Alzheimer disease is a neurological disorder that is characterized by the presence of fibrils and oligomers composed of the amyloid ␤ (A␤) peptide. In models of Alzheimer disease, overexpression of molecular chaperones, specifically heat shock protein 70 (Hsp70), suppresses phenotypes related to A␤ aggregation. These observations led to the hypothesis that chaperones might interact with A␤ and block self-association. However, although biochemical evidence to support this model has been collected in other neurodegenerative systems, the interaction between chaperones and A␤ has not been similarly explored. Here, we examine the effects of Hsp70/40 and Hsp90 on A␤ aggregation in vitro. We found that recombinant Hsp70/40 and Hsp90 block A␤ self-assembly and that these chaperones are effective at substoichiometric concentrations (ϳ1:50). The antiaggregation activity of Hsp70 can be inhibited by a nonhydrolyzable nucleotide analog and encouraged by pharmacological stimulation of its ATPase activity. Finally, we were interested in discerning what type of amyloid structures can be acted upon by these chaperones. To address this question, we added Hsp70/40 and Hsp90 to pre-formed oligomers and fibrils. Based on thioflavin T reactivity, the combination of Hsp70/40 and Hsp90 caused structural changes in oligomers but had little effect on fibrils. These results suggest that if these chaperones are present in the same cellular compartment in which A␤ is produced, Hsp70/40 and Hsp90 may suppress the early stages of self-assembly. Thus, these results are consistent with a model in which pharmacological activation of chaperones might have a favorable therapeutic effect on Alzheimer disease.The amyloid diseases are a collection of protein misfolding disorders associated with the formation of distinctive fibrils (reviewed in Refs. 1-5). Alzheimer disease is one of the most common amyloid diseases, and it is characterized by fibrils composed of A␤, 2 a 39 -43-amino acid proteolytic fragment of the amyloid precursor protein (reviewed in Ref. 6). Upon release from amyloid precursor protein, A␤ becomes enriched in ␤-sheet structure and acquires the propensity to self-assemble (7). Initial theories to explain the pathology of AD focused on the involvement of the visually striking fibrils, but more recent evidence has strongly supported a role for soluble oligomers (reviewed in Refs. 3 and 8 -10). A␤ oligomers can be prepared in vitro (11,12), biosynthesized by cultured cells (13,14), or collected from AD tissues (15), and in all cases, these structures are highly neurotoxic. Despite these important observations, numerous questions about the basis of disease are unanswered. For example, although there is active research in this area (8,11,16), the number of A␤ monomers present in an oligomer has not been fully described. Moreover, the subcellular site(s) of oligomer production and the conditions that lead to their assembly are still debated; fibrils are found in extracellular space, but there is growing evidence that oligomers may be pr...

Heat shock protein 70 (Hsp70) is a highly conserved molecular chaperone that plays multiple roles in protein homeostasis. In these various tasks, the activity of Hsp70 is shaped by interactions with co-chaperones, such as Hsp40. The Hsp40 family of co-chaperones binds to Hsp70 through a conserved J-domain, and these factors stimulate ATPase and protein-folding activity. Using chemical screens, we identified a compound, 115-7c, which acts as an artificial co-chaperone for Hsp70. Specifically, the activities of 115-7c mirrored those of a Hsp40; the compound stimulated the ATPase and protein-folding activities of a prokaryotic Hsp70 (DnaK) and partially compensated for a Hsp40 loss-of-function mutation in yeast. Consistent with these observations, NMR and mutagenesis studies indicate that the binding site for 115-7c is adjacent to a region on DnaK that is required for J-domain-mediated stimulation. Interestingly, we found that 115-7c and the Hsp40 do not compete for binding but act in concert. Using this information, we introduced additional steric bulk to 115-7c and converted it into an inhibitor. Thus, these chemical probes either promote or inhibit chaperone functions by regulating Hsp70-Hsp40 complex assembly at a native protein-protein interface. This unexpected mechanism may provide new avenues for exploring how chaperones and co-chaperones cooperate to shape protein homeostasis.Heat shock protein 70 (Hsp70) is a member of a ubiquitously expressed family of molecular chaperones that are involved in protein homeostasis. In its role as a mediator of protein fate, this chaperone has been linked to multiple tasks, including roles in de novo protein folding, subcellular trafficking, protein disaggregation, proteasome-mediated degradation, and autophagy (1-6). In addition, Hsp70 has been linked to numerous diseases, especially cancer and disorders of protein folding (7). Thus, there is interest in better understanding the biology of Hsp70 in order to test its potential as a therapeutic target (8).To accomplish its various chaperone functions, Hsp70 physically interacts with the exposed hydrophobic residues of polypeptides via its C-terminal substrate-binding domain (SBD). NIH-PA Author ManuscriptHydrolysis of ATP in the adjacent, N-terminal nucleotide-binding domain (NBD) propagates an allosteric change to the SBD, resulting in an approximately 10-fold enhancement in substrate affinity (9-12). These findings suggest an important role for the nucleotide state in controlling the interactions of Hsp70 with misfolded substrates. Consistent with the proposed importance of nucleotide turnover, a family of essential cochaperones, the Hsp40s, is known to tightly regulate the ATPase rate of Hsp70. These cochaperones are defined by the presence of a conserved, 60 amino acid J-domain. Interaction of the J-domain with the NBD of a Hsp70 stimulates its ATP hydrolysis and favors tight association with bound substrates. For example, the J-domain containing co-chaperone, DnaJ, stimulates the nucleotide hydrolysis rate o...

