10-Formyltetrahydrofolate dehydrogenase (FDH) catalyzes an NADP+-dependent dehydrogenase reaction resulting in conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. This reaction is a result of the concerted action of two catalytic domains of FDH, the amino-terminal hydrolase domain and the carboxyl-terminal aldehyde dehydrogenase domain. In addition to participation in the overall FDH mechanism, the C-terminal domain is capable of NADP+-dependent oxidation of short chain aldehydes to their corresponding acids. We have determined the crystal structure of the C-terminal domain of FDH and its complexes with oxidized and reduced forms of NADP. Compared to other members of the ALDH family, FDH demonstrates a new mode of binding of the 2'-phosphate group of NADP via a water-mediated contact with Gln600 that may contribute to the specificity of the enzyme for NADP over NAD. The structures also suggest how Glu673 can act as a general base in both acylation and deacylation steps of the reaction. In the apo structure, the general base Glu673 is positioned optimally for proton abstraction from the sulfur atom of Cys707. Upon binding of NADP+, the side chain of Glu673 is displaced from the active site by the nicotinamide ring and contacts a chain of highly ordered water molecules that may represent a pathway for translocation of the abstracted proton from Glu673 to the solvent. When reduced, the nicotinamide ring of NADP is displaced from the active site, restoring the contact between Cys707 and Glu673 and allowing the latter to activate the hydrolytic water molecule in deacylation.

Hyperhomocyst(e)inemia is a metabolic derangement that is linked to the distribution of folate pools, which provide one-carbon units for biosynthesis of purines and thymidylate and for remethylation of homocysteine to form methionine. In humans, methionine synthase deficiency results in the accumulation of methyltetrahydrofolate at the expense of folate derivatives required for purine and thymidylate biosynthesis. Complete ablation of methionine synthase activity in mice results in embryonic lethality. Other mouse models for hyperhomocyst(e)inemia have normal or reduced levels of methyltetrahydrofolate and are not embryonic lethal, although they have decreased ratios of AdoMet/AdoHcy and impaired methylation. We have constructed a mouse model with a gene trap insertion in the Mtrr gene specifying methionine synthase reductase, an enzyme essential for the activity of methionine synthase. This model is a hypomorph, with reduced methionine synthase reductase activity, thus avoiding the lethality associated with the absence of methionine synthase activity. Mtrr gt/gt mice have increased plasma homocyst(e)ine, decreased plasma methionine, and increased tissue methyltetrahydrofolate. Unexpectedly, Mtrr gt/gt mice do not show decreases in the AdoMet/AdoHcy ratio in most tissues. The different metabolite profiles in the various genetic mouse models for hyperhomocysteinemia may be useful in understanding biological effects of elevated homocyst(e)ine.

FDH (10-formyltetrahydrofolate dehydrogenase) is strongly downregulated in tumors while its elevation suppresses proliferation of cancer cells and induces p53-dependent apoptosis. We have previously shown that FDH induces phosphorylation of p53 at Ser6, which is a required step in the activation of apoptosis. In the present study, we report that FDH-induced p53 phosphorylation is carried out by JNK1 and JNK2 (c-Jun N-terminal kinases) working in concert. We have demonstrated that FDH induces phosphorylation of JNK1 and JNK2, while treatment of FDH-expressing cells with JNK inhibitor SP600125, as well as knockdown of JNK1 or JNK2 by siRNA, prevents phosphorylation of p53 at Ser6 and protects cells from apoptosis. Interestingly, the knockdown of JNK1 abolished phosphorylation of JNK2 in response to FDH, while knockdown of JNK2 did not prevent JNK1 phosphorylation. Pull-down assay with the p53-specific antibody has shown that JNK2, but not JNK1, is physically associated with p53. Our studies revealed a novel mechanism in which phosphorylation of JNK2 is mediated by JNK1 before phosphorylation of p53, and then p53 is directly phosphorylated by JNK2 at Ser6.

Real-time PCR performed on an array of human tissues hasshown that although cytosolic FDH mRNA is highest in liver, kidney, and pancreas, mtFDH mRNA is most highly expressed in pancreas, heart, and brain. In contrast to the cytosolic enzyme, which is not detectable in cancer cells, the presence of mtFDH was demonstrated in several human cancer cell lines by conventional and real-time PCR and by Western blot. Analysis of genomes of different species indicates that the mitochondrial enzyme is a later evolutionary product when compared with the cytosolic enzyme. We propose that this novel mitochondrial enzyme is a likely source of CO 2 production from 10-formyltetrahydrofolate in mitochondria and plays an essential role in the distribution of one-carbon groups between the cytosolic and mitochondrial compartments of the cell.

