The role of molecular chaperones in human misfolding diseases

FEBS Letters 583 (2009) 2647–2653

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The role of molecular chaperones in human misfolding diseases Sarah A. Broadley, F. Ulrich Hartl * Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany

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Article history: Received 2 April 2009 Accepted 17 April 2009 Available online 24 April 2009 Edited by Per Hammarström Keywords: Molecular chaperone Protein misfolding Heat shock protein Neurodegeneration

a b s t r a c t Human misfolding diseases arise when proteins adopt non-native conformations that endow them with a tendency to aggregate and form intra- and/or extra-cellular deposits. Molecular chaperones, such as Hsp70 and TCP-1 Ring Complex (TRiC)/chaperonin containing TCP-1 (CCT), have been implicated as potent modulators of misfolding disease. These chaperones suppress toxicity of disease proteins and modify early events in the aggregation process in a cooperative and sequential manner reminiscent of their functions in de novo protein folding. Further understanding of the role of Hsp70, TRiC, and other chaperones in misfolding disease is likely to provide important insight into basic pathomechanistic principles that could potentially be exploited for therapeutic purposes. Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction A protein must fold into a specific three-dimensional structure in order to acquire its functionally active, native state. Although all information required for achieving the native state is encoded in the amino acid sequence, the concentrated milieu of the cellular environment presents challenges to the folding process that are overcome with the help of cellular machinery termed molecular chaperones. The cooperative action of molecular chaperones and the input of metabolic energy allow proteins to avoid aggregation and efficiently reach their native states in vivo [1,2]. However, proteins may misfold or unfold in the face of certain stresses, such as changes in the cellular environment due to ageing or temperature fluctuation, genetic mutation, or exposure to amino acid analogues. The resulting increase in the burden of misfolded or unfolded proteins is normally counterbalanced by quality control machinery, including chaperones that are activated through the cytosolic stress pathway, or heat shock response. This pathway, which is mediated by heat shock transcription factors including HSF-1, results in nearly-instantaneous induction of expression of genes encoding chaperones (or heat shock proteins) [3]. Stress-activated chaperones act as essential modulators of protein homeostasis to minimize aberrantly folded species by promoting their productive folding or degradation.

Abbreviations: Hsp, heat shock protein; polyQ, polyglutamine; TRiC, TCP-1 Ring Complex; CCT, chaperonin containing TCP-1 * Corresponding author. Fax: +49 (0)89 8578 2240. E-mail address: [email protected] (F.U. Hartl).

Unchecked protein aggregation and misfolding are now recognized as the root cause of a large and diverse collection of diseases termed ‘protein misfolding’ or ‘protein conformational’ disease [4]. These diseases, which include amyotrophic lateral sclerosis (ALS), Alzheimer’s, Parkinson’s, Huntington’s and other polyglutamine diseases, arise when certain proteins adopt non-native conformations that endow them with a tendency to aggregate and form intra- and/or extra-cellular deposits. In all these cases, protein misfolding results in ‘gain-of-function’ proteotoxicity, whereby misfolding confers newly-gained cytotoxicity onto the disease protein, e.g. by promoting inappropriate interactions that are detrimental to the cell (see discussion below). Misfolding and aggregation of the disease protein may also confer some degree of loss of function, which may additionally contribute to disease pathogenesis [5,6]. The aggregation process associated with misfolding disease results in the formation of homotypic fibrillar aggregates with amyloid-like structure, as defined by a ‘cross-b’ core with extensive bsheet structure, detergent insolubility, as well as other biochemical characteristics. Fibrillar aggregates may participate in heterotypic interactions that result in sequestration of cellular proteins in disease-specific deposits (e.g. inclusion bodies, plaques, or Lewy bodies). These deposits, and the fibrillar aggregates therein, likely represent final manifestations of a multi-step, and perhaps multipathway, aggregation process involving a wide range of stable as well as metastable intermediates [7–9]. Aggregation intermediates, or ‘pre-fibrillar’ aggregates, include monomeric and oligomeric species that are soluble in nature. Abundant evidence points towards pre-fibrillar monomers or oligomers as primary toxic agents underlying misfolding disease, while fibrillar

