Methylenetetrahydrofolate Dehydrogenase Research Articles

Controlling supplies for DNA and RNA synthesis

Metabolism The mTORC1 protein kinase complex regulates anabolic metabolism and coordinates cellular signals that promote growth with availability of required precursor metabolites. Signaling through mTORC1 controls pyrimidine synthesis. Ben-Sahra et al. found that mTORC1 also functions by a different mechanism to regulate purine biosynthesis, thus generating precursors for the synthesis of RNA and DNA (see the Perspective by Ma and Jones). Signaling by mTORC1 caused accumulation of the transcription factor ATF4, which enhances production of the enzyme methylenetetrahydrofolate dehydrogenase 2, thus leading to increased production of the purine nucleotides needed for cell growth. Science , this issue p. [728][1]; see also p. [670][2] [1]: /lookup/doi/10.1126/science.aad0489 [2]: /lookup/doi/10.1126/science.aaf1929

Is MTHFD1 polymorphism rs 2236225 (c.1958G>A) associated with the susceptibility of NSCL/P? A systematic review and meta-analysis.

To investigate the association between the methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) polymorphism rs 2236225 (c.1958G>A) and susceptibility to non-syndromic cleft of the lip and/or palate (NSCL/P). An extensive literature review has been conducted using PubMed, Web of Science, Cochrane Library, Google Scholar, the China National Knowledge Infrastructure (CNKI), and Wanfang Database for eligible researches. The terms for searching were "cleft lip OR cleft palate OR CLP OR CL/P OR oral facial cleft OR OFC" AND "methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1 OR methenyltetrahydrofolate cyclohydrolase formyltetrahydrofolate synthetase OR MTHFD1 OR MTHFD". Two independent researchers screened, evaluated and extracted the data of included studies. The pooled odds ratios (OR) with 95% confidence intervals (95% CI) were calculated by random effects model under five gene models. Subgroup, sensitivity analysis and publication bias were also assessed. Ten case-control studies have been included in the systematic review and eight studies have been considered for the meta-analysis. Overall, the MTHFD1 polymorphism rs2236225 and the risk of NSCL/P showed pooled OR (95% CI) of 1.02 (0.86-1.21) under allelic model. A higher degree of heterogeneity was observed in Asian countries (I (2) = 75.6%) compared to non-Asian countries (I (2) = 48.9%). Similar consequence appeared in the subgroup of children (I (2) = 78.6%) compared with that of mothers (I (2) = 0.0%). There was no significant difference in the publication bias by the Begg's funnel plot (P = 0.711) and Egger's regression test (P = 0.746). Our assessment suggested there was no significant association between the MTHFD1 polymorphism rs 2236225 (c.1958G>A) and the susceptibility to NSCL/P. Further investigations using a large sample size and a more advanced technique should be adopted to reach a more precise conclusion in the future.

LncRNA Taurine-Upregulated Gene 1 Promotes Cell Proliferation by Inhibiting MicroRNA-9 in MCF-7 Cells.

PurposeThis study was designed to investigate the role of taurine-upregulated gene 1 (TUG1) in MCF-7 breast cancer cells and the molecular mechanism involved in the regulation of microRNA-9 (miR-9).MethodsThe expression of TUG1 in breast cancer tissues and cells was evaluated using quantitative reverse transcription polymerase chain reaction. Cell viability was examined using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay; cell cycle progression and apoptosis were analyzed using flow cytometry. A dual luciferase reporter assay was used to detect the relationship between TUG1 and miR-9. The expression of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) was measured by western blot.ResultsHigher expression of TUG1 was observed in breast cancer tissues and cell lines than in the corresponding controls. TUG1 knockdown reduced proliferation, suppressed cell cycle progression, and promoted apoptosis of MCF-7 cells. The dual luciferase reporter assay showed that TUG1 could negatively regulate the expression of miR-9. MiR-9 inhibition abrogated the effect of TUG1 knockdown on the proliferation, cell cycle progression, and apoptosis of MCF-7 cells. TUG1 positively regulated the expression of MTHFD2 in breast cancer cells.ConclusionTUG1 knockdown was significantly associated with decreased cell proliferation and it promoted apoptosis of breast cancer cells through the regulation of miR-9.

