This project focuses on N-acetylglutamate (NAG) synthase (NAGS) and its function in urea cycle, which disposes of ammonia produced by protein catabolism. Current treatments for patients with urea cycle disorders (UCD) focus on reduction of blood ammonia levels through protein restricted diet and activation of alternative pathways for ammonia removal. Despite these treatments UCD patients experience hyperammonemia. NAG is an essential allosteric activator of the rate-limiting enzyme of ureagenesis – carbamyl phosphate synthetase 1 (CPS1). Therefore, NAGS could regulate ureagenesis through supplying variable amounts of NAG to modulate CPS1 activity and urea production. While NAGS activity is necessary for ureagenesis, it is not known whether binding of L-arginine to NAGS in vivo regulates urea production. Understanding of this pathway for control of ureagenesis will provide new targets for therapies aimed at stimulating urea production either during hyperammonemic crisis, or for long-term management of patients' ammonia removal, or both. We will use mouse models of UCD, AAV-based gene transfer, measurements of ureagenesis and visualization of enzymes in vivo to test our overarching hypothesis that activation of NAGS by L-arginine and localized interactions of NAGS with CPS1 and ornithine transcarbamylase (OTC) modulate ureagenesis by regulating the amount of NAG provided to activate CPS1. The following specific aims will test this hypothesis: Aim 1: Is L-arginine binding to NAGS in vivo the trigger for dietary protein-dependent increase in ureagenesis? We will test the hypothesis that in vivo activation of NAGS by L-arginine regulates ureagenesis by modulating amount of NAG needed for CPS1 activity by: A. Assessing if decreased stability of NAGS enzyme that cannot bind L- arginine reduces production of NAG and urea; B. Determine if dietary L-arginine binds to NAGS in vivo to stimulate urea production; and C. Examine the role of L-arginine effect on NAGS in the adaptation of urea cycle enzymes to high protein diet. Aim 2. Examine short-term regulation of urea cycle flux by a dynamic urea cycle enzyme super-complex formation between the mitochondrial urea cycle proteins. We will test the hypothesis that formation of urea cycle enzyme super-complex is a short-term regulatory mechanism that enables efficient ureagenesis on demand by: A. Structural modeling and biochemical analysis to decipher 3D organization of the NAGS-CPS1-OTC super-complex in basal and high protein intake states; B. Employing in situ super-resolution microscopy to visualize and compare the nanoscale organization of NAGS-CPS1-OTC super-complex at the hepatocyte inner mitochondrial membrane in healthy and disease models; and C. Monitoring assembly and activity of the NAGS-CPS1-OTC complex based on substrate channeling. These studies will directly address for the first time whether NAGS regulates ureagenesis in vivo, determine the responsible mechanisms, explain how high protein diet e...