0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.04.029

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aggregates or their deposits may be inert or even protective [10– 15]. For instance, in a neuronal cell model of Huntington’s disease, cell death correlates with increased levels of diffuse polyglutamine (polyQ)-expanded huntingtin, the disease protein associated with Huntington’s disease; whereas inclusion body formation predicts improved survival [16]. Proteinaceous deposits are also not associated with toxicity in Parkinson’s and Alzheimer’s disease [16–18]. Furthermore, soluble oligomeric forms of polyQ-expansion proteins, Ab and a-synuclein, the proteins associated with Alzheimer’s and Parkinson’s disease, respectively, were found to cause toxicity when added exogenously to cells [15]. Importantly, microinjection of several different polyQ conformers into mammalian cells indicate that a soluble b-sheet monomer is cytotoxic [10]. However, rather than a single toxic conformer, it is perhaps more likely that a range of soluble intermediates exert toxicity, as determined by a host of factors, including subcellular location, structural, and functional properties of the disease protein [19]. Toxic oligomeric intermediates in misfolding disease may share structural features critical to the pathogenic process. The anti-oligomer antibody, A11, recognizes a sequence-independent structural element common to toxic oligomers comprised of Ab, asynuclein, prion, and polyQ proteins. A11 was found to block toxicity of all these oligomers, indicating that the recognized structural epitope common to these oligomers is likely directly involved in aberrant interactions that lead to toxicity [15]. Furthermore, AFM analyses indicate that polyQ-expanded huntingtin, asynuclein, and Ab share a propensity to form spherical or ring-like aggregates that may be on or off-pathway to fibril formation [14,20]. These shared structural features may indicate a common pathogenic mechanism underlying misfolding diseases. Numerous hypotheses have been put forward to account for neurodegeneration in misfolding disease. Prominent among these is the ‘‘amyloid pore” hypothesis that poses that ring-like oligomeric intermediates exposing hydrophobic regions cause toxicity by forming pores in cellular membranes [20,21]. Prefibrillar species of a-synuclein and other proteins including Ab and polyQ peptides have been found to permeabilize membranes under experimental conditions, lending support to this hypothesis [20,21]. A second disease mechanism emphasizes aberrant interactions of soluble intermediates with cellular proteins. Such interactions may inactivate essential cellular factors like TATA-binding protein, or alter heteroprotein complexes, as has been shown for polyQ-expansion proteins [5,13,22]. Finally, deficient or non-productive association of misfolded disease proteins with quality control machinery may cause general perturbation of cellular protein homeostasis leading to pathogenesis [23]. The strongest evidence to support this hypothesis comes from the observation that components of the quality control system are among the most potent modifiers of disease phenotypes. For the remainder of this review we will focus on two chaperone classes that have been strongly implicated as modulators of misfolding disease.

2. Chaperones as molecular defenders against misfolding disease 2.1. The Hsp70 system Members of the Hsp70 family of molecular chaperones function in co- and post-translational folding and the quality control of misfolded proteins [1,2]. More specifically, Hsp70s participate in folding and assembly of newly synthesized proteins into macromolecular complexes; aggregation prevention; dissolution and refolding of aggregated proteins; as well as protein degradation [24]. Hsp70s have an N-terminal ATP-binding domain (NBD) and a Cterminal substrate-binding domain (SBD) which are both critical

