REVIEW ARTICLE -misfolding diseases and -based therapeutic approaches Tapan K. Chaudhuri and Subhankar Paul

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

Keywords A large number of neurodegenerative diseases in humans result from pro- chaperone-based therapeutic approaches; tein misfolding and aggregation. Protein misfolding is believed to be the chemical and pharmacological chaperones; primary cause of Alzheimer’s disease, Parkinson’s disease, Huntington’s molecular chaperones; protein disease, Creutzfeldt–Jakob disease, cystic fibrosis, Gaucher’s disease and conformational diseases; protein misfolding and aggregation many other degenerative and neurodegenerative disorders. Cellular mole- cular chaperones, which are ubiquitous, stress-induced , and newly Correspondence found chemical and pharmacological chaperones have been found to be T. K. Chaudhuri, Department of Biochemical effective in preventing misfolding of different disease-causing proteins, Engineering and Biotechnology, Indian essentially reducing the severity of several neurodegenerative disorders and Institute of Technology Delhi, Hauz Khas, many other protein-misfolding diseases. In this review, we discuss the prob- New Delhi 110016, India able mechanisms of several protein-misfolding diseases in humans, as well Fax: +91 11 2658 2282 Tel: +91 11 2659 1012 as therapeutic approaches for countering them. The role of molecular, E-mail: [email protected] chemical and pharmacological chaperones in suppressing the effect of pro- tein misfolding-induced consequences in humans is explained in detail. (Received 3 January 2006, revised 10 Febru- Functional aspects of the different types of chaperones suggest their uses as ary 2006, accepted 14 February 2006) potential therapeutic agents against different types of degenerative diseases, including neurodegenerative disorders. doi:10.1111/j.1742-4658.2006.05181.x

In order to be functionally active, a protein has to ubiquitous protein, the molecular chaperones [3]. acquire a unique 3D conformation via a complicated Molecular chaperones assist other proteins to achieve folding pathway, which is described by the primary a functionally active 3D structure and thus prevent the amino acid sequence and the local cellular environment formation of a misfolded or aggregated structure, [1]. is vital for a living organism essentially enhancing folding efficiency by influencing because it adds flesh to the skeleton. A small the kinetics of the process and inhibiting events that error in the folding process results in a misfolded lead to unproductive end points (e.g. aggregation). structure, which can sometimes be lethal [2]. However, Chaperones are located at various points in the cell within the cellular environment, which is highly vis- and interact with nascent polypeptides during synthesis cous, many proteins cannot fold properly by them- and translocation to different cellular compartments. selves and require the assistance of a special kind of Chaperones are able to distinguish between the native

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

FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS 1331 Protein-misfolding diseases T. K. Chaudhuri and S. Paul and non-native states of targeted proteins, but how inactive [cystic fibrosis transmembrane regulator they discriminate between correctly and incorrectly (CFTR) protein]. In other cases, however, the mutations folded proteins and how they selectively retain and tar- are relatively minor and the resulting proteins show only get the latter for degradation is yet to be understood. a partial loss of normal activity. Despite having partial Proteins that are not able to achieve the native state, biological activity, these mutant proteins are not deliv- due either to an unwanted mutation in their amino acid ered to their correct location, either inside the cell or in sequence or simply because of an error in the folding the extracellular space. One example of disease invol- process, are recognized as misfolded and subsequently ving abnormal protein trafficking is a1-antitrypsin defi- targeted to a degradation pathway. This is referred to ciency [9]. In almost all cases of protein misfolding- as a protein ‘quality control’ (QC) system and is com- mediated disorders, mutation in the gene (encoding the posed of two components: molecular chaperones and disease-causing protein) is very common. However, the the ubiquitin proteasome system (UPS) [4]. The QC more frequent amyloid-related neurodegenerative dis- system plays a critical role in cell function and survival. eases are characterized by the appearance of a toxic A special class of chaperone, for example, calnexin, function caused by the misfolded proteins [10]. forms part of the ‘quality control monitors’ that recog- One or more of a chaperone’s activities result in the nize and target abnormally folded proteins for rapid prevention ⁄ suppression of a few devastating neurode- degradation [5]. One class of QC chaperone associated generative diseases. Reduction in the intracellular level with the endoplasmic reticulum (ER), e.g. calnexin and of chaperones results in an increase in abnormally calreticulin, BiP and ERp 57 [6], is able to recognize folded proteins inside the cell [5]. Therefore, toxicity in misfolded proteins and help their retention in the ER, different neurodegenerative disorders may result from allowing only correctly folded proteins to reach the an imbalance between normal chaperone capacity and cytosol [5]. One very strong and crucial aspect of QC in the production of misfolded protein species. Increased the cell is the ubiquitin proteasome pathway (UPP). chaperone expression can suppress the neurotoxicity Studies suggest that disturbance in or impairment of caused by protein misfolding, suggesting that chaper- the UPP, which may be induced by the accumulation ones could be used as possible therapeutic agents [11]. of misfolded proteins in the ER or loss of function of Natural, chemical or pharmacological chaperones have the enzymes involved in the ubiquitin conjugation and been shown to be promising agents for the control of deconjugation pathway, leads to altered UPS function, many protein conformational disorders (PCD). These which positively affects the accumulation of protein diseases include CF, AD, PD and HD, as well as sev- aggregates in the cell [4]. The formation of oligomers eral forms of prion diseases. Here, we discuss the and aggregates occurs in the cell when a critical concen- causes of protein misfolding, aggregation and amyloid tration of misfolded protein is reached. Aggregated formation in the cell, and the use of different proteins inside the cell often lead to the formation of chaperones as therapeutic agents against various an amyloid-like structure, which eventually causes dif- protein-misfolding disorders. ferent types of degenerative disorders and ultimately cell death [4]. Protein misfolding and aggregation In almost all protein-misfolding disorders, an error in cause several diseases folding occurs because of either an undesirable muta- tion in the polypeptide or, in a few cases, some less- Protein misfolding and its pathogenic consequences known reason. The harmful effect of the misfolded have become an important issue over the last two dec- protein may be due to: (a) loss of function, as observed ades. According to the prion researcher Susan Lind- in cystic fibrosis (CF) and a1-antitrypsin deficiency; or quist, ‘protein misfolding could be involved in up to (b) deleterious ‘gain of function’ as seen in many neuro- half of all human diseases’ [12]. Protein misfolding is degenerative diseases such as Alzheimer’s disease (AD), also responsible for many p53-mediated cancers, which Parkinson’s disease (PD) and Huntington’s disease are also the result of incorrect protein folding. Many (HD), in which protein misfolding results in the forma- cancers and other protein-misfolding disorders are tion of harmful amyloid [7]. Protein aggregates are caused by mutations in proteins (Table 1) that are key sometimes converted to a fibrillar structure containing a regulators of growth and differentiation. Structural large number of intermolecular hydrogen bonds which changes in a few proteins subsequently lead to aggre- is highly insoluble. These are commonly called amyloids gated masses, which occasionally result in neuro- and their accumulation occasionally results in a plaque- toxicity and cell death. Hooper [13] reported that like structure [8]. In some cases, the mutations are so aggregated ⁄ misfolded proteins become neurotoxic (e.g. severe that they render the gene product biologically prion protein in mad cow disease; MCD) because of

