REVIEW ARTICLE Protein-misfolding diseases and chaperone-based therapeutic approaches Tapan K. Chaudhuri and Subhankar Paul Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, New Delhi, India In order to be functionally active, a protein has to acquire a unique 3D conformation via a complicated folding pathway, which is described by the primary amino acid sequence and the local cellular environment [1]. Protein folding is vital for a living organism because it adds flesh to the gene skeleton. A small error in the folding process results in a misfolded structure, which can sometimes be lethal [2]. However, within the cellular environment, which is highly vis- cous, many proteins cannot fold properly by them- selves and require the assistance of a special kind of ubiquitous protein, the molecular chaperones [3]. Molecular chaperones assist other proteins to achieve a functionally active 3D structure and thus prevent the formation of a misfolded or aggregated structure, essentially enhancing folding efficiency by influencing the kinetics of the process and inhibiting events that lead to unproductive end points (e.g. aggregation). Chaperones are located at various points in the cell and interact with nascent polypeptides during synthesis and translocation to different cellular compartments. Chaperones are able to distinguish between the native Keywords chaperone-based therapeutic approaches; chemical and pharmacological chaperones; molecular chaperones; protein conformational diseases; protein misfolding and aggregation Correspondence T. K. Chaudhuri, Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India Fax: +91 11 2658 2282 Tel: +91 11 2659 1012 E-mail: tapan@dbeb.iitd.ac.in (Received 3 January 2006, revised 10 Febru- ary 2006, accepted 14 February 2006) doi:10.1111/j.1742-4658.2006.05181.x A large number of neurodegenerative diseases in humans result from pro- tein misfolding and aggregation. Protein misfolding is believed to be the primary cause of Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Creutzfeldt–Jakob disease, cystic fibrosis, Gaucher’s disease and many other degenerative and neurodegenerative disorders. Cellular mole- cular chaperones, which are ubiquitous, stress-induced proteins, and newly found chemical and pharmacological chaperones have been found to be effective in preventing misfolding of different disease-causing proteins, essentially reducing the severity of several neurodegenerative disorders and many other protein-misfolding diseases. In this review, we discuss the prob- able mechanisms of several protein-misfolding diseases in humans, as well as therapeutic approaches for countering them. The role of molecular, chemical and pharmacological chaperones in suppressing the effect of pro- tein misfolding-induced consequences in humans is explained in detail. Functional aspects of the different types of chaperones suggest their uses as potential therapeutic agents against different types of degenerative diseases, including neurodegenerative disorders. Abbreviations AD, Alzheimer’s disease; ADH, antidiuretic hormone; AVP, arginine vasopressin; BSE, bovine spongiform encephalopathy; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane regulator; CJD, Creutzfeldt–Jacob disease; DMSO, dimethyl sulfoxide; ER, endoplasmic reticulum; FAP, familial amyloid polyneuropathy; GD, Gaucher’s disease; GSH-MEE, glutathione monoethyl ester; HbS, hemoglobin S; HD, Huntington’s disease; HSP, heat shock protein; MCD, mad cow disease; MJD, Machado-Joseph disease; NAC, N-acetyl- L-cysteine; NDI, nephrogenic diabetes insipidus; NOV, N-octyl-h-valienamine; PCD, protein conformational disease; PD, Parkinson’s disease; PGD, polyglutamine disease; RP, retinitis pigmentosa; SCA, spinocerebeller ataxia; SSA, senile systemic amyloidosis; TMAO, trimethylamine- N-oxide; UPP, ubiquitin proteasome pathway. FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS 1331 and non-native states of targeted proteins, but how they discriminate between correctly and incorrectly folded proteins and how they selectively retain and tar- get the latter for degradation is yet to be understood. Proteins that are not able to achieve the native state, due either to an unwanted mutation in their amino acid sequence or simply because of an error in the folding process, are recognized as misfolded and subsequently targeted to a degradation pathway. This is referred to as a protein ‘quality control’ (QC) system and is com- posed of two components: molecular chaperones and the ubiquitin proteasome system (UPS) [4]. The QC system plays a critical role in cell function and survival. A special class of chaperone, for example, calnexin, forms part of the ‘quality control monitors’ that recog- nize and target abnormally folded proteins for rapid degradation [5]. One class of QC chaperone associated with the endoplasmic reticulum (ER), e.g. calnexin and calreticulin, BiP and ERp 57 [6], is able to recognize misfolded proteins and help their retention in the ER, allowing only correctly folded proteins to reach the cytosol [5]. One very strong and crucial aspect of QC in the cell is the ubiquitin proteasome pathway (UPP). Studies suggest that disturbance in or impairment of the UPP, which may be induced by the accumulation of misfolded proteins in the ER or loss of function of the enzymes involved in the ubiquitin conjugation and deconjugation pathway, leads to altered UPS function, which positively affects the accumulation of protein aggregates in the cell [4]. The formation of oligomers and aggregates occurs