Protein-Misfolding Diseases and Chaperone-Based 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
Keywords chaperone-based therapeutic approaches; chemical and pharmacological chaperones; molecular chaperones; protein conformational diseases; protein misfolding and aggregation Correspondence T. K. Chaudhuri, Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India Fax: +91 11 2658 2282 Tel: +91 11 2659 1012 E-mail: tapan@dbeb.iitd.ac.in (Received 3 January 2006, revised 10 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 Alzheimers disease, Parkinsons disease, Huntingtons disease, CreutzfeldtJakob disease, cystic brosis, Gauchers 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 esh 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 efciency by inuencing 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, Alzheimers disease; ADH, antidiuretic hormone; AVP, arginine vasopressin; BSE, bovine spongiform encephalopathy; CF, cystic brosis; CFTR, cystic brosis transmembrane regulator; CJD, CreutzfeldtJacob disease; DMSO, dimethyl sulfoxide; ER, endoplasmic reticulum; FAP, familial amyloid polyneuropathy; GD, Gauchers disease; GSH-MEE, glutathione monoethyl ester; HbS, hemoglobin S; HD, Huntingtons 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, Parkinsons disease; PGD, polyglutamine disease; RP, retinitis pigmentosa; SCA, spinocerebeller ataxia; SSA, senile systemic amyloidosis; TMAO, trimethylamineN-oxide; UPP, ubiquitin proteasome pathway.
FEBS Journal 273 (2006) 13311349 2006 The Authors Journal compilation 2006 FEBS
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Protein-misfolding diseases
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 brosis (CF) and a1-antitrypsin deciency; or (b) deleterious gain of function as seen in many neurodegenerative diseases such as Alzheimers disease (AD), Parkinsons disease (PD) and Huntingtons disease (HD), in which protein misfolding results in the formation of harmful amyloid [7]. Protein aggregates are sometimes converted to a brillar 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 brosis 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 trafcking is a1-antitrypsin deciency [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 chaperones 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
Table 1. Mutation observed in different disease causing proteins. CF, cystic brosis; NDI, nephrogenic diabetes insipidus; PD, Parkinsons disease; AD, Alzheimers disease; HD, Huntingtons disease; SCA, spinocerebellar ataxia. Disease CF a-Antitrypsin deciency NDI Proteins affected CFTR a-Antitrypsin Aquaporin-2 V2asopressin 1 a-Galactosidase A p-53 a-synuclein Amyloid precursor protein Huntingtin Ataxin Mutations mutated gene DF508 D342K T126M, A147T, R187C R187C D6264, 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 Ref. [20] [21] [22]
Fabry Cancer PD AD aHD SCA
[23] [24] [16] [25] [25] [25]
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
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 [2729]. 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) [3032]. 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 efciency 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 bril-like structure called amyloid via an intermolecular interaction [33,34].
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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 modication 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 brillar deposits are also seen in patients, e.g. intracellular bundles of neurobrillary tangles in AD [4043]. 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 brils, these are polymerized to cross b-sheet structures with the b-strands arranged perpendicular to the long axis of the ber.
-helix
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 neurobrilary 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 rst dened by the presence of NFTs caused by the protein
-helix
-helix
-sheet
A B
-sheet
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) nal misfolded structure of polypeptide chain contains mostly b-pleated sheets.
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I: Dimerization
II: Oligomerization
Monomer
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-
feldtJakob 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; 1015% 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 brils, 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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Normal cellular prion protein are infected by Scrapie prion molecule PrPSc
Newly converted prions again infect other normal cellular prions PrPSc PrPSc PrPSc
All the normal cellular functional prion molecules converted into transmissible form
PrPSc PrPSc
PrPSc PrPSc
PrPC PrPC (i)
PrPC PrPC
PrPC (ii)
PrPC
PrPSc
(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. Protein responsible Huntingtin Androgen receptor Normal No. of repeats 1134 1133 No. repeats in mutant protein 40120 4062
Disorder Huntington Spinal and bulbar muscular atrophy Spinocerebellar ataxia Type 1 Type 2 Type 3 Type 6 Type 7 DentatorubropallidoLuysian atrophy
Ref. [45,7578] [79]
Ataxin 1 Ataxin 2 Ataxin 3 Ataxin 6 Ataxin 7 Atrophin 1
2536 1524 1336 416 735 725
4181 3559 6282 2127 37130 4985
[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. MachadoJoseph 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 [6874].
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
identied, 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 conrmed as a trafcking 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 deciency 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 deciency. 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-specic 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
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 [9294]. 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 ber formation. Mutant hemoglobin S (HbS) also leads to a stable ber-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 identied; 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 identied, 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 identied [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 deciency of galactosidase A activity in lysosomes, resulting in an accumulation of glycosphingolipid globotriosylceramide (Gb3). The majority of
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Table 3. Classication 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 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 Designation AL AH ATTR Ab2M ApoA-I ApoA-II ApoA-IV Alys AANF Ains Acys IAPP AGel AFib Diffusion G, L G, L G G G, L G G L L L L G Syndrome Isolated or associated with myeloma Isolated Familial amyloid neuropathy Hemodialysis amyloidosis Familial systemic amyloidosis
Familial systemic amyloidosis Iatrogenic Thyroid medullary cancer Diabetes type 2 islets of Langerhans, 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 ERs protein quality control and degraded before sorting into lysosomes. Fabry disease is specic 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]. Deciency 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 brillar 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 deciency causes the disease. In a third case, even if the brils 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)
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[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 classied 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 specic 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
Table 4. Proteins involved in different human diseases caused by misfolding, aggregation and trafcking [5,26]. Proteins 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 Disease Sickle cell anemia Cystic brosis Creutzfeld Jakob disease Scrapie (Mad Cow Disease), Familial insomnia Huntingtons disease Alzheimers disease Gauchers disease Parkinsons disease Nephrogenic diabetes insipidus Transthyretin amyloidoses Machado-Joseph atrophy Retinitis pigmentosa aB1B-Antitrypsin Fabry Cancer Cause Aggregation Trafcking Aggregation Ref. [96] [89] [110]
Aggregation Aggregation Trafcking Aggregation Trafcking Aggregation Trafcking Trafcking aggregation Trafcking Trafcking
[45,7578] [46] [103,105] [51] [97,98] [6774] [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, 110112]. 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
inefcient disaggregation, which is generally limited to small aggregates of luciferase and a-galactosidase. Their ndings 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
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DNA RNA
Ribosome CHIP Ubiquitin conjugation (A) Ubiquitinated protein

