Project Summary Coronary heart disease, also called Ischemic Heart Disease (IHD), is the leading cause of death worldwide. Repeated episodes of cardiac stress in IHD patients leads to an increase in misfolded proteins in their hearts. In response to this acute onset of cellular stress, heat shock proteins (HSP)s are upregulated in myocardiocytes and help restore protein homeostasis (proteostasis). Heat shock cognate 71 kDa (HSC70) is an essential, HSP70 chaperone that is located in the cytosol in the absence of cellular stress. This ATP-dependent chaperone actively refolds, degrades, and disaggregates proteins. HSC70 is also critical for autophagy, a cellular recycling/degradation process that protects the heart from misfolded proteins following ischemia. Impaired HSC70 function has been implicated in heart disease, yet despite the critical role of HSC70 in cellular physiology, the molecular mechanisms that regulate nuclear transport and govern the disparate functions of HSC70 remain unknown. Protein AMPylation recently emerged as a distinct PTM that regulates HSP70 family chaperones through the addition of adenosine monophosphate (AMP) to threonine residues. This process is catalyzed by the mammalian AMPylase, HYPE. My preliminary work revealed that HYPE AMPylates HSC70 on two sites, and increased cellular AMPylation levels prevent the nuclear-cytosolic shuttling of HSC70 as well as its ATPase activity. I also found that, similar to decreased levels of the HSC70 ortholog HSP-1, increased AMPylation inhibits autophagosome formation in C. elegans. These data suggest that HYPE-mediated AMPylation of HSC70 inhibits its critical functions associated with maintaining proteostasis, including nuclear-cytosolic shuttling, protein folding, and autophagy. The goals of this proposal are to 1) define the functional impact of HYPE-mediated AMPylation on HSC70 and 2) determine the impact of AMPylation on HSC70’s nuclear-cytosolic shuttling mechanism. Ultimately, we aim to define how a novel post-translational modification (PTM), AMPylation, regulates HSC70 function in proteostasis and cardiovascular diseases. Advancing our knowledge of the mechanisms that control HSC70 function is critical to identify novel avenues to therapeutic strategies that capitalize on restoring HSC70 regulation.