Abstract A14: Role of mitochondrial folate metabolism in cancer cell growth and proliferation

Abstract Introduction: Increased expression of mitochondrial folate enzymes, especially mitochondrial methylenetetrahydrofolate dehydrogenase (MTHFD2), is one of the most consistent gene expression changes in human tumors. Folate metabolism is a major consumer of serine, the primary donor of one-carbon units. One-carbon units generated from serine in the mitochondrion can be transferred into the cytosol as formate for use in cytosolic biosynthesis. Using genetics and quantitative metabolic methods, we examined the role of mitochondrial folate metabolism in cell growth in culture and in xenografted tumors. Methods: Using CRISPR-Cas9 technology, we created deletion mutant cell lines for enzymes in folate metabolism. Cells deleted for MTHFD2 were grown as xenografts in mice. Metabolites from cultured cells and xenografts were analyzed by liquid chromatography mass spectrometry (LC-MS). Results: Mitochondrial folate metabolism is not required for growth in standard tissue culture media conditions. The reversal of the direction of cytosolic one-carbon flux enables rapid cell growth of MTHFD2 mutants albeit with metabolic hallmarks of 10-formyltetrahydrofolate deficiency. However, MTHFD2 deletion cells are, as previously shown, glycine auxotrophs and under those conditions show metabolic hallmarks of oxidative stress. Exogenous formate rescues the 10-formyltetrahydrofolate deficiency but not the glycine auxotrophy. Additionally, we find these cells are also growth impaired under low serine conditions. Importantly, compared to paired wild type tumors, growth of MTHFD2 mutant subcutaneous xenografts was markedly reduced. Conclusions: Mitochondrial folate metabolism is conditionally required for rapid cell growth. High expression of mitochondrial folate genes in cancer may function to meet requirements for one-carbon units, glycine, or redox homeostasis under nutrient stress. Citation Format: Gregory S. Ducker, Mark B. Esposito, Yibin Kang, Joshua D. Rabinowitz. Role of mitochondrial folate metabolism in cancer cell growth and proliferation. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A14.

The preliminary study on the function of methylenetetrahydrofolate dehydrogenase 2 in hepatocellular carcinoma

To investigate the expression of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) in hepatocellular carcinoma (HCC) tissues and its correlation with clinical parameters. Fluorogenic quantitative-polymerase chain reaction (FQ-PCR) was used to measure the MTHFD2 mRNA expression. The MTHFD2 protein expression was detected by immunohistochemical staining in cancerous tissues and adjacent noncancerous counterparts. The relationship of MTHFD2 expression, clinicopathological parameters and the prognosis of hepatocellular carcinoma was subsequently analysed. The MTHFD2 mRNA expression in cancerous tissues was higher than that in adjacent noncancerous counterparts (31/47) (P<0.05). The positive rate of MTHFD2 protein in cancerous tissues was significantly higher than that in adjacent noncancerous counterparts (69.5% vs. 33.9%) (P<0.05). MTHFD2 overexpression was found to correlate with clinical pathological parameters such as tumor metastasis, recurrence and poor prognosis (P<0.05). The patients with overexpressed MTHFD2 had shorter tumor-free survival time. Overexpression of MTHFD2 in HCC may be a risk factor of tumor metastasis and recurrence. MTHFD2 could be a new biomarker for prognosis of HCC.

