N-acetylglutamate Synthase: Structure, Function & Defects

项目描述（申请人提供）：本项目的重点是N-乙酰谷氨酸合酶（NAGS）基因及其催化N-乙酰谷氨酸（NAG）形成的酶产物的调控。氨甲酰磷酸合成酶1（CPS 1）通常被认为是尿素生成的第一个和限速酶，它需要NAG作为一个重要的变构激活剂。因此，肝脏和小肠上皮细胞线粒体基质中NAG的水平在调节尿素产生中起重要作用。该NAGS/NAG系统是高氨血症新治疗的新兴靶点。在之前的资助期间开发的新工具将使我们能够解决有关该系统的许多重要问题，其临床相关性，以及如何利用它来开发管理和治疗高血氨症的新方法。该项目的具体目标是：目标1 -表征NAGS的转录调控及其在人类疾病中的作用。在这个目标中，我们将阐明NAGS基因的两个保守的非编码序列在其转录调控中的功能重要性。这将通过研究肝和肠来源的细胞，并通过使用生物信息学，报告，染色质免疫沉淀和DNA下拉测定来实现。目的2 -确定L-精氨酸激活哺乳动物NAGS的结构基础和生理作用。该目的将探索（a）通过使用具有和不具有结合底物的哺乳动物NAGS的X射线晶体学，在蛋白质水平上L-精氨酸对NAGS活性的正调节作用的结构和机制相关性，以及（B）通过转染对精氨酸不敏感的和敏感的NAGS转基因，在koNAGS小鼠中对尿素生成和NAGS的精氨酸活化的其他代谢参数的体内作用。目的3 -表征NAGS和CPS 1的N-末端之间的结构和功能相互作用。这一目标将使用X射线晶体学来确定NAGS-CPS 1相互作用的结构基础，并将研究用各种NAGS突变体转染的koNAGS小鼠，以确定氮代谢的改变，这将揭示这种相互作用的功能重要性。目的4 -确定N-氨甲酰谷氨酸（NCG）转运至肝细胞和肠细胞线粒体的机制。该目标将使用亲和标记和siRNA敲低方法来鉴定参与将NCG递送至肝脏和肠细胞线粒体的转运蛋白。总的来说，该项目使用新的研究工具来获得对系统的新见解，该系统可用于更好地诊断和治疗NAGS缺乏症和其他与高氨血症相关的疾病。

DESCRIPTION (provided by applicant): 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.