for chaperone function. Non-native substrates with exposed hydrophobic stretches within an accessible polypeptide backbone associate transiently with Hsp70 via its SBD. ATP binding to the NBD triggers opening of the SBD binding pocket, decreasing affinity for polypeptide substrates, accelerating both on and off rates. Reciprocally, substrate binding induces ATP hydrolysis, ‘closing’ the SDB and thus stabilizing the substrate-Hsp70 complex [1,2]. It is this cycle of rapid but controlled binding and release of the substrate that fosters folding and assembly with partner proteins while preventing aggregation of substrates; however, detailed mechanistic understanding of how Hsp70 accomplishes these feats is not yet available [24]. Generally, it is assumed that an unfolded protein partitions to the native state upon release from Hsp70; rebinding of Hsp70 to slow-folding intermediates shields them from intermolecular interactions, thereby ‘holding’ them in a folding-competent state and preventing aggregation [1,3,24]. Numerous hypotheses have been put forth to explain the molecular mechanism of Hsp70-induced structural conversion of substrate proteins. For example, an ‘entropic pulling’ mechanism has been proposed, whereby Hsp70 binding stabilizes peptide segments in an unfolded state, causing local unfolding, thereby facilitating disaggregation and allowing refolding upon Hsp70 release [25]. Co-factors, such as the nucleotide exchange factors (NEFs) and co-chaperones, are crucial regulatory components of the Hsp70 cycle that confer versatility and specificity to the Hsp70 chaperone machine [1,2]. The Hsp40 co-chaperone targets substrates to Hsp70 while stimulating ATP hydrolysis; NEFs like Bag-1 (BCL2associated athanogene 1) and Hsp110 reinitiate the Hsp70 cycle by facilitating ADP release and rebinding of ATP [24]. Bag-1 has the additional ability to bind to the 26S proteasome [26]. Another BAG isoform, the Bag-3 co-chaperone, links Hsp70 to the macroautophagic degradation pathway during the ageing process [27]. CHIP (carboxy terminus of HSC70-interacting protein), a co-chaperone of Hsp70 that also has E3 ubiquitin ligase activity, cooperates with Bag-1, and possibly Bag-3, in order to facilitate degradation of terminally misfolded substrate proteins [28,29]. Studies indicating that mutations in Hsp70 co-factors are lethal [30–32] or may be associated with neurodegenerative disease [33] underscore the importance of regulating the Hsp70 cycle. Hsp70 has been extensively implicated in the pathogenesis of misfolding disease [14]. Numerous studies initially found that Hsp70, other chaperones, and components of the ubiquitin–proteasome system associate with inclusion bodies/plaques characteristic of misfolding diseases, indicating a general activation of the cellular quality control machinery in an attempt to circumvent the accumulation of misfolded species [34]. Further analyses in a polyglutamine cell culture model using fluorescence imaging revealed that Hsp70 rapidly associates and dissociates with aggregates in a manner similar to interactions between Hsp70 and unfolded substrates [35]. For unclear reasons, though actively engaged in the task of refolding, the Hsp70 system is ultimately unable to refold disease proteins, causing perturbation of protein homeostasis associated with disease onset. Several hypotheses account for this apparent imbalance between the production of misfolded proteins and Hsp70 activity. The capacity of the Hsp70 system, and the cellular folding environment in general, could simply be overwhelmed by increasing amounts of misfolded disease proteins [34,36]. This would be particularly relevant in certain neuronal cell types, which appear to be unable to induce Hsp70 expression above basal levels under stress [37,38]. Progressive reduction in protein levels and/or activity of Hsp70 and other components of the quality control network may exacerbate this imbalance, permitting further accumulation of toxic misfolded proteins. Such reduction could be due to the ageing process, as transcription of Hsp70 decreases during ageing of the human brain [39]. The DNA binding activity of the HSF-1 transcription factor likewise de-