in the cell when a critical concen- tration of misfolded protein is reached. Aggregated proteins inside the cell often lead to the formation of an amyloid-like structure, which eventually causes dif- ferent types of degenerative disorders and ultimately cell death [4]. In almost all protein-misfolding disorders, an error in folding occurs because of either an undesirable muta- tion in the polypeptide or, in a few cases, some less- known reason. The harmful effect of the misfolded protein may be due to: (a) loss of function, as observed in cystic fibrosis (CF) and a1-antitrypsin deficiency; or (b) deleterious ‘gain of function’ as seen in many neuro- degenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and Huntington’s disease (HD), in which protein misfolding results in the forma- tion of harmful amyloid [7]. Protein aggregates are sometimes converted to a fibrillar structure containing a large number of intermolecular hydrogen bonds which is highly insoluble. These are commonly called amyloids and their accumulation occasionally results in a plaque- like structure [8]. In some cases, the mutations are so severe that they render the gene product biologically inactive [cystic fibrosis transmembrane regulator (CFTR) protein]. In other cases, however, the mutations are relatively minor and the resulting proteins show only a partial loss of normal activity. Despite having partial biological activity, these mutant proteins are not deliv- ered to their correct location, either inside the cell or in the extracellular space. One example of disease invol- ving abnormal protein trafficking is a 1 -antitrypsin defi- ciency [9]. In almost all cases of protein misfolding- mediated disorders, mutation in the gene (encoding the disease-causing protein) is very common. However, the more frequent amyloid-related neurodegenerative dis- eases are characterized by the appearance of a toxic function caused by the misfolded proteins [10]. One or more of a chaperone’s activities result in the prevention ⁄ suppression of a few devastating neurode- generative diseases. Reduction in the intracellular level of chaperones results in an increase in abnormally folded proteins inside the cell [5]. Therefore, toxicity in different neurodegenerative disorders may result from an imbalance between normal chaperone capacity and the production of misfolded protein species. Increased chaperone expression can suppress the neurotoxicity caused by protein misfolding, suggesting that chaper- ones could be used as possible therapeutic agents [11]. Natural, chemical or pharmacological chaperones have been shown to be promising agents for the control of many protein conformational disorders (PCD). These diseases include CF, AD, PD and HD, as well as sev- eral forms of prion diseases. Here, we discuss the causes of protein misfolding, aggregation and amyloid formation in the cell, and the use of different chaperones as therapeutic agents against various protein-misfolding disorders. Protein misfolding and aggregation cause several diseases Protein misfolding and its pathogenic consequences have become an important issue over the last two dec- ades. According to the prion researcher Susan Lind- quist, ‘protein misfolding could be involved in up to half of all human diseases’ [12]. Protein misfolding is also responsible for many p53-mediated cancers, which are also the result of incorrect protein folding. Many cancers and other protein-misfolding disorders are caused by mutations in proteins (Table 1) that are key regulators of growth and differentiation. Structural changes in a few proteins subsequently lead to aggre- gated masses, which occasionally result in neuro- toxicity and cell death. Hooper [13] reported that aggregated ⁄ misfolded proteins become neurotoxic (e.g. prion protein in mad cow disease; MCD) because of Protein-misfolding diseases T. K. Chaudhuri and S. Paul 1332 FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS an inhibition of proteasome function. Csermely [14] suggested a ‘chaperone overload’ hypothesis, which explains that with aging, there is an overburden of accumulated misfolded protein that prevents molecular chaperones from repairing phenotypically silent muta- tions which might cause disease. It has been shown that the yield of correctly folded protein obtained from in vitro refolding is low due to the formation of ther- modynamically stable folding intermediates. These conformations are called ‘dead-end’ conformations and are ‘off-pathway’ intermediates, they generally lead to the formation of insoluble aggregates [15] that may eventually causes different degenerative diseases. Clas- sic examples of these degenerative diseases are CF, which is caused by the deletion of a single residue phenylalanine in the CFTR protein, and sickle cell anemia, which originated due to a mutation in hemo- globin. A common feature of almost all protein conforma- tional diseases is the formation of an aggregate caused by destabilization of the a-helical structure and the simultaneous formation of a b-sheet [16]. These b- sheets are formed between alternating peptide strands. Linkages between these strands result from hydrogen bonding between their aligned pleated structures. Such b-linkages [17] with a pleated strand from one mole- cule being inserted into a pleated sheet of the next lead to hydrogen-bond formation between molecules [18]. The prerequisites for b-linkage formation are the pres- ence of a donor peptide sequence that can adopt a pleated structure and a b sheet that can act as an acceptor for the