Yeast prions and Hsp70 chaperones

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Organisms encode multiple Hsp70s to regulate abundance of Hsp70 in accordance with need in response to changing environments and to provide a range of distinct Hsp70 functions for carrying out many different tasks within cells and across cell types. We constructed a yeast system to evaluate Hsp70s from any source and have found that each of the four functionally redundant essential yeast Hsp70s possess distinct activities. We also developed our system to investigate functions of the two Hsp90 paralogs. We are continuing to use this system to investigate how Hsp70s and Hsp90s within and across species act in cellular PQC processes or influence propagation of amyloid in vivo. The many ways that prion phenotypes change when activities or abundance of Hsp70s or their co-chaperones are altered provides a sensitive way to investigate even subtle functional distinctions among highly redundant Hsp70s, and a useful approach to uncover the underlying mechanisms. The large number of co-chaperones that act on different steps of the Hsp70 reaction cycle can cooperate to provide both a broad range of function and fine tuning of Hsp70 activity to specify its proper functions in defined roles in cells. We discovered that a human J-domain protein regulator of Hsp70 (DnaJB6b) protects cells from a latent toxicity of PSI+ prions that propagate as the amyloid form of translation factor Sup35. This prion toxicity is uncovered when the non-essential substrate-binding activity of yeast J-domain protein Sis1 or ability of Sis1 to interact with Hsp70 is disrupted. Our findings show that protection by DnaJB6b requires coincides with an increase in Sup35 solubility, which suggests it reduces incorporation of the essential Sup35 protein into insoluble amyloid aggregates. We further found DnaJB6b protects cells independently of interaction with Hsp70 implying that it acts directly on amyloid to limit its propagation in cells. This behavior is different from the way we earlier showed Sis1 protects from amyloid by cooperating with Hsp70 to recover Sup35 from prion aggrgegates or to moderate toxic depletion of Sup35 into amyloid. Hsp70 cooperates with Hsp90 to promote the proper folding and functions of many "client" proteins that regulate various fundamental cellular processes. Altering abundance or function of Hsp70 and Hsp90 can lessen pathology in models of protein folding disorders, while in the same models reducing activity of either chaperone can cause or exacerbate pathology. These protein chaperones therefore are promising therapeutic candidates for amyloid and other protein folding disorders and they are being evaluated intensively as drug targets. We are using yeast, human and disease organism paralogs of Hsp70 and Hsp90 to study specific and conserved functional interactions of these chaperones. Using human Hsp90s alpha and beta (hHsp90), which function in place of the yeast Hsp90s, we have identified specific sites on these paralogs that are important for regulating functional output of interactions with Hsp70 and other co-chaperones. We also discovered that a mutation in Hsp90 that destroys its ability to hydrolyze ATP does not prevent either hHsp90 from supporting growth of evolutionarily distant cells. Similarly mutated versions of Hsp90s from three disease organisms also retained ability to support cell growth. Using biophysical and biochemical assays we showed how the mutation affects conformational dynamics of Hsp90 upon binding of ATP or ADP. Together our findings connect how specific changes in the Hsp90 reaction cycle relate to its functions in vivo, and provide a framework for understanding the molecular mechanics needed to specify action of these Hsp90s under different physiological and environmental conditions. Additionally, our findings showing ATP hydrolysis is dispensable for essential and non-essential Hsp90 functions is obligating the field to reconsider the role of ATP in Hsp90 activity. Our work toward understanding what underlies specificity in activities of functionally redundant Hsp70s and Hsp90s provide insight for new approaches to modify Hsp90 activity in specific ways. This understanding can help guide decisions about which Hsp70 or Hsp90-family members would be most useful for such applications, or point to potential problems that could arise due to differences in ways that different Hsp70 or Hsp90 paralogs respond to specific compounds. Overall our work provides insight into functions of protein quality control factors that can help guide strategies for using chaperones as targets for therapy in protein folding disorders.