Amyloid in neurodegenerative diseases: friend or foe?

Accumulation of amyloid-like aggregates is a hallmark of numerous neurodegenerative disorders such as Alzheimer's and polyglutamine disease. Yet, whether the amyloid inclusions found in these diseases are toxic or cytoprotective remains unclear. Various studies suggest that the toxic culprit in the amyloid folding pathway is actually a soluble oligomeric species which might interfere with normal cellular function by a multifactorial mechanism including aberrant protein-protein interactions. Molecular chaperones suppress toxicity of amyloidogenic proteins by inhibiting aggregation of non-native disease substrates and targeting them for refolding or degradation. Paradoxically, recent studies also suggest a protective action of chaperones in their promotion of the assembly of large, tightly packed, benign aggregates that sequester toxic protein species.     

Chaperones in control of protein disaggregation

The chaperone protein network controls both initial protein folding and subsequent maintenance of proteins in the cell. Although the native structure of a protein is principally encoded in its amino-acid sequence, the process of folding in vivo very often requires the assistance of molecular chaperones. Chaperones also play a role in a post-translational quality control system and thus are required to maintain the proper conformation of proteins under changing environmental conditions. Many factors leading to unfolding and misfolding of proteins eventually result in protein aggregation. Stress imposed by high temperature was one of the first aggregation-inducing factors studied and remains one of the main models in this field. With massive protein aggregation occurring in response to heat exposure, the cell needs chaperones to control and counteract the aggregation process. Elimination of aggregates can be achieved by solubilization of aggregates and either refolding of the liberated polypeptides or their proteolysis. Here, we focus on the molecular mechanisms by which heat-shock protein 70 (Hsp70), Hsp100 and small Hsp chaperones liberate and refold polypeptides trapped in protein aggregates.     

Role of sHsps in organizing cytosolic protein aggregation and disaggregation

Small heat shock proteins (sHsps) exhibit an ATP-independent chaperone activity to prevent the aggregation of misfolded proteins in vitro. The seemingly conflicting presence of sHsps in insoluble protein aggregates in cells obstructs a precise definition of sHsp function in proteostasis networks. Recent findings specify sHsp activities in protein quality control systems. The sHsps of yeast, Hsp42 and Hsp26, interact with early unfolding intermediates of substrates, keeping them in a ready-to-refold conformation close to the native state. This activity facilitates substrate refolding by ATP-dependent Hsp70-Hsp100 disaggregating chaperones. Hsp42 can actively sequester misfolded proteins and promote their deposition at specific cellular sites. This aggregase activity represents a cytoprotective protein quality control strategy. The aggregase function of Hsp42 controls the formation of cytosolic aggregates (CytoQs) under diverse stress regimes and can be reconstituted in vitro, demonstrating that Hsp42 is necessary and sufficient to promote protein aggregation. Substrates sequestered at CytoQs can be dissociated by Hsp70-Hsp100 disaggregases for subsequent triage between refolding and degradation pathways or are targeted for destruction by selective autophagy termed proteophagy.     

Molecular chaperones

Chaperones are unique remodeling proteins that participate in a great number of intracellular processes and are involved in the correction of protein structure, the prevention of the aggregation of misfolded proteins, the destruction of protein aggregates, and also the unfolding of native protein targets for their translocation across a membrane. In addition to this, chaperones assist in the dismantling of active oligomers into inactive unfolded monomers for their subsequent proteolytic degradation and the assembly of folded subunits into protein assemblies and specific complexes. Data on the structure and functioning of molecular chaperones from five basic families are summarized in the review.     