extra strand [19]. It is not clear whether misfolding triggers protein aggregation or protein oligomerization induces con- formational changes [26]. Based on the kinetic modeling of protein aggregation, it has been proposed that the critical event in PCD is the formation of pro- tein oligomers that can then act as seeds to induce protein misfolding [27–29]. In this model, misfolding occurs as a consequence of aggregation (polymeriza- tion hypothesis) [26], which follows a crystallization- like process dependent on nucleus formation. The alternative model suggests that the underlying protein is stable in both the folded and misfolded forms in solution (conformational hypothesis) [30–32]. This hypothesis proposes that spontaneous or induced conformational changes result in formation of the mis- folded protein, which may or may not form an aggre- gate. But in this hypothesis the critical question is what factors are responsible for changes in conforma- tion without the induction of aggregates. Studies have described several factors that play a crucial role, such as mutation in the gene, which destabilizes the correct structure. For example, mutation is common in all neurodegenerative disorders, which reduces the folding efficiency by changing the proper folding energetic. Induced protein misfolding has been described as being responsible for all familial diseases. In addition to mutation, other environmental stresses such as oxida- tive stress, alkalosis, acidosis, pH shift and osmotic shock are able to change the structure of a protein without involving aggregates. In a third hypothesis, the native protein conforma- tion is changed to an amyloidogenic intermediate, which is not stable in the cellular environment. This intermediate has many exposed hydrophobic regions and therefore develops small oligomers, mainly com- posed of b sheets, via intermolecular interactions. These small oligomers form an ordered fibril-like structure called amyloid via an intermolecular interaction [33,34]. Table 1. Mutation observed in different disease causing proteins. CF, cystic fibrosis; NDI, nephrogenic diabetes insipidus; PD, Parkinson’s disease; AD, Alzheimer’s disease; HD, Huntington’s disease; SCA, spinocerebellar ataxia. Disease Proteins affected Mutations ⁄ mutated gene Ref. CF CFTR DF508 [20] a-Antitrypsin deficiency a-Antitrypsin D342K [21] NDI Aquaporin-2 ⁄ V2asopressin 1 T126M, A147T, R187C R187C ⁄ D62–64, L59P, L83Q, Y128S, S16L, A294P, P322H, R337X [22] Fabry a-Galactosidase A R301Q, Q279E [23] Cancer p-53 R175, G245, R248, R249, R273 and R282 [24] PD a-synuclein A53T, A30P [16] AD a- Amyloid precursor protein AD 1, AD 2, AD 3, AD 4 Tau, preselinin 1 and 2, a-macroglobulin [25] HD Huntingtin HD [25] SCA Ataxin SCA [25] T. K. Chaudhuri and S. Paul Protein-misfolding diseases FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS 1333 Protein aggregation is an inevitable consequence of a cellular existence and these aggregates are oligomeric complexes of non-native conformers that arise from intermolecular interactions among structured and kin- etically trapped intermediates in the protein folding or assembly pathway [35,36]. Protein aggregation is facili- tated by partial unfolding during thermal and oxida- tive stress and by alterations in the primary structure caused by mutation, RNA modification or transla- tional misincorporation [36,37]. Protein aggregates can be either structured (e.g. amyloid) or amorphous. In either case, they are insoluble and metabolically stable in the physiological environment [38]. For various dis- eases associated with protein misfolding, one or more proteins are converted from the native structure to an aggregated mass, which is commonly called an ‘amy- loid’. The net accumulation of toxic protein aggregates in the cell depends on the stability, compactness and hydrophobic exposure of the aggregates, as well as on the rate of protein synthesis in the cell [39]. The accu- mulation of toxic aggregates in the cell depends on chaperone expression and protease networks [39]. Environmental stress may induce the synthesis of higher levels of chaperones and proteases in the cell, which can better remove toxic aggregates [39]. Fibrillar amyloids are commonly extracellular, but intracellular fibrillar deposits are also seen in patients, e.g. intracel- lular bundles of neurofibrillary tangles in AD [40–43]. Although the initial process might be different in dif- ferent diseases, a common trend is that during the for- mation of aggregates, a-helical domains disappear, leading to an increase of b-sheet-dominated secondary structure (Fig. 1) [44]. Recently, many other physiolo- gical disorders have been recognized as being caused by the formation of protein aggregation, which subse- quently forms a plaque-like structure containing a large number of amyloid fibrils, these are polymerized to cross b-sheet structures with the b-strands arranged perpendicular to the long axis of the fiber. Toxic amyloid formation causes many human neurodegenerative disorders Neurodegenerative disorders that are chronic and pro- gressive are characterized by the selective and symmet- rical loss of neurons in motor, sensory or cognitive systems. The most common feature of all the neuro- degenerative disorders is the occurrence of brain lesions, formed by the intra- or extracellular accumula- tion of misfolded, aggregated or ubiquitinated proteins [4]. Proteins associated with some neurodegenerative diseases like AD, PD and HD, are tau ⁄ b-amyloid (Ab), a-synuclein and huntingtin, respectively [8]. For AD, PD and CJD a few cases are familial or inherited but the remainder are sporadic in nature. AD is a progressive degenerative disease of the brain in the elderly which clouds memory and causes impaired behavior [45]. The neuropathological features of this devastating disease are the extracellular depos- ition of Ab and neurofibrilary tangles (NFT) in the brain. A central process of AD is the cleavage of a 42 amino acid b-amyloid peptide from an otherwise nor- mal membrane precursor protein [46,47]. The main pro- tein is a membrane protein called amyloid precursor protein, which after being cleaved by b-secretase produ- ces a b-amyloid precursor peptide fragment, this is further cleaved by another protease b-secretase to pro- duce Ab-42 instead of Ab-40, which is amyloidogenic. It is thought that cellular degradation of Ab-42 is the normal fate of this peptide fragment when produced in small amounts under normal conditions, however, in some lesser known conditions it forms extracellular aggregates and subsequently generates amyloid plaques. Studies have reported that impairment of the UPS may be involved in this disorder [16]. An increase in neuro- toxicity has been generated by dimer and oligomer for- mation (Fig. 2) of the Ab fragment [48]. According to many scientists, AD should be first defined by the presence of NFTs caused by the protein α-helix α-helix β-sheet β-sheet α-helix AB C Fig. 1. During amyloid formation most of the a-helical structures in the polypeptide chain of a native protein are converted into b-pleated sheets. (A) Native polypeptide chain composed of mainly a-helical secondary structure. (B) Misfolding causes conversion of a-helical structure to b-pleated sheets and (C) final misfolded structure of polypeptide chain contains mostly b-pleated sheets. Protein-misfolding diseases T. K. Chaudhuri and S. Paul 1334 FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS tau. NFTs are aggregations of the microtubular pro- tein tau, which are found to be hyperphosphorylated in the neuronal cells of AD patients. Although, tau polymer formation is a hallmark of other degenerative disorders, such as corticobasal degeneration, progres- sive supranuclear palsy and pick disease [49], all differ from AD in that they lack Ab plaque deposition [50]. In contrast to AD, it is believed that in PD, protein accumulates in the intracellular space [51]. PD is the second most common, late-onset neurodegenerative disorder, and is characterized by muscular rigidity, postural instability and resting tremor. It is a slow pro- gressive disorder and the pathology of PD involves the degeneration of dopaminergic neurons in the substan- tia nigra and the deposition of intracytoplasmic inclu- sion bodies called Lewy bodies in brain cells. The exact mechanism by which these cells are lost is not known. Heritable forms of PD are caused by gene mutations. To date, three genes encoding a-synuclein, parkin and ubiquitin C-terminal hydrolase L1 protein have been shown to be associated with familial forms of PD [52]. All three proteins are present in Lewy bod- ies in sporadic PD [53] and in dementia with Lewy bodies [54]. Two missense mutations in the gene enco- ding a-synuclein are linked to dominantly inherited PD, thereby directly implicating a-synuclein in the pathogenesis of the disease. Recent studies suggest that the intracellular accumulation of a-synuclein [55] leads to mitochondrial dysfunction [56], oxidative stress [57,58] and caspase degradation [59] accentuated by mutations associated with familial parkinsonism [60,61]. The prion protein, which is thought to be respon- sible for causing a disease in cattle, called bovine spongiform encephalopathy (BSE, or ‘mad cow dis- ease’), and a disease in humans, called variant Creutz- feldt–Jakob disease (vCJD) [62] is thought to undergo a conformational change in which a helices of the wild- type protein PrP C are converted into b-sheet-dominant PrP Sc , resulting in misfolding and aggregation [63,64]. CJD is inherited as an autosomal dominant disorder and the most common human prion disease, the spor- adic form, accounts for  85% of cases;  10–15% of cases are familial. Sporadic CJD results from the endogenous generation of prions. In general, familial CJD has an earlier age-of-onset and a longer clinical course than sporadic CJD. Fatal familial insomnia is the strangest phenotype of familial prion diseases. The symptoms are dominated by progressive insomnia, autonomic dysfunction and dementia. In the case of infectious prion disease, the infectious scrapie protein (PrP Sc ) drives the conversion of cellular PrP C into disease-causing PrP Sc (Fig. 3) [63]. The normal prion protein is protease sensitive, soluble, and has a high a-helix content, but its normal function is unknown. The disease-causing prion protein (the transmissible isoform) is protease resistant and insoluble, forms amyloid fibrils, and has a high b-sheet content. Studies have reported that prion protein PrP Sc has a neuro- protective function and the defective prion can induce normal as well as huntingtin protein to change confor- mation, which later form aggregates [63,65,66]. In some human disorders, protein misfolding takes place due to repetition of glutamine in the polypeptide chain, which is called polyglutamine disease (PGD). This disorder is progressive, inherited, either auto- somal dominant ⁄ X-linked and appears in mid-life lead- ing to severe neuronal dysfunction and neuronal cell death [67]. In all of these diseases, the CAG trinucleo- tides, which code