1332 FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS T. K. Chaudhuri and S. Paul Protein-misfolding diseases

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

Disease Proteins affected Mutations ⁄ mutated gene Ref.

CF CFTR DF508 [20] a-Antitrypsin deficiency a-Antitrypsin D342K [21] NDI Aquaporin-2 ⁄ V2asopressin T126M, A147T, R187C [22] R187C ⁄ D62–64, L59P, L83Q, 1 Y128S, S16L, A294P, P322H, R337X Fabry a-Galactosidase A R301Q, Q279E [23] Cancer p-53 R175, G245, R248, R249, [24] R273 and R282 PD a-synuclein A53T, A30P [16] AD Amyloid precursor protein AD 1, AD 2, AD 3, AD 4 Tau, preselinin 1 and 2, [25] a- a-macroglobulin HD Huntingtin HD [25] SCA Ataxin SCA [25]

an inhibition of proteasome function. Csermely [14] modeling of protein aggregation, it has been proposed suggested a ‘chaperone overload’ hypothesis, which that the critical event in PCD is the formation of pro- explains that with aging, there is an overburden of tein oligomers that can then act as seeds to induce accumulated misfolded protein that prevents molecular protein misfolding [27–29]. In this model, misfolding chaperones from repairing phenotypically silent muta- occurs as a consequence of aggregation (polymeriza- tions which might cause disease. It has been shown tion hypothesis) [26], which follows a crystallization- that the yield of correctly folded protein obtained from like process dependent on nucleus formation. in vitro refolding is low due to the formation of ther- The alternative model suggests that the underlying modynamically stable folding intermediates. These protein is stable in both the folded and misfolded conformations are called ‘dead-end’ conformations and forms in solution (conformational hypothesis) [30–32]. are ‘off-pathway’ intermediates, they generally lead to This hypothesis proposes that spontaneous or induced the formation of insoluble aggregates [15] that may conformational changes result in formation of the mis- eventually causes different degenerative diseases. Clas- folded protein, which may or may not form an aggre- sic examples of these degenerative diseases are CF, gate. But in this hypothesis the critical question is which is caused by the deletion of a single residue what factors are responsible for changes in conforma- phenylalanine in the CFTR protein, and sickle cell tion without the induction of aggregates. Studies have anemia, which originated due to a mutation in hemo- described several factors that play a crucial role, such globin. as mutation in the gene, which destabilizes the correct A common feature of almost all protein conforma- structure. For example, mutation is common in all tional diseases is the formation of an aggregate caused neurodegenerative disorders, which reduces the folding by destabilization of the a-helical structure and the efficiency by changing the proper folding energetic. simultaneous formation of a b-sheet [16]. These b- Induced protein misfolding has been described as being sheets are formed between alternating peptide strands. responsible for all familial diseases. In addition to Linkages between these strands result from hydrogen mutation, other environmental stresses such as oxida- bonding between their aligned pleated structures. Such tive stress, alkalosis, acidosis, pH shift and osmotic b-linkages [17] with a pleated strand from one mole- shock are able to change the structure of a protein cule being inserted into a pleated sheet of the next lead without involving aggregates. to hydrogen-bond formation between molecules [18]. In a third hypothesis, the native protein conforma- The prerequisites for b-linkage formation are the pres- tion is changed to an amyloidogenic intermediate, ence of a donor peptide sequence that can adopt a which is not stable in the cellular environment. This pleated structure and a b sheet that can act as an intermediate has many exposed hydrophobic regions acceptor for the extra strand [19]. and therefore develops small oligomers, mainly com- It is not clear whether misfolding triggers protein posed of b sheets, via intermolecular interactions. These aggregation or protein oligomerization induces con- small oligomers form an ordered fibril-like structure formational changes [26]. Based on the kinetic called amyloid via an intermolecular interaction [33,34].

FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS 1333 Protein-misfolding diseases T. K. Chaudhuri and S. Paul

