Pharmacological Modulation of Hsp70 to Selectively Remove Misfolded Neuronal Nitric Oxide Synthase

Abstract ID 23589 Poster Board 332 The accumulation of misfolded proteins can result in various diseases, including several neurodegenerative disorders. Critical proteins that are associated with the onset of some of these disorders are client proteins of the heat shock protein (Hsp) 90 and Hsp70 chaperone system that selectively regulates their stability and degradation. Previously, we’ve determined the opposing roles Hsp90 and Hsp70 have on client protein stability using neuronal nitric oxide synthase (nNOS), a well-established client protein. Hsp90 stabilizes misfolded client proteins and prevents their ubiquitination, whereas Hsp70 enhances their ubiquitination and degradation. Thus, the Hsp90/70 chaperone system is a promising target for the development of treatments for these disorders. Over 20 inhibitors of Hsp90 have been developed but all clinical trials to date have failed, in large part, due to toxicity issues. In that Hsp90 inhibition may target functional client proteins in near native states, we hypothesize that targeting Hsp70 may be more selective for misfolded proteins. In the current study, we have developed a cellular model stably expressing native nNOS or C331A mutant of nNOS, which represents a slightly misfolded but functionally active nNOS, to better test this hypothesis. We showed that Hsp70 modulators, such as YM-1 and JG-98, caused a time- and dose- dependent decrease in C331A mutant of nNOS while the native nNOS was not affected under the same conditions. The Hsp90 inhibitor, radicicol, was not as selective as loss of both C331A nNOS and wild type nNOS were observed. These results suggest that Hsp70 modulation selectively removes the misfolded client protein while sparing the natively folded or near native nNOS proteins. Future studies will focus on developing and characterizing other Hsp70 modulators to identify small molecules that may better enhance the ubiquitination and degradation of misfolded nNOS. This was supported in whole or in part, by National Institutes of Health Grant GM077420 and the University of Michigan Medical School’s Protein Folding Diseases Initiative. AMG is a trainee of the University of Michigan, Training Program in Translational Science (NIH T32-GM141840) and a Department of Pharmacology Lucchesi Fellow.