Genetic variants in one-carbon folate metabolism have been identified as risk factors for disease because they may impair the production or use of one-carbon folates required for nucleotide synthesis and methylation. p.R653Q (1958G>A) is a single-nucleotide polymorphism (SNP) in the 10-formyltetrahydrofolate (formylTHF) synthetase domain of the trifunctional enzyme MTHFD1; this domain produces the formylTHF which is required for the de novo synthesis of purines. Approximately 20% of Caucasians are homozygous for the Q allele. MTHFD1 p.R653Q has been proposed as a risk factor for neural tube defects (NTDs), congenital heart defects (CHDs) and pregnancy losses. We have generated a novel mouse model in which the MTHFD1 synthetase activity is inactivated without affecting protein expression or the other activities of this enzyme. Complete loss of synthetase activity (Mthfd1S(-/-)) is incompatible with life; embryos die shortly after 10.5 days gestation, and are developmentally delayed or abnormal. The proportion of 10-formylTHF in the plasma and liver of Mthfd1S(+/-) mice is reduced (P < 0.05), and de novo purine synthesis is impaired in Mthfd1S(+/-) mouse embryonic fibroblasts (MEFs, P < 0.005). Female Mthfd1S(+/-) mice had decreased neutrophil counts (P < 0.05) during pregnancy and increased incidence of developmental defects in embryos (P = 0.052). These findings suggest that synthetase deficiency may lead to pregnancy complications through decreased purine synthesis and reduced cellular proliferation. Additional investigation of the impact of synthetase polymorphisms on human pregnancy is warranted.

A folate enzyme, FDH (10-formyltetrahydrofolate dehydrogenase; EC 1.5.1.6), is not a typical tumour suppressor, but it has two basic characteristics of one, i.e. it is down-regulated in tumours and its expression is selectively cytotoxic to cancer cells. We have recently shown that ectopic expression of FDH in A549 lung cancer cells induces G1 arrest and apoptosis that was accompanied by elevation of p53 and its downstream target, p21. It was not known, however, whether FDH-induced apoptosis is p53-dependent or not. In the present study, we report that FDH-induced suppressor effects are strictly p53-dependent in A549 cells. Both knockdown of p53 using an RNAi (RNA interference) approach and disabling of p53 function by dominant-negative inhibition with R175H mutant p53 prevented FDH-induced cytotoxicity in these cells. Ablation of the FDH-suppressor effect is associated with an inability to activate apoptosis in the absence of functional p53. We have also shown that FDH elevation results in p53 phosphorylation at Ser-6 and Ser-20 in the p53 transactivation domain, and Ser-392 in the C-terminal domain, but only Ser-6 is strictly required to mediate FDH effects. Also, translocation of p53 to the nuclei and expression of the pro-apoptotic protein PUMA (Bcl2 binding component 3) was observed after induction of FDH expression. Elevation of FDH in p53 functional HCT116 cells induced strong growth inhibition, while growth of p53-deficient HCT116 cells was unaffected. This implies that activation of p53-dependent pathways is a general downstream mechanism in response to induction of FDH expression in p53 functional cancer cells.

FDH (10-formyltetrahydrofolate dehydrogenase, the product of the ALDH1L1 gene), a major folate-metabolizing enzyme in the cytosol, is involved in the regulation of cellular proliferation. We have previously demonstrated that FDH is strongly and ubiquitously down-regulated in malignant human tumors and cancer cell lines. Here, we report that promoter methylation is a major mechanism controlling FDH levels in human cancers. A computational analysis has identified an extensive CpG island in the ALDH1L1 promoter region. It contains 96 CpG pairs and covers the region between −525 and +918 bp of the ALDH1L1 gene including the promoter, the entire exon 1, and a part of intron 1 immediately downstream of the exon. Bisulfite sequencing analysis revealed extensive methylation of the island (76%-95% of CpGs) in cancer cell lines. In agreement with these findings, treatment of FDHdeficient A549 cells with the methyltransferase inhibitor 5-aza-2′-deoxycytidine restored FDH expression. Analysis of the samples from patients with lung adenocarcinomas demonstrated methylation of the ALDH1L1 CpG island in tumor samples and a total lack of methylation in respective normal tissues. The same phenomenon was observed in liver tissues: the CpG island was methylation free in DNA extracted from normal hepatocytes but was extensively methylated in a hepatocellular carcinoma. Levels of ALDH1L1 mRNA and protein correlated with the methylation status of the island, with tumor samples demonstrating down-regulation of expression or even complete silencing of the gene. Our studies have also revealed that exon 1 significantly increases transcriptional activity of ALDH1L1 promoter in a luciferase reporter assay. Interestingly, the exon is extensively methylated in samples with a strongly down-regulated or silenced ALDH1L1 gene. Keywords ALDH1L1, CpG island methylation, folate metabolism, lung adenocarcinomaOriginal Article

The importance of proper consumption of dietary folate for human health has been highlighted by an extensive number of publications over several decades. Fortification of grain products with folic acid was initiated with the specific intent to prevent neural tube defects, and the scope of this endeavor is unique in that its target population (women of the periconceptional period) is many times smaller than the population it affects (everyone who ingests fortified grain products). Folate fortification has been wildly successful in terms of its goal; since its inception, the incidence of neural tube defects has markedly decreased. In the wake of this public health triumph, it is important to catalogue both the serendipitous benefits and potential side effects of folic acid supplementation. The vitamin is generally regarded as a harmless nutrient based on studies evaluating the safe upper limits of folate intake. In recent years, however, a concern has been raised with respect to a potential downside to folate supplementation; namely, its proposed ability to enhance proliferation of malignant tumors. The current review summarizes the available literature on the effects of folate supplementation and the molecular mechanisms by which high doses of folate may have negative consequences on human health, especially with regard to cancer.