Molecular Regulation of Ureagenesis

尿素生成的分子调控

• 批准号: 7384805
• 负责人: Ljubica Morizono Caldovic
• 金额: $ 10.52万
• 依托单位: CHILDREN'S RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7384805
• 关键词: Ammonia; Animals; Bioinformatics; Blood; Cleaved cell; Densitometry; Development; Dietary Proteins; Drosophila genus; Endopeptidases; Enzymes; Gene Expression; Genes; Goals; Homologous Gene; Individual; Intake; Intervention; Lead; Liver; Liver Mitochondria; Mass Spectrum Analysis; Measures; Messenger RNA; Mitochondria; Modification; Molecular; Mus; Nitrogen; Organism; Patients; Peptide Hydrolases; Play; Post-Translational Protein Processing; Process; Proteins; Proteolytic Processing; RNA; Regulation; Relative (related person); Repression; Role; Signal Pathway; Signaling Molecule; Testing; Time; Two-Dimensional Gel Electrophoresis; Urea; Variant; career; hepatic ureagenesis; response; tool; urea cycle

DESCRIPTION (provided by applicant): This is a revised career development proposal, aimed at understanding molecular regulation of the urea cycle. The urea cycle functions in the liver to convert neuro-toxic ammonia into non-toxic urea. One potential strategy to alleviate the effects of high blood ammonia is to increase patients' innate ability to produce urea by raising the expression of urea cycle enzymes. Such interventions require an understanding of the regulation of ureagenesis at the molecular level. The activities of all urea cycle enzymes coordinately change in response to varying nitrogen loads. However, it appears that different mechanisms regulate gene expression of individual urea cycle genes. The goal of this project is to understand the regulation of ureagenesis at the molecular level. The specific aims of this project are: 1) To determine the functional and structural changes in liver NAGS in response to changing dietary protein intake. My hypothesis is that the ratio of two NAGS variants in the liver mitochondria is determined by the organism's nitrogen load. Furthermore, changes of the ratio of two NAGS variants reflect a post- translational mechanism for regulation of hepatic ureagenesis in response to varying nitrogen load. This hypothesis will be tested by measuring the relative amounts of the two NAGS variants in the livers from mice exposed to different nitrogen load, and identification of mitochondrial protease(s) capable of processing NAGS. 2) To identify signaling pathways that respond to changes in nitrogen loads using transcriptional profiling. My hypothesis is that change in nitrogen loads lead to changes in expression levels of signaling molecules that result in either activation or repression of urea cycle genes. This hypothesis will be tested by measuring changes of mRNA levels in response to varying nitrogen load by transcriptional profiling of RNA using Affymetrix microarrays. 3) To identify proteins whose amounts and post- translational modifications change in response to changing nitrogen loads. My hypothesis is that changes in nitrogen loads triggers changes in amounts and/or post-translational modifications of proteins in one or more signaling pathways, resulting in change of expression and activity of urea cycle enzymes. This hypothesis will be tested by measuring changes in protein levels and their modifications in response to varying nitrogen loads using two- dimensional gel electrophoresis, densitometry and mass spectrometry.

描述（由申请人提供）： 这是修订后的职业发展提案，旨在了解尿素循环的分子调控。尿素循环在肝脏中发挥作用，将神经毒性氨转化为无毒尿素。减轻高血氨影响的一种潜在策略是通过提高尿素循环酶的表达来增强患者产生尿素的先天能力。此类干预措施需要在分子水平上了解尿素生成的调节。所有尿素循环酶的活性随着不同的氮负荷而协调变化。然而，似乎有不同的机制调节各个尿素循环基因的基因表达。该项目的目标是在分子水平上了解尿素生成的调节。该项目的具体目标是： 1) 确定肝脏 NAGS 因膳食蛋白质摄入量变化而发生的功能和结构变化。我的假设是肝脏线粒体中两种 NAGS 变体的比例是由生物体的氮负荷决定的。此外，两个 NAGS 变体比例的变化反映了响应不同氮负荷调节肝尿素生成的翻译后机制。该假设将通过测量暴露于不同氮负荷的小鼠肝脏中两种 NAGS 变体的相对量以及鉴定能够处理 NAGS 的线粒体蛋白酶来检验。 2) 使用转录分析来识别响应氮负荷变化的信号通路。我的假设是氮负荷的变化会导致信号分子表达水平的变化，从而导致尿素循环基因的激活或抑制。该假设将通过使用 Affymetrix 微阵列对 RNA 进行转录分析来测量 mRNA 水平随氮负荷变化的变化而得到检验。 3) 鉴定其数量和翻译后修饰随氮负荷变化而变化的蛋白质。我的假设是，氮负荷的变化会引发一种或多种信号传导途径中蛋白质的数量和/或翻译后修饰的变化，从而导致尿素循环酶的表达和活性的变化。该假设将通过使用二维凝胶电泳、光密度测定和质谱测量蛋白质水平的变化及其响应不同氮负荷的修饰来检验。