HUMAN MITOCHONDRIAL CI-TETRAHYDROFOLATE SYNTHASE

人线粒体CI-四氢叶酸合成酶

• 批准号: 7011129
• 负责人: DEAN R APPLING
• 金额: $ 5.2万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-25 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7011129
• 关键词: aminohydrolases; carbon nitrogen ligase; cell line; choline deficiency; complementary DNA; congenital nervous system disorder; dietary proteins; dihydrofolate reductase; enzyme activity; enzyme complex; folate; folate deficiency; gene environment interaction; gene expression; genetic polymorphism; genetic screening; homocystinuria; human genetic material tag; laboratory mouse; mitochondria; neural plate /tube; nutrition related tag; protein localization; tetrahydrofolates; vitamin metabolism

The objective of this proposal is to characterize the mammalian mitochondrial trifunctional enzyme, C1-THF synthase, and determine what role it plays in the metabolism of folate- mediated one-carbon units. Folate metabolism is essential in all cells, and mitochondria play a critical role in these pathways. This is reflected in human diseases associated with mitochondrial defects, such as the mitochondrial myopathies and nonketotic hyperglycinemia, as well as the recently recognized connection between homocysteine and mitochondrial one-carbon metabolism. Elevated plasma homocysteine is now recognized as a major independent risk factor for cardiovascular disease, a leading cause of mortality in the U.S. We have carried out extensive studies on these compartmentalized pathways in yeast, but little is known about the enzymes and their regulation in mammals. Using molecular tools made possible by the Human Genome Project, we are now able to study the mitochondrial pathway in humans and other mammals. The Specific Aims are to: (1) Clone and express a cDNA encoding the human mitochondrial C1-THF synthase; (2) Purify and characterize the human enzyme; (3) Examine the expression and nutritional regulation of mitochondrial C1-THF synthase in human and mouse; and (4) Determine whether mutations in mitochondrial C1-THF synthase are related to neural tube defects or homocysteinemia. The experimental design includes complementation of yeast mutants with the human cDNA and expression in CHO cells to confirm its localization to mitochondria. The protein will be purified for analysis of its kinetics and substrate specificity. Tissue distribution in humans will be deduced from measurement of transcript and protein levels in various human tissues. Nutritional regulation studies will be performed in mice, including response to choline or folate deficiency. Metabolic interactions with serine hydroxymethyltransferase and glycine cleavage will be studied by NMR methods. PCR will be used to screen DNAs from patients with NTD or homocysteinemia for polymorphisms in the gene. These studies will add to our knowledge of the normal function of the mitochondrial pathway and should lead to a better understanding of how defects in this pathway contribute to human disease related to homocysteine metabolism.

本提案的目的是表征哺乳动物线粒体三功能酶 C1-THF 合酶，并确定它在叶酸介导的一碳单位代谢中所起的作用。 叶酸代谢在所有细胞中都是必需的，线粒体在这些途径中发挥着关键作用。这反映在与线粒体缺陷相关的人类疾病中，例如线粒体肌病和非酮症高甘氨酸血症，以及最近认识到的同型半胱氨酸和线粒体一碳代谢之间的联系。血浆同型半胱氨酸升高现已被认为是心血管疾病的主要独立危险因素，心血管疾病是美国死亡的主要原因。我们对酵母中的这些分隔途径进行了广泛的研究，但对哺乳动物中的酶及其调节知之甚少。利用人类基因组计划提供的分子工具，我们现在能够研究人类和其他哺乳动物的线粒体途径。 具体目标是： (1) 克隆并表达编码人线粒体C1-THF合酶的cDNA； (2) 人体酶的纯化和表征； (3) 检测人和小鼠线粒体C1-THF合酶的表达和营养调节； (4)确定线粒体C1-THF合酶突变是否与神经管缺陷或同型半胱氨酸血症有关。 实验设计包括酵母突变体与人类 cDNA 的互补以及在 CHO 细胞中的表达，以确认其定位于线粒体。 该蛋白质将被纯化以分析其动力学和底物特异性。 人类的组织分布可以通过测量各种人体组织中的转录物和蛋白质水平来推断。 营养调节研究将在小鼠中进行，包括对胆碱或叶酸缺乏的反应。 将通过 NMR 方法研究与丝氨酸羟甲基转移酶和甘氨酸裂解的代谢相互作用。 PCR 将用于筛选 NTD 或同型半胱氨酸血症患者的 DNA 中的基因多态性。 这些研究将增加我们对线粒体途径正常功能的了解，并有助于更好地了解该途径的缺陷如何导致与同型半胱氨酸代谢相关的人类疾病。