RESUMO
One-carbon units, critical intermediates for cell growth, may be produced by a variety of means, one of which is via the production of formate. Excessive formate accumulation, known as formate overflow and a characteristic of oxidative cancer, has been observed in cancer cells. However, the basis for this high rate of formate production is unknown. We examined the effect of elevated expression of oncogenic Ras (RasV12), on formate production in NIH-3T3 cells (mouse fibroblasts) cultured with either labelled 13C-serine or 13C-glycine. Formate accumulation by the fibroblasts transformed by RasV12 was increased two-threefold over those by vector control (Babe) cells. The production of formate exceeded the rate of utilization in both cell types. 13C-formate was produced almost exclusively from the #3 carbon of 13C-serine. Virtually no labelled formate was produced from either the #2 carbon of serine or the #2 carbon of glycine. The increased formate production by RasV12 cells was associated with increased mRNA abundances for enzymes of formate production in both the mitochondria and the cytosol. Thus, we find the oncogenic RasV12 significantly increases formate overflow and may be one way for tumor cells to produce one-carbon units required for enhanced proliferation of these cells and/or for other processes which have not been identified.
Assuntos
Formiatos/metabolismo , Genes ras , Proteínas ras , Aminoidrolases/genética , Aminoidrolases/metabolismo , Animais , Formiato-Tetra-Hidrofolato Ligase/genética , Formiato-Tetra-Hidrofolato Ligase/metabolismo , Regulação da Expressão Gênica , Glicina/metabolismo , Glicina Hidroximetiltransferase/genética , Glicina Hidroximetiltransferase/metabolismo , Meteniltetra-Hidrofolato Cicloidrolase/genética , Meteniltetra-Hidrofolato Cicloidrolase/metabolismo , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Camundongos , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Células NIH 3T3 , RNA Mensageiro/genética , Serina/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Background: Suboptimal folate intake, a risk factor for birth defects, is common even in areas with folate fortification. A polymorphism in methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), R653Q (MTHFD1 c.1958 G > A), has also been associated with increased birth defect risk, likely through reduced purine synthesis. Objective: We aimed to determine if the interaction of MTHFD1 synthetase deficiency and low folate intake increases developmental abnormalities in a mouse model for MTHFD1 R653Q. Methods: Female Mthfd1S+/+ and Mthfd1S+/- mice were fed control or low-folate diets (2 and 0.3 mg folic acid/kg diet, respectively) before mating and during pregnancy. Embryos and placentas were examined for anomalies at embryonic day 10.5. Maternal 1-carbon metabolites were measured in plasma and liver. Results: Delays and defects doubled in litters of Mthfd1S+/- females fed low-folate diets compared to wild-type females fed either diet, or Mthfd1S+/- females fed control diets [P values (defects): diet 0.003, maternal genotype 0.012, diet × maternal genotype 0.014]. These adverse outcomes were associated with placental dysmorphology. Intrauterine growth restriction was increased by embryonic Mthfd1S+/- genotype, folate deficiency, and interaction of maternal Mthfd1S+/- genotype with folate deficiency (P values: embryonic genotype 0.045, diet 0.0081, diet × maternal genotype 0.0019). Despite a 50% increase in methylenetetrahydrofolate reductase expression in low-folate maternal liver (P diet = 0.0007), methyltetrahydrofolate concentration decreased 70% (P diet <0.0001) and homocysteine concentration doubled in plasma (P diet = 0.0001); S-adenosylmethionine decreased 40% and S-adenosylhomocysteine increased 20% in low-folate maternal liver (P diet = 0.002 and 0.0002, respectively). Conclusions: MTHFD1 synthetase-deficient mice are more sensitive to low folate intake than wild-type mice during pregnancy. Reduced purine synthesis due to synthetase deficiency and altered methylation potential due to low folate may increase pregnancy complications. Further studies and individualized intake recommendations may be required for women homozygous for the MTHFD1 R653Q variant.
