Molecular Regulation of Ureagenesis

描述(由申请人提供)： 这是一份经过修订的职业发展建议，旨在了解尿素循环的分子调控。尿素循环在肝脏中发挥作用，将神经毒性的氨转化为无毒的尿素。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提高患者产生尿素的先天能力。这样的干预需要在分子水平上了解尿失禁的调节。所有尿素循环酶的活性随着氮素负荷的变化而协调变化。然而，似乎不同的机制调节个别尿素循环基因的基因表达。这个项目的目标是在分子水平上了解尿失禁的调节。这个项目的具体目标是：1)确定肝脏NAG的功能和结构变化，以响应饮食蛋白质摄入量的变化。我的假设是，肝脏线粒体中两个NAG变异体的比例由生物体的氮负荷决定。此外，两个NAG变异体比率的变化反映了翻译后调节肝脏尿失禁的机制，以响应不同的氮负荷。这一假说将通过测量暴露在不同氮负荷下的小鼠肝脏中两种NAG变体的相对数量，以及鉴定能够处理NAG的线粒体蛋白酶(S)来验证。2)利用转录图谱确定对氮负荷变化作出反应的信号通路。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。这一假设将通过使用Affymetrix微阵列对RNA的转录图谱进行测量，来测量mRNA水平对不同氮负荷的反应变化，从而验证这一假说。3)确定其数量和翻译后修饰随着氮负荷的变化而变化的蛋白质。我的假设是，氮负荷的变化触发了一个或多个信号通路中蛋白质的数量和/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。这一假说将通过二维凝胶电泳法、密度测量法和质谱仪测量蛋白质水平的变化及其对不同氮负荷的修改来验证。

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.