N-acetylglutamate Synthase: Structure, Function & Defects

N-乙酰谷氨酸合成酶：结构、功能

基本信息

批准号：
9261515
负责人：
Ljubica Morizono Caldovic
金额：
$ 35.88万
依托单位：
CHILDREN'S RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-08-15 至 2019-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9261515
关键词：
Acetyl Coenzyme A Address Affect Affinity Chromatography Affinity Labels Ammonia Arginine Binding Binding Sites Biochemical Bioinformatics Biological Assay Biology Blood Brain Brain Injuries Carbamyl Phosphate Cell membrane Cells Cloning Co-Immunoprecipitations Crystallization DNA Defect Development Diagnosis Distal Dose Elements Enzymes Epithelial Epithelial Cells Evolution Funding Gene Proteins Genes Genetic Transcription Genotype Glutamates Hepatocyte Homology Modeling Human Hyperammonemia Intestines Introns Investigation Knockout Mice Knowledge Life Ligands Ligase Liver Liver Mitochondria Liver diseases Mammals Mass Spectrum Analysis Mediating Metabolic Methods Mitochondria Mitochondrial Matrix Molecular Diagnosis Mus N acetyl L glutamate N-carbamylglutamate N-terminal Nitrogen Nucleic Acid Regulatory Sequences Patients Phenotype Physiological Play Post-Translational Regulation Production Proteins Regulation Reporter Research Resistance Role Small Interfering RNA Structure System Tertiary Protein Structure Testing Toxic effect Transcriptional Regulation Transfection Transgenes Untranslated RNA Urea Work X-Ray Crystallography affinity labeling analog chromatin immunoprecipitation clinically relevant human disease improved in vivo inhibitor/antagonist insight knock-down metabolic phenotype mouse model mutant new therapeutic target nitrogen metabolism novel novel strategies polyproline promoter protein protein interaction public health relevance three dimensional structure tool transcription factor

项目摘要

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.

描述（由申请人提供）：该项目重点关注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。虽然氨甲酰磷酸合成酶 1 (CPS1) 通常被称为尿素生成的第一个限速酶，但它需要 NAG 作为必需的变构激活剂。因此，肝脏和小肠上皮细胞线粒体基质中NAG的水平在调节尿素生成中发挥重要作用。该 NAGS/NAG 系统是高氨血症新治疗的新兴靶标。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、其临床相关性以及如何利用它来开发管理和治疗高氨血症的新方法的许多重要问题。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。为此，我们将阐明 NAGS 基因的两个保守非编码序列在其转录调控中的功能重要性。这将通过研究肝脏和肠道来源的细胞并使用生物信息学、报告基因、染色质免疫沉淀和 DNA 下拉测定来实现。目标 2 - 确定 L-精氨酸激活哺乳动物 NAGS 的结构基础和生理作用。该目标将通过使用有或没有结合底物的哺乳动物 NAGS 的 X 射线晶体学，探索 (a) L-精氨酸在蛋白质水平上对 NAGS 活性的正调节作用的结构和机制相关性，以及 (b) 对尿素生成的体内影响以及通过转染精氨酸不敏感和敏感 NAGS 转基因在 koNAGS 小鼠中精氨酸激活 NAGS 的其他代谢参数。目标 3 - 表征 NAGS 的 N 末端和 CPS1 之间的结构和功能相互作用。该目标将使用 X 射线晶体学来确定 NAGS-CPS1 相互作用的结构基础，并且将研究用各种 NAGS 突变体转染的 koNAGS 小鼠，以确定氮代谢的变化，从而揭示这种相互作用的功能重要性。目标 4 - 确定 N-氨甲酰谷氨酸 (NCG) 递送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 敲除方法来识别参与将 NCG 递送至肝和肠细胞线粒体的转运蛋白。总体而言，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 缺乏症以及与高氨血症相关的其他疾病。

项目成果

期刊论文数量（9）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Gene delivery corrects N-acetylglutamate synthase deficiency and enables insights in the physiological impact of L-arginine activation of N-acetylglutamate synthase.

基因传递纠正了 N-乙酰谷氨酸合酶缺陷，并能够深入了解 L-精氨酸激活 N-乙酰谷氨酸合酶的生理影响。

DOI：
发表时间：
2021
期刊：
影响因子：
4.6
作者：
Sonaimuthu, P;Senkevitch, E;Haskins, N;Uapinyoying, P;McNutt, M;Morizono, H;Tuchman, M;Caldovic, L
通讯作者：
Caldovic, L

Transcriptional regulation of N-acetylglutamate synthase.

N-乙酰谷氨酸合酶的转录调控。

DOI：
发表时间：
2012
期刊：
影响因子：
3.7
作者：
Heibel, Sandra Kirsch;Lopez, Giselle Yvette;Panglao, Maria;Sodha, Sonal;Mariño;Tuchman, Mendel;Caldovic, Ljubica
通讯作者：
Caldovic, Ljubica

N-Acetylglutamate Synthase Deficiency Due to a Recurrent Sequence Variant in the N-acetylglutamate Synthase Enhancer Region.

由于 N-乙酰谷氨酸合酶增强子区域中的重复序列变异导致 N-乙酰谷氨酸合酶缺陷。

DOI：
发表时间：
2018
期刊：
影响因子：
4.6
作者：
Williams, Monique;Burlina, Alberto;Rubert, Laura;Polo, Giulia;Ruijter, George J G;van den Born, Myrthe;Rüfenacht, Véronique;Haskins, Nantaporn;van Zutven, Laura J C M;Tuchman, Mendel;Saris, Jasper J;Häberle, Johannes;Caldovic, Ljubica
通讯作者：
Caldovic, Ljubica

A novel bifunctional N-acetylglutamate synthase-kinase from Xanthomonas campestris that is closely related to mammalian N-acetylglutamate synthase.

一种来自野油菜黄单胞菌的新型双功能 N-乙酰谷氨酸合酶激酶，与哺乳动物 N-乙酰谷氨酸合酶密切相关。