Systematic Analysis of the Regulation of Hsp90 by its cofactors

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Protein homeostasis in the cell is regulated by a wide array of molecular systems consisting of molecular chaperones and proteases. These systems ensure that proteins fold to their native state and maintain their proper conformation throughout their lifetime in the cell. The chaperone surveillance systems also ensure that misfolded proteins and short-lived regulatory proteins are targeted for degradation. Hsp90 is one of the major and most highly conserved chaperones of the cell that functions to assist proteins in attaining their active conformation. Hsp90 has also been found to be involved in protein degradation. It is an ATP-dependent chaperone that forms a constitutive dimer, which exhibits multiple conformations regulated by ATP binding and hydrolysis. The in vivo and in vitro activity of Hsp90 is controlled by a large number of cofactors that target the chaperone to specific substrates. Well-established Hsp90 cofactors in yeast include: Sti1 (Hop), Sba1 (p23), Cdc37 (p50), Cpr6, Cpr7, Cns1, Aha1, Hch1, and Ppt1. Hsp90 forms specific complexes with these cofactors and target substrates. Knowledge of the composition and relative function of each of these different Hsp90-cofactor complexes is still fragmentary and has come largely from reconstitution experiments using partially purified proteins. As a result, the mechanism of Hsp90 function remains elusive due to the dynamic and heterogeneous nature of the chaperone-cofactor-substrate complexes. In this grant application, we propose to carry out a systematic analysis using proteomic and biochemical approaches in order to understand how substrate specificity is established by the Hsp90-cofactor system and, consequently, how the different cofactors regulate Hsp90 activity. To this end, several approaches will be undertaken. (1) Systematic analysis of interactors common to Hsp90 and its cofactors under normal and 'stress' growth conditions will be carried out. (2) The interaction studies will be complimented by extensive computational interrogation of the data to reveal common features that dictate the interaction of a substrate with a specific Hsp90-cofactor complex. (3) Follow up biochemical studies will be carried out to verify the results from the bioinformatic analysis.