Abstract
Heat Shock Protein 90 (Hsp90) is an essential molecular chaperone that maintains the stability of up to 15% of structurally diverse client proteins, many implicated in cancer development and progression. Hsp90 collaborates with cochaperones in an ATP-dependent process to identify, fold, and mature client proteins. Clients recognized by Hsp70 chaperones are transferred to Hsp90 via cochaperones such as Hop/Sti1 or p50/Cdc37 during the “loading” phase. During the closing phase, ATP-binding induces conformational changes in Hsp90 towards a closed conformation, stabilized by other cochaperones such as p23/Sba1. During the ‘reopening’ phase, hydrolysis of ATP is a driver towards client maturation and eventually release from Hsp90 complex. The N-terminal (N-) domain of Hsp90 contains the ATP-binding pocket that drives conformational cycling between open and closed states essential for client maturation. Current inhibitors target this site, broadly suppressing Hsp90 activity and degrading both oncogenic and essential clients, limiting therapeutic potential. Understanding how specific cochaperones or conformational states contribute to client specificity is therefore critical for developing selective strategies that modulate, rather than abolish, Hsp90 function. Our lab recently identified mutations in Hsp90 that interfere with each of the three phases outlined above, alter interactions with its cochaperones or Hsp70 chaperone and selectively disrupts the activity of three diverse client proteins. Because Hsp90 supports cancer-relevant signaling pathways, it has long been pursued as a chemotherapeutic target, yet it remains unclear whether perturbing specific steps in the Hsp90 cycle or individual cochaperone pathways can remodel the proteome in a selective rather than globally toxic manner. We lack a proteome-wide view of how the kinase-focused cochaperone Cdc37 contributes to proteostasis under acute stress and whether its influence extends beyond canonical kinase clients. This dissertation investigates how perturbations in the Hsp90 system reshape the proteome. First, we characterized the effects of Hsp90 mutations that disrupt distinct steps of the folding cycle, revealing that individual conformational states support overlapping yet distinct client subsets. Second, we performed an analysis of the cdc37-S14A cochaperone mutant, which impairs phosphorylation required for Hsp90–kinase complex formation, revealing the impacts extending beyond kinases to non-kinase regulators of stress adaptation and metabolism. Together, these studies demonstrate that Hsp90–cochaperone networks define client specificity through coordinated conformational and regulatory mechanisms. By integrating data-independent acquisition mass spectrometry (DIA-MS) with yeast genetics, this work provides a framework for selectively perturbing Hsp90–cochaperone interactions to reveal client dependencies and modulate proteostasis with greater precision.