Protein aggregation is an inevitable consequence of Toxic amyloid formation causes many a cellular existence and these aggregates are oligomeric human neurodegenerative disorders complexes of non-native conformers that arise from intermolecular interactions among structured and kin- Neurodegenerative disorders that are chronic and pro- etically trapped intermediates in the protein folding or gressive are characterized by the selective and symmet- assembly pathway [35,36]. Protein aggregation is facili- rical loss of neurons in motor, sensory or cognitive tated by partial unfolding during thermal and oxida- systems. The most common feature of all the neuro- tive stress and by alterations in the primary structure degenerative disorders is the occurrence of brain caused by mutation, RNA modification or transla- lesions, formed by the intra- or extracellular accumula- tional misincorporation [36,37]. Protein aggregates can tion of misfolded, aggregated or ubiquitinated proteins be either structured (e.g. amyloid) or amorphous. In [4]. Proteins associated with some neurodegenerative either case, they are insoluble and metabolically stable diseases like AD, PD and HD, are tau ⁄ b-amyloid in the physiological environment [38]. For various dis- (Ab), a-synuclein and huntingtin, respectively [8]. For eases associated with protein misfolding, one or more AD, PD and CJD a few cases are familial or inherited proteins are converted from the native structure to an but the remainder are sporadic in nature. aggregated mass, which is commonly called an ‘amy- AD is a progressive degenerative disease of the brain loid’. The net accumulation of toxic protein aggregates in the elderly which clouds memory and causes in the cell depends on the stability, compactness and impaired behavior [45]. The neuropathological features hydrophobic exposure of the aggregates, as well as on of this devastating disease are the extracellular depos- the rate of protein synthesis in the cell [39]. The accu- ition of Ab and neurofibrilary tangles (NFT) in the mulation of toxic aggregates in the cell depends on brain. A central process of AD is the cleavage of a 42 chaperone expression and protease networks [39]. amino acid b-amyloid peptide from an otherwise nor- Environmental stress may induce the synthesis of mal membrane precursor protein [46,47]. The main pro- higher levels of chaperones and proteases in the cell, tein is a membrane protein called amyloid precursor which can better remove toxic aggregates [39]. Fibrillar protein, which after being cleaved by b-secretase produ- amyloids are commonly extracellular, but intracellular ces a b-amyloid precursor peptide fragment, this is fibrillar deposits are also seen in patients, e.g. intracel- further cleaved by another protease b-secretase to pro- lular bundles of neurofibrillary tangles in AD [40–43]. duce Ab-42 instead of Ab-40, which is amyloidogenic. Although the initial process might be different in dif- It is thought that cellular degradation of Ab-42 is the ferent diseases, a common trend is that during the for- normal fate of this peptide fragment when produced in mation of aggregates, a-helical domains disappear, small amounts under normal conditions, however, in leading to an increase of b-sheet-dominated secondary some lesser known conditions it forms extracellular structure (Fig. 1) [44]. Recently, many other physiolo- aggregates and subsequently generates amyloid plaques. gical disorders have been recognized as being caused Studies have reported that impairment of the UPS may by the formation of protein aggregation, which subse- be involved in this disorder [16]. An increase in neuro- quently forms a plaque-like structure containing a toxicity has been generated by dimer and oligomer for- large number of amyloid fibrils, these are polymerized mation (Fig. 2) of the Ab fragment [48]. to cross b-sheet structures with the b-strands arranged According to many scientists, AD should be first perpendicular to the long axis of the fiber. defined by the presence of NFTs caused by the protein

α-helix α-helix α-helix

β-sheet β-sheet 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.

1334 FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS T. K. Chaudhuri and S. Paul Protein-misfolding diseases

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 pro- feldt–Jakob disease (vCJD) [62] is thought to undergo tein tau, which are found to be hyperphosphorylated a conformational change in which a helices of the wild- in the neuronal cells of AD patients. Although, tau type protein PrPC are converted into b-sheet-dominant polymer formation is a hallmark of other degenerative PrPSc, resulting in misfolding and aggregation [63,64]. disorders, such as corticobasal degeneration, progres- CJD is inherited as an autosomal dominant disorder sive supranuclear palsy and pick disease [49], all differ and the most common human prion disease, the spor- from AD in that they lack Ab plaque deposition [50]. adic form, accounts for  85% of cases;  10–15% of In contrast to AD, it is believed that in PD, protein cases are familial. Sporadic CJD results from the accumulates in the intracellular space [51]. PD is the endogenous generation of prions. In general, familial second most common, late-onset neurodegenerative CJD has an earlier age-of-onset and a longer clinical disorder, and is characterized by muscular rigidity, course than sporadic CJD. Fatal familial insomnia is postural instability and resting tremor. It is a slow pro- the strangest phenotype of familial prion diseases. The gressive disorder and the pathology of PD involves the symptoms are dominated by progressive insomnia, degeneration of dopaminergic neurons in the substan- autonomic dysfunction and dementia. In the case of tia nigra and the deposition of intracytoplasmic inclu- infectious prion disease, the infectious scrapie protein sion bodies called Lewy bodies in brain cells. The (PrPSc) drives the conversion of cellular PrPC into exact mechanism by which these cells are lost is not disease-causing PrPSc (Fig. 3) [63]. The normal prion known. Heritable forms of PD are caused by gene protein is protease sensitive, soluble, and has a high mutations. To date, three encoding a-synuclein, a-helix content, but its normal function is unknown. parkin and ubiquitin C-terminal hydrolase L1 protein The disease-causing prion protein (the transmissible have been shown to be associated with familial forms isoform) is protease resistant and insoluble, forms of PD [52]. All three proteins are present in Lewy bod- amyloid fibrils, and has a high b-sheet content. Studies ies in sporadic PD [53] and in dementia with Lewy have reported that prion protein PrPSc has a neuro- bodies [54]. Two missense mutations in the gene enco- protective function and the defective prion can induce ding a-synuclein are linked to dominantly inherited normal as well as huntingtin protein to change confor- PD, thereby directly implicating a-synuclein in the mation, which later form aggregates [63,65,66]. pathogenesis of the disease. Recent studies suggest that In some human disorders, protein misfolding takes the intracellular accumulation of a-synuclein [55] leads place due to repetition of glutamine in the polypeptide to mitochondrial dysfunction [56], oxidative stress chain, which is called polyglutamine disease (PGD). [57,58] and caspase degradation [59] accentuated by This disorder is progressive, inherited, either auto- mutations associated with familial parkinsonism somal dominant ⁄ X-linked and appears in mid-life lead- [60,61]. ing to severe neuronal dysfunction and neuronal cell The prion protein, which is thought to be respon- death [67]. In all of these diseases, the CAG trinucleo- sible for causing a disease in cattle, called bovine tides, which code for phenylalanine in the coding spongiform encephalopathy (BSE, or ‘mad cow dis- regions of genes, are thought to be translated into ease’), and a disease in humans, called variant Creutz- polyglutamine (polyQ) tracts. As a result, the protein

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

Normal cellular Newly converted All the normal cellular prion protein are prions again infect functional prion infected by Scrapie other normal molecules converted prion molecule cellular prions into transmissible form

PrPSc PrPSc PrPSc PrPSc PrPSc PrPSc PrPSc PrPSc PrPC PrPC PrPSc PrPC PrPC PrPC PrPC

(i) (ii) (iii)

Fig. 3. Propagation of PrPSc takes place through the interaction of PrPSc with normal cellular protein PrPC. Binding between PrPSc and PrPC induces conformational change in PrPC protein that results in the formation of PrPSc, which form aggregates through intermolecular 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.

Protein Normal No. No. repeats in Disorder responsible of repeats mutant protein Ref.