Targeting MTHFD2 in Acute Myeloid Leukemia

Alterations in differentiation pathways contribute to the development of acute myeloid leukemia (AML). Differentiation therapy with all-trans retinoic acid (ATRA) has dramatically altered the treatment of acute promyelocytic leukemia, transforming it from a nearly fatal disease to a curable one. We set out to identify cellular pathways that contribute to AML differentiation, with the goal of identifying new therapeutic targets. We analyzed gene expression data from AML cell lines treated with phorbol 12-myristate 13-acetate (PMA), ATRA, Vitamin D, the BET inhibitor JQ1 and the DOT1L inhibitor EPZ00477, treatments known to induce AML differentiation and impair growth. Folate-mediated one-carbon metabolism was one of only three metabolic pathways altered by these compounds, with expression of MTHFD2 consistently downregulated with each compound.MTHFD2 is an NAD-dependent, bi-functional mitochondrial methylenetetrahydrofolate dehydrogenase and cyclohydrolase. It is differentially expressed in embryonic and transformed tissues and is upregulated in myeloid progenitors. MTHFD2 is the most differentially expressed metabolic enzyme in cancer cells versus normal cells, including normal proliferating cells. We thus investigated the role of MTHFD2 in myeloid malignancy. First, we demonstrated using ChIP-qPCR, MYC knockdown and MYC inhibition with a BET inhibitor, that MYC directly regulates MTHFD2 expression in AML. Knockdown of MTHFD2 with two shRNAs confirmed to have on-target activity, induced myeloid differentiation in AML cell lines, as measured by Cd11b expression, morphologic changes and induction of a previously validated AML differentiation gene expression signature. MTHFD2 knockdown decreased cell growth in AML cell lines, as well as decreased colony formation in methylcellulose in both AML cell lines and primary patient blasts. AML cells transduced with these two MTHFD2-directed shRNAs demonstrated attenuated growth in an orthotopic mouse model of AML. Furthermore, three MTHFD2-directed shRNAs prolonged survival in an MLL-AF9 mouse leukemia model. Additionally, using a doxycycline inducible shRNA system, we demonstrated that two miR30-shRNAs directed against MTHFD2 decreased AML burden in mice with established disease and prolonged survival.To identify biomarkers of response to MTHFD2 suppression, we used single sample Gene Set Enrichment Analysis (ssGSEA) to identify primary patient AML samples enriched for gene expression signatures of folate-mediated one-carbon metabolism and MTHFD2. We found in both independent data sets that the cluster of patients enriched for expression of the one-carbon folate pathway gene signatures was also enriched for patients with FLT3-ITD mutations, a subset of AML with a particularly poor prognosis. In addition, in an shRNA screen targeting 11,194 genes and performed in 216 cancer cell lines, including 17 AML lines, FLT3-ITD was a biomarker of response to MTHFD2 knockdown. We next validated that while MTHFD2 suppression induced measureable differentiation in all six AML cell lines examined, it induced the most robust induction of apoptosis in FLT3-ITD mutant AML. The mitochondrial one-carbon folate pathway is thought to contribute to cellular oxidative balance by providing reducing power in the form of NAD(P)H, and suppression of MTHFD2 was previously shown to increase ROS levels. Indeed, suppression of MTHFD2 led to a marked increase in ROS in the FLT3-ITD positive AML cell lines in which apoptosis was induced.In summary, a decrement in MTHFD2 expression was found at the center of multiple AML perturbations that impair AML growth and induce differentiation. Our data support MTHFD2 as an AML dependency and FLT3-ITD as a potential biomarker of response. We thus provide critical preclinical evidence for targeting of MTHFD2 as a therapeutic strategy in AML. DisclosuresStone:Celgene: Consultancy; Merck: Consultancy. DeAngelo:Celgene: Consultancy; Pfizer: Consultancy; Incyte: Consultancy; Agios: Consultancy; Novartis: Consultancy; Ariad: Consultancy; Bristol Myers Squibb: Consultancy; Amgen: Consultancy. Stegmaier:Novartis Pharmaceuticals: Consultancy.

Physiological role of FolD (methylenetetrahydrofolate dehydrogenase), FchA (methenyltetrahydrofolate cyclohydrolase) and Fhs (formyltetrahydrofolate synthetase) from Clostridium perfringens in a heterologous model of Escherichia coli.

Most organisms possess bifunctional FolD [5,10-methylenetetrahydrofolate (5,10-CH2-THF) dehydrogenase-cyclohydrolase] to generate NADPH and 10-formyltetrahdrofolate (10-CHO-THF) required in various metabolic steps. In addition, some organisms including Clostridium perfringens possess another protein, Fhs (formyltetrahydrofolate synthetase), to synthesize 10-CHO-THF. Here, we show that unlike the bifunctional FolD of Escherichia coli (EcoFolD), and contrary to its annotated bifunctional nature, C. perfringens FolD (CpeFolD) is a monofunctional 5,10-CH2-THF dehydrogenase. The dehydrogenase activity of CpeFolD is about five times more efficient than that of EcoFolD. The 5,10-methenyltetrahydrofolate (5,10-CH+-THF) cyclohydrolase activity in C. perfringens is provided by another protein, FchA (5,10-CH+-THF cyclohydrolase), whose cyclohydrolase activity is ∼ 10 times more efficient than that of EcoFolD. Kinetic parameters for CpeFhs were also determined for utilization of all of its substrates. Both CpeFolD and CpeFchA are required to substitute for the single bifunctional FolD in E. coli. The simultaneous presence of CpeFolD and CpeFchA is also necessary to rescue an E. coli folD deletion strain (harbouring CpeFhs support) for its formate and glycine auxotrophies, and to alleviate its susceptibility to trimethoprim (an antifolate drug) or UV light. The presence of the three clostridial proteins (FolD, FchA and Fhs) is required to maintain folate homeostasis in the cell.

