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The molecular bases of inherited urea cycle disorders and ureagenesis regulation

遗传性尿素循环障碍和尿素生成调节的分子基础

基本信息

批准号：
7886489
负责人：
Mendel Tuchman
金额：
$ 33.27万
依托单位：
CHILDREN'S RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-06-16 至 2012-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7886489
关键词：
Adult Affinity Chromatography Arginine Biochemical Biochemistry Biology Carbamyl Phosphate Carrier Proteins Catalysis Cells Complex Defect Dependency Disease Environment Enzyme Kinetics Escherichia coli Evolution Functional disorder Funding Genes Genotype Glutamates Heterozygote Human Hyperammonemia Immunoprecipitation Impairment In Vitro Inborn Genetic Diseases Individual Inherited Insecta Intake Intestines Investigation Ligands Ligase Liver Mass Spectrum Analysis Measures Methodology Methods Mitochondria Modeling Molecular Motion Mus Mutation Mutation Spectra N acetyl L glutamate N-carbamylglutamate N-terminal Neuraxis Nitrogen Ornithine Ornithine Carbamoyltransferase Patients Peptide Hydrolases Peptide Leader Sequences Phenotype Physiological Process Property Protein-Restricted Diet Proteins Recombinants Regulation Relative (related person)Research Research Personnel Role Secondary to Small Intestines Solutions Structure Substrate Specificity System Tertiary Protein Structure Tissues Transfection Translational Research Variant Western Blotting Writing Yeast Model System Yeasts analog analytical ultracentrifugation base cofactor crosslink ent-kaurene synthetase A healthy volunteer hepatic ureagenesis human NAT7 protein improved in vivo ketotic hyperglycinemia methylmalonic aciduria mouse argA protein mutant novel novel therapeutic intervention ornithine transporter ornithinemia pathogen programs protein protein interaction research study response stable isotope three dimensional structure tool transcarbamylase urea cycle

项目摘要

DESCRIPTION (provided by applicant): This is a continuing proposal to study the mechanism and regulation of ureagenesis and its inherited disorders. Our previous studies have elucidated the correlations between sequence, structure and function of ornithine transcarbamylase (OTC) and the spectrum of mutation and genotype/phenotype relationships. A yeast model of the human ornithine transporter has been created and the wild type and prevalent mutant characterized in the mitochondrial environment. The mouse and human N-acetylglutamate synthase (NAGS) genes were identified and cloned and mutations causing hyperammonemia were identified. Recently, we found evidence for multiple NAGS proteins in the liver and for in vivo and in vitro interactions between NAGS and carbamyl phosphate synthetase I (CPSI). We also showed that N-carbamylglutamate (NCG) restores ureagenesis to normal in NAGS deficient patients. This proposal focuses on the mechanisms by which ureagenesis is regulated by NAGS protein processing, NAGS-CPS interactions and N- acetylglutamate's role in modulating nitrogen flux through the urea cycle. The specific aims are.to: 1. Investigate functional and structural changes in NAGS proteins of liver and intestine in response to changing nitrogen load. 2. Characterize the interactions between NAGS and CPSI. 3. Study the effect of the NAG analog NCG on ureagenesis in healthy volunteers and patients with urea cycle disorders and organic acidemias using stable isotope enrichment methods. The N-termini of the variably processed NAGS proteins will be determined. The amounts of the different NAGS proteins and how they change in response to nitrogen intake will be determined using western blotting and mass-spectrometry. The interactions between CPSI and the "mature" and "conserved" NAGS proteins, as well as the variable domain peptide (VDP) will be characterized by immunoprecipitation, protein affinity chromatography, analytical ultracentrifugation, protein cross-linking and mass spectrometry, and the effects on catalysis will be studied by enzyme kinetics experiments. In a study involving healthy volunteers, in vivo, steady state, stable isotope enrichment methodology will be used to explore the role of NAG in short-term regulation of ureagenesis by measuring ureagenesis before and after NCG administration on low vs. high protein intake. The same methodology will then be used to examine the effect of NCG on ureagenesis in patients with primary NAG deficiency (NAGS deficiency), NAG dependency (CPSI deficiency) and secondary NAG deficiency (propionic, methylmalonic acidemia).