for phenylalanine in the coding regions of genes, are thought to be translated into polyglutamine (polyQ) tracts. As a result, the protein II: OligomerizationI: Dimerization Tetramer: Forming aggregate MonomerDimerMonomer Fig. 2. Protein oligomerization. Misfolded monomers forming aggregate through intermolecular hydrogen bonding interaction leading to b-sheet formation. T. K. Chaudhuri and S. Paul Protein-misfolding diseases FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS 1335 product, now containing an usually long string of glu- tamine residues, appears to misfold and form large detergent-insoluble aggregates within the nucleus or cytoplasm, thereby leading to the eventual demise of the effected neuron [5]. To date eight different inher- ited neurodegenerative diseases (Table 2) have been found to be due to expansion of glutamine repeats in the affected proteins. HD is the most frequent of them. Machado–Joseph disease ⁄ spinocerebellar ataxia-3 (MJD ⁄ SCA-3) is another inherited neurodegenerative disorder caused by expansion of the polyglutamine stretch in the MJD gene-encoded protein ataxin-3. The truncated form of mutated ataxin-3 causes aggregation and cell death in vitro and in vivo. In vitro cellular models and transgenic animals have been created and analyzed with the truncated ataxin-3 with an expanded polyglutamine stretch, in which polyglutamine-contain- ing aggregates and cell death were invariably observed [68–74]. Protein misfolding and loss of function leads to several lethal diseases CF is characterized by thick mucous secretions in the lung and intestines [8]. Amino acid sequence analysis of CFTR protein has shown that the protein resides within membranes, contains 12 potential transmem- brane domains, two nucleotide-binding domains, and a highly charged hydrophilic region, which has been shown to act as a regulatory domain [5]. Although many mutations in the CFTR sequence have been Normal cellular prion protein are infected by Scrapie prion molecule PrP Sc PrP C PrP C PrP C PrP C (i) Newly converted prions again infect other normal cellular prions All the normal cellular functional prion molecules converted into transmissible form PrP C PrP C PrP Sc PrP Sc PrP Sc PrP Sc PrP Sc PrP Sc PrP Sc PrP Sc (ii) (iii) Fig. 3. Propagation of PrP Sc takes place through the interaction of PrP Sc with normal cellular protein PrP C . Binding between PrP Sc and PrP C induces conformational change in PrP C protein that results in the formation of PrP Sc , which form aggregates through intermolecular associ- ation. (i) Transmissible isoform of one prion protein molecule infects other normal cellular prion molecules. (ii) Infection causes induction in conformation of normal prions that converts them to transmissible prion molecules, which again start infecting other normal prion molecules. (iii) All the cellular normal prions are transformed into disease causing scrapie prion proteins. Table 2. Neurodegenerative diseases caused by repetition of CAG codon which encodes glutamine in the polypeptide chain of the respon- sible proteins. Disorder Protein responsible Normal No. of repeats No. repeats in mutant protein Ref. Huntington Huntingtin 11–34 40–120 [45,75–78] Spinal and bulbar muscular atrophy Androgen receptor 11–33 40–62 [79] Spinocerebellar ataxia Type 1 Ataxin 1 25–36 41–81 [80] Type 2 Ataxin 2 15–24 35–59 [81] Type 3 Ataxin 3 13–36 62–82 [82,83] Type 6 Ataxin 6 4–16 21–27 [84] Type 7 Ataxin 7 7–35 37–130 [85] Dentatorubropallido- Luysian atrophy Atrophin 1 7–25 49–85 [86] Protein-misfolding diseases T. K. Chaudhuri and S. Paul 1336 FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS identified, one in particular has been noted in over 705 patients examined, in this mutation deletion of three nucleotides coding for a phenylalanine residue at posi- tion 508 (DF508 CFTR) took place within a polypep- tide of 1480 amino acids [87]. The DF508 allele of CFTR has been confirmed as a trafficking mutation that blocks maturation of the protein in the ER and targets it for premature proteolysis [88]. The clinical importance of this mutation becomes evident when considering that it accounts for 70% of patients diag- nosed with CF [89]. The most common and severe form of a1-antitrypsin deficiency is caused by the Z mutation, a single base substitution (Gul342-Lys) in the a1-antitrypsin gene. Misfolding of proteins during synthesis can initiate an ordered polymerization, which leads to aggregation of the protein within the cell. This slows the rate of pro- tein folding in the cell, allowing the accumulation of an intermediate, which then polymerizes [90], impeding its release and leading to plasma deficiency. The a1- antitrypsin is a serpin – an inhibitor of proteolytic enzymes with serine at the active site, which, on bind- ing to its target proteinase(s), undergoes a conforma- tional change. It is known that serpin polymerization involves the interaction of one serpin molecule with the b-sheet of another molecule of the same type; extensive knowledge of this mechanism may help in the development of b-strand blockers to prevent self- association of these proteins [91]. The tumor suppressor protein p53, which is a sequence-specific transcription factor whose function is to maintain genome integrity, presents a classic exam- ple of a protein misfolding-mediated disorder. Inacti- vation of p53 by mutation is a key molecular event, and is detected in > 50% of all human cancers [24]. The p53 tumor suppressor is one of our defenses against uncontrolled cell growth which leads to