Investigating Hsp90 Mechanism Using Client Reporters and Comprehensive Mutational Scanning

Heat shock protein 90 (Hsp90) is an essential chaperone in eukaryotes that helps many kinases and other signaling proteins to function. However, the mechanism by which Hsp90 binds to and activates its substrates or client proteins is poorly understood. Through its client proteins, Hsp90 has broad impacts on proteotoxic stress response, transcriptional activity, and epigenetic gene regulation. In cancer, many of the mutant proteins that promote the metastasis of cancer cells are unstable, making them highly dependent on Hsp90 chaperone activity for function. An improved understanding of how different client proteins bind to and are activated by Hsp90 may help in developing approaches to inhibit the cancer promoting activity without compromising its essential function. In our study, we aim to gain insight into how Hsp90 interacts with four of its clients including Heat Shock Factor 1 (Hsf1), Hog1 kinase, Glucocorticoid Receptor (GR), and the phosphatase, Calcineurin. We have built GFP reporters in yeast for each of these four Hsp90 clients. These reporters will be used to study how all possible point mutants of Hsp90 activate the four different clients using previously generated comprehensive mutant libraries. These studies will provide mechanistic insights into commonalities and distinctions in how Hsp90 binds to and activates different clients.