E1 E3 E2
ATP
Ubiquitin
Native Porotein (C)
(B)
Misfolded protein
Impaired ubiquiti n
(L)
(D) Aggregate/Fibrillar amyloid
Misfolded protein (E)
(F)
(M)
(K) (J)
26S P ro te o s o m e
Partially folded protein
(I) (O) Hsp60 Ubiquitin Hsp104 Hsp90 Hsp40 Hsp70 E1 E2 E3 Amyloidoses (Familial amyloid neuropathy) Cause several neurodegenerative diseases and lead cell demise like Alzheimer disease, Parkinson disease Gain of toxicity (N)
impaired proteasome
(H)
Misfolded protein
(G)
Degraded protein
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 specic mutation or cellular stress. (D) In the rst 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) Proteasomes action degrades misfolded protein into small peptides and ubiquitin is regenerated. (H) Impaired proteasome system couldnt 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 brillar 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] rst 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
Step1
Step2
Step3
ATP ClpB
ATP DnaK/DnaJ/ GrpE Loose aggregate
ATP GroEL/ GroES
Matured aggregate
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 nal 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 y 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 inefciently 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 proteins lethal conformation is to inuence
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 trafcking 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
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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 efciency of a1-AT in cell lines and transgenic animals. Although these compounds do not bind to a1-AT specically, they increase the secretion efciency of variants of a1-AT that are normally retained in the ER. Glycerol and 4-phenylbutyric acid accomplish a1-AT hypersecretion ability without inuencing the secretion efciency 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 specically 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-bril formation might be a reasonable therapeutic strategy because familial mutations that lead to an increase in Ab concentration or to its aggregation increase neuropathology [137139]. Peptidomimetics, based on the peptide LVFFA from Ab, modied 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 prole 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 specicity 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 inuence 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 specicity 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-
Table 5. Mutational effect in human proteins corrected by molecular, chemical and pharmacological chaperones [5]. HD, Huntingtons disease; MJD, Machado-Joseph disease; AD, Alzheimers disease; PD, Parkinsons disease; CF, cystic brosis; NDI, nephrogenic diabetes insipidus; CJD, Creuzfeld-Jacob diease; GD, Gauchers disease. Type of chaperone Molecular Chemical Pharmacological Chaperone name Hsp70, Hsp104, Hsp40 DMSO, glycerol, TMAO SR121463A, VPA985, 1-deoxy-galactonojirimycin CP31398, CP257042, amyloidoses, capsaicin, cycloporin, Vinblatin, verapamil Disease 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 specic 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 specic and have the same function as a chaperone, hence they are referred to as pharmacological chaperones. Loo & Clarke functionally characterized articial 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 specic 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 specic pathway. It might be that the proteasome pathway is not efcient 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 specic site inside the cell where they function causes disease. Good examples of this are CF and a-antitrypsin deciency 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
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Table 6. Mutations rescued by chemical and pharmacological chaperones. CF, cystic brosis; NDI, nephrogenic diabetes insipidus. Mutations recovered DF508 D342K T126M, A147t, r187c T126M, A147T, R187C,R187C D6264,l59p, l83q, Y128S, S16L, A294P, P322H, R337X R301Q, Q279E L173A, L175S, V249S, M273H
Disease CF a-Antitrypsin deciency NDI NDI
Protein CFTR a-Antitrypsin Aquaporin-2 Aquaporin-2 V2 vasopressin
Agents used Glycerol, DMSO, TMAO Glycerol Glycerol, DMSO, TMAO SR121463A, VPA985
Ref. [20] [21] [22] [146]
Fabry Cancer
a-Galactosidase A p53
1-Deoxy galactonojirimycin CP31398, CP257042
[23] [24]
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 ies 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 difcult. 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 ies 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 benets are yet to be reported. In order to have chaperone treatment it is worth knowing the exact mechanism of aggregate
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formation in the underlying protein. In future, an understanding of the causes of protein aggregation and the genetic and environmental susceptibility of a specic 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 y 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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Protein-misfolding diseases and chaperone-based therapeutic approaches