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creases with age in rat hepatocytes, causing a continuous decline in the ability to induce expression of genes encoding chaperones during the cytosolic stress response [40,41]. Consistently, stress-induced expression of Hsp70 is weakened in senescent fibroblasts [42]. If an age-related decrease in chaperone activity indeed contributes to disease, slowing the ageing process should postpone or even prevent disease onset, as has been suggested by several studies in which aggregation-mediated proteotoxicity was ameliorated by delaying the ageing process in Caenorhabditis elegans models of Huntington’s and Alzheimer’s diseases [43–45]. Alternatively, disease processes themselves might cause, or worsen, chaperone deficiency. Inclusions have been proposed to sequester Hsp70 and other proteins in a non-functional state, inhibiting their essential function in cellular processes [13,34,46]. Additionally, studies report that several cellular models of misfolding diseases do not promptly activate the cytosolic stress response upon overexpression of disease proteins [47–51]. It is unclear why cells might fail to activate this critical pathway during disease pathogenesis; however, this defect might be linked to the known entrapment and inactivation of key transcription factors in inclusions associated with several misfolding diseases [13,52–55]. Along similar lines, it was recently shown that sequestration of NF-Y, a regulator of Hsp70 transcription, in huntingtin inclusions reduces Hsp70 expression in several Huntington’s disease models [53]. Regardless of whether ageing and/or disease-related processes are responsible, levels of Hsp70 are decreased in neurons most severely affected by disease, arguing that the Hsp70 system has a key role in regulating neuronal susceptibility to degeneration [49,50,56]. Counterbalancing the accumulation of misfolded proteins by overexpressing Hsp70 and/or its co-chaperones suppresses aggregation and toxicity in models of misfolding disease [14,19,57]. For instance, increased Hsp70 levels caused reduced aggregation and toxicity of tau and Ab, respectively, two components associated with Alzheimer’s disease [58–60]. Similarly, overexpression of Hsp70 reduces toxicity and accumulation of a-synuclein in high molecular weight and detergent-insoluble deposits [61]. Increased expression of Hsp70 likewise reduced apoptosis and the formation of co-aggregates between the prion disease protein, PrP, and the cell death regulator, Bcl-2 [62]. Numerous studies have also shown that Hsp70 overexpression reduces polyQ toxicity; however, results are mixed as to whether Hsp70 reduces polyQ inclusion body formation [14]. In yeast, overexpression of Hsp70 (Ssa1) and Hsp40 (Ydj1) reduces the accumulation of detergent-insoluble fibrillar aggregates and instead promotes smaller amorphous aggregates [63], and mutations in these genes were found to specifically inhibit the accretion of aggregates [64]. Overexpression of Hsp70 co-factors also impacts misfolding disease proteins. Hsp40 suppresses polyQ inclusion formation and toxicity in a variety of model systems [65–68]. CHIP suppressed toxicity of a-synuclein and polyQ proteins, possibly by enhancing ubiquitination and degradation of oligomers [69–72], although in at least one instance CHIP reduced the solubility and thus enhanced the aggregation of polyQ-expanded ataxin-1 [73]. CHIP likewise enhances cell survival and accelerates tau and Ab removal probably by the ubiquitin–proteasome system [74,75]. Bag-1, which associates with inclusions via interactions with Hsp70, alleviates toxicity caused by polyQ-expanded huntingtin fragments [76]. Hsp110 interacts with and suppresses aggregation of the mutant Cu/Zn superoxide dismutase associated with ALS [77,78], and furthermore suppresses the aggregation and toxicity of polyQ-expanded androgen receptor, the cause of Kennedy’s disease [79]. Results of in vitro work provide some insight into the mechanism of action of Hsp70 against misfolding and thus toxicity of disease proteins. Purified Hsp70 acts preferentially on Ab, huntingtin, and a-synuclein pre-fibrillar species (i.e. monomers or oligomers,

