Transcriptional Regulation of N-Acetylglutamate Synthase and its Clinical Relevance

The urea cycle converts ammonia, the toxic byproduct of protein metabolism, to non-toxic urea utilizing six enzymes in mammalian liver hepatocytes. In the mitochondria, the enzyme N-acetylglutamate synthase (NAGS), produces the essential allosteric activator, N-acetylglutamate (NAG), for the first enzyme of the cycle, carbamylphosphate synthetase 1 (CPS1). Ammonia sequestered by CPS1 is condensed with bicarbonate and phosphate from ATP to produce to carbamylphosphate, which is then condensed with ornithine to produce citrulline by ornithine transcarbamylase (OTC). Citrulline is then transported to the cytoplasm where it is further converted by the distal urea cycle enzymes argininosuccinate synthetase, argininosuccinate lyase, and arginase 1 to the end product, urea. Prior to sequestration of ammonia, signaling pathways sense dietary protein load. Analysis was conducted to determine the effect of protein composition on metabolic signaling and nutrient sensing pathways AMPK, mTOR, and eIF2, promote transcriptional activation of urea cycle genes and translation of urea cycle proteins. Future studies will determine the importance of these pathways on the urea cycle and whether post-translational control mechanisms, such as phosphorylation, also respond to protein in the diet. Since NAGS plays an important role in controlling the rate of urea production by activating the rate limiting CPS1, its regulatory mechanisms control ureagenesis flux. This project elucidated the regulatory domains of NAGS including a promoter which contains multiple tissue- and species- specific transcription initiation sites and binding sites for transcription factors CREB and Sp1. It also found and characterized an enhancer 3kb upstream of the translational start site which confers liver specificity and has binding sites for NF-Y and HNF-1. These transcription factors are regulated by glucocorticoid and glucagon hormone signaling pathways and are also regulated in a tissue selective manner. A deficiency of NAGS in humans and mice leads to high ammonia, low urea, and high glutamine levels in the plasma, and can be overcome by treatment with N-carbamylglutamate (NCG). This project identified a patient with NAGS deficiency and hyperammonemia caused by a deleterious mutation in the HNF-1 binding site within the enhancer of the NAGS gene, which was identified and characterized by this research. This mutation caused decreased transcription of NAGS due to reduced HNF-1 binding. Subsequently, this work found disease-causing mutations in the promoter region of OTC which have been shown to interfere with HNF-4 binding. The knowledge garnered by this project significantly increases our understanding of the regulation of urea cycle RNA and protein expression and their role in disease pathophysiology. Understanding of these mechanisms will lead to improved diagnoses and continued development of effective treatments for people with urea cycle disorders and hyperammonemia