tumor proliferation. Under normal conditions there is a low level of p53 tumor suppressor protein in the cell, how- ever, when DNA damage is sensed, p53 levels rise and initiate protective measures. p53 protein binds to many regulatory sites in the genome and begins production of proteins that halt cell division until the damage is repaired. If the damage is too severe, p53 initiates the process of programmed cell death, or apoptosis, which directs the cell to commit suicide, permanently remov- ing the damage. The human p53 suppressor gene is mutated with high frequency in cancers [91]. Most of these are missense mutations, affecting residues that are critical for maintaining the structural fold of this highly conserved DNA-binding protein, changing the information in the DNA at one position and causing the cell to produce p53 protein with an error through swapping an incorrect amino acid at one point in its polypeptide chain. In these mutants, the normal func- tion of p53 is lost and the protein is unable to prevent multiplication in the damaged cell [92–94]. Sickle cell anemia is a genetic disorder in which the amino acid valine at the sixth position of the b-globin chain is replaced by glutamine. Galkin and Vekilov [95] have reported that this mutation promotes inter- molecular bonding among adjacent hemoglobin mole- cules and results in stable long polymer fiber formation. Mutant hemoglobin S (HbS) also leads to a stable fiber-like structure while HbS is in deoxy state. This polymerization changes the shape and rigidity of red blood cells and triggers abnormality. Lot of b-plea- ted sheet accumulates as ‘amyloid plaques’. Nephrogenic diabetes insipidus (NDI) is a disorder known to be caused by misfolding of one hormonal protein, antidiuretic hormone, also known as vasopres- sin. NDI is characterized by an inability of the kidneys to remove water from the urinea and by resistance of the kidneys to the action of arginine vasopressin [96]. Wildin et al. [97] reported that a mutation in the AVPR2 gene, which encodes arginine vasopressin, is most common in NDI. More than 70 different muta- tions have been identified; the majority are missense and nonsense mutations. Furthermore, 18 frameshift mutations due to nucleotide deletions or insertions (up to 35 bp) and four large deletions have been reported. Retinitis pigmentosa (RP) is the most common cause of inherited blindness with over 25 genetic loci identi- fied, it is characterized by night-blindness and loss of peripheral vision, followed by loss of central vision. Mutations in the gene encoding rhodopsin have been identified [98] and more than 100 mutations have now been described that account for  15% of all inherited human retinal degenerations. The failure of rhodopsin to translocate to the outer segment per se does not appear to be enough to cause RP; rather, it would appear that misfolded rhodopsin acquires a ‘gain of function’ that leads to cell death. The nature of this gain of function is unclear, but may be related to sat- uration of normal protein processing, transport and degradation. In transfected cells, rhodopsin with muta- tions in the intradiscal, transmembrane and cytoplas- mic domains fails to translocate to the plasma membrane, and accumulates in the ER and Golgi. Hence these mutant proteins fail to translocate because of misfolding and this causes the disorder [99]. Another protein conformational disorder is Fabry disease, which is a lysosomal storage disorder, caused by a deficiency of galactosidase A activity in lyso- somes, resulting in an accumulation of glycosphingo- lipid globotriosylceramide (Gb3). The majority of T. K. Chaudhuri and S. Paul Protein-misfolding diseases FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS 1337 cardiac Fabry patients have missense mutations in the a-Gal A gene (GLA), although alternative splicing mutations and small deletions have also been observed [100,101]. Such mutant enzymes appear to be misfold- ed, recognized by the ER’s protein quality control and degraded before sorting into lysosomes. Fabry disease is specific for those missense mutations that cause mis- folding of a-Gal A. GD is an inherited lipid-storage disorder. It is caused by mutation in the gene encoding acid b-glu- cosidase (GlcCerase) [102], an enzyme that participates in the degradation of glycosphingolipids [103]. Symp- toms may have neurological discrepancy or may be non-neurological [104]. Deficiency of this enzyme cau- ses accumulation of glucocerebrosides in macrophage lysosome. In very few cases, GD is caused by mutation in the saposin C domain of the gene prosaposin, which controls the optimum activity of GlcCerase by enco- ding a protein saposin C [102]. Amyloidoses In all the above cases either misfolded proteins form fibrillar aggregates which become toxic and lead to cell death (all neurodegenrative diseases) or, in other cate- gory of disease, misfolded proteins are directed to the proteasome pathway for degradation (proteolysis), and protein deficiency causes the disease. In a third case, even if the fibrils themselves are not toxic, the ready autolinkage of proteins and polypeptides by b-strand bonding involves risks of further linkage to give insol- uble macrostructures [105,106], these macrostructures are deposited in the tissues and cause disease (Table 3) [107]. Different amyloidosis may be heterogeneous in nature but all have common properties in that they all bind the dye Congo red that intercalates between their b strands [108]. Amyloidosis is classified according to clinical