Comprehensive characterization of the Hsp70 interactome reveals novel client proteins and interactions mediated by posttranslational modifications.

Hsp70 interactions are critical for cellular viability and the response to stress. Previous attempts to characterize Hsp70 interactions have been limited by their transient nature and the inability of current technologies to distinguish direct versus bridged interactions. We report the novel use of cross-linking mass spectrometry (XL-MS) to comprehensively characterize the Saccharomyces cerevisiae (budding yeast) Hsp70 protein interactome. Using this approach, we have gained fundamental new insights into Hsp70 function, including definitive evidence of Hsp70 self-association as well as multipoint interaction with its client proteins. In addition to identifying a novel set of direct Hsp70 interactors that can be used to probe chaperone function in cells, we have also identified a suite of posttranslational modification (PTM)-associated Hsp70 interactions. The majority of these PTMs have not been previously reported and appear to be critical in the regulation of client protein function. These data indicate that one of the mechanisms by which PTMs contribute to protein function is by facilitating interaction with chaperones. Taken together, we propose that XL-MS analysis of chaperone complexes may be used as a unique way to identify biologically important PTMs on client proteins.

Chemogenomic screening identifies the Hsp70 co-chaperone DNAJA1 as a hub for anticancer drug resistance.

Heat shock protein 70 (Hsp70) is an important molecular chaperone that regulates oncoprotein stability and tumorigenesis. However, attempts to develop anti-chaperone drugs targeting molecules such as Hsp70 have been hampered by toxicity issues. Hsp70 is regulated by a suite of co-chaperone molecules that bring "clients" to the primary chaperone for efficient folding. Rather than targeting Hsp70 itself, here we have examined the feasibility of inhibiting the Hsp70 co-chaperone DNAJA1 as a novel anticancer strategy. We found DNAJA1 to be upregulated in a variety of cancers, suggesting a role in malignancy. To confirm this role, we screened the NIH Approved Oncology collection for chemical-genetic interactions with loss of DNAJA1 in cancer. 41 compounds showed strong synergy with DNAJA1 loss, whereas 18 dramatically lost potency. Several hits were validated using a DNAJA1 inhibitor (116-9e) in castration-resistant prostate cancer cell (CRPC) and spheroid models. Taken together, these results confirm that DNAJA1 is a hub for anticancer drug resistance and that DNAJA1 inhibition is a potent strategy to sensitize cancer cells to current and future therapeutics. The large change in drug efficacy linked to DNAJA1 suggests a personalized medicine approach where tumor DNAJA1 status may be used to optimize therapeutic strategy.

Oligomerization of Hsp70: Current Perspectives on Regulation and Function.

The Hsp70 molecular chaperone in conjunction with Hsp90 and a suite of helper co-chaperones are required for the folding and subsequent refolding of a large proportion of the proteome. These proteins are critical for cell viability and play major roles in diseases of proteostasis which include neurodegenerative diseases and cancer. As a consequence, a large scientific effort has gone into understanding how chaperones such as Hsp70 function at the in vitro and in vivo level. Although many chaperones require constitutive self-interaction (dimerization and oligomerization) to function, Hsp70 has been thought to exist as a monomer, especially in eukaryotic cells. Recent studies have demonstrated that both bacterial and mammalian Hsp70 can exist as a dynamic pool of monomers, dimer, and oligomers. In this mini-review, we discuss the mechanisms and roles of Hsp70 oligomerization in Hsp70 function, as well as thoughts on how this integrates into well-established ideas of Hsp70 regulation.