Assuntos
Anormalidades Congênitas/etiologia , Deficiência de Ácido Fólico/complicações , Ácido Fólico/administração & dosagem , Formiato-Tetra-Hidrofolato Ligase/deficiência , Genótipo , Meteniltetra-Hidrofolato Cicloidrolase/deficiência , Metilenotetra-Hidrofolato Desidrogenase (NADP)/deficiência , Enzimas Multifuncionais/deficiência , Polimorfismo Genético , Complicações na Gravidez/etiologia , Animais , Metilação de DNA , Dieta , Modelos Animais de Doenças , Feminino , Desenvolvimento Fetal , Retardo do Crescimento Fetal/etiologia , Ácido Fólico/sangue , Deficiência de Ácido Fólico/sangue , Deficiência de Ácido Fólico/genética , Deficiência de Ácido Fólico/metabolismo , Formiato-Tetra-Hidrofolato Ligase/genética , Formiato-Tetra-Hidrofolato Ligase/metabolismo , Ligases , Fígado/metabolismo , Meteniltetra-Hidrofolato Cicloidrolase/genética , Meteniltetra-Hidrofolato Cicloidrolase/metabolismo , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Camundongos , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Placenta , Gravidez , Complicações na Gravidez/sangue , Complicações na Gravidez/genética , Complicações na Gravidez/metabolismo , Prenhez , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Tetra-Hidrofolatos/sangueRESUMO
The common R653Q variant (â¼20% homozygosity in Caucasians) in the synthetase domain of the folate-metabolizing enzyme MTHFD1 reduces purine synthesis. Although this variant does not appear to affect risk for colorectal cancer, we questioned whether it would affect growth of colorectal tumors. We induced tumor formation in a mouse model for MTHFD1-synthetase deficiency (Mthfd1S+/- ) using combined administration of azoxymethane (AOM) and dextran sodium sulfate (DSS) in male and female wild-type and Mthfd1S+/- mice. Tumor size was significantly smaller in MthfdS+/- mice, particularly in males. A reduction in the proliferation of MthfdS+/- mouse embryonic fibroblast cell lines, compared with wild-type lines, was also observed. Tumor number was not influenced by genotype. The amount of inflammation observed within tumors from male Mthfd1S+/- mice was lower than that in wild-type mice. Gene expression analysis in tumor adjacent normal (pre-neoplastic) tissue identified several genes involved in proliferation (Fosb, Fos, Ptk6, Esr2, Atf3) and inflammation (Atf3, Saa1, TNF-α) that were downregulated in MthfdS+/- males. In females, MthfdS+/- genotype was not associated with these gene expression changes, or with differences in tumor inflammation. These findings suggest that the mechanisms directing tumor growth differ significantly between males and females. We suggest that restriction of purine synthesis, reduced expression of genes involved in proliferation, and/or reduced inflammation lead to slower tumor growth in MTHFD1-synthetase deficiency. These findings may have implications for CRC tumor growth and prognosis in individuals with the R653Q variant. © 2016 Wiley Periodicals, Inc.
Assuntos
Aminoidrolases/deficiência , Neoplasias Colorretais/patologia , Formiato-Tetra-Hidrofolato Ligase/deficiência , Meteniltetra-Hidrofolato Cicloidrolase/deficiência , Metilenotetra-Hidrofolato Desidrogenase (NADP)/deficiência , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Antígenos de Histocompatibilidade Menor/genética , Complexos Multienzimáticos/deficiência , Enzimas Multifuncionais/deficiência , Polimorfismo de Nucleotídeo Único , Animais , Azoximetano/efeitos adversos , Proliferação de Células , Células Cultivadas , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/genética , Sulfato de Dextrana/efeitos adversos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , CamundongosRESUMO
Methylenetetrahydrofolate dehydrogenase (NAD(P)+ dependent) 2, methenyltetrahydrofolate cyclohydrolase (MTHFD2) is a mitochondrial enzyme involved in folate metabolism. A number of recent studies have highlighted this enzyme as being highly expressed in many solid tumors, including breast cancer, and to be correlated with poor survival. However, the metabolic functions of MTHFD2 in cancer cells have not been well-defined. To investigate the function of MTHFD2 in breast cancer cells, we generated and characterized MCF-7 cells with stable suppression of MTHFD2 expression using a combination of cellular assays and metabolic profiling. Loss of MTHFD2 caused MCF7 cells to become glycine auxotrophs, that is, reliant on exogenous glycine, and more sensitive to exogenous folate depletion. Another prominent metabolic alteration observed as a consequence of MTHFD2 suppression was a more glycolytic phenotype, consistent with widespread modifications of cellular metabolism. Collectively, these data suggest that targeting MTHFD2 activity is likely to influence multiple metabolic pathways in breast cancer and could be combined with a range of antimetabolite therapies.