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic lateral sclerosis, and α-synuclein, implicated in Parkinson’s disease, are toxic and form cytoplasmic aggregates in yeast. These features recapitulate protein pathologies observed in patients with these disorders. Thus, yeast are an ideal platform for isolating toxicity suppressors from libraries of protein variants. We are interested in applying protein disaggregases to eliminate misfolded toxic protein conformers. Specifically, we are engineering Hsp104, a hexameric AAA+ protein from yeast that is uniquely capable of solubilizing both disordered aggregates and amyloid and returning the proteins to their native conformations. While Hsp104 is highly conserved in eukaryotes and eubacteria, it has no known metazoan homologue. Hsp104 has only limited ability to eliminate disordered aggregates and amyloid fibers implicated in human disease. Thus, we aim to engineer Hsp104 variants to reverse the protein misfolding implicated in neurodegenerative disorders. We have developed methods to screen large libraries of Hsp104 variants for suppression of proteotoxicity in yeast. As yeast are prone to spontaneous nonspecific suppression of toxicity, a two-step screening process has been developed to eliminate false positives. Using these methods, we have identified a series of potentiated Hsp104 variants that potently suppress the toxicity and aggregation of TDP-43, FUS, and α-synuclein. Here, we describe this optimized protocol, which could be adapted to screen libraries constructed using any protein backbone for suppression of toxicity of any protein that is toxic in yeast.     

A chaperone pathway in protein disaggregation. Hsp26 alters the nature of protein aggregates to facilitate reactivation by Hsp104

Cellular protein folding is challenged by environmental stress and aging, which lead to aberrant protein conformations and aggregation. One way to antagonize the detrimental consequences of protein misfolding is to reactivate vital proteins from aggregates. In the yeast Saccharomyces cerevisiae, Hsp104 facilitates disaggregation and reactivates aggregated proteins with assistance from Hsp70 (Ssa1) and Hsp40 (Ydj1). The small heat shock proteins, Hsp26 and Hsp42, also function in the recovery of misfolded proteins and prevent aggregation in vitro, but their in vivo roles in protein homeostasis remain elusive. We observed that after a sublethal heat shock, a majority of Hsp26 becomes insoluble. Its return to the soluble state during recovery depends on the presence of Hsp104. Further, cells lacking Hsp26 are impaired in the disaggregation of an easily assayed heat-aggregated reporter protein, luciferase. In vitro, Hsp104, Ssa1, and Ydj1 reactivate luciferase:Hsp26 co-aggregates 20-fold more efficiently than luciferase aggregates alone. Small Hsps also facilitate the Hsp104-mediated solubilization of polyglutamine in yeast. Thus, Hsp26 renders aggregates more accessible to Hsp104/Ssa1/Ydj1. Small Hsps partially suppress toxicity, even in the absence of Hsp104, potentially by sequestering polyglutamine from toxic interactions with other proteins. Hence, Hsp26 plays an important role in pathways that defend cells against environmental stress and the types of protein misfolding seen in neurodegenerative disease.     

Chaperones Hsp70 and Hsp40 suppress aggregate formation and apoptosis in cultured neuronal cells expressing truncated androgen receptor protein with expanded polyglutamine tract

Spinal and bulbar muscular atrophy (SBMA) is one of a group of human inherited neurodegenerative diseases caused by polyglutamine expansion. We have previously demonstrated that the SBMA gene product, the androgen receptor protein, is toxic and aggregates when truncated. Heat shock proteins function as molecular chaperones, which recognize and renaturate misfolded protein (aggregate). We thus assessed the effect of a variety of chaperones in a cultured neuronal cell model of SBMA. Overexpression of chaperones reduces aggregate formation and suppresses apoptosis in a cultured neuronal cell model of SBMA to differing degrees depending on the chaperones and their combinations. Combination of Hsp70 and Hsp40 was the most effective among the chaperones in reducing aggregate formation and providing cellular protection, reflecting that Hsp70 and Hsp40 act together in chaperoning mutant and disabled proteins. Although Hdj2/Hsdj chaperone has been previously reported to suppress expanded polyglutamine tract-formed aggregate, Hsdj/Hdj2 showed little effect in our system. These findings indicate that chaperones may be one of the key factors in the developing of CAG repeat disease and suggested that increasing expression level or enhancing the function of chaperones will provide an avenue for the treatment of CAG repeat disease.     

