Affinity Purification Probes of Potential Use To Investigate the Endogenous Hsp70 Interactome in Cancer

ABSTRACT: Heat shock protein 70 (Hsp70) is a family of proteins with key roles in regulating malignancy. Cancer cells rely on Hsp70 to inhibit apoptosis, regulate senescence and autophagy, and maintain the stability of numerous onco-proteins. Despite these important biological func-tions in cancer, robust chemical tools that enable the analysis of the Hsp70-regulated proteome in a tumor-by-tumor manner are yet unavailable. Here we take advantage of a recently reported Hsp70 ligand to design and develop an affinity purification chemical toolset for potential use in the investigation of the endogenous Hsp70-interacting proteome in cancer. We demonstrate that these tools lock Hsp70 in complex with onco-client proteins and effectively isolate Hsp70 complexes for identification through biochemical techniques. Using these tools we provide proof-of-concept analyses that glimpse into the complex roles played by Hsp70 in maintaining a multitude of cell-specific malignancy-driving proteins. The heat shock protein 70 family members (Hsp70s) areimportant cancer chaperones. They are abundantly ex-pressed in malignant tumors of various origins, and their ex-pression correlates with increased cell proliferation, poor differentiation, metastases, resistance to therapies, and poo