The 90 kDa heat shock proteins (Hsp90) belong to a family of chaperones that regulate intracellular functions and are required for the refolding of denatured proteins following heat shock as well as the conformational maturation of a large number of key proteins involved in cellular processes. This proposal outlines a novel proteomics approach toward the identification of Hsp90 client proteins, cochaperones, immunophilins, and multiprotein complexes that fold nascent polypeptides. Geldanamycin derivatives will be synthesized to contain both photolabile and nonphotolabile biotin linkers, which are expected to bind to the N-terminal ATP binding region of Hsp90 and stabilize the complex association of Hsp90, client proteins, cochaperones, and immunophilins. In the presence of ubiquitination and protease inhibitors, these Hsp90/biotinylated GDA complexes will be removed from crude cellular lysate and the multiprotein components analyzed. The identification of proteins and multiprotein complexes that are bound to Hspg0 will provide insight into the role Hsp90 plays in the maturation of regulatory pathways by the identification of Hsp90 associated proteins and could provide additional targets for cancer chemotherapy. The identification of Hsp90 client proteins will provide evidence for the rational design of future inhibitors that are selective against individual client proteins and help determine the ramifications of inhibition of the entire Hsp90 process. A derivative of geldanamycin (17-AAG) has entered clinical trials, however, it is unlikely to proceed to phase II studies due to cytotoxicity unrelated to the inhibition of Hsp90. Using the multicomponent assay described in this proposal, it is possible to identify proteins other than Hsp90 to which geldanamycin binds, providing additional controls for the development of future Hsp90 inhibitors. Using similar techniques, Hsp90 dependent proteins from various cancer cell lines will be profiled. Protein profiling of Hsp90 dependent proteins will provide additional insight into the identification of proteins that are under or overexpressed in different cancer cell lines for future applications of Hsp90 inhibition.
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