Interplay between protein homeostasis networks in protein aggregation and proteotoxicity

The misfolding and aggregation of disease proteins is characteristic of numerous neurodegenerative diseases. Particular neuronal populations are more vulnerable to proteotoxicity while others are more apt to tolerate the misfolding and aggregation of disease proteins. Thus, the cellular environment must play a significant role in determining whether disease proteins are converted into toxic or benign forms. The endomembrane network of eukaryotes divides the cell into different subcellular compartments that possess distinct sets of molecular chaperones and protein interaction networks. Chaperones act as agonists and antagonists of disease protein aggregation to prevent the accumulation of toxic intermediates in the aggregation pathway. Interacting partners can also modulate the conformation and localization of disease proteins and thereby influence proteotoxicity. Thus, interplay between these protein homeostasis network components can modulate the self‐association of disease proteins and determine whether they elicit a toxic or benign outcome. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 229–236, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Heat shock protein 70 prevents both tau aggregation and the inhibitory effects of preexisting tau aggregates on fast axonal transport

Aggregation and accumulation of the microtubule-associated protein tau are associated with cognitive decline and neuronal degeneration in Alzheimer's disease and other tauopathies. Thus, preventing the transition of tau from a soluble state to insoluble aggregates and/or reversing the toxicity of existing aggregates would represent a reasonable therapeutic strategy for treating these neurodegenerative diseases. Here we demonstrate that molecular chaperones of the heat shock protein 70 (Hsp70) family are potent inhibitors of tau aggregation in vitro, preventing the formation of both mature fibrils and oligomeric intermediates. Remarkably, addition of Hsp70 to a mixture of oligomeric and fibrillar tau aggregates prevents the toxic effect of these tau species on fast axonal transport, a critical process for neuronal function. When incubated with preformed tau aggregates, Hsp70 preferentially associated with oligomeric over fibrillar tau, suggesting that prefibrillar oligomeric tau aggregates play a prominent role in tau toxicity. Taken together, our data provide a novel molecular basis for the protective effect of Hsp70 in tauopathies.     

The therapeutic potential of chemical chaperones in protein folding diseases

Several neurodegenerative diseases are caused by defects in protein folding, including Alzheimer, Parkinson, Huntington, and prion diseases. Once a disease-specific protein misfolds, it can then form toxic aggregates which accumulate in the brain, leading to neuronal dysfunction, cell death, and clinical symptoms. Although significant advances have been made toward understanding the mechanisms of protein aggregation, there are no curative treatments for any of these diseases. Since protein misfolding and the accumulation of aggregates are the most upstream events in the pathological cascade, rescuing or stabilizing the native conformations of proteins is an obvious therapeutic strategy. In recent years, small molecules known as chaperones have been shown to be effective in reducing levels of misfolded proteins, thus minimizing the accumulation of aggregates and their downstream pathological consequences. Chaperones are classified as molecular, pharmacological, or chemical. In this mini-review we summarize the modes of action of different chemical chaperones and discuss evidence for their efficacy in the treatment of protein folding diseases in vitro and in vivo.     

Active solubilization and refolding of stable protein aggregates by cooperative unfolding action of individual hsp70 chaperones

Hsp70 is a central molecular chaperone that passively prevents protein aggregation and uses the energy of ATP hydrolysis to solubilize, translocate, and mediate the proper refolding of proteins in the cell. Yet, the molecular mechanism by which the active Hsp70 chaperone functions are achieved remains unclear. Here, we show that the bacterial Hsp70 (DnaK) can actively unfold misfolded structures in aggregated polypeptides, leading to gradual disaggregation. We found that the specific unfolding and disaggregation activities of individual DnaK molecules were optimal for large aggregates but dramatically decreased for small aggregates. The active unfolding of the smallest aggregates, leading to proper global refolding, required the cooperative action of several DnaK molecules per misfolded polypeptide. This finding suggests that the unique ATP-fueled locking/unlocking mechanism of the Hsp70 chaperones can recruit random chaperone motions to locally unfold misfolded structures and gradually disentangle stable aggregates into refoldable proteins.     