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rather than fibrillar aggregates) to modulate the aggregation process [8,11,13,80,81]. Hsp70 effectively inhibits the aggregation of Ab and a-synuclein species even at substoichiometric levels, suggesting that Hsp70 can recognize multimeric protein assemblies [80,81]. In certain cases, the influence of Hsp70 on aggregation requires its ATPase activity, and its efficacy is enhanced by the cochaperone Hsp40, highlighting the importance of ATP-dependent cycles of substrate binding and release [8,13,63,80]. On the other hand, several studies indicate that Hsp70 can strongly inhibit asynuclein aggregation in vitro by binding to cytotoxic prefibrillar species, even in the absence of Hsp40 or other co-factors [11,81]. However, Hsp70 function in vivo is likely coupled to co-chaperones and other co-factors for assistance in binding to aggregating substrates and facilitating their degradation or deposition into inclusion bodies or other assemblies [34]. Substantial progress has been made regarding the impact of Hsp70 on the aggregation of polyQ-expanded huntingtin in vitro. In an ATP-dependent manner, Hsp70/Hsp40 interfere with an intramolecular conformational change in polyQ-expanded huntingtin that occurs immediately upon initiation of conditions that favor aggregation, perhaps while huntingtin is still monomeric [13]. Consistently, Hsp70 together with Hsp40 was found to stabilize a monomeric huntingtin conformation, and by doing so, prevent the accumulation of spherical and ring-like oligomers that likely represent toxic species on- or offpathway for fibril formation [8]. As a result of the action of Hsp70/Hsp40 as well as other chaperones, mutant huntingtin is deviated from the potentially toxic, fibrillar aggregation pathway and instead accumulates in amorphous aggregates, or other benign conformers (see discussion below) [8,63,82]. Sequestered in these conformers, mutant huntingtin may no longer participate in heterotypic interactions known to inactivate essential cellular machinery, such as polyQ-containing transcription factors [13]. It is tempting to speculate about the molecular mechanism of Hsp70 action on misfolded disease proteins by analogy to its role in de novo folding (Fig. 1). Considering that Hsp70 generically recognizes exposed hydrophobic regions in newly-synthesized substrates, it is probable that Hsp70 binds via its SBD to such regions that may be exposed in a range of prefibrillar species, from monomer to early oligomer [8,11,80,81]. Hsp70 is likely unable to bind productively to hydrophobic regions that are embedded in fibrils occurring late in the aggregation process, and therefore fibrils are not readily modified by Hsp70 action [8,11,80,81]. ATP-dependent binding of Hsp70 to monomers/oligomers may induce a structural change. Binding and release cycles would convert the structure of the disease protein to one less likely to self-assemble on a toxic aggregation pathway, and instead more likely to partition to a non-toxic conformation (e.g. amorphous aggregates, Fig. 1), or one that is more easily degraded. Recognition and modification of toxic pre-fibrillar species by Hsp70 is likely a critical component of the cellular defense against protein misfolding disease. Other cellular roles of Hsp70, for instance its known role in facilitating degradation of misfolded substrates, likely provide additional neuroprotective effects [14]. 2.2. Chaperonins Chaperonins are a structurally conserved class of molecular chaperones that are divided into two subgroups [1]. Group I chaperonins include the extensively characterized Escherichia coli GroEL system as well as mitochondrial Hsp60, both of which are essential for maintaining cellular protein homeostasis. Inactivation of mitochondrial Hsp60 via the V98I point mutation severely perturbs this balance in certain cell types, causing hereditary spastic paraplegia (SPG13), a late-onset neurodegenerative disease associated with progressive paraparesis of the lower limbs [83,84]. The Group II chaperonins, as represented by cytosolic TRiC (TCP-1 Ring Com-

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Toxic conformers?

200 kDa soluble intermediates?

TRiC ?

(iv.)

Benign conformers

(i.)

(iii.)

40 70

?

(ii.)

TRiC

unstable soluble oligomer

monomeric intermediate

amorphous inclusions

soluble oligomers (500 kDa?)

Fig. 1. A speculative model depicting the role of Hsp70/Hsp40 and TRiC/CCT as modulators of aggregation and toxicity of aberrantly folding disease proteins. Newly synthesized (or proteolytically generated) disease polypeptides expose hydrophobic regions (blue colored segment). (i) Hsp70/Hsp40 bind to these regions and stabilize the unfolded substrate. Upon release, partial refolding may allow the polypeptide to achieve a conformation that may be more conducive to interaction with downstream chaperones, such as TRiC/CCT. (ii) TRiC/CCT may bind to exposed b-strands (green colored segments) and induce a conformational change, leading to the stable formation of benign, soluble oligomers in the 500 kDa size range, as has been suggested by studies with polyQ proteins. Whether this applies to other proteins of misfolding disease remains unknown. (iii) When TRiC/CCT levels are limiting, the action of Hsp70/Hsp40 leads to the accumulation of benign amorphous aggregates. (iv) When overall chaperone levels are inadequate, the newly synthesized (or proteolytically generated) disease polypeptide undergoes an intramolecular compaction in the earliest steps of the aggregation process, generating a b-structured (green segments) monomer with exposed hydrophobic regions (blue segment). This monomer undergoes fibrillization, a process that may be inhibited by TRiC/CCT. By virtue of its exposed hydrophobic regions, this monomer may also form 200 kDa soluble ring-like or spherical intermediates, here depicted as off-pathway for fibril formation, that exert toxicity by interacting with essential cellular machinery or generating membrane pores.

