Mammalian Mitochondrial One-Carbon Metabolism

这项提议的目标是确定哺乳动物线粒体中所涉及的酶。 一碳代谢，并确定这些酶如何共同发挥作用 支持线粒体一碳代谢。叶酸代谢在ALL中是必不可少的 细胞和线粒体在这些途径中起着关键作用。这一点反映在人类身上 与叶酸和同型半胱氨酸代谢相关的疾病(例如心血管疾病 疾病和神经管缺陷)，以及最近发现的 同型半胱氨酸和线粒体一碳代谢。血浆浓度升高 同型半胱氨酸现在被认为是心血管疾病的主要独立危险因素 疾病，在美国是主要的死亡原因。我们进行了广泛的研究 关于酵母中这些分隔的途径，但我们对 哺乳动物线粒体的一碳途径还远未完成。使用分子 由人类基因组计划提供的工具，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) 鉴定哺乳动物的MTHFD2L基因和酶；(2)检测 线粒体Mthfd1l和MTHFD2L的定位及代谢作用 线粒体一碳代谢中的酶；以及(3)产生靶向性 构建了未来生产Mthfd1l和MTHFD2L基因敲除小鼠的结构。这个 实验设计包括分离人和啮齿动物的cDNA并在 中国仓鼠卵巢细胞确认线粒体定位。MTHFD2L 将对蛋白质进行纯化，以分析其动力学和底物专一性。这个 该基因在人类和小鼠组织以及小鼠胚胎中的表达谱将是 检查过了。我们将利用培养细胞中的siRNA敲除来研究 线粒体一碳代谢中的MTHFD2L酶。靶向载体 旨在产生有条件的Mthfd1l和MTHFD2L基因敲除小鼠品系 是被建造的。然而，实际生产的基因敲除鼠标品系超出了 这个为期两年的项目的范围。从这些研究中获得的信息将填补一个巨大的 我们对叶酸和哺乳动物线粒体一碳的认识差距 新陈代谢，并可能最终建议更好的治疗人类疾病，如 心血管疾病和神经管缺陷。

The goal of this proposal is to identify the enzymes involved in mammalian mitochondrial one-carbon metabolism and to determine how these enzymes function together to support mitochondrial one-carbon metabolism. Folic acid metabolism is essential in all cells, and mitochondria play a critical role in these pathways. This is reflected in human diseases associated with folate and homocysteine metabolism (e.g. cardiovascular disease and neural tube defects), and in 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 our understanding of the mitochondrial one-carbon pathway in mammals is far from complete. 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) Characterize the mammalian MTHFD2L gene and enzyme; (2) Examine the mitochondrial localization and metabolic roles of the MTHFD1L and MTHFD2L enzymes in mitochondrial one-carbon metabolism; and (3) Generate targeting constructs for future production of MTHFD1L and MTHFD2L knockout mice. The experimental design includes isolation of human and rodent cDNAs and expression in Chinese hamster ovary cells to confirm localization to mitochondria. The MTHFD2L protein will be purified for analysis of its kinetics and substrate specificity. The expression profile of the gene in human and mouse tissues and mouse embryos will be examined. We will utilize siRNA knockdowns in cultured cells to study the role of the MTHFD2L enzyme in mitochondrial one-carbon metabolism. Targeting vectors designed to produce conditional MTHFD1L and MTHFD2L knockout mouse strains will be constructed. However, actual production of the knockout mouse lines is beyond the scope of this two-year project. The information gained from these studies will fill a huge gap in our understanding of folic acid and mammalian mitochondrial one-carbon metabolism, and may ultimately suggest better treatments for human disorders such as cardiovascular disease and neural tube defects.