Mammalian Mitochondrial One-Carbon Metabolism

哺乳动物线粒体一碳代谢

基本信息

批准号：
7930555
负责人：
DEAN R APPLING
金额：
$ 31.36万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-15 至 2012-07-31
项目状态：
已结题

项目摘要

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.

该提议的目的是确定与哺乳动物线粒体有关的酶单碳代谢，并确定这些酶如何共同发挥作用支持线粒体一碳代谢。叶酸代谢在所有人中都是必不可少的细胞和线粒体在这些途径中起关键作用。这反映在人类与叶酸和同型半胱氨酸代谢相关的疾病（例如心血管疾病和神经管缺陷），在最近公认的联系同型半胱氨酸和线粒体一碳代谢。血浆升高同型半胱氨酸现在被认为是心血管的主要独立危险因素疾病是美国死亡率的主要原因，我们进行了广泛的研究在酵母中的这些隔离途径上，但我们对哺乳动物中的线粒体一碳途径远非完整。使用分子人类基因组项目使工具成为可能，我们现在能够研究人类和其他哺乳动物的线粒体途径。具体目的是：（1）表征哺乳动物MTHFD2L基因和酶；（2）检查 MTHFD1L和MTHFD2L的线粒体定位和代谢作用线粒体一碳代谢中的酶；（3）生成目标未来生产MTHFD1L和MTHFD2L敲除小鼠的构造。这实验设计包括隔离人和啮齿动物cDNA以及在中国仓鼠卵巢细胞确认在线粒体上定位。 MTHFD2L 蛋白质将被纯化以分析其动力学和底物特异性。这基因在人和小鼠组织以及小鼠胚胎中的表达谱将是检查。我们将利用培养细胞中的siRNA敲低来研究线粒体一碳代谢中的MTHFD2L酶。定位向量设计用于产生有条件的MTHFD1L和MTHFD2L敲除小鼠菌株被构造。但是，淘汰鼠标线的实际生产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充满大量我们对叶酸和哺乳动物线粒体一碳的理解差距新陈代谢，最终可能提出更好的人类疾病治疗心血管疾病和神经管缺陷。