Assuntos
Aminoidrolases/deficiência , Glicólise , Metaboloma , Meteniltetra-Hidrofolato Cicloidrolase/deficiência , Metilenotetra-Hidrofolato Desidrogenase (NADP)/deficiência , Enzimas Multifuncionais/deficiência , Ácido Fólico/metabolismo , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glicina/metabolismo , Humanos , Células MCF-7 , Redes e Vias Metabólicas , Proteínas de NeoplasiasRESUMO
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 â¼ 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.
Assuntos
Clostridium perfringens/enzimologia , Escherichia coli/enzimologia , Formiato-Tetra-Hidrofolato Ligase/metabolismo , Meteniltetra-Hidrofolato Cicloidrolase/metabolismo , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Sequência de Aminoácidos , Clostridium perfringens/química , Clostridium perfringens/genética , Escherichia coli/química , Escherichia coli/genética , Formiato-Tetra-Hidrofolato Ligase/química , Formiato-Tetra-Hidrofolato Ligase/genética , Cinética , Meteniltetra-Hidrofolato Cicloidrolase/química , Meteniltetra-Hidrofolato Cicloidrolase/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/química , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
Bioconversion of the C1 compounds into value-added products is one of the CO2-reducing strategies. In particular, because CO2 can be easily converted into formate, the efficient and direct bioconversion of CO2 through formate assimilation is attracting attention. The tetrahydrofolate (THF) cycle is the highly efficient reconstructed formate assimilation pathway, and 5,10-methenyltetrahydrofolate cyclohydrolase (FchA) is an essential enzyme involved in the THF cycle. In this study, a kinetic analysis of FchA from Methylobacterium extorquens AM1 (MeFchA) was performed and revealed that the enzyme has much higher cyclization than hydrolyzation activity, making it an optimal enzyme for formate assimilation. The crystal structure of MeFchA in the apo- and the THF-complexed forms was also determined, revealing that the substrate-binding site of the enzyme has three differently charged regions to stabilize the three differently charged moieties of the formyl-THF substrate. The residues involved in the substrate binding were also verified through site-directed mutagenesis. This study provides a biochemical and structural basis for the molecular mechanism underlying formate assimilation.
Assuntos
Meteniltetra-Hidrofolato Cicloidrolase , Methylobacterium extorquens , Sítios de Ligação , Cinética , Meteniltetra-Hidrofolato Cicloidrolase/metabolismo , Methylobacterium extorquens/genética , Methylobacterium extorquens/metabolismo , Mutagênese Sítio-DirigidaRESUMO
The causal metabolic pathways underlying associations between folate and risk for colorectal cancer (CRC) have yet to be established. Folate-mediated one-carbon metabolism is required for the de novo synthesis of purines, thymidylate and methionine. Methionine is converted to S-adenosylmethionine (AdoMet), the major one-carbon donor for cellular methylation reactions. Impairments in folate metabolism can modify DNA synthesis, genomic stability and gene expression, characteristics associated with tumorigenesis. The Mthfd1 gene product, C1-tetrahydrofolate synthase, is a trifunctional enzyme that generates one-carbon substituted tetrahydrofolate cofactors for one-carbon metabolism. In this study, we use Mthfd1(gt/+) mice, which demonstrate a 50% reduction in C1-tetrahydrofolate synthase, to determine its influence on tumor development in two mouse models of intestinal cancer, crosses between Mthfd1(gt/+) and Apc(min)(/+) mice and azoxymethane (AOM)-induced colon cancer in Mthfd1(gt/+) mice. Mthfd1 hemizygosity did not affect colon tumor incidence, number or load in Apc(min/+) mice