PROJECT SUMMARY Although secreted molecular chaperone heat shock protein 90 (eHSP90) has been implicated in tumor cell invasion through binding and stabilizing extracellular matrix protease client MMP2, our limited understanding of the regulatory mechanisms that control the eHSP90 chaperone system is a major drawback for designing successful HSP90-targeted therapies. Our long-term goal is to develop therapeutic strategies that prevent tumor cell invasion by targeting the extracellular HSP90 chaperone machinery. Our overall objective is to elucidate how eHSP90 and its regulators are mechanistically modulated, individually and collectively, and in the presence of their extracellular inhibitors. Our central hypothesis is that extracellular ATP, and tyrosine phosphorylation of eHSP90 co-chaperone TIMP2, modulate the chaperoning of client MMP2, and affect HSP90 binding to extracellular inhibitors. This hypothesis is based on our recent published work and preliminary studies showing that eHSP90 binds and hydrolyzes ATP, and that co-chaperone TIMP2 enhances HSP90 binding to ATP and its pharmacologic inhibitors. Our rationale is that by elucidating mechanisms of eHSP90 chaperone regulation, we will design better therapies for diseases that implicate the eHSP90 machinery. We plan to test our central hypothesis by pursuing the following three specific aims: 1) Investigate the role of ATPase activity in the regulation of extracellular HSP90 (eHSP90); 2) Determine the impact of tyrosine phosphorylation of TIMP2 on its co-chaperone function; and 3) Elucidate the role of TIMP2 in enhancing eHSP90 binding to extracellular inhibitors. Under the first aim, approaches to be used are already established in PI's laboratory and include purification of intracellular and extracellular HSP90 from human cell cultures, isothermal calorimetry to determine affinity towards an ATP analog, ATPase assays to measure ATPase activity, enzyme kinetics to determine proteolytic activity, and in vitro and in vivo protein-protein interactions in the presence and following depletion of extracellular ATP in genetic knock out cell cultures. Under the second aim, phosphomimetic or non-phosphorylatable TIMP2 mutants designed in the PI's laboratory will be used, CRISPR knock out cell lines for c-Src kinase, TIMP2 and MMP2 will be employed for protein-protein interactions and to determine impact on MMP2 activity. Under the third aim, the role of TIMP2, non-phosphorylatable and phophomimetic mutants in enhanced binding of eHSP90 to its extracellular inhibitors will be evaluated in drug binding assays performed in vitro and extracellularly in vivo, using human cell lines and siRNA technology. The impact of TIMP2, and eHSP90 inhibitors on MMP2 mediated proteolysis will be evaluated in matrix degradation studies. The research approach is innovative, in the PI's opinion, because it represents a new and substantive departure from the status quo by shifting focus towards targeting chaperone-regulat...