The long-term goal of this project is to investigate the regulation of hepatic folate-dependent one-carbon (1-C) metabolism; specifically to investigate the supply and oxidation of 1-C units from serine, glycine, choline, histidine and formate. A model is proposed for the unidirectional flow of 1-C units generated from serine, glycine and choline in mitochondria via 10-formyltetrahydrofolate (10-HCO-H4PteGlu) and delivered to the cytosol as formate, in adult liver. The key enzyme in the mitochondrial conversion of folate-linked 1-C units to formate is a mitochondrial form of C1-tetrahydrofolate synthase (mC1-THFS), and enzyme that has not been purified or characterized from mitochondria. Formate is the predominant source of 1-C units in cytosol and is assimilated into the cytosolic folate pool by conversion to 10-HCO-H4PteGlu and conversion to other folate forms by cytosolic (c) C1-THFS. Cytosolic and mitochondrial 10- HCO-H4PteGlu pools are reservoirs of 1-C units that supply the biosynthetic folate-dependent reactions or are oxidized to H4PteGlu and CO2 by isozymes of 10-formyltetrahydrofolate dehydrogenase (FDH). NEUT2 homozygous mice lack both cytosolic and mitochondrial isozymes of FDH and are unable to oxidize 1-C units as 10-HCO-H4PteGlu to CO2 and H4PteGlu. Lack of FDH in homozygous NEUT2 mice results in expanded 10-HCO-H4PteGlu pools and diminished H4PteGlu pools. The changes in the H4PteGlu pools correlates with changes in the protein levels of liver cC1-THFS. It is hypothesized that expression of cC1-THFS is regulated at the transcriptional level by the cytosolic level of H4PteGlu. NEUT2 mice offer a unique opportunity for the study of 1-C metabolism in a system where 1-C units as, 10-HCO-H4PteGlu, cannot be oxidized.
The specific aims are; 1) purification, characterization and cloning of mC1-THFS; 2) investigation of the promoter region of the cC1-THFS gene and 3), cloning and function of mFDH.
| Cook, R J (2001) Disruption of histidine catabolism in NEUT2 mice. Arch Biochem Biophys 392:226-32 |
| Cook, R J; Champion, K M; Giometti, C S (2001) Methanol toxicity and formate oxidation in NEUT2 mice. Arch Biochem Biophys 393:192-8 |
| Kim, S; Park, G H; Joo, W A et al. (1998) Identification of protein-arginine N-methyltransferase as 10-formyltetrahydrofolate dehydrogenase. J Biol Chem 273:27374-82 |
| Krupenko, S A; Wagner, C; Cook, R J (1997) Expression, purification, and properties of the aldehyde dehydrogenase homologous carboxyl-terminal domain of rat 10-formyltetrahydrofolate dehydrogenase. J Biol Chem 272:10266-72 |
| Krupenko, S A; Wagner, C; Cook, R J (1997) Domain structure of rat 10-formyltetrahydrofolate dehydrogenase. Resolution of the amino-terminal domain as 10-formyltetrahydrofolate hydrolase. J Biol Chem 272:10273-8 |
| Cook, R J (1997) Use of 10-formyl-5,8-dideazafolate as substrate for rat 10-formyltetrahydrofolate dehydrogenase. Methods Enzymol 281:129-34 |
