This project focuses on the regulation of the N-acetylglutamate synthase (NAGS) gene and its enzyme product which catalyzes the formation of N-acetylglutamate (NAG). While carbamyl phosphate synthetase 1 (CPS1) is often referred to as the first and rate-limiting enzyme of ureagenesis, it requires NAG as an essential allosteric activator. Therefore, the levels of NAG in the mitochondrial matrix of liver and small intestinal epithelial cells play an important role in regulating urea production. This NAGS/NAG system is an emerging target for new treatment of hyperammonemia. New tools that were developed during the previous funding periods will allow us to address many important questions about this system, its clinical relevance, and how it can be exploited to develop new approaches for managing and treating hyperammonemia.
The specific aims of this project are to:
Aim 1 - Characterize the transcriptional regulation of NAGS and its role in human disease. In this aim we will elucidate the functional importance of two conserved non-coding sequences of the NAGS gene in the regulation of its transcription. This will be accomplished by studying liver and intestine derived cells and by using bioinformatics, reporter, chromatin immunoprecipitation and DNA-pull-down assays.
Aim 2 - Determine the structural basis and physiological role of mammalian NAGS activation by L-arginine.
This aim will explore (a) structural and mechanistic correlates of the positive regulatory effect of L-arginine on NAGS activity at the protein level by using x-ray crystallography of mammalian NAGS with and without bound substrates and (b) in vivo effect on ureagenesis and other metabolic parameters of arginine activation of NAGS in koNAGS mouse by transfection with arginine-insensitive and sensitive NAGS transgenes.
Aim 3 - Characterize the structural and functional interactions between the N-terminus of NAGS and CPS1.
This aim will use X-ray crystallography to determine the structural basis of NAGS-CPS1 interaction, and the koNAGS mouse transfected with various NAGS mutants will be studied to identify alterations in nitrogen metabolism that will reveal the functional importance of this interaction.
Aim 4 - Determine the mechanism of N-carbamylglutamate (NCG) delivery to hepatocyte and intestinal cell mitochondria.
This aim will use affinity labeling and siRNA knock down methods to identify transporters that are involved in delivery of NCG to liver and intestinal cell mitochondria. Overall, the project uses new tools of research to gain new insights into a system that can be exploited for better diagnosis and treatment of NAGS deficiency and other conditions associated with hyperammonemia.
This project is dedicated to the investigation of an important gene and protein (NAGS) that controls how much nitrogen we eliminate from our bodies. It is important to know this since one of the main problems in liver disease is the inability to eliminate toxic nitrogen (ammonia) which can damage the brain. We will study liver and intestinal cells, and a mouse model that does not have the NAGS gene and protein, to allow us to better understand this system and how it works. The results from this project should allow the development of new treatments for elevated ammonia in the blood to protect the brain from its toxic effects.
|Ah Mew, Nicholas; McCarter, Robert; Daikhin, Yevgeny et al. (2014) Augmenting ureagenesis in patients with partial carbamyl phosphate synthetase 1 deficiency with N-carbamyl-L-glutamate. J Pediatr 165:401-403.e3|
|Caldovic, Ljubica; Haskins, Nantaporn; Mumo, Amy et al. (2014) Expression pattern and biochemical properties of zebrafish N-acetylglutamate synthase. PLoS One 9:e85597|
|Zhao, Gengxiang; Jin, Zhongmin; Allewell, Norma M et al. (2013) Crystal structure of the N-acetyltransferase domain of human N-acetyl-L-glutamate synthase in complex with N-acetyl-L-glutamate provides insights into its catalytic and regulatory mechanisms. PLoS One 8:e70369|
|Zhao, Gengxiang; Allewell, Norma M; Tuchman, Mendel et al. (2013) Structure of the complex of Neisseria gonorrhoeae N-acetyl-L-glutamate synthase with a bound bisubstrate analog. Biochem Biophys Res Commun 430:1253-8|
|Zhao, Gengxiang; Haskins, Nantaporn; Jin, Zhongmin et al. (2013) Structure of N-acetyl-L-glutamate synthase/kinase from Maricaulis maris with the allosteric inhibitor L-arginine bound. Biochem Biophys Res Commun 437:585-90|
|Heibel, Sandra Kirsch; Lopez, Giselle Yvette; Panglao, Maria et al. (2012) Transcriptional regulation of N-acetylglutamate synthase. PLoS One 7:e29527|
|Senkevitch, Emilee; Cabrera-Luque, Juan; Morizono, Hiroki et al. (2012) A novel biochemically salvageable animal model of hyperammonemia devoid of N-acetylglutamate synthase. Mol Genet Metab 106:160-8|
|Heibel, Sandra K; Ah Mew, Nicholas; Caldovic, Ljubica et al. (2011) N-carbamylglutamate enhancement of ureagenesis leads to discovery of a novel deleterious mutation in a newly defined enhancer of the NAGS gene and to effective therapy. Hum Mutat 32:1153-60|
|Nissim, Itzhak; Horyn, Oksana; Nissim, Ilana et al. (2011) Down-regulation of hepatic urea synthesis by oxypurines: xanthine and uric acid inhibit N-acetylglutamate synthase. J Biol Chem 286:22055-68|
|Shi, Dashuang; Li, Yongdong; Cabrera-Luque, Juan et al. (2011) A novel N-acetylglutamate synthase architecture revealed by the crystal structure of the bifunctional enzyme from Maricaulis maris. PLoS One 6:e28825|
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