Huntington Huntingtin 11–34 40–120 [45,75–78] Spinal and bulbar Androgen receptor 11–33 40–62 [79] muscular atrophy Spinocerebellar ataxia Type 1 Ataxin 1 25–36 41–81 [80] Type 2 Ataxin 2 15–24 35–59 [81] Type 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- Atrophin 1 7–25 49–85 [86] Luysian atrophy product, now containing an usually long string of glu- analyzed with the truncated ataxin-3 with an expanded tamine residues, appears to misfold and form large polyglutamine stretch, in which polyglutamine-contain- detergent-insoluble aggregates within the nucleus or ing aggregates and cell death were invariably observed cytoplasm, thereby leading to the eventual demise of [68–74]. the effected neuron [5]. To date eight different inher- ited neurodegenerative diseases (Table 2) have been Protein misfolding and loss of function found to be due to expansion of glutamine repeats in leads to several lethal diseases the affected proteins. HD is the most frequent of them. CF is characterized by thick mucous secretions in the Machado–Joseph disease ⁄ spinocerebellar ataxia-3 lung and intestines [8]. Amino acid sequence analysis (MJD ⁄ SCA-3) is another inherited neurodegenerative of CFTR protein has shown that the protein resides disorder caused by expansion of the polyglutamine within membranes, contains 12 potential transmem- stretch in the MJD gene-encoded protein ataxin-3. The brane domains, two nucleotide-binding domains, and truncated form of mutated ataxin-3 causes aggregation a highly charged hydrophilic region, which has been and cell death in vitro and in vivo. In vitro cellular shown to act as a regulatory domain [5]. Although models and transgenic animals have been created and many mutations in the CFTR sequence have been

1336 FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS T. K. Chaudhuri and S. Paul Protein-misfolding diseases identified, one in particular has been noted in over 705 swapping an incorrect amino acid at one point in its patients examined, in this mutation deletion of three polypeptide chain. In these mutants, the normal func- nucleotides coding for a phenylalanine residue at posi- tion of p53 is lost and the protein is unable to prevent tion 508 (DF508 CFTR) took place within a polypep- multiplication in the damaged cell [92–94]. tide of 1480 amino acids [87]. The DF508 allele of Sickle cell anemia is a genetic disorder in which the CFTR has been confirmed as a trafficking mutation amino acid valine at the sixth position of the b-globin that blocks maturation of the protein in the ER and chain is replaced by glutamine. Galkin and Vekilov targets it for premature proteolysis [88]. The clinical [95] have reported that this mutation promotes inter- importance of this mutation becomes evident when molecular bonding among adjacent hemoglobin mole- considering that it accounts for 70% of patients diag- cules and results in stable long polymer fiber nosed with CF [89]. formation. Mutant hemoglobin S (HbS) also leads to a The most common and severe form of a1-antitrypsin stable fiber-like structure while HbS is in deoxy state. deficiency is caused by the Z mutation, a single base This polymerization changes the shape and rigidity of substitution (Gul342-Lys) in the a1-antitrypsin gene. red blood cells and triggers abnormality. Lot of b-plea- Misfolding of proteins during synthesis can initiate an ted sheet accumulates as ‘amyloid plaques’. ordered polymerization, which leads to aggregation of Nephrogenic diabetes insipidus (NDI) is a disorder the protein within the cell. This slows the rate of pro- known to be caused by misfolding of one hormonal tein folding in the cell, allowing the accumulation of protein, antidiuretic hormone, also known as vasopres- an intermediate, which then polymerizes [90], impeding sin. NDI is characterized by an inability of the kidneys its release and leading to plasma deficiency. The a1- to remove water from the urinea and by resistance of antitrypsin is a serpin – an inhibitor of proteolytic the kidneys to the action of arginine vasopressin [96]. enzymes with serine at the active site, which, on bind- Wildin et al. [97] reported that a mutation in the ing to its target proteinase(s), undergoes a conforma- AVPR2 gene, which encodes arginine vasopressin, is tional change. It is known that serpin polymerization most common in NDI. More than 70 different muta- involves the interaction of one serpin molecule with tions have been identified; the majority are missense the b-sheet of another molecule of the same type; and nonsense mutations. Furthermore, 18 frameshift extensive knowledge of this mechanism may help in mutations due to nucleotide deletions or insertions (up the development of b-strand blockers to prevent self- to 35 bp) and four large deletions have been reported. association of these proteins [91]. Retinitis pigmentosa (RP) is the most common cause The tumor suppressor protein p53, which is a of inherited blindness with over 25 genetic loci identi- sequence-specific transcription factor whose function is fied, it is characterized by night-blindness and loss of to maintain genome integrity, presents a classic exam- peripheral vision, followed by loss of central vision. ple of a protein misfolding-mediated disorder. Inacti- Mutations in the gene encoding rhodopsin have been vation of p53 by mutation is a key molecular event, identified [98] and more than 100 mutations have now and is detected in > 50% of all human cancers [24]. been described that account for  15% of all inherited The p53 tumor suppressor is one of our defenses human retinal degenerations. The failure of rhodopsin against uncontrolled cell growth which leads to tumor to translocate to the outer segment per se does not proliferation. Under normal conditions there is a low appear to be enough to cause RP; rather, it would level of p53 tumor suppressor protein in the cell, how- appear that misfolded rhodopsin acquires a ‘gain of ever, when DNA damage is sensed, p53 levels rise and function’ that leads to cell death. The nature of this initiate protective measures. p53 protein binds to many gain of function is unclear, but may be related to sat- regulatory sites in the genome and begins production uration of normal protein processing, transport and of proteins that halt cell division until the damage is degradation. In transfected cells, rhodopsin with muta- repaired. If the damage is too severe, p53 initiates the tions in the intradiscal, transmembrane and cytoplas- process of programmed cell death, or apoptosis, which mic domains fails to translocate to the plasma directs the cell to commit suicide, permanently remov- membrane, and accumulates in the ER and Golgi. ing the damage. The human p53 suppressor gene is Hence these mutant proteins fail to translocate because mutated with high frequency in cancers [91]. Most of of misfolding and this causes the disorder [99]. these are missense mutations, affecting residues that Another protein conformational disorder is Fabry are critical for maintaining the structural fold of this disease, which is a lysosomal storage disorder, caused highly conserved DNA-binding protein, changing the by a deficiency of galactosidase A activity in lyso- information in the DNA at one position and causing somes, resulting in an accumulation of glycosphingo- the cell to produce p53 protein with an error through lipid globotriosylceramide (Gb3). The majority of

FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS 1337 Protein-misfolding diseases T. K. Chaudhuri and S. Paul

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

Precursor protein Designation Diffusion Syndrome

Immunoglobulin light chain 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 IAPP L Diabetes type 2 islets of Langerhans, Insulinoma Gelsolin AGel G Familial Fibrinogen A a AFib – Nephropathy, hyperpathy cardiac Fabry patients have missense mutations in the [107]. Different amyloidosis may be heterogeneous in a-Gal A gene (GLA), although alternative splicing nature but all have common properties in that they all mutations and small deletions have also been observed bind the dye Congo red that intercalates between their [100,101]. Such mutant enzymes appear to be misfold- b strands [108]. ed, recognized by the ER’s protein quality control and Amyloidosis is classified according to clinical symp- degraded before sorting into lysosomes. Fabry disease toms and biochemical type of amyloid protein is specific for those missense mutations that cause mis- involved. Many amyloidoses are multisystemic, gener- folding of a-Gal A. alized or diffuse but a few are also localized. They GD is an inherited lipid-storage disorder. It is mainly affect kidneys, heart, gastrointestinal tract, caused by mutation in the gene encoding acid b-glu- liver, skin, peripheral nerve and eyes. It is a slowly cosidase (GlcCerase) [102], an enzyme that participates progressive disease that can lead to morbidity and in the degradation of glycosphingolipids [103]. Symp- death. Amyloid deposits are extracellular and not toms may have neurological discrepancy or may be metabolized or cleared by the body, thus the deposits non-neurological [104]. Deficiency of this enzyme cau- eventually impair the function of the organ where they ses accumulation of glucocerebrosides in macrophage accumulate. lysosome. In very few cases, GD is caused by mutation Table 4 shows the causes of different disorders by in the saposin C domain of the gene prosaposin, which specific disease-causing proteins and Fig. 4 shows the controls the optimum activity of GlcCerase by enco- possible fate of misfolded proteins through the path- ding a protein saposin C [102]. 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- Amyloidoses ative disorders. In all the above cases either misfolded proteins form fibrillar aggregates which become toxic and lead to cell Molecular chaperones can prevent death (all neurodegenrative diseases) or, in other cate- protein misfolding and aggregation gory of disease, misfolded proteins are directed to the proteasome pathway for degradation (proteolysis), and Large multidomain proteins have been found to protein deficiency causes the disease. In a third case, form a misfolded structure and aggregated mass during even if the fibrils themselves are not toxic, the ready in vitro refolding [109]. The cellular environment is autolinkage of proteins and polypeptides by b-strand crowded with proteins and other macromolecules, and bonding involves risks of further linkage to give insol- so the chance of a newly synthesized unfolded protein uble macrostructures [105,106], these macrostructures forming aggregates is greater in vivo than in vitro. are deposited in the tissues and cause disease (Table 3) Cellular molecular chaperones are proteins that change

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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 Scrapie (Mad Cow Disease), F 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] aB1B-Antitrypsin aB1B-Antitrypsin Trafficking ⁄ aggregation [90] a-Galactosidase Fabry Trafficking [101,102] P53 Cancer Trafficking [92] this equation by selectively recognizing and binding to inefficient disaggregation, which is generally limited to the exposed hydrophobic surfaces of a non-native small aggregates of luciferase and a-galactosidase. protein via non-covalent interactions, thus inhibiting Their findings have been supported by evidence that irreversible aggregation of those proteins in vivo [5] both chaperones collaborate in the cellular acquisition and in vitro. of thermotolerance [115]. It has been reported that the Molecular chaperones are composed of several dis- yeast non-Mendelian factor [psi+], which is analogous tinct classes of sequence-conserved proteins, most of to mammalian prions, is propagated at when there are which are stress inducible like heat shock proteins intermediate amounts of the chaperone protein Hsp104 (Hsp). Major classes of these Hsp are Hsp100 (in and overproduction or inactivation of Hsp104 caused E. coli, ClpA ⁄ B ⁄ X, HslU), (in E. coli, HtpG), loss of [psi+] [116]. These results suggest that chaper- (in E. coli, DnaK), Hsp60 (in E. coli, GroEL) ones are crucial in prion disease progression and that a and the small Hsps (in E. coli, IbpA ⁄ B). These mole- certain level of chaperone expression can rid cells of cular chaperones have important damage-control prions without affecting their viability. Control of the functions during and following stress. Under in vitro expression level of Hsp104 may provide a therapy conditions, many chaperones, such as E. coli IbpB, against prion disease. In addition, Hsp104, along with DnaK, DnaJ, GroEL, HtpG and SecB, and proteases Hsp70, has been shown to be responsible for solubiliz- such as DegP, HslU and Ion can bind chemically ing prion-like aggregates in Saccharomyces cerevisiae unfolded polypeptides and prevent aggregation [21, [116,117]. Many other positive responses have been 110–112]. They are also involved in aggregate solubili- reported on cellular chaperone-mediated disaggrega- zation. Stable aggregates are resistant to most ATPase tion in vivo. A classic experiment was performed chaperone systems when functioning individually, for by Goloubinoff et al., who proved the phenomena of example GroELS, Hsp90, ClpB, and low concentra- in vitro reactivation and disaggregation of stable aggre- tions of DnaK. Skowyra et al. [113] observed that the gates of malate dehydrogenase by ClpB together with DnaK chaperone system might reactivate some forms DnaK, DnaJ and GrpE (KJE), and further explained of protein aggregate. It has been observed that the mechanism of the whole disaggregation process Hsp100, which includes Ipb, ClpA, HslU and ClpX in (Fig. 5) [118]. E. coli, has disaggregation activity [114]. ClpA and Mogk, Tomoyasu and colleagues [110,119] showed ClpX have been shown to destabilize some native that, in E. coli, stable protein aggregates rapidly disap- protein structures, allowing them through the central pear from the insoluble fraction following chaperone cavity into the ClpP for proteolysis [114]. action during a short recovery period. Under normal Schrimer et al. have shown that Hsp70 and Hsp100 conditions, chaperones repair the conformational function in combination to reactivate many protein defects of some mutated proteins, thus reducing their aggregates [114]. They also showed that Hsp104 phenotypic effects and dampening genome cleansing cooperates with Hsp70 and Hsp40 in a slow and (elimination of damaged genes from the gene pool of a

FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS 1339 Protein-misfolding diseases T. K. Chaudhuri and S. Paul

DNA Ribosome RNA CHIP Ubiquitin conjugation Ubiquitinated Ubiquitin protein ATP (A) Misfolded Native protein E1 Porotein (B) E3

(C) E2 I u m b i p q a u i i Misfolded r t e

(D) i d (L) protein n Aggregate/Fibrillar amyloid (E) (F) (M) (K) 2 (J) P 6 r o S t e o

Partially s o

folded m e protein (I)

impaired (O) proteasome Hsp60 (N) Ubiquitin (H) Hsp104 (G) Hsp90 Misfolded protein Degraded Hsp40 Gain of protein toxicity Hsp70 E1 Amyloidoses (Familial amyloid E2 neuropathy) Cause several neurodegenerative E3 diseases and lead cell demise like Loss of protein function cause Alzheimer disease, Parkinson disease several diseases like cystic fibrosis

Fig. 4. The fate of cellular misfolded protein is shown. (A) Nascent polypeptide chain is converted into folded protein. (B) Polypetide chain reaches misfolded structure. (C) Native protein molecule is converted into misfolded structure due to specific mutation or cellular stress. (D) In the first step Hsp 40 ⁄ 70 ⁄ 90 facilitate to direct them to the proteasomal pathway and the second step is ubiquitination of misfolded pro- tein assisted by E1 (ubiquitin activating enzyme), E2 (ubiquitin conjugating enzyme) & E3 (). (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. population, which normally takes place via natural aggregation of polyglutamine-containing proteins. It selection). Sherman & Goldberg [120] first reported has been reported that Hsp70 and Hsp40 chaperone that Hsp70 and Hsp40 molecular chaperones prevent family members act together. The chaperone complex