T. K. Chaudhuri and S. Paul

Protein misfolding and aggregation cause several diseases

T. K. Chaudhuri and S. Paul

Fabry Cancer PD AD aHD SCA

[23] [24] [16] [25] [25] [25]

T. K. Chaudhuri and S. Paul

Toxic amyloid formation causes many human neurodegenerative disorders

T. K. Chaudhuri and S. Paul

Tetramer: Forming aggregate

T. K. Chaudhuri and S. Paul

PrPC PrPC (i)

Ref. [45,7578] [79]

Ataxin 1 Ataxin 2 Ataxin 3 Ataxin 6 Ataxin 7 Atrophin 1

2536 1524 1336 416 735 725

4181 3559 6282 2127 37130 4985

[80] [81] [82,83] [84] [85] [86]

Protein misfolding and loss of function leads to several lethal diseases

T. K. Chaudhuri and S. Paul

T. K. Chaudhuri and S. Paul

Molecular chaperones can prevent protein misfolding and aggregation

T. K. Chaudhuri and S. Paul

T. K. Chaudhuri and S. Paul

Ribosome CHIP Ubiquitin conjugation (A) Ubiquitinated protein

Native Porotein (C)

(D) Aggregate/Fibrillar amyloid

Misfolded protein (E)

Partially folded protein

Loss of protein function cause several diseases like cystic fibrosis

T. K. Chaudhuri and S. Paul

ATP DnaK/DnaJ/ GrpE Loose aggregate

ATP GroEL/ GroES

Partially folded chain

T. K. Chaudhuri and S. Paul

T. K. Chaudhuri and S. Paul

T. K. Chaudhuri and S. Paul

Disease CF a-Antitrypsin deciency NDI NDI

Protein CFTR a-Antitrypsin Aquaporin-2 Aquaporin-2 V2 vasopressin

Ref. [20] [21] [22] [146]

1-Deoxy galactonojirimycin CP31398, CP257042

T. K. Chaudhuri and S. Paul

T. K. Chaudhuri and S. Paul

T. K. Chaudhuri and S. Paul

T. K. Chaudhuri and S. Paul

T. K. Chaudhuri and S. Paul

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