Molecular Regulation of Ureagenesis

描述（由申请人提供）： 这是一个修订的职业发展建议，旨在了解尿素循环的分子调控。尿素循环在肝脏中起作用，将神经毒性氨转化为无毒尿素。减轻高血氨影响的一个潜在策略是通过提高尿素循环酶的表达来增加患者产生尿素的先天能力。这种干预措施需要在分子水平上了解尿素生成的调控。所有尿素循环酶的活性响应于不同的氮负荷而协调变化。然而，似乎不同的机制调节单个尿素循环基因的基因表达。本项目的目标是在分子水平上了解尿素生成的调控。本研究的具体目的是：1）研究肝脏NAGS在蛋白质摄入量变化时的功能和结构变化。我的假设是，肝脏线粒体中两种NAGS变体的比例是由有机体的氮负荷决定的。此外，两种NAGS变体的比率的变化反映了响应于不同氮负荷调节肝尿素生成的翻译后机制。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，并鉴定能够加工NAGS的线粒体蛋白酶来检验该假设。2)利用转录谱分析确定响应氮负荷变化的信号通路。我的假设是，氮负荷的变化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。将通过使用Affytron微阵列通过RNA的转录谱来测量响应于不同氮负荷的mRNA水平的变化来检验该假设。3)确定蛋白质的数量和翻译后修饰的变化，以响应不断变化的氮负荷。我的假设是，氮负荷的变化触发一个或多个信号通路中蛋白质的量和/或翻译后修饰的变化，导致尿素循环酶的表达和活性的变化。将通过使用二维凝胶电泳、光密度测定法和质谱法测量蛋白质水平的变化及其响应于不同氮负荷的修饰来检验该假设。

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.