Abstract 73: Mitochondrial 10-formyltetrahydrofolate dehydrogenase: A novel enzyme in folate metabolism

Abstract

Abstract Cytosolic 10-formyltetrahydrofolate dehydrogenase (FDH, Aldh1L1) is an important regulator of intracellular folate pools, which displays antiproliferative effects in cancer cells. We have identified a gene at the chromosome locus 12q24.11 in the human genome, the product of which has 87% sequence similarity with cytosolic FDH. This protein has an extra amino-terminal sequence of 22 amino acid residues enriched in arginines, which is predicted to be a mitochondrial translocation signal. The mitochondrial targeting function of the leader has been confirmed in Cos7 cells: green fluorescent protein (GFP)-tagged at the amino-terminus with the leader, localizes to mitochondria. Transfection of Cos7 or A549 cell lines with a construct, in which GFP has been introduced between the leader sequence and the rest of the putative mitochondrial FDH (mtFDH), has also shown mitochondrial localization, suggesting that the identified gene encodes a mitochondrial enzyme. To evaluate the abundance of mtFDH, we have measured its mRNA levels in a wide array of human tissues by real-time PCR, and compared them to the levels of mRNA that encode cytosolic FDH. While cytosolic FDH mRNA is highest in liver, kidney and pancreas, mtFDH mRNA is most highly expressed in pancreas, heart and brain, but not in liver or kidney. In contrast to the cytosolic enzyme, which is non detectable in human cancer cell lines, the presence of mtFDH mRNA was demonstrated in A549 and PC3 cells by conventional and real-time PCR. The presence of the endogenous enzyme in mitochondria of A549 cells has been further confirmed using specific polyclonal antibody generated against purified recombinant mtFDH. We have also shown that recombinant mtFDH, similar to the cytosolic enzyme, catalyzes NADP+-dependent oxidation of the 10-formyltetrahydrofolate to tetrahydrofolate and CO2. Thus, the enzyme is a likely source of CO2 production in mitochondria and we propose that it plays an essential role in distribution of one-carbon groups between cytosolic and mitochondrial compartments of the cell. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 73.