1340 FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS T. K. Chaudhuri and S. Paul Protein-misfolding diseases

Step1Step2 Step3

ATP ATP ATP

ClpB DnaK/DnaJ/ GroEL/ GrpE GroES

Matured Loose aggregate Partially Native aggregate folded chain

Fig. 5. Protein disaggregation process in E.coli by chaperones [121]. Step1: ClpB chaperone preproceses the aggregate and produces a loose structure having more hydrophobic surfaces (HS) exposed to the solvent. Step2: Dnak binds to those newly exposed HS along with co-chaperones DnaJ and GrpE, disaggregates the loose aggregates and refolds them partially. Step3: GroEL and GroES assist final refolding from monomeric partially folded form to native state. system is ubiquitous from bacteria to mammals. The the protein folding environment inside the cell. In Hsp40 family regulates the chaperone activity of the order to test this idea, DF508 CFTR protein was tested Hsp family by upregulating their ATPase activity for its ability to fold at 37 C and < 30 C. It was [121]. A classic in vivo experiment was performed on observed that at the higher temperature part of the Drosophila melanogaster [8] in which human Hsp70 newly synthesized protein was misfolded and degraded, was overexpressed. Complete suppression of the polyQ whereas at the lower temperature a portion formed the neurodegenerative disorder, which causes an external native structure. This helped in the discovering of eye defect, took place. Overexpression of Hsp70 sup- some chemical compounds that stabilize proteins pressed neurodegeneration and increased the lifespan against thermal denaturation and might help to correct of the fruit fly by twofold. The same experiment was folding defects. These compounds were collectively later performed in mammals. In a mouse model, called chemical chaperones [123]. Recent studies sug- Hsp70 was overexpressed and found to be effective for gest that chemical chaperones are effective in inhibiting type 1 spinocerebellar ataxin (SCA1) disease [103]. It the formation of misfolded structure and subsequent has also been suggested that the chaperones Hsp70 amyloid formation [124]. They are low molecular mass and Hsp40 may play a role in some human neurode- compounds known to stabilize protein conformation gerarative disorders like PD and AD [122]. Hsp70 against thermal and chemical denaturation [9]. Chem- molecular chaperone was reported to mitigate dopam- ical chaperones have been shown to reverse the inergic neuron loss induced by a-synuclein protein in intracellular retention of several different misfolded in vivo studies in a Drosophila model. Overexpression proteins such as CFTR [21,22], a-antitrypsin [125], of Hsp70 also suppresses a-synuclein neurotoxicity. aquaporin-2 [22], vasopressin V2 receptor [95], a-galac- Many wild-type proteins fold inefficiently in the ER, tosidase A [99], p53 and P-glycoprotein [126]. even with the help of Hsp40, Hsp70 and Hsp90 which Recently, some of these compounds have been shown facilitate the refolding or retrotranslocation of misfold- in cell culture models to correct folding and trafficking ed proteins back into the cytoplasm, where they are defects in DF508 CFTR in CF [20], the prion protein degraded by the proteasome [7]. PrP [127], and temperature-sensitive mutants of the tumor suppressor protein p53, the viral oncogene pro- tein pp60 and the ubiquitin-activating enzyme E1 The role of chemical and pharmaco- [128]. Glycerol is an example of a chemical chaperone logical chaperones in rescuing protein and enhances protein stability by decreasing the sol- conformational defects vent-accessible surface area of the protein [129,130]. It thus increases the rate of in vitro protein folding [131] Chemical chaperones and enhances the rate of oligomeric assembly [132]. Another strategy to prevent misfolding or correct a Although chemical chaperones have not been tested in mutant protein’s lethal conformation is to influence 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 inverso peptidomimetics, block both Ab seeding and and colleagues [104] have shown that N-octyl-h-valien- growth. Unfortunately, the pharmacological profile of amine (NOV) can be used as a carbohydrate mimic these compounds is far from ideal. Nonpeptidic, aro- that would act as an inhibitor of b-glucosidase and matic-rich Ab aggregation inhibitors have also been could be used in the suppression of GD, in which a reported by many pharmaceutical and biotechnology defect in b-glucosidase (b-Glu) leads to a misfolded companies. But their lack of specificity is a problem. conformation. Sawkar and colleagues [133] reported In CF, the DF508 mutation can be rescued by treat- that a subset of N-alkylated deoxynorjirimycin and ing cells with chemical chaperones like glycerol, simpler six-member ring N-heterocycles increase the dimethyl sulfoxide (DMSO), trimethylamine-N-oxide activity of b-glucosidase within human cells, this is the (TMAO) and deuterated water [140,141]. Mutation in best compound discovered to date. This may be a con- DF508 position leads to the appearance of small num- venient alternative to intravenous enzyme-replacement ber of chloride channels in the plasma membrane. therapy. However, relevant transgenic animal models These can be recovered by incubating cells at a higher to test this approach are not yet available. temperature. Glycerol, DMSO and TMAO mimic the The failure to secrete a1-AT into the circulation same act and thus rescue the mutation. The therapeu- allows neutrophil elastase to degrade lung parenchyma, tic effect of chemical chaperones has been studied on causing emphysema. Burrows and colleagues [125] have MJD, in which organic solvent DMSO, cellular osmo- shown that osmolytes such as 4-phenylbutyric acid and lytes glycerol and TMAO were used. Using an in vitro glycerol increase the secretion efficiency of a1-AT in cell culture system, the same effect has been observed cell lines and transgenic animals. Although these com- when chemical chaperones were used. These reagents pounds do not bind to a1-AT specifically, they increase include the organic solvent DMSO and cellular osmo- the secretion efficiency of variants of a1-AT that are lytes glycerol and TMAO, these are called chemical normally retained in the ER. Glycerol and 4-phenylbu- chaperones because of their influence on protein con- tyric acid accomplish a1-AT hypersecretion ability formation [127]. Application of these three chemical without influencing the secretion efficiency of other chaperones has been shown to be useful in suppressing proteins or decreasing proteasome activity. the polyglutamine diseases and preventing cell death. Chemical chaperones have also been tried as thera- Because DMSO was found to be an antioxidant [142], peutic agents in prion disease. A variety of compounds two others, N-acetyl-l-cysteine (NAC) and glutathione including anthracyclines, porphyrins and diazo dyes monoethyl ester (GSH-MEE) [143], were used to check block prion replication when administered with PrPSc, whether the antioxidant property of DMSO has any the aggregated infectious form of the prion protein, in effect on the reduction in aggregate formation. How- animal models [134,135]. Unfortunately, this is not a ever, NAC and GSH-MEE did not prevent protein clinically relevant model for therapeutic intervention, aggregation, and so DMSO acts on the polypeptide because subclinical disease exists for months in mice chain in a similar way to glycerol and TMAO [124]. and years in humans. However, quinacrine has been clinically approved, and clinical trials are being carried Pharmacological chaperones out to test the usefulness of this molecule in patients with CJD. Recently, Vogtherr et al. [136] used NMR The use of chemical chaperones in reversing mutant spectroscopy and chemical shift titration to show that protein folding are well documented, but their use quinacrine binds specifically to PrPC the normally requires high concentrations, at least micromolar level. folded cellular isoform of the prion protein. On bind- Although, small molecular mass molecules, including ing with PrPC, quinacrine stabilizes the conformation osmolytes like glycerol, have been used to reverse of the protein and hence the conversion of PrPC to misfolding in several disease-causing proteins, their PrPSc can be prevented, this in turn suppresses the pro- lack of specificity means that they are a far from prac- gression of CJD. tical therapeutic approach in humans. In AD, no effective therapy using a chaperone sys- Because stability of the protein molecule can be tem has been found. Inhibition of Ab-fibril formation achieved by binding to substrates molecules, Loo & might be a reasonable therapeutic strategy because Clarke examined the idea of using different substrates familial mutations that lead to an increase in Ab con- of P-glycoprotein in cells expressing mutant P-glyco- centration or to its aggregation increase neuropatholo- protein, which causes retention in the ER and subse- gy [137–139]. Peptidomimetics, based on the peptide quent degradation of the protein. Their work proved LVFFA from Ab, modified at the N- or C-terminus, effective from a therapeutic point of view because and the all-d (right-handed) version and several retro- synthesis of this mutant protein in the presence of sub-

1342 FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS T. K. Chaudhuri and S. Paul Protein-misfolding diseases

Table 5. Mutational effect in human proteins corrected by molecular, chemical and pharmacological chaperones [5]. HD, Huntington’s dis- ease; MJD, Machado-Joseph disease; AD, Alzheimer’s disease; PD, Parkinson’s disease; CF, cystic fibrosis; NDI, nephrogenic diabetes insipidus; CJD, Creuzfeld-Jacob diease; GD, Gaucher’s disease.

Type of chaperone Chaperone name Disease

Molecular Hsp70, Hsp104, Hsp40 HD, MJD, AD, PD Chemical DMSO, glycerol, TMAO AD, PD, CF, NDI, atherosclerosis, CJD, HD Pharmacological SR121463A, VPA985, GD, NDI, Transthyretin 1-deoxy-galactonojirimycin congenital hypogonadotropic CP31398, CP257042, hypogonadism amyloidoses, capsaicin, cycloporin, Vinblatin, verapamil strate molecules resulted in mature and active P-glyco- mentally and reported to reverse the mutational effect protein, and based on the same type of approach, of the protein conformation and suppress the pheno- other similar molecules were found which are collec- type are shown in Tables 5 and Table 6. tively named as pharmacological chaperones. Their use is needed only at the micro molar level in the cell to Conclusions prevent misfolding of mutant proteins. Pharmacological chaperones have proved very From the discussion on the mechanisms of different effective in rescuing a few receptor proteins from pro- protein misfolding disorders, it is clear that a nascent teasomal degradation. Pharmacological ligands act by polypeptide chain can become misfolded due to a spe- binding to specific conformations of receptor proteins cific gene mutation, which takes place in almost all and stabilizing them. Selective VB2B receptor antago- familial neurodegenerative diseases, or a matured nists, which are retained in the ER and are responsible native protein can also achieve a misfolded conforma- for NDI, were assessed to reveal whether they facilitate tion inside the cell, an example is the cause of prion the folding of mutant VB2B receptor protein. Biosyn- disease. The fates of these misfolded proteins in var- thesis of mutant VB2B receptors was monitored in the ious disorders are different, in one class of diseases presence of the selective V2 receptor nonpeptide antag- misfolded proteins interact further with each other onist SR121463A. Morello et al. [144] proposed a through intermolecular interaction and form structured model for the mode of action of pharmacological aggregates thus gaining toxicity. Neurodegenerative chaperones. Small nonpeptide VB2B receptor antago- disorders are good examples of this specific pathway. nists permeate the cell and bind to unstable folding It might be that the proteasome pathway is not effi- intermediates of the mutant receptors; this would sta- cient enough to degrade these misfolded proteins prior bilize a conformation of the receptor that allows its to aggregation because of impairment of the UPS. In release from the ER quality control system. The stabil- another case, misfolded proteins are directed to the ized receptor proteins would then be targeted to the UPP with the help of many other chaperones in addi- cell surface where they bind AVP and promote signal tion to , and are consequently degraded by transduction upon dissociation from the antagonist. the action of proteasome. Hence these proteins cannot