Potentiated Hsp104 variants suppress toxicity of diverse neurodegenerative disease-linked proteins

Protein misfolding is implicated in numerous lethal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and Parkinson disease (PD). There are no therapies that reverse these protein-misfolding events. We aim to apply Hsp104, a hexameric AAA+ protein from yeast, to target misfolded conformers for reactivation. Hsp104 solubilizes disordered aggregates and amyloid, but has limited activity against human neurodegenerative disease proteins. Thus, we have previously engineered potentiated Hsp104 variants that suppress aggregation, proteotoxicity and restore proper protein localization of ALS and PD proteins in Saccharomyces cerevisiae, and mitigate neurodegeneration in an animal PD model. Here, we establish that potentiated Hsp104 variants possess broad substrate specificity and, in yeast, suppress toxicity and aggregation induced by wild-type TDP-43, FUS and α-synuclein, as well as missense mutant versions of these proteins that cause neurodegenerative disease. Potentiated Hsp104 variants also rescue toxicity and aggregation of TAF15 but not EWSR1, two RNA-binding proteins with a prion-like domain that are connected with the development of ALS and frontotemporal dementia. Thus, potentiated Hsp104 variants are not entirely non-specific. Indeed, they do not unfold just any natively folded protein. Rather, potentiated Hsp104 variants are finely tuned to unfold proteins bearing short unstructured tracts that are not recognized by wild-type Hsp104. Our studies establish the broad utility of potentiated Hsp104 variants.KEY WORDS: FUS, Hsp104, TDP-43, α-synuclein, Disaggregase, Neurodegeneration     

Bistability explains threshold phenomena in protein aggregation both in vitro and in vivo

Neurodegenerative disease can originate from the misfolding and aggregation of proteins, such as Amyloid-beta, SOD1, or Huntingtin. Fortunately, all cells possess protein quality control machinery that sequesters misfolded proteins, either refolding or degrading them, before they can self-associate into proteotoxic oligomers and aggregates. This activity is largely performed by the stress response chaperones (i.e., Hsp70). However, the expression level of molecular chaperones varies widely among cell types. To understand the potential consequence of this variation, we studied the process of protein aggregation in the presence of molecular chaperones using mathematical modeling. We demonstrate that protein aggregation, in the presence of molecular chaperones, is a bistable process. Bistability in protein aggregation offers an explanation for threshold transitions to high aggregate concentration, which are observed both in vitro and in vivo. Additionally, we show that slight variations in chaperone concentration, due to natural fluctuations, have important consequences in a bistable system for the onset of protein aggregation. Therefore, our results offer a possible theoretical explanation for neuronal vulnerability observed in vivo and the onset of neurodegenerative phenotypes in neurons lacking an effective heat-shock response.     

ClpB/Hsp100 proteins and heat stress tolerance in plants

High-temperature stress can disrupt cellular proteostasis, resulting in the accumulation of insoluble protein aggregates. For survival under stressful conditions, it is important for cells to maintain a pool of native soluble proteins by preventing and/or dissociating these aggregates. Chaperones such as GroEL/GroES (Hsp60/Hsp10) and DnaK/DnaJ/GrpE (Hsp70/Hsp40/nucleotide exchange factor) help cells minimize protein aggregation. Protein disaggregation is accomplished by chaperones belonging to the Caseinolytic Protease (Clp) family of proteins. ClpB/Hsp100 proteins are strikingly ubiquitous and are found in bacteria, yeast and multi-cellular plants. The expression of these proteins is regulated by heat stress (HS) and developmental cues. Bacteria and yeast contain one and two forms of ClpB proteins, respectively. Plants possess multiple forms of these proteins that are localized to different cellular compartments (i.e. cytoplasm/nucleus, chloroplast or mitochondria). Overwhelming evidence suggests that ClpB/Hsp100 proteins play decisive roles in cell adaptation to HS. Mutant bacteria and yeast cells lacking active ClpB/Hsp100 proteins are critically sensitive to high-temperature stress. Likewise, Arabidopsis, maize and rice mutants lacking cytoplasmic ClpB proteins are very sensitive to heat. In this study, we present the structural and functional attributes of plant ClpB forms.     

