The molecular bases of inherited urea cycle disorders and ureagenesis regulation

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www.example.com：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。

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).