Heart disease and resultant heart failure remain a leading cause of death and disability in humans, with heart failure being the most expensive single diagnosis in US healthcare. Protein degradation by the ubiquitin-proteasome system (UPS) is pivotal to protein quantity and quality control in the cell. UPS dysfunction especially proteasome functional insufficiency and increased proteotoxic stress (IPTS) play a major pathogenic role in the progression from a large subset of heart disease to heart failure and, accordingly, proteasome enhancement promises to become a new strategy to battle heart disease with IPTS. Developing more effective therapeutics to enhance the proteasome, however, requires better understanding how proteasome function is regulated. Recent advance in cell biology excitingly unravels the proteasome as a nodal point for controlling UPS proteolytic function. For example, it is recently reported that the 19S proteasome subunit RPN6/PSMD11 can be specifically phosphorylated at Ser14 by cAMP-dependent kinase (PKA), resulting in marked increases in the proteolytic function of the proteasome in cultured cells. We have reported that cGMP-dependent kinase (PKG) activates the proteasome, facilitates proteasomal degradation of misfolded proteins and thereby protects against cardiac IPTS but the proteasome phosphosite(s) of PKG remains undefined. Phosphodiesterase (PDE) 1, an enzyme known to degrade both cGMP and cAMP and thereby suppress PKG and PKA, is upregulated in mouse and human failing hearts. We have discovered that PDE1 is increased in mouse hearts with IPTS and PDE1 inhibition (PDE1i) improves UPS performance in a PKA- and PKG-dependent manner and confers striking therapeutic benefits to a bona fide mouse model of heart disease with IPTS. We newly created the Rpn6S14A and Rpn6S14D gene knock-in mice and confirmed that proteasome activation by PKA is specifically blocked and mimicked in them, respectively. Our pilot studies reveal that elevating cGMP but not cAMP further increases myocardial 26S proteasome activities in the Rpn6S14D mice. Hence, we propose and this project will test the novel hypothesis that distinctive but complementary molecular mechanisms are taken by PKG and PKA to activate the proteasome such that dual activation of PKG and PKA can complementarily enhance proteasome proteolytic function and thereby protect against cardiac IPTS. We will identify proteasome phosphosite(s) responsible for the proteasome activation by PKG using tandem mass spectrometry based proteomics combined with biochemical and genetic interrogations, determine the role of proteasome phosphoregulation by PKA in PDE1i-induced protection against cardiac IPTS, and test the effects of PKG and PKA duo-activation on UPS performance and protection against cardiac IPTS. Completion of this project will not only delineate the molecular mechanism by which PKG regulates the proteasome but also provide comprehensive experimental basis for developing PKG and PKA duo-activation as a novel strategy to treat heart disease with IPTS, a large subset of heart disease in humans.
Despite recent advances in both basic research and clinical management, heart disease and resultant heart failure remain the leading cause of death and disability in the US and even for the entire mankind. This research project will test a potential new therapeutic strategy by exploitation of how heart muscle cells control their protein breakdown pathways to increase their ability to remove unwanted and unneeded proteins in the cell during diseased states. This will help deepen our understanding of the molecular mechanisms underlying the progression from a large subset of heart diseases to heart failure and contribute to the search for new strategies to prevent or more effectively treat this common and yet life-threatening disorder.
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