Amyloid oligomer conformation in a group of natively folded proteins

Recent in vitro and in vivo studies suggest that destabilized proteins with defective folding induce aggregation and toxicity in protein-misfolding diseases. One such unstable protein state is called amyloid oligomer, a precursor of fully aggregated forms of amyloid. Detection of various amyloid oligomers with A11, an anti-amyloid oligomer conformation-specific antibody, revealed that the amyloid oligomer represents a generic conformation and suggested that toxic beta-aggregation processes possess a common mechanism. By using A11 antibody as a probe in combination with mass spectrometric analysis, we identified GroEL in bacterial lysates as a protein that may potentially have an amyloid oligomer conformation. Surprisingly, A11 reacted not only with purified GroEL but also with several purified heat shock proteins, including human Hsp27, 40, 70, 90; yeast Hsp104; and bovine Hsc70. The native folds of A11-reactive proteins in purified samples were characterized by their anti-beta-aggregation activity in terms of both functionality and in contrast to the beta-aggregation promoting activity of misfolded pathogenic amyloid oligomers. The conformation-dependent binding of A11 with natively folded Hsp27 was supported by the concurrent loss of A11 reactivity and anti-beta-aggregation activity of heat-treated Hsp27 samples. Moreover, we observed consistent anti-beta-aggregation activity not only by chaperones containing an amyloid oligomer conformation but also by several A11-immunoreactive non-chaperone proteins. From these results, we suggest that the amyloid oligomer conformation is present in a group of natively folded proteins. The inhibitory effects of A11 antibody on both GroEL/ES-assisted luciferase refolding and Hsp70-mediated decelerated nucleation of Abeta aggregation suggested that the A11-binding sites on these chaperones might be functionally important. Finally, we employed a computational approach to uncover possible A11-binding sites on these targets. Since the beta-sheet edge was a common structural motif having the most similar physicochemical properties in the A11-reactive proteins we analyzed, we propose that the beta-sheet edge in some natively folded amyloid oligomers is designed positively to prevent beta aggregation.     

Proteinaceous infectious behavior in non-pathogenic proteins is controlled by molecular chaperones

External stresses or mutations may cause labile proteins to lose their distinct native conformations and seek alternatively stable aggregated forms. Molecular chaperones that specifically act on protein aggregates were used here as a tool to address the biochemical nature of stable homo- and hetero-aggregates from non-pathogenic proteins formed by heat-stress. Confirmed by sedimentation and activity measurements, chaperones demonstrated that a single polypeptide chain can form different species of aggregates, depending on the denaturing conditions. Indicative of a cascade reaction, sub-stoichiometric amounts of one fast-aggregating protein strongly accelerated the conversion of another soluble, slow-aggregating protein into insoluble, chaperone-resistant aggregates. Chaperones strongly inhibited seed-induced protein aggregation, suggesting that they can prevent and cure proteinaceous infectious behavior in homo- and hetero-aggregates from common and disease-associated proteins in the cell.     

