The molecular bases of inherited urea cycle disorders and ureagenesis regulation

描述（由申请人提供）：这是一个持续的项目，旨在研究尿潴留及其遗传性疾病的机制和调控。我们之前的研究已经阐明了鸟氨酸转氨基甲酰基酶（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。已经建立了人类鸟氨酸转运蛋白的酵母模型，并在线粒体环境中鉴定了野生型和流行突变体。对小鼠和人n -乙酰谷氨酸合成酶（NAGS）基因进行了鉴定和克隆，并鉴定出引起高氨血症的突变。最近，我们在肝脏中发现了多种NAGS蛋白，并在体内和体外发现了NAGS与氨甲酰磷酸合成酶I （CPSI）相互作用的证据。我们还发现n -氨甲酰谷氨酸（NCG）可使NAGS缺陷患者的尿原性恢复正常。本文主要研究了NAGS蛋白加工、NAGS- cps相互作用以及N-乙酰谷氨酸在尿素循环中调节氮通量的作用对尿素发生的调控机制。具体目标是。: 1。研究肝脏和肠道NAGS蛋白在氮负荷变化下的功能和结构变化。2. 描述NAGS和CPSI之间的相互作用。3. 采用稳定同位素富集方法研究NAG类似物NCG对健康志愿者和尿素循环障碍及有机酸血症患者尿生成的影响。可变加工的NAGS蛋白的n端将被确定。不同的NAGS蛋白的数量以及它们如何随着氮的摄入而变化，将使用western blotting和质谱法来确定。利用免疫沉淀、蛋白质亲和层析、分析超离心、蛋白质交联和质谱等技术表征CPSI与“成熟”和“保守”的NAGS蛋白以及可变结构域肽（VDP）之间的相互作用，并通过酶动力学实验研究CPSI对催化作用的影响。在一项涉及健康志愿者的研究中，将采用体内稳态、稳定同位素富集方法，通过测量低蛋白摄入和高蛋白摄入NCG前后的尿床情况，探讨NAG在短期调节尿床中的作用。同样的方法将被用于检查NCG对原发性NAG缺乏症（NAG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）患者的尿原形成的影响。

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).