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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.

这项建议的目标是确定参与哺乳动物线粒体的酶，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。叶酸代谢是必不可少的，细胞和线粒体在这些途径中发挥关键作用。这反映在人类与叶酸和同型半胱氨酸代谢相关的疾病（例如心血管疾病疾病和神经管缺陷），并在最近认识到之间的联系同型半胱氨酸和线粒体一碳代谢。升高的血浆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，在美国死亡率的主要原因，我们已经进行了广泛的研究酵母中这些分隔的途径，但我们对哺乳动物的线粒体一碳途径远未完成。使用分子通过人类基因组计划的工具，我们现在能够研究线粒体途径在人类和其他哺乳动物。具体目标是：（1）表征哺乳动物MTHFD 2L基因和酶;（2）检查 MTHFD 1 L和MTHFD 2L的线粒体定位和代谢作用线粒体一碳代谢中的酶;和（3）产生靶向用于将来产生MTHFD 1 L和MTHFD 2L敲除小鼠的构建体。的实验设计包括分离人和啮齿动物的cDNA，中国仓鼠卵巢细胞，以确认定位于线粒体。MTHFD2L 将纯化蛋白质以分析其动力学和底物特异性。的该基因在人类和小鼠组织以及小鼠胚胎中的表达谱将被考察我们将在培养的细胞中利用siRNA敲低来研究siRNA在细胞中的作用。线粒体一碳代谢中的MTHFD 2L酶。靶向载体旨在产生条件性MTHFD 1 L和MTHFD 2L敲除小鼠品系，被建造。然而，敲除小鼠品系的实际生产超出了这个为期两年的项目。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理解存在差距代谢，并可能最终提出更好的治疗人类疾病，如心血管疾病和神经管缺陷。