The N-end rule pathway relates the in vivo half-life of a protein to the identity of its N terminal residue. In this pathway, a substrate bearing N-degron is recognized and ubiquitylated by a family of E3 ubiquitin ligases named UBR proteins. The N-end rule pathway is implicated in various physiological and pathological processes including cardiac development and angiogenesis. It has been previously shown that mice lacking ATE1, which mediates N-terminal arginylation, die during embryogenesis associated with various defects in cardiovascular development. The goal of my graduate research was to understand the function of the N-end rule pathway in cardiovascular development, signaling, and homeostasis. In my first project, I employed a genome-wide functional proteomic approach to identify physiological substrates of ATE1, that potentially underlie the above cardiovascular phenotypes. I found that RGS4, RGS5, and RGS16 are in vivo substrates of the N-end rule pathway, the first to be identified in mammals. These RGS proteins, emerging regulators for cardiovascular G protein signaling, were degraded through sequential N-terminal modifications including N-terminal exposure of their Cys 2, its oxidation, and arginylation. In the second project, to understand the physiological meaning of ATE1-mediated RGS proteolysis in cardiac development and signaling, I characterized ATE1-/- mice and embryonic cardiomyocytes with an emphasis on GPCR signaling. I found that cell-autonomous function of ATE1 regulates the proliferation of cardiomyocytes and the homeostasis of Gq-dependent cardiac signaling. In the third project, I explored a model of heterovalent interaction by developing RF-C11, a small molecule inhibitor of the N-end rule pathway. Its two heterovalent ligands were designed to cooperatively target two cognate sites of N-recognins. RF-C11 showed higher inhibitory efficiency than its homovalent controls, providing molecular basis of designing multivalent inhibitors for specific intracellular pathways. Moreover, the treatment of RF-C11 reduced cardiac proliferation and hypertrophy in cardiomyocytes, unveiling a previously unknown function of the pathway in cardiac proliferation and signaling. In summary, my graduate research contributes to comprehensive understanding of the function of the N-end rule pathway in the cardiovascular system