plex, also called CCT for chaperonin-containing TCP1), have been implicated more extensively in neurodegenerative disease. The main cellular function of TRiC is to promote the folding of newlysynthesized polypeptides, which may be presented by Hsp70 and/or the cochaperone prefoldin [1,85]. For this reason, TRiC is not induced by stress but is instead transcriptionally and functionally linked to protein synthesis [86]. TRiC is a large complex composed of eight homologous subunits arranged in two octameric rings, stacked back-to-back, that form a cage for protein folding to occur unimpaired by aggregation. Whereas all TRiC subunits have essentially identical ATPase domains, their polypeptide-binding regions have significantly diverged during evolution to create substrate binding specificity [87]. As a result, a diverse set of polypeptide substrates, estimated to be 5–10% of newly synthesized cytoplasmic proteins, including actin and tubulin, flux through TRiC [1,88,89]. PolyQ-expanded huntingtin has also been identified as a TRiC substrate in recent studies [82,90,91]. An RNA interference screen for suppressors of polyQ aggregation in C. elegans identified six of eight TRiC subunits, initially implicating TRiC as an in vivo modulator of polyQ aggregation [92]. Several studies went on to show that TRiC partially colocalizes with huntingtin aggregates and remodeled their morphology while reducing cell death [82,91]. It was also shown that overexpression of subunit 1 of TRiC was effective at inhibiting huntingtin aggregation and increasing viability

[90]. However, knock-down of another subunit (subunit 6), which impairs the function of the TRiC complex as a whole, increases huntingtin aggregation and toxicity [85,91]. These results along with others from yeast studies argue that TRiC as a fully assembled complex exerts neuroprotective effects by modulating huntingtin aggregation [82,91]. TRiC substrates tend to be large, hydrophobic proteins with regions of b-strand propensity that are inherently aggregation-prone [88,93–96]. Consistently, polyQ-expanded huntingtin, as well as all other amyloidogenic proteins, tend to form fibrillar aggregates possessing a ‘cross-b’ core with extensive b-sheet structure [4]. Moreover, the aggregation pathway of these proteins begins by a rapid conformational transition from native monomer into a compact b-sheet structure necessary for the formation of mature amyloid fibrils [13,34,82]. It is therefore plausible that TRiC regulates the conformation of huntingtin and possibly other amyloidogenic proteins by binding directly to b-sheet structures formed after aggregation initiation, potentially directly after synthesis or proteolytic generation of aggregation prone fragments (Fig. 1). Optimal protection by TRiC requires prior processing by the Hsp70 machinery in a cooperative reaction similar to de novo folding of certain substrates [1,82]. TRiC/Hsp70 were found to act in concert on huntingtin monomers or small oligomers early in the aggregation pathway to promote the formation of non-pathogenic oligomers of 500 kDa and reduce levels of 200 kDa oligomers

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that are reactive with the A11 antibody and appear to be associated with polyQ toxicity [82]. Chaperone-assisted folding and aggregation reactions mediated by Hsp70/TRiC therefore share intriguing features. In both cases, Hsp70 first interacts with the substrate to stabilize it in a conformation conducive to interaction with TRiC, the downstream factor which subsequently promotes folding to the native or non-pathogenic state. Furthermore, in both de novo folding and aggregation reactions, Hsp70/TRiC act at critical early steps to ensure productive (or protective) folding of the polypeptide substrate. Thus, basic principles of chaperone cooperation between Hsp70 and TRiC used in de novo protein folding are also employed in the cellular defense mechanism against amyloidogenic proteins.