These antagonists are VB2B specific and have the same be secreted from the ER, but are degraded and function as a chaperone, hence they are referred to as their disappearance from the specific site inside the pharmacological chaperones. cell where they function causes disease. Good Loo&Clarkefunctionallycharacterized artificialmuta- examples of this are CF and a-antitrypsin deficiency tions of the multidrug resistance 1 gene (ABCB1), which disorders. codes for P-glycoprotein 1, an energy-dependent trans- Whatever the reason for a protein not achieving its porter at the plasma membrane that interacts with a functional form, it is the conformational defect that wide variety of cytotoxic agents [126]. Morello et al. leads to disease. Therapy should therefore aim to inhi- [144] proved that selective V2 receptor antagonists bit and ⁄ or reverse conformational changes in the (SR121463A, VPA985) can permeate the cell surface protein molecules responsible. In most PCDs the mis- and facilitate the folding of mutant V2 receptors which folded protein is rich in b sheet, and therapy are retained in the ER and cause NDI. should involve designing a peptide to prevent and Different molecular, chemical and pharmacological reverse b-sheet formation. It might be possible to cor- chaperones, which have been already studied experi- rect these diseases by persuading the misfolded proteins

FEBS Journal 273 (2006) 1331–1349 ª 2006 The Authors Journal compilation ª 2006 FEBS 1343 Protein-misfolding diseases T. K. Chaudhuri and S. Paul

Table 6. Mutations rescued by chemical and pharmacological chaperones. CF, cystic fibrosis; NDI, nephrogenic diabetes insipidus.

Mutations Disease Protein recovered Agents used Ref.

CF CFTR DF508 Glycerol, DMSO, TMAO [20] a-Antitrypsin deficiency a-Antitrypsin D342K Glycerol [21] NDI Aquaporin-2 T126M, Glycerol, DMSO, TMAO [22] A147t, r187c NDI Aquaporin-2 ⁄ T126M, A147T, SR121463A, VPA985 [146] V2 vasopressin R187C,R187C ⁄ D62–64,l59p, l83q, Y128S, S16L, A294P, P322H, R337X Fabry a-Galactosidase A R301Q, Q279E 1-Deoxy galactonojirimycin [23] Cancer p53 L173A, L175S, CP31398, CP257042 [24] V249S, M273H to fold correctly. There is no doubt that chaperones formation in the underlying protein. In future, an play a critical role in controlling protein misfolding and understanding of the causes of protein aggregation and thus reducing the threat of associated neurodegenera- the genetic and environmental susceptibility of a speci- tive diseases. Many questions remain regarding their fic individual may provide a better opportunity for mode of action in suppressing and correcting the mis- effective therapeutic intervention. folding of disease-causing proteins. In MJD and SCA1 In a few cases, drugs may boost chaperone activa- disease Hsp70 and Hsp40 have been shown to be highly tion ⁄ upregulation, which would then prevent protein effective in suppressing the degeneration of polyQ- misfolding. In 2001, Wanker and colleagues (Annual mediated disorders and increasing the lifespan of fruit Conference of the Genetics Society of Australia, July flies and mice. However, for other major neurodegener- 2004) at the Max Planck Institute found that geldana- ative diseases like AD and PD the result of the same mycin, an antibiotic, activated a heat shock response experiment is not known. It has been shown that a and inhibited huntingtin aggregation in a cell culture combination of heat shock proteins Hsp70 and Hsp40 model of HD. This was promising but aggregates, is most effective in inhibiting huntingtin aggregation although not normal, are not a primary problem in in vitro and in a mammalian cell culture model system. HD. However, Nancy Bonini [12] has shown that this In some cases, the actual relationship between the dis- antibiotic not only prevented protein aggregation in a ease and its phenotype is not known [10]. However, fruit fly model of neurodegeneration but also stopped experimental data suggest that correction of protein the degeneration. Geldanamycin may be a treatment misfolding constitutes a viable therapeutic strategy for for HD. This unpublished result was also discussed diseases caused by protein misfolding. Most of these at the Annual Conference of the Genetics Society of genetic disorders are progressive, and treatment is Australia (July 2004). therefore difficult. However, for some diseases, a grow- It may be possible that in the near future molecular, ing number of treatment options such as drugs, antioxi- chemical and pharmacological chaperones might dants, cell transplantation, surgery, rehabilitation change the mode of treatment and open a new door in procedures and preimplantation diagnosis are available clinical research into the neurodegenerative diseases. [52]. In most cases, they have proved to be risk worthy and having little adverse effect, whereas overexpressed References molecular chaperone-induced therapy has been shown to be highly effective in fruit flies and even mammals 1 Anfinsen CB (1973) Principles that govern the folding like the mouse [103,120]. Chemical chaperones like of protein chains. Science 181, 223–230. DMSO and TMAO have been studied in vitro, and 2 Ellis RJ & Pinheiro TJ (2002) Danger – misfolding pro- showed reduced cytotoxicity and cell death, which has teins. Nature 416, 483–484. been reported to be a good therapeutic strategy [124]. 3 Kinjo AR & Takada S (2003) Competition between Chaperone treatment in humans and its benefits are yet protein folding and aggregation with molecular chaper- to be reported. In order to have chaperone treatment it ones in crowded solutions: insight from mesoscopic is worth knowing the exact mechanism of aggregate simulations. Biophys J 85, 3521–3531.

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