The broad objectives of this proposal are to understand the metabolic role of one of the most abundant folate enzymes, FDH. FDH converts 10-formyltetrahydrofolate (10-fTHF) to tetrahydrofolate in an NADP-dependent dehydrogenase reaction or in an NADP-independent hydrolase reaction. Our recent studies have demonstrated that FDH possesses tumor suppressor-like activity: it is strongly and ubiquitously down regulated in tumors and induces apoptosis in FDH-deficient cancer cells. Therefore, we proposed that the enzyme is one of the intrinsic mechanisms that protect against excessive and uncontrolled cellular proliferation. Since the FDH substrate, 10-fTHF, formylates methionyl-tRNA, presumably a required step in initiation of translation in mitochondria, we propose that FDH regulates protein biosynthesis in mitochondria through the control of intracellular 10-fTHF levels. We further hypothesize that the product of the FLJ38508 gene (locus 12q23.3) is a mitochondria! FDH, and that the cytosolic and mitochondria! enzymes regulate distribution of 10-fTHF between cytosolic and mitochondrial compartments directing 10-fTHF to the de novo purine biosynthesis or mitochondrial protein biosynthesis pathway. We also propose that FDH hydrolase reaction occurs in vivo in mitochondria and that the biological role of this reaction is to supply formate for biosynthesis of 10-fTHF in cytosol. The following specific aims are proposed to test the hypotheses. (1) Manipulate the levels of 10-fTHF, through FDH expression, to establish its importance in control of protein biosynthesis in mitochondria. (2) Explore the role of the mitochondrial FDH in cellular function. (3) Investigate whether FDH- catalyzed 10-fTHF hydrolase reaction occurs in vivo. Cultured mammalian cells with different supplementation of folate and purines will be used as a model in this proposal. FDH expression in mammalian cells, measurement of folate and nucleotide pools, analysis of folate enzymes, assays of ATP production and protein expression in mitochondria, assays of apoptosis and mitochondrial integrity, enzyme activity assays, immunochemical methods, siRNA techniques will be used to achieve the goals of the project. The well-known role of folate in prevention of megaloblastic anemia, vascular disease, neural tube birth defects and cancer, as well as crucial role of mitochondria in regulation of apoptosis, and growing body of evidence for mitochondrial basis of many diseases make these studies particularly relevant.
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