Proteomic Analysis of One-carbon Metabolism-related Marker in Liver of Rat Offspring

Maternal food intake has a significant effect on the fetal environment, and an inadequate maternal diet may result in intrauterine growth restriction. Intrauterine growth restriction newborn rat pups nursed by normal diet-fed dams exhibited rapid catch-up growth, which plays a critical role in the risk for metabolic and cardiovascular disease in later life. Specifically, one-carbon metabolism in the liver plays a critical role in placental and fetal growth. Impaired functioning of one-carbon metabolism is associated with increased homocysteine levels. In this study, we applied a comprehensive proteomic approach to identify differential expression of proteins related to one-carbon metabolism in the livers of rat offspring as an effect of maternal food restriction during gestation. Data are available via ProteomeXchange with identifier PXD002578. We determined that betaine-homocysteine S-methyltransferase 1, methylenetetrahydrofolate dehydrogenase 1, and ATP synthase subunit beta mitochondrial (ATP5B) expression levels were significantly reduced in the livers of rat offspring exposed to maternal food restriction during gestation compared with in the offspring of rats fed a normal diet (p < 0.05). Moreover, the expression levels of betaine-homocysteine S-methyltransferase 1, methylenetetrahydrofolate dehydrogenase 1, and ATP synthase subunit beta mitochondrial were negatively correlated with serum homocysteine concentration in male offspring exposed to maternal food restriction during gestation and normal diet during lactation. However, in female offspring only expression levels of methylenetetrahydrofolate dehydrogenase 1 were negatively correlated with homocysteine concentration. This study shows that maternal food restriction during late gestation and normal diet during lactation lead to increased homocysteine concentration through disturbance of one-carbon metabolism in the livers of male offspring. This suggests that male offspring have an increased gender-specific susceptibility to disease in later life through fetal programming.

Mitochondrial Methylenetetrahydrofolate Dehydrogenase (MTHFD2) Overexpression Is Associated with Tumor Cell Proliferation and Is a Novel Target for Drug Development.

Rapidly proliferating tumors attempt to meet the demands for nucleotide biosynthesis by upregulating folate pathways that provide the building blocks for pyrimidine and purine biosynthesis. In particular, the key role of mitochondrial folate enzymes in providing formate for de novo purine synthesis and for providing the one-carbon moiety for thymidylate synthesis has been recognized in recent studies. We have shown a significant correlation between the upregulation of the mitochondrial folate enzymes, high proliferation rates, and sensitivity to the folate antagonist methotrexate (MTX). Burkitt lymphoma and diffuse large-cell lymphoma tumor specimens have the highest levels of mitochondrial folate enzyme expression and are known to be sensitive to treatment with MTX. A key enzyme upregulated in rapidly proliferating tumors but not in normal adult cells is the mitochondrial enzyme methylenetetrahydrofolate dehydrogenase (MTHFD2). This perspective outlines the rationale for specific targeting of MTHFD2 and compares known and generated crystal structures of MTHFD2 and closely related enzymes as a molecular basis for developing therapeutic agents against MTHFD2. Importantly, the development of selective inhibitors of mitochondrial methylenetetrahydrofolate dehydrogenase is expected to have substantial activity, and this perspective supports the investigation and development of MTHFD2 inhibitors for anticancer therapy.

Paternal transmission of MTHFD1 G1958A variant predisposes to neural tube defects in the offspring.

This study aimed to evaluate the role of methylenetetrahydrofolate dehydrogenase (MTHFD1) G1958A variant (rs2236225) as a 'maternal, paternal, or embryonic' genetic risk factor for neural tube defect (NTD) susceptibility. It also estimated differential associations based on type of NTD, offspring sex, maternal-paternal-offspring genotype incompatibility, and parent-of-origin effects (POE) using both case-control and family-based approach. In addition, genotype impact on serum folate levels was also assessed. The study population (n=900) consisted of 120 NTD case-parent triads (n=120×3=360) and 180 healthy control-parent triads (n=180×3=540) from South India. Umbilical cord tissues were collected from those with NTD and control newborn infants, and blood samples from case and control parents. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism. Statistical analysis used were SPSS, transmission disequilibrium test and POE. Serum folate levels were estimated using enzyme-linked immunosorbent assay. In the case-control study, those with the MTHFD1 G1958A variant were associated with around twofold risk of anencephaly (p=0.01) and spina bifida (p<0.01). Among parents, fathers were associated with around twofold risk of having an offspring with anencephaly (p<0.01). Considering offspring sex, the A allele in single or double dose conferred around two- to fourfold risk of anencephaly (p=0.01), spina bifida (p<0.01), and encephalocele (p<0.05) in females only. Maternal AA genotype was not associated independently but conferred threefold risk when combined with paternal GA genotype (p=0.01). Transmission disequilibrium and POE were not observed in controls (p>0.05) but revealed excess total (odds ratio [OR]=2.21; p<0.01) and paternal transmission (OR=7.00; p<0.01) of the G1958A allele to those with spina bifida, which remained the same for female cases (total transmission OR=3.00, p=0.01; paternal transmission OR=12.00, p<0.01). Increased serum folate levels were observed in case fathers with GA and AA genotypes than control fathers (p<0.01). Our research provides the first evidence supporting a paternal, rather than a maternal, transmission bias of MTHFD1 G1958A variant for NTD susceptibility in the offspring.