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Nine neurodegenerative disorders are caused by the abnormal expansion of polyglutamine (polyQ) regions within distinct proteins. Genetic and biochemical evidence has documented that the molecular chaperone, heat shock protein 70 (Hsp70), modulates polyQ toxicity and aggregation, yet it remains unclear how Hsp70 might be used as a potential target in polyQ-related diseases. We have utilized a pair of membrane-permeable compounds that tune the activity of Hsp70 by either stimulating or by inhibiting its ATPase functions. Using these two pharmacological agents in both yeast and PC12 cell models of polyQ aggregation and toxicity, we were surprised to find that stimulating Hsp70 solubilized polyQ conformers and simultaneously exacerbated polyQ-mediated toxicity. By contrast, inhibiting Hsp70’s ATPase activity protected against polyQ toxicity and promoted aggregation. These findings clarify Hsp70’s role as a possible drug target in polyQ disorders and suggest that Hsp70 uses ATP hydrolysis to help partition polyQ proteins into structures with varying levels of proteotoxicity. Our results thus support an emerging concept in which certain kinds of polyQ aggregates may be protective, while more soluble polyQ species are toxic.

In Saccharomyces cerevisae, expanded polyglutamine (polyQ) fragments are assembled into discrete cytosolic aggregates in a process regulated by the molecular chaperones Hsp26, Hsp70, Hsp90, and Hsp104. To better understand how the different chaperones might cooperate during polyQ aggregation, we used sequential immunoprecipitations and mass spectrometry to identify proteins associated with either soluble (Q25) or aggregation-prone (Q103) fragments at both early and later times after induction of their expression. We found that Hsp26, Hsp70, Hsp90, and other chaperones interact with Q103, but not Q25, within the first 2 h. Further, Hsp70 and Hsp90 appear to be partially released from Q103 prior to the maturation of the aggregates and before the recruitment of Hsp104. To test the importance of this seemingly ordered process, we used a chemical probe to artificially enhance Hsp70 binding to Q103. This treatment retained both Hsp70 and Hsp90 on the polyQ fragment and, interestingly, limited subsequent exchange for Hsp26 and Hsp104, resulting in incomplete aggregation. Together, these results suggest that partial release of Hsp70 may be an essential step in the continued processing of expanded polyQ fragments in yeast.The heat shock proteins (HSPs) 3 are abundant molecular chaperones that have been shown to be important for maintaining proteostasis under both normal conditions and during times of cellular stress (1-3). Together, these factors play multiple roles in quality control, especially related to polypeptide synthesis, folding, and turnover (4 -7). Moreover, HSPs are emerging as drug targets in cancer, neurodegenerative disorders and other diseases (8, 9). Thus, there is interest in better understanding how they coordinate protein quality control decisions.The major families of HSPs are named based on their approximate kDa molecular weights, including Hsp60, Hsp70, Hsp90,