Date of Award

Winter 2023

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

First Advisor

Feixia Chu

Second Advisor

Krisztina Varga

Third Advisor

Sarah Walker

Abstract

Heat shock protein 90 (Hsp90) is a highly conserved and essential molecular chaperone in eukaryotes. Through interactions with various co-chaperones, Hsp90 is able to adapt to meet the specific needs of a structurally and functionally diverse, but specific set of clienteles. The diversity of its clients makes it a key regulator of nearly all cellular processes, and it serves as a central hub for many different signaling networks. One of the major functions of the Hsp90 chaperone machinery is in the modulation of protein homeostasis, a protective function often exploited in cancer cells in order to buffer oncogenic mutations and the overexpression of numerous oncoproteins, thus linking Hsp90 to tumorigenesis. Inhibitors of Hsp90 have entered clinical trials displaying distinct pharmacological efficacies and toxicological profiles. In this study we first investigate the posttranslational and conformational states of Hsp90 targeted by either PU-H71 or geldanamycin (GA) using mass spectrometry. Quantitative analysis suggests that PU-H71 binds to Hsp90 in a multi-chaperone complex with an adopted closed-like conformation, while GA binds to uncomplexed Hsp90 in an open-like conformation. Additionally, phosphorylation was found to be enriched in the acidic linker region of Hsp90 targeted by PU-H71. Mutational analysis revealed that phosphorylation of S226/S255 promotes association of Hsp90 with epichaperome related cochaperones (Cdc37, FKBP4, and Aha1). Further investigation revealed that S255 phosphorylation was particularly important for promoting ESC self-renewal providing a possible biochemical mechanism by which epichaperome formation is promoted and linked to the maintenance of ESCs. In order to expand the scope of our analysis when evaluating Hsp90 complexes and the PTMs that regulate them we then developed a novel method for purification and separation of Hsp90 complexes prior to MS-based proteomics analysis. This new method involved expression of an affinity tagged Hsp90 containing a recognizable proteolytic sequence, which allows capture and separation of Hsp90 complexes under native conditions. We demonstrate that this technique we termed as a native elution allows for detailed characterization of Hsp90 complexes, previously unattainable. Co-chaperones and clients acquire distinct distribution profiles based off the Hsp90 complexes they associate with. Furthermore, subunits of large protein complexes appear with very distinctive distribution profiles generally with significant enrichment in only a few of the high-molecular weight species. Lastly, we identified multiple Hsp90 PTMs across various Hsp90 complexes. As PTMs remain an important determinant of Hsp90 inhibitor sensitivity, the quantitative data will be used for continued bioinformatic analyses of these modifications, in which a functional relationship between modification(s) and Hsp90-bound proteins will be mapped.

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