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.
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| Prasannan, Priya; Pike, Schuyler; Peng, Kun et al. (2003) Human mitochondrial C1-tetrahydrofolate synthase: gene structure, tissue distribution of the mRNA, and immunolocalization in Chinese hamster ovary calls. J Biol Chem 278:43178-87 |
| Tibbetts, Anne S; Oesterlin, Lena; Chan, Sherwin Y et al. (2003) Mammalian mitochondrial initiation factor 2 supports yeast mitochondrial translation without formylated initiator tRNA. J Biol Chem 278:31774-80 |