Altered folate metabolism modifies cell proliferation and progesterone secretion in human placental choriocarcinoma JEG-3 cells.

Folate is an essential B vitamin required for de novo purine and thymidylate synthesis, and for the remethylation of homocysteine to form methionine. Folate deficiency has been associated with placenta-related pregnancy complications, as have SNP in genes of the folate-dependent enzymes, methionine synthase (MTR) and methylenetetrahydrofolate dehydrogenase 1 (MTHFD1). We aimed to determine the effect of altered folate metabolism on placental cell proliferation, viability and invasive capacity and on progesterone and human chorionic gonadotropin (hCG) secretion. Human placental choriocarcinoma (JEG-3) cells cultured in low folic acid (FA) (2 nM) demonstrated 13% (P<0.001) and 26% (P<0.001) lower proliferation, 5.5% (P=0.025) and 7.5% (P=0.004) lower invasion capacity, and 5 to 7.5% (P=0.004-0.025) lower viability compared with control (20 nM) or supplemented (100 nM) cells, respectively. FA concentration had no effect on progesterone or hCG secretion. Small interfering RNA (siRNA) knockdown of MTR gene and protein expression resulted in 17.7% (P<0.0001) lower proliferation and 61% (P=0.014) higher progesterone secretion, but had no effect on cell invasion and hCG secretion. siRNA knockdown of MTHFD1 gene expression in the absence of detectable changes in protein expression resulted in 10.3% (P=0.001) lower cell proliferation, but had no effect on cell invasion and progesterone or hCG secretion. Our data indicate that impaired folate metabolism can result in lower trophoblast proliferation, and could alter viability, invasion capacity and progesterone secretion, which may explain in part the observed associations between folate and placenta-related complications.

Folate-mediated one-carbon metabolism genes and interactions with nutritional factors on colorectal cancer risk: Women's Health Initiative Observational Study.

Investigations of folate-mediated one-carbon metabolism (FOCM) genes and gene-nutrient interactions with respect to colorectal cancer (CRC) risk are limited to candidate polymorphisms and dietary folate. This study comprehensively investigated associations between genetic variants in FOCM and CRC risk and whether the FOCM nutrient status modified these associations. Two hundred eighty-eight candidate and tagging single-nucleotide polymorphisms (SNPs) in 30 FOCM genes were genotyped for 821 incident CRC case-control matched pairs in the Women's Health Initiative Observational Study cohort. FOCM biomarkers (red blood cell [RBC] folate, plasma folate, pyridoxal-5'-phosphate [PLP], vitamin B12, and homocysteine) and self-reported alcohol consumption were measured at the baseline. Conditional logistic regression was implemented; effect modification was examined on the basis of known enzyme-nutrient relations. Statistically significant associations were observed between CRC risk and functionally defined candidate SNPs of methylenetetrahydrofolate dehydrogenase 1 (MTHFD1; K134R), 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR; P450R), and PR domain containing 2 with ZNF domain (PRDM2; S450N) and a literature candidate SNP of thymidylate synthase (TYMS; g.676789A>T; nominal P < .05). In addition, suggestive associations were noted for tagging SNPs in cystathionine-β-synthase (CBS), dihydrofolate reductase (DHFR), DNA (cytosine-5-)-methyltransferase 3β (DNMT3B), methionine adenosyltransferase I α (MAT1A), MTHFD1, and MTRR (nominal P < .05; adjusted P, not significant). Significant interactions between nutrient biomarkers and candidate polymorphisms were observed for 1) plasma/RBC folate and folate hydrolase 1 (FOLH1), paraoxonase 1 (PON1), transcobalamin II (TCN2), DNMT1, and DNMT3B; 2) plasma PLP and TYMS TS3; 3) plasma B12 and betaine-homocysteine S-methyltransferase 2 (BHMT2); and 4) homocysteine and methylenetetrahydrofolate reductase (MTHFR) and alanyl-transfer RNA synthetase (AARS). Genetic variants in FOCM genes are associated with CRC risk among postmenopausal women. FOCM nutrients continue to emerge as effect modifiers of genetic influences on CRC risk.