Critical role of the proline-rich region in Huntingtin for aggregation and cytotoxicity in yeast

Nine neurodegenerative diseases, such as Huntington, are caused by a polyglutamine (poly(Q)) expansion in otherwise unrelated proteins. Although poly(Q) expansion causes aggregation of the affected proteins, the protein context might determine the selective neuronal vulnerability found in each disease. Here we have report that, although expression of Huntingtin derivatives with a pathological poly(Q) expansion are innocuous in yeast, deletion of the flanking proline-rich region alters the shape and number of poly(Q) inclusions and unmasks toxic properties. Strikingly, deletion of Hsp104 increases the size of inclusions formed by expanded poly(Q) lacking the proline-rich region and abolishes toxicity. Overexpression of the chaperones Hsp104 or Hsp70 rescues growth defects in affected cells without resolving inclusions. However, aggregates formed by nontoxic Huntingtin derivatives or by toxic derivatives cured by chaperones are physically distinct from aggregates formed by toxic proteins. This study identifies the proline-rich region in Huntingtin as a profound cis-acting modulator of expanded poly(Q) toxicity and distinguishes between aggregates of toxic or non-toxic proteins.     

Small heat shock proteins, protein degradation and protein aggregation diseases

Small heat shock proteins have been characterized in vitro as ATP-independent molecular chaperones that can prevent aggregation of un- or mis-folded proteins and assist in their refolding with the help of ATP-dependent chaperone machines (e.g., the Hsp70 proteins). Comparison of the functionality of the 10 human members of the small HSPB family in cell models now reveals that some members function entirely differently and independently from Hsp70 machines. One member, HSPB7, has strong activities to prevent toxicity of polyglutamine-containing proteins in cells and Drosophila, and seems to act by assisting the loading of misfolded proteins or small protein aggregates into autophagosomes.     

Implications of protein structure instability: from physiological to pathological secondary structure

Proteins are folded during their synthesis; this process may be spontaneous or assisted. Both phenomena are carefully regulated by the "housekeeping" mechanism and molecular chaperones to avoid the appearance of misfolded proteins. Unfolding process generally occurs during physiological degradation of protein, but in some specific cases it results from genetic or environmental changes and does not correspond to metabolic needs. The main outcome of these phenomena is the appearance of nonfunctional pathologically unfolded proteins with a strong tendency to aggregation. Moreover, for some of these unfolded proteins, the agglomeration that follows initial proteins association may give rise to highly structured soluble aggregates. These aggregates have been identified as the main cause of the so-called amyloidosis or amyloid diseases, such as Alzheimer's, Parkinson's, and Creutzfeldt-Jakob diseases, and type II diabetes mellitus. Although some common mechanisms of amyloid protein aggregation have been identified, the roles of the environmental conditions inducing amyloidosis remain to be clarified. In this review, we will summarize recent studies identifying the origin of amyloid nucleation and will try to predict the therapeutic prospects that may be opened by elucidation of the amyloidosis mechanisms.     

Hsp110 Is a Bona Fide Chaperone Using ATP to Unfold Stable Misfolded Polypeptides and Reciprocally Collaborate with Hsp70 to Solubilize Protein Aggregates

Structurally and sequence-wise, the Hsp110s belong to a subfamily of the Hsp70 chaperones. Like the classical Hsp70s, members of the Hsp110 subfamily can bind misfolding polypeptides and hydrolyze ATP. However, they apparently act as a mere subordinate nucleotide exchange factors, regulating the ability of Hsp70 to hydrolyze ATP and convert stable protein aggregates into native proteins. Using stably misfolded and aggregated polypeptides as substrates in optimized in vitro chaperone assays, we show that the human cytosolic Hsp110s (HSPH1 and HSPH2) are bona fide chaperones on their own that collaborate with Hsp40 (DNAJA1 and DNAJB1) to hydrolyze ATP and unfold and thus convert stable misfolded polypeptides into natively refolded proteins. Moreover, equimolar Hsp70 (HSPA1A) and Hsp110 (HSPH1) formed a powerful molecular machinery that optimally reactivated stable luciferase aggregates in an ATP- and DNAJA1-dependent manner, in a disaggregation mechanism whereby the two paralogous chaperones alternatively activate the release of bound unfolded polypeptide substrates from one another, leading to native protein refolding.