Project Summary: Abdominal aortic aneurysm (AAA) is a life-threatening disease that afflicts ~1.5 million Americans, particularly in the elderly populations. Despite its high mortality (80-90%) upon rupture, there is a lack of effective drug therapies for clinical management of AAA. Surgical repairs remain the only effective options; however, not only are they considered traumatic and risky, but also not indicated for the majority of newly diagnosed AAA patients. Therefore, there is a pressing unmet need to develop non-surgical strategies to contain AAA progression. Dietary restrictions of amino acids emerge as promising avenues to combat cardiovascular diseases. For the first time, our study demonstrated the potential of dietary methionine or leucine restrictions in inhibiting the development of AAA in a rat model. It is well established that such dietary restrictions trigger the amino acid starvation response, a potent protective mechanism driven by the metabolic stress kinase general control nondepressible 2 (GCN2). Indeed, in our preliminary study, silencing GCN2 in rat abdominal aorta nullified the benefits of methionine restriction against AAA, suggesting a protective role of GCN2 in reinstating aortic homeostasis. Activating GCN2 through methionine restriction protected smooth muscle cells (SMCs) from undergoing degeneration, which is one of the key determinants of AAA pathogenesis; conversely, reduced GCN2 activity was robustly observed in clinical specimens of AAA, and GCN2 silencing effectively compromised the protection against SMC degeneration. Finally, we serendipitously identified GCN2 to be citrullinated by peptidyl arginine deiminase 3 (PADI3) at its 1475 arginine (R1475) residue in the starvation-sensing domain. This post-translational modification (PTM) of GCN2 was increased in AAA and correlated with repressed GCN2 activity. Collectively, these preliminary results lead to our central hypothesis: GCN2 plays a pivotal role in protecting against SMC degeneration and AAA formation, and its activity is negatively modulated by PADI3-mediated citrullination; GCN2-activating strategies offer a new paradigm for effective prevention and intervention of AAA. In Aim 1, we will dissect the role of GCN2 in safeguarding SMC homeostasis as well as its downstream signaling using human and murine SMCs. In Aim 2, we will characterize a new PTM regulation that negatively modulates GCN2 activity and delineate the biological consequences of GCN2 citrullination in SMC. In Aim 3, we will determine the mechanistic and therapeutic implications of GCN2 activation in AAA, utilizing both transgenic (SMC-specific GCN2 knockout mice) and dietary intervention approaches (methionine restriction for treatment of pre-existing AAA lesions). The successful completion of the proposed studies will not only uncover an intrinsic protective mechanism against SMC degeneration and AAA pathogenesis, but also may provide a new paradigm through GCN2-activating dietary restr...