Is MTHFD1 polymorphisms rs 2236225 (c.1958G&gt;A) associated with the susceptibility of NSCL/P? A systematic review and meta-analysis

To investigate the association between the methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) polymorphism rs 2236225 (c.1958G>A) and susceptibility to non-syndromic cleft of the lip and/or palate (NSCL/P). An extensive literature review has been conducted using PubMed, Web of Science, Cochrane Library, Google Scholar, the China National Knowledge Infrastructure (CNKI), and Wanfang Database for eligible researches. The terms for searching were "cleft lip OR cleft palate OR CLP OR CL/P OR oral facial cleft OR OFC" AND "methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1 OR methenyltetrahydrofolate cyclohydrolase formyltetrahydrofolate synthetase OR MTHFD1 OR MTHFD". Two independent researchers screened, evaluated and extracted the data of included studies. The pooled odds ratios (OR) with 95% confidence intervals (95% CI) were calculated by random effects model under five gene models. Subgroup, sensitivity analysis and publication bias were also assessed. Ten case-control studies have been included in the systematic review and eight studies have been considered for the meta-analysis. Overall, the MTHFD1 polymorphism rs2236225 and the risk of NSCL/P showed pooled OR (95% CI) of 1.02 (0.86-1.21) under allelic model. A higher degree of heterogeneity was observed in Asian countries (I (2) = 75.6%) compared to non-Asian countries (I (2) = 48.9%). Similar consequence appeared in the subgroup of children (I (2) = 78.6%) compared with that of mothers (I (2) = 0.0%). There was no significant difference in the publication bias by the Begg's funnel plot (P = 0.711) and Egger's regression test (P = 0.746). Our assessment suggested there was no significant association between the MTHFD1 polymorphism rs 2236225 (c.1958G>A) and the susceptibility to NSCL/P. Further investigations using a large sample size and a more advanced technique should be adopted to reach a more precise conclusion in the future.

Role of Trifunctional Enzyme MTHFD1 in Maintenance of DNA Stability

MTHFD1 is a trifunctional enzyme possessing methyleneTHF dehydrogenase (D), methenylTHF cyclohydrolase (C) and formylTHF synthetase (S) activities. We have recently demonstrated that MTHFD1 translocates to the nucleus in S phase where it plays a key enzymatic role providing one‐carbon units for dTMP synthesis. An inborn error of metabolism associated with mutations in the human methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) gene has been recently identified. The proband presented with severe combined immune deficiency (SCID), megaloblastic anemia and neurologic abnormalities. In this study, we examined the impact of MTHFD1 loss‐of‐function on DNA stability. Patient fibroblasts exhibited increased DNA damage relative to control fibroblasts. γH2AX levels, a marker of double‐strand breaks in DNA, were markedly increased in MTHFD1‐deficient patient fibroblasts relative to both control lines as well as the adenosine deaminase deficient fibroblast lines. γH2AX staining increased in patient fibroblasts when cultured in folate‐deficient medium as compared to fibroblasts cultured in folate‐replete medium, indicating that the MTHFD1‐deficient fibroblasts are sensitized to folate deficiency‐induced DNA damage.

