases, or a matured
native protein can also achieve a misfolded conformation inside the cell, an example is the cause of prion
disease. The fates of these misfolded proteins in various 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 efficient 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 addition 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 inhibit and ⁄ or reverse conformational changes in the
protein molecules responsible. In most PCDs the misfolded 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 correct these diseases by persuading the misfolded proteins
FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS
1343
Protein-misfolding diseases
T. K. Chaudhuri and S. Paul
Table 6. Mutations rescued by chemical and pharmacological chaperones. CF, cystic fibrosis; NDI, nephrogenic diabetes insipidus.
Disease
Protein
CF
a-Antitrypsin deficiency
NDI
CFTR
a-Antitrypsin
Aquaporin-2
NDI
Aquaporin-2 ⁄
V2 vasopressin
Fabry
Cancer
a-Galactosidase A
p53
Mutations
recovered
DF508
D342K
T126M,
A147t, r187c
T126M, A147T,
R187C,R187C ⁄
D62–64,l59p, l83q,
Y128S, S16L,
A294P,
P322H, R337X
R301Q, Q279E
L173A, L175S,
V249S, M273H
to fold correctly. There is no doubt that chaperones
play a critical role in controlling protein misfolding and
thus reducing the threat of associated neurodegenerative diseases. Many questions remain regarding their
mode of action in suppressing and correcting the misfolding of disease-causing proteins. In MJD and SCA1
disease Hsp70 and Hsp40 have been shown to be highly
effective in suppressing the degeneration of polyQmediated disorders and increasing the lifespan of fruit
flies and mice. However, for other major neurodegenerative 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 disease 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 growing number of treatment options such as drugs, antioxidants, 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
1344
Agents used
Ref.
Glycerol, DMSO, TMAO
Glycerol
Glycerol, DMSO, TMAO
[20]
[21]
[22]
SR121463A, VPA985
[146]
1-Deoxy galactonojirimycin
CP31398, CP257042
[23]
[24]
formation in the underlying protein. In future, an
understanding of the causes of protein aggregation and
the genetic and environmental susceptibility of a specific individual may provide a better opportunity for
effective therapeutic intervention.
In a few cases, drugs may boost chaperone activation ⁄ 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 geldanamycin, 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.
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