symp- toms and biochemical type of amyloid protein involved. Many amyloidoses are multisystemic, gener- alized or diffuse but a few are also localized. They mainly affect kidneys, heart, gastrointestinal tract, liver, skin, peripheral nerve and eyes. It is a slowly progressive disease that can lead to morbidity and death. Amyloid deposits are extracellular and not metabolized or cleared by the body, thus the deposits eventually impair the function of the organ where they accumulate. Table 4 shows the causes of different disorders by specific disease-causing proteins and Fig. 4 shows the possible fate of misfolded proteins through the path- way where they are processed by a different chaperone system, UPS, and subsequently reach their destination by gain or loss of function leading to several degener- ative disorders. Molecular chaperones can prevent protein misfolding and aggregation Large multidomain proteins have been found to form a misfolded structure and aggregated mass during in vitro refolding [109]. The cellular environment is crowded with proteins and other macromolecules, and so the chance of a newly synthesized unfolded protein forming aggregates is greater in vivo than in vitro. Cellular molecular chaperones are proteins that change Table 3. Classification of amyloidoses and name of precursor proteins and nomenclature [109a]. Amyloidoses that affect central nervous system are not considered here. G, generalized; L, localized. Precursor protein Designation Diffusion Syndrome Immunoglobulin light chain AL G, L Isolated or associated with myeloma Immunoglobulin heavy chain AH G, L Isolated Transthyretin ATTR G Familial amyloid neuropathy Familial cardiac amyloid b-2-microglobulin Ab2M G Hemodialysis amyloidosis Prostatic amyloid Apolipoprotein A-I ApoA-I G, L Familial systemic amyloidosis Apolipoprotein A-I ApoA-II G Apolipoprotein A-IV ApoA-IV Lysozyme Alys G Familial systemic amyloidosis Atrial natriuretic factor AANF L Insulin Ains L Iatrogenic Cystatin Acys L Thyroid medullary cancer Amylin Insulinoma IAPP L Diabetes type 2 islets of Langerhans, Gelsolin AGel G Familial Fibrinogen A a AFib – Nephropathy, hyperpathy Protein-misfolding diseases T. K. Chaudhuri and S. Paul 1338 FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS this equation by selectively recognizing and binding to the exposed hydrophobic surfaces of a non-native protein via non-covalent interactions, thus inhibiting irreversible aggregation of those proteins in vivo [5] and in vitro. Molecular chaperones are composed of several dis- tinct classes of sequence-conserved proteins, most of which are stress inducible like heat shock proteins (Hsp). Major classes of these Hsp are Hsp100 (in E. coli, ClpA ⁄ B ⁄ X, HslU), Hsp90 (in E. coli, HtpG), Hsp70 (in E. coli, DnaK), Hsp60 (in E. coli, GroEL) and the small Hsps (in E. coli , IbpA ⁄ B). These mole- cular chaperones have important damage-control functions during and following stress. Under in vitro conditions, many chaperones, such as E. coli IbpB, DnaK, DnaJ, GroEL, HtpG and SecB, and proteases such as DegP, HslU and Ion can bind chemically unfolded polypeptides and prevent aggregation [21, 110–112]. They are also involved in aggregate solubili- zation. Stable aggregates are resistant to most ATPase chaperone systems when functioning individually, for example GroELS, Hsp90, ClpB, and low concentra- tions of DnaK. Skowyra et al. [113] observed that the DnaK chaperone system might reactivate some forms of protein aggregate. It has been observed that Hsp100, which includes Ipb, ClpA, HslU and ClpX in E. coli, has disaggregation activity [114]. ClpA and ClpX have been shown to destabilize some native protein structures, allowing them through the central cavity into the ClpP for proteolysis [114]. Schrimer et al. have shown that Hsp70 and Hsp100 function in combination to reactivate many protein aggregates [114]. They also showed that Hsp104 cooperates with Hsp70 and Hsp40 in a slow and inefficient disaggregation, which is generally limited to small aggregates of luciferase and a-galactosidase. Their findings have been supported by evidence that both chaperones collaborate in the cellular acquisition of thermotolerance [115]. It has been reported that the yeast non-Mendelian factor [psi+], which is analogous to mammalian prions, is propagated at when there are intermediate amounts of the chaperone protein Hsp104 and overproduction or inactivation of Hsp104 caused loss of [psi+] [116]. These results suggest that chaper- ones are crucial in prion disease progression and that a certain level of chaperone expression can rid cells of prions without affecting their viability. Control of the expression level of Hsp104 may provide a therapy against prion disease. In addition, Hsp104, along with Hsp70, has been shown to be responsible for solubiliz- ing prion-like aggregates in Saccharomyces cerevisiae [116,117]. Many other positive responses have been reported on cellular chaperone-mediated disaggrega- tion in vivo. A classic experiment was performed by Goloubinoff et al., who proved the phenomena of in vitro reactivation and disaggregation of stable aggre- gates of malate dehydrogenase by ClpB together with DnaK, DnaJ and GrpE (KJE), and further explained the mechanism of the whole disaggregation process (Fig. 5) [118]. Mogk, Tomoyasu and colleagues [110,119] showed that, in E. coli, stable protein aggregates rapidly disap- pear from the insoluble fraction following chaperone action during a short recovery period. Under normal conditions, chaperones repair the