MTHFD1 and Nuclear Thymidylate Biosynthesis

Thymidylate (dTMP) is the only DNA nucleotide formed at the sites of DNA replication. dTMP is synthesized by a multi‐enzyme complex, which includes serine hydroxymethylase 1 (SHMT1) and methylenetetrahydrofolate dehydrogenase 1 (MTHFD1). When dTMP synthesis is impaired, dU is incorporated into DNA, which can lead to DNA strand breaks. SHMT1 has been shown to act as a scaffold to anchor the thymidylate biosynthesis complex to the nuclear lamina and MTHFD1 supplies folate‐activated single carbons for dTMP synthesis. Both proteins have been shown to be SUMOylated in vitro and in vivo. Protein SUMOylation serves many functions including trafficking proteins to the nucleus, protecting proteins from ubiquitin dependent proteasome degradation, and assembling proteins into complexes. It is hypothesized that the non‐covalent interaction between SUMOylated proteins and SUMO interacting motifs (SIMs) on other proteins facilitate multiprotein complex formation and stabilization. In this study, MTHFD1 is shown to localize to the nucleus during S‐phase in HeLa cells and when MTHFD1 is mutated at its SUMOylation site, it's nuclear localization is impaired. Furthermore, we have identified a SIM on SHMT1 that may function in complex formation and may be regulated by CK2 phosphorylation. We also demonstrate that arsenic exposure affects SHMT1 and MTHFD1 SUMOylation, causes genome instability, and that the effects of folate‐depletion and arsenic on genome stability are additive. Collectively, these studies reveal the factors that regulate the metabolic complex responsible for dTMP biosynthesis to maintain genome stability.

The Role of Serine Hydroxymethyltransferase and Methylene‐Tetrahydrofolate Dehydrogenase in Genome Stability

The conversion of tetrahydrofolate (THF) to 5,10‐methyleneTHF is catalyzed by the isozymes serine hydroxymethyltransferase 1 and 2 (SHMT1, SHMT2) and the trifunctional enzyme methylenetetrahydrofolate dehydrogenase 1 (MTHFD1). 5,10‐methyleneTHF is a cofactor for the enzyme thymidylate synthase (TYMS) in the conversion of deoxyuridylate (dUMP) to thymidylate (dTMP) via one‐carbon metabolism. De novo dTMP synthesis is critical to prevent uracil misincoporation and double stranded breaks (DSBs). The objective of this experiment is to observe the effects of SHMT and MTHFD1 knockdown and arsenic exposure on the thymidylate biosynthesis pathway and genome stability. Reduced SHMT1, SHMT2, and MTHFD1 expression, either alone or in combination, led to a greater percentage of average nuclear area displaying the signal for γH2AX phosphorylation, indicating that knockdown of these proteins increases the extent of DSBs in DNA of affected cells. Arsenic induced uracil incorporation into DNA and increased the extent of genome instability without affecting de novo purine biosynthesis. These data indicate that both SHMT and MTHFD1 contribute to maintenance of genome stability. Supported by Public Health Services grant HD059120 to PJS.

Effects of Mthfd1 Gene on Mouse Behavior

Methylenetetrahydrofolate dehydrogenase 1 (Mthfd1) gene encodes the trifunctional C1 tetrahydrofolate (C1THF) enzyme that plays an important role in the folate-mediated one-carbon metabolism. A polymorphism of this gene (1958G>A; rs2236225) has been associated in humans with neural tube defects and with neuropsychiatric disorders such as bipolar disorder, depression, and schizophrenia. This gene has been shown to be essential in mice and moderate deficiency has been associated with impaired fetal growth. To understand the effects of moderate C1THF deficiency on cognition and behavior, we are running a battery of behavioral tests at adulthood on Mthfd1gt/+ and Mthfd1+/+ mice. We are assessing both male and female mice for locomotor activity (open field), sensorimotor function (rotarod), anxiety (open field and light-dark box), novel object and social investigation, and spatial memory (Morris Water maze). Our initial results show no difference between the heterozygous mice and wild type litter mates on locomotor activity and anxiety, as assessed in the open field. We also did not observe a difference in the novel object investigation. These findings and the results that will be obtained when the tests will be complete, will give us a picture of the role of Mthfd1 gene on behavior. This research is funded from Nutrition Research Institute, UNC Chapel Hill.

Effect of genetic polymorphisms involved in folate metabolism on the concentration of serum folate and plasma total homocysteine (p-tHcy) in healthy subjects after short-term folic acid supplementation: a randomized, double blind, crossover study.

