Mammalian Mitochondrial One-Carbon Metabolism

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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.