Despite promising data in vitro, chaperone inhibitors have met with limited success in clinical trials due to inherent toxicity of a drug that targets an essential cellular protein. Small molecule inhibitors of chaperones have been developed and assessed for their ability to inhibit cancer cell proliferation in vivo and in vitro. Cancer cells require Hsp70 to maintain the function of unstable oncoproteins and as such are “addicted” to chaperone function and Hsp70 is often found to be overexpressed in breast and prostate cancers. Many housekeeping proteins require Hsp70 for stability, making Hsp70 essential for cell viability (2). While Hsp70 assists both in the folding of newly synthesized proteins and denatured proteins (“clients”), it also targets damaged proteins for degradation by the proteasomal system. Heat Shock Protein 70 (Hsp70) is a well-conserved, highly expressed molecular chaperone protein. Taken together, this work suggests a novel anticancer strategy-inhibition of RNR by targeting Hsp70 co-chaperone function. Mammalian cells lacking HDJ2 were significantly more sensitive to RNR inhibiting drugs such as hydroxyurea, gemcitabine and triapine. Perturbation of the Ssa1-Ydj1 interaction through mutation or Hsp70-HDJ2 via the small molecule 116-9e compromised RNR function, suggesting chaperone dependence of this novel role. Suggesting broad conservation from yeast to human, HDJ2 binds R2B and regulates RNR stability in human cells. Ydj1 bound the RNR subunit Rnr2 and cells lacking Ydj1 showed a destabilized RNR complex. Ydj1, an important cytoplasmic Hsp70 co-chaperone was identified to be required for growth on HU. To identify co-chaperone(s) involved in RNR activity, 28 yeast co-chaperone knockout mutants were screened for sensitivity to the RNR-perturbing agent Hydroxyurea. While previous studies have shown the anticancer target ribonucleotide reductase (RNR) is a client of Hsp70, the regulatory co-chaperones involved remain to be determined. Hsp70 is regulated by a suite of co-chaperone molecules that assist in Hsp70-client interaction and stimulate the intrinsic ATPase activity of Hsp70. Hsp70 is a well-conserved molecular chaperone involved in the folding, stabilization, and eventual degradation of many “client” proteins.
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