Data on the effect of combined genetic polymorphisms, involved in folate metabolism, on the concentration of serum folate after folic acid supplementation are scarce. Therefore, we investigated the impact of seven gene polymorphisms on the concentration of serum folate and p-tHcy in healthy subjects after short-term folic acid supplementation. In a randomized, double blind, crossover study, apparently healthy subjects were given either 0.8mg folic acid per day (n=46) or placebo (n=45) for 14days. The washout period was 14days. Fasting blood samples were collected on day 1, 15, 30 and 45. Data on subjects on folic acid supplementation (n=91) and on placebo (n=45) were used for the statistical analysis. The concentration of serum folate increased higher in subjects with higher age (53.5±7.0years) than in subjects with lower age (24.3±3.2years) after folic acid supplementation (p=0.006). The baseline concentration of serum folate in subjects with polymorphism combination, reduced folate carrier protein, RFC1-80 GA and methylenetetrahydrofolate reductase, MTHFR677 CT+TT, was lower than RFC1-80 AA and MTHFR677 CT+TT (p=0.002). After folic acid supplementation, a higher increase in the concentration of serum folate was detected in subjects with polymorphism combination RFC1-80 GA and MTHFR677 CC than RFC1-80 GG and MTHFR CT+TT combination (p<0.0001). The baseline concentration of plasma total homocysteine (p-tHcy) was altered by combined polymorphisms in genes associated with folate metabolism. After folic acid supplementation, in subjects with combined polymorphisms in methylenetetrahydrofolate dehydrogenase, MTHFD1-1958 and MTHFR-677 genes, the concentration of p-tHcy was changed (p=0.002). The combination of RFC1-80 and MTHFR-677 polymorphisms had a profound affect on the concentration of serum folate in healthy subjects before and after folic acid supplementation.

Folate metabolism gene polymorphisms and risk for down syndrome offspring in Turkish women.

Down syndrome (DS) is the most common chromosomal abnormality. Many studies have assessed the association between maternal gene polymorphisms involved in folate metabolism and the risk of having a DS offspring, but data are conflicting. Six common polymorphisms in folate-metabolizing genes were analayzed to determine possible risk factors for a child to be born having DS (DS mothers); these samples were taken from 47 Turkish mothers having DS children (case group) and 49 control mothers. Investigated polymorphisms include methylenetetrahydrofolate reductase (MTHFR) C677T (rs1801133), A1298C (rs1801131), methionine synthase reductase (MTRR) A66G (rs1801394), methylenetetrahydrofolate dehydrogenase (MTHFD1) G1958A (rs2236225), reduced folate carrier (RFC1) A80G (rs1051266), and cystathionine β-synthase (CBS) 844ins68. The frequency of the MTHFR 677C allele in DS mothers (79.8%) was significantly higher than in controls (66.3%), with a 0.499-fold increased risk of having a DS offspring (p=0.038 and 95% confidence interval [CI], 0.259-0.961). Mothers with the MTHFD1 1958A allele had a 1.880-fold increased risk of having a child with DS (p=0.031 and 95% CI, 1.060-3.335). No significant association was found for the other polymorphic variants in this study. Gene-gene interactions were not statistically significant. Polymorphic variants of the enzymes involved in folate metabolism may play an important role in determining the susceptibility of having a DS offspring. The gene-nutrition, gene-gene interactions and ethnicity are important variables to be considered in future studies.

Potential functional gene diversity involved in methanogenesis and methanogenic community structure in Indian buffalo (Bubalus bubalis) rumen.

Understanding the methanogen community structure and methanogenesis from Bubalus bubalis in India may be beneficial to methane mitigation. Our current understanding of the microbial processes leading to methane production is incomplete, and further advancement in the knowledge of methanogenesis pathways would provide means to manipulate its emission in the future. In the present study, we evaluated the methanogenic community structure in the rumen as well as their potential genes involved in methanogenesis. The taxonomic and metabolic profiles of methanogens were assessed by shotgun sequencing of rumen metagenome by Ion Torrent semiconductor sequencing. The buffalo rumen contained representative genera of all the families of methanogens. Members of Methanobacteriaceae were found to be dominant, followed by Methanosarcinaceae, Methanococcaceae, Methanocorpusculaceae, and Thermococcaceae. A total of 60 methanogenic genera were detected in buffalo rumen. Methanogens related to the genera Methanobrevibacter, Methanosarcina, Methanococcus, Methanocorpusculum, Methanothermobacter, and Methanosphaera were predominant, representing >70 % of total archaeal sequences. The metagenomic dataset indicated the presence of genes involved in the methanogenesis and acetogenesis pathways, and the main functional genes were those of key enzymes in the methanogenesis. Sequences related to CoB--CoM heterodisulfide reductase, methyl coenzyme M reductase, f420-dependent methylenetetrahydromethanopterin reductase, and formylmethanofuran dehydrogenase were predominant in rumen. In addition, methenyltetrahydrofolate cyclohydrolase, methylenetetrahydrofolate dehydrogenase, 5,10-methylenetetrahydrofolate reductase, and acetyl-coenzyme A synthetase were also recovered.