Enzymatic and Biochemical Analysis of the Hsp90 Chaperone: Regulation by Co-chaperones, SUMOylation, and Interdomain Dynamics

• Author / Creator

  Wolmarans, Annemarie

• Hsp90 is an essential eukaryotic molecular chaperone that plays a critical role in protein folding. It regulates the stability, maturation, and activation of numerous client proteins, many of which are involved in oncogenesis. It has been well established that Hsp90 is tightly regulated by co-chaperone proteins and post-translational modifications (PTMs) in the context of an ATP-driven, functional cycle. Although Hsp90 is a homodimeric ATPase, growing evidence suggest that it functions as an asymmetric machine, where each subunit becomes individually functionalized when co-chaperones and clients bind, or when PTMs occur. Much remains to be elucidated about how these asymmetric interactions and modifications influence Hsp90 function. The goal of my thesis was to define a biochemical and mechanical model for the asymmetric interactions of co-chaperones with Hsp90 and how they regulate the ATPase activity of Hsp90. Various Hsp90 mutants were used in the context of homodimers and heterodimers in ATPase assay to conduct in-depth analyses on how co-chaperones regulate Hsp90 activity. Specifically, I demonstrated that co-chaperones exert different effects on Hsp90 depending on which subunit they bind and proposed a model outlining the mechanism of Aha1p-mediated stimulation of Hsp90 ATPase activity. How asymmetric SUMOylation of Hsp90 influence co-chaperone regulation of the ATPase activity of Hsp90 was also addressed. This was achieved by developing a novel strategy to chemically couple SUMO to Hsp90 in vitro. My analysis revealed that chemically SUMOylated Hsp90 recapitulates the selectivity of co-chaperone binding that is observed in vivo, mainly Aha1p-recruitment, but also demonstrated that SUMOylation impairs Sba1p regulation. Lastly, I investigated the conformational dynamics of Hsp90 by restricting interdomain rearrangements of Hsp90 using linker truncation mutants. These analyses brought novel insight into which Hsp90 conformations favor Aha1p and/or Sba1p binding. My work provides a framework to integrate subunit-specific interactions of co-chaperones and PTMs to further elucidate how the functional cycle of Hsp90 is regulated. It is crucial to understand how Hsp90 functions at a molecular level to advance the development of therapeutics that target the Hsp90 system.

• Subjects / Keywords

  • Hsp90
  • SUMOylation
  • Aha1
  • ATPase

• Graduation date

  Fall 2017

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3Q52FV7T

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.