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dc.contributor.advisorDimitra Bourboulia
dc.contributor.authorBaker-Williams, Alexander J.
dc.date.accessioned2021-08-06T17:41:41Z
dc.date.available2021-08-06T17:41:41Z
dc.date.issued2021
dc.identifier.urihttp://hdl.handle.net/20.500.12648/2041
dc.description.abstractHeat Shock Protein- 90 (HSP90) is an essential molecular chaperone. HSP90 relies on its intrinsic ATPase activity as well as interactions with co-chaperone proteins to chaperone its clients. HSP90 is also an extracellular protein, performing both a signaling and chaperoning role. Extracellular client, matrix metalloproteinase-2 (MMP2) relies on HSP90 for its stability. MMP2 mediates extracellular matrix remodeling through its gelatinolytic activity. MMP2 activity is also tightly regulated by its endogenous inhibitor, the Tissue Inhibitor of Metalloproteinase-2 (TIMP2). At present how HSP90 performs its chaperoning role in the extracellular matrix is uncertain. In this thesis, I describe that TIMP2 acts as the first bona fide extracellular co-chaperone of eHSP90, and show that TIMP2 is a stress inducible protein. I describe how TIMP2 directly interacts with HSP90, and how TIMP2 decelerates the HSP90 ATPase cycle. TIMP2 also sensitizes HSP90 to both ATP and N-terminal pharmaceuticals. Overall, TIMP2 acts as both a scaffold and a disruptor of the client/chaperone relationship between MMP2 and HSP90, performing both a HSP90 co-chaperone and MMP2 inhibitor role, non-mutually exclusively. The activatory co-chaperone AHA1 competes with TIMP2 for HSP90 binding. TIMP2 and AHA1 are able to form two independent ternary complexes with MMP2 and HSP90; as a result, the TIMP2 complex is MMP2 proteolytically inactive and the AHA1, active. This competition is further described in vivo where it can be inhibited by both _AHA1 antibodies and TIMP2 AHA1 antibodies and TIMP2 exogenous protein treatments, whilst induced following AHA1 protein and _AHA1 antibodies and TIMP2 TIMP2 antibody treatments. Finally, the role of phos-Y-TIMP2 was examined in relation to its interaction with HSP90. To address this, a novel methodology to purify hTIMP2 from E.Coli without previously necessary refolding strategies in a scale-able manner suitable for therapeutic TIMP2 treatments, was developed. Wild type recombinant human TIMP2 and phospho-mutants Y90E, Y90F, and TE (Y62E, Y90E, Y165E) were purified, were inhibitory towards MMP2, and modulated TIMP2 interaction with HSP90. Taken together, I demonstrate how extracellular HSP90 is regulated by co-chaperones to facilitate the chaperoning of pro-invasive client, MMP2. It further shows ways in which we can manipulate this system to promote an inactive MMP2 protease, a key strategy in cancer therapeutics.
dc.rightsAttribution-NonCommercial-NoDerivatives 4.0 International
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subjectExtracellular
dc.subjectCo-chaperone
dc.subjectECM
dc.subjectChaperone
dc.titleIdentification of TIMP2 as the first secretory co-chaperone of eHSP90
dc.typeDissertation
dc.description.institutionUpstate Medical University
dc.description.departmentBiochemistry & Molecular Biology
dc.description.degreelevelPhD


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