3. Concluding remarks Based on aforementioned principles of de novo folding and protein aggregation, a speculative model of chaperone-mediated aggregation in misfolding disease is proposed (Fig. 1). Newly synthesized or proteolytically generated disease proteins may expose hydrophobic regions, which are soon bound by the Hsp70/Hsp40 machinery early in the aggregation process. Upon release from Hsp70, refolding may bury hydrophobic regions and/or allow the disease polypeptide to achieve a conformation that may be more conducive to interaction with downstream chaperones. According to studies with polyQ-expansion proteins, the disease polypeptide is then either deposited into benign amorphous inclusions or further modified by the TRiC chaperonin [63,82]. TRiC may bind to exposed b-strands and induce a conformational change in the disease protein that promotes its stable accumulation in soluble oligomers of 500 kDa, as has been shown for polyQ-expanded huntingtin fragments [13]. Whether this part of the pathway applies to other disease proteins is currently unknown. When overall chaperone levels are inadequate, the newly synthesized (or proteolytically generated) disease polypeptide undergoes an intramolecular structural change in the earliest steps of the aggregation process [13], generating a b-structured monomer with exposed hydrophobic regions. By virtue of these hydrophobic regions, the monomer may accumulate in ring-like or spherical intermediates on- or off-pathway for fibril formation, that exert toxicity by generating membrane pores or inactivating essential cellular factors [8,13,82]. Such structures have been reported in models of Huntington’s, Parkinson’s, and Alzheimer’s disease and proposed to be primary agents of toxicity in the pathogenesis of misfolding disease [14]. Monomers may also accumulate in long fibrillar structures in a process that may be inhibited by TRiC, as has been described for polyQ aggregation [82]. Amyloid fibrils that form late in the aggregation process collect in inclusions, plaques, or Lewy bodies in brain tissue and are not modified readily by chaperone function [8,11,13,80,81]. Given the protective effects of overexpressing chaperones in models of misfolding disease, it is probable that drug-mediated enhancement of chaperone levels would be a effective strategy for delaying or even preventing misfolding disease. Promising results have been reported for geldanamycin (GA) and its derivatives 17-AAG and 17-DMAG, which inhibit Hsp90 by binding to its Nterminal ATP-binding pocket, resulting in HSF-1 stimulation and induction of stress protein expression [57,97]. These inhibitors are broadly effective against aggregation and toxicity in various model systems of misfolding disease [17,50,98–100]. However, a possible problem with developing these inhibitors for human use is their potential to elicit side effects, which stem from inhibition of Hsp90 [57]. Indeed, results from clinical trials evaluating the effect of geldanamycin analogues on cancer suggest no efficacy at doses with acceptable toxicity [101,102]. Another Hsp90 inhibitor,

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novobiocin, may prove to be a more useful therapeutic agent. Novobiocin, a coumarin-containing DNA gyrase inhibitor, binds instead to the C-terminal domain of Hsp90 and causes no apparent cytotoxicity at doses found to elevate Hsp70 expression. Novobiocin and its derivatives were reported to suppress Ab neurotoxicity, suggesting they may be optimal candidates for clinical testing with patients with misfolding disease [103]. Conditions such as caloric restriction, which stimulate HSF-1 through other mechanisms may also prove useful. Stress due to moderate caloric restriction induces known beneficial effects, like life-span extension, in part by activating the deacetylase SIRT1 [104]. It has recently come to light that SIRT1 activates HSF1, thereby enhancing the transcription of target chaperone genes such as Hsp70 upon exposure to stress [105]. Caloric restriction or chemical activators of SIRT1 (e.g. by resveratrol) likely provide a means to enhance levels of Hsp70 and other chaperones while promoting longevity and potentially delaying or preventing disease onset. Caloric restriction and resveratrol indeed improve phenotypes in misfolding disease models, and their effect on Alzheimer’s disease patients is currently being evaluated in clinical trials [104]. Other drug candidates that stimulate HSF-1 include arimoclomol and celastrol, both of which were found to improve misfolding disease phenotypes in model systems [106,107]. These and other methods that result in induction of expression of Hsp70 and other chaperones associated with the cytosolic stress response hold great promise as therapeutic agents. In principle, drug-mediated induction of the TRiC chaperonin would be another worthy therapeutic strategy; however, almost nothing is known about the regulation of the TRiC chaperonin and therefore efforts in this direction have so far not been fruitful. While it remains to be seen whether patients will eventually benefit from these types of approaches, a better understanding of the role of Hsp70, TRiC, and other chaperones in misfolding disease is likely to provide important insight into basic pathomechanistic principles.

4. Conflict of interest statement The authors declare no competing financial interests. Acknowledgements Work in the authors’ laboratory was supported by the Max Planck Society, the Deutsche Forschungsgemeinschaft (SFB 596), the Ernst-Jung Foundation and the Körber Foundation.

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