描述（由申请人提供）：这是一项研究尿素生成及其遗传性疾病的机制和调控的持续提案。我们以前的研究已经阐明了鸟氨酸转氨甲酰酶（OTC）的序列、结构和功能与突变谱和基因型/表型关系之间的相关性。已经建立了人鸟氨酸转运蛋白的酵母模型，并且野生型和普遍突变体在线粒体环境中表征。对小鼠和人的N-乙酰谷氨酸合酶（NAGS）基因进行了鉴定和克隆，并鉴定了引起高氨血症的突变。最近，我们发现了多种NAGS蛋白在肝脏和NAGS和氨甲酰磷酸合成酶I（CPSI）之间的体内和体外相互作用的证据。我们还发现，N-氨甲酰谷氨酸盐（NCG）恢复尿素生成正常的NAGS缺乏患者。该建议的重点是通过NAGS蛋白质加工、NAGS-CPS相互作用和N-乙酰谷氨酸盐在调节通过尿素循环的氮通量中的作用来调节尿素生成的机制。具体目标are.to：1.研究肝脏和肠道NAGS蛋白质响应氮负荷变化的功能和结构变化。2.描述NAGS和CPSI之间的相互作用。3.用稳定同位素富集法研究NAG类似物NCG对健康志愿者和尿素循环障碍和有机酸中毒患者的尿素生成的影响。将测定经酶处理的NAGS蛋白的N-末端。不同NAGS蛋白质的量以及它们如何响应氮摄入而变化将使用蛋白质印迹法和质谱法来确定。CPSI和“成熟”和“保守”NAGS蛋白，以及可变结构域肽（VDP）之间的相互作用将通过免疫沉淀，蛋白质亲和层析，分析超离心，蛋白质交联和质谱法进行表征，并将通过酶动力学实验研究对催化的影响。在一项涉及健康志愿者的研究中，将使用体内、稳态、稳定同位素富集方法，通过在低蛋白质摄入量与高蛋白质摄入量下测量NCG给药前后的尿素生成，探索NAG在短期调节尿素生成中的作用。然后将使用相同的方法来检查NCG对原发性NAG缺乏症（NAGS缺乏症）、NAG依赖症（CPSI缺乏症）和继发性NAG缺乏症（丙酸、甲基丙二酸血症）患者的尿素生成的影响。

项目成果

期刊论文数量（27）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Blood levels of ammonia and nitrogen scavenging amino acids in patients with inherited hyperammonemia.

DOI：
10.1006/mgme.1998.2783
发表时间：
1999
期刊：
Molecular genetics and metabolism
影响因子：
3.8
作者：
M. Tuchman;M. Yudkoff
通讯作者：
M. Tuchman;M. Yudkoff

Identification of 'private' mutations in patients with ornithine transcarbamylase deficiency.

鸟氨酸转氨甲酰酶缺乏症患者的“私人”突变的鉴定。

DOI：
10.1023/a:1005301513465
发表时间：
1997
期刊：
Journal of inherited metabolic disease
影响因子：
4.2
作者：
Tuchman,M;Morizono,H;Rajagopal,BS;Plante,RJ;Allewell,NM
通讯作者：
Allewell,NM

Inversion of allosteric effect of arginine on N-acetylglutamate synthase, a molecular marker for evolution of tetrapods.

DOI：
10.1186/1471-2091-9-24
发表时间：
2008-09-18
期刊：
BMC biochemistry
影响因子：
0
作者：
Haskins N;Panglao M;Qu Q;Majumdar H;Cabrera-Luque J;Morizono H;Tuchman M;Caldovic L
通讯作者：
Caldovic L

Polymorphisms in the human ornithine transcarbamylase gene useful for allele tracking. Mutations in brief no. 193. Online.

人鸟氨酸转氨甲酰酶基因的多态性可用于等位基因追踪。