conformational defects of some mutated proteins, thus reducing their phenotypic effects and dampening genome cleansing (elimination of damaged genes from the gene pool of a Table 4. Proteins involved in different human diseases caused by misfolding, aggregation and trafficking [5,26]. Proteins Disease Cause Ref. Hemoglobin Sickle cell anemia Aggregation [96] CFTR protein Cystic fibrosis Trafficking [89] Prion protein (PrP) Creutzfeld Jakob disease Aggregation [110] S F Scrapie (Mad Cow Disease), Familial insomnia Huntingtin Huntington’s disease Aggregation [45,75–78] b-amyloid protein Alzheimer’s disease Aggregation [46] b-glucosidase Gaucher’s disease Trafficking [103,105] a-Synuclein Parkinson’s disease Aggregation [51] V2 vasopressin receptor Nephrogenic diabetes insipidus Trafficking [97,98] Transthyretin Transthyretin amyloidoses Aggregation [67–74] M Machado-Joseph atrophy Rhodopsin Retinitis pigmentosa Trafficking [99] aB 1B -Antitrypsin aB 1B -Antitrypsin Trafficking ⁄ aggregation [90] a-Galactosidase Fabry Trafficking [101,102] P53 Cancer Trafficking [92] T. K. Chaudhuri and S. Paul Protein-misfolding diseases FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS 1339 population, which normally takes place via natural selection). Sherman & Goldberg [120] first reported that Hsp70 and Hsp40 molecular chaperones prevent aggregation of polyglutamine-containing proteins. It has been reported that Hsp70 and Hsp40 chaperone family members act together. The chaperone complex (B) (D) (L) (H) (M) (J) Loss of protein function cause several diseases like cystic fibrosis (E) (F) (I) (K) Gain of toxicity Cause several neurodegenerative diseases and lead cell demise like Alzheimer disease, Parkinson disease Degraded protein S 62 e m o so e t o r P Native Porotein Ubiquitin Aggregate/Fibrillar amyloid (N) (G) Hsp60 Ubiquitin Hsp104 Hsp90 Hsp40 Hsp70 DNA RNA Ribosome (A) (C) E1 E2 E3 ATP Ubiquitin conjugation E1 E2 E3 Ubiquitinated protein Partially folded protein CHIP d e r i a p m I n i t i uq i b u Misfolded protein Misfolded protein impaired proteasom e Misfolded protein Amyloidoses (Familial amyloid neuropathy) (O) Fig. 4. The fate of cellular misfolded protein is shown. (A) Nascent polypeptide chain is converted into folded protein. (B) Polypetide chain reaches misfolded structure. (C) Native protein molecule is converted into misfolded structure due to specific mutation or cellular stress. (D) In the first step Hsp 40 ⁄ 70 ⁄ 90 facilitate to direct them to the proteasomal pathway and the second step is ubiquitination of misfolded pro- tein assisted by E1 (ubiquitin activating enzyme), E2 (ubiquitin conjugating enzyme) & E3 (ubiquitin ligase). (E) Due to the damage of ubiquitin enzymes, misfolded protein is directed to the aggregation pathway. (F) Misfolded protein enters into the proteasome system with the help of ubiquitin complex. (G) Proteasome’s action degrades misfolded protein into small peptides and ubiquitin is regenerated. (H) Impaired pro- teasome system couldn’t degrade misfolded protein. (I, J) The misfolded protein forms aggregate. (K) Cellular Hsp104 disaggregates the compact aggregates and develop partially folded monomer with the assistance of Hsp70. (L) Partially folded protein is converted into native protein by the action of Hsp60 chaperones. (M) Hsp104 and Hsp70 chaperones can directly convert compact aggregate into native mono- meric protein. (N) Aggregates or fibrillar amyloid may further interact each other to form plaque like structure and accumulates in the differ- ent cellular space and becomes toxic and this toxicity formation cause amyloidosis class of disorders. (O) Non-toxic matured amyloid cause Amyloidoses type disorders. Protein-misfolding diseases T. K. Chaudhuri and S. Paul 1340 FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS [...]... 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,... 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. .. 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... 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 1341 Protein-misfolding diseases T K Chaudhuri and S Paul in vitro and the response was satisfactory Hou Lin and colleagues [104] have... 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. .. 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... Alpha-synuclein and Parkinson’s disease: selective neurodegenerative effect of alphasynuclein fragment on dopaminergic neurons in vitro and in vivo Ann Neurol 47, 632–640 Kanda S, Bishop J, Eglitis MA, Yang Y & Mouradian MM (2000) Enhanced vulnerability to oxidative stress by a-synuclein mutations and C-terminal truncation Neuroscience 97, 279–284 Prusiner SB (2001) Neurodegenerative diseases and prions... Kisilevsky R, Westaway D & Fraser PE (2001) Assembly of Alzheimer’s amyloid-b fibrils and approaches for therapeutic intervention Amyloid 8, 10–19 Howlett DS et al (2002) Assembly of Alzheimer’s amyloid aggregation Curr Top Med Chem 2, 417–423 Powell K & Zeitlin PL (2002) Therapeutic approaches to repair defects in DF508 CFTR folding and cellular targeting Adv Drug Deliv Rev 54, 1395–1408 Howard M & Welch WJ... 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... 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 . ARTICLE Protein-misfolding diseases and chaperone-based therapeutic approaches Tapan K. Chaudhuri and Subhankar Paul Department of Biochemical Engineering and. in understanding the pathogenesis of polyglutamine diseases: involvement of molecular chaperones and Protein-misfolding diseases T. K. Chaudhuri and S. Paul 1346