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 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 all of the functionally redundant essential yeast Hsp70s possess distinct activities. We are continuing to use this system to investigate how Hsp70s within and across species influence propagation of amyloid in vivo and act in cellular protein quality control (PQC) processes. The wide range of responses that prions have to alterations of Hsp70s and their co-chaperones provides a sensitive way to investigate even subtle functional distinctions among highly redundant Hsp70s and an approach to uncover the underlying mechanisms. Hsp70s act by binding and releasing client substrate proteins. Co-chaperones interact with Hsp70s to regulate their activity. 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. After Hsp70 binds client proteins, nucleotide exchange factors (NEFs) facilitate release of the substrates and restarting of the binding cycle. Hsp70 cooperates with Hsp90 to promote 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 chaperone activity 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 and increasingly as a drug target. We are studying functional interactions of Hsp70 with Hsp90 using both yeast and human proteins. Our findings have identified specific sites on human Hsp90s that are important for regulating interactions with Hsp70 and other co-chaperones. We also identified a site on Hsp90 that revealed how subtle structural differences determine functional distinctions of the two highly homologous, but functionally distinct, human Hsp90s. We identified changes at additional sites on human Hsp90 that improve or inhibit its ability to function in vivo. We used a biophysical assay to monitor how these changes affect confromational dynamics of Hsp90 during its reaction cycle that connected the ways the changes affected these dynamics to functions in vivo. Our findings provide a basis for understanding the molecular mechanics needed to specify action of these Hsp90s under different physiological and environmental conditions. They also provide insight for new approaches to modify Hsp90 activity in specific ways. Our work toward understanding what underlies specificity in activities of functionally redundant Hsp70s and Hsp90s can help guide decisions about which Hsp70 or Hsp90-family members would be most useful for such applications or identify potential problems that could arise due to ways different Hsp70s or Hsp90s respond differently 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.