REGULATION OF FOLATE CATABOLISM

Cornell University Ithaca, Ithaca NY

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Abstract

DESCRIPTION: The catabolism of cellular folate is not well understood and has been assumed to be a non enzymatic process. However, a number of studies have shown that cellular folate catabolism is increased during periods of fetal development and as a result of chronic alcohol exposure which suggests that folate catabolism is regulated. The applicant has recently identified an enzyme in human cell lines that specifically cleaves 5-formyltetrahydrofolate (5-formylTHF) to p-aminobenzoylglutamate. Also, he has purified this enzyme from rat liver. He proposes to study the role of this 5-formylTHF hydrolyase in maintaining intracellular folate concentrations and the regulation of this enzyme. The specific aims are: 1) to clone and characterize the cDNA from both human and rat cDNA libraries and to overexpress, purify, and characterize the enzyme in both eukaryotic and prokaryotic systems; 2) the human gene will be cloned, sequenced, and its chromosomal location determined. The promoter region will be analyzed for consensus DNA regulatory elements and the contribution of the elements to regulation of expression will be determined; 3) cell culture models will be developed to study the role of 5-formylTHF catabolism in regulating intracellular folate concentrations by overexpressing the human 5-formylTHF hydrolyase gene in both the sense and antisense orientation. This will allow the effects of variable hydrolyase activity on folate accumulation and one-carbon metabolism to be determined; and 4) the role of the hydrolyase, cytoplasmic serine hydroxymethyltransferase (cSHMT), and 5,10-methenylTHF synthetase activities in the increased folate catabolism seen in pregnancy will be studied in a rat model using antibodies and cDNA probes to these enzymes in order to determine their level of transcription and expression in maternal and fetal tissues. These studies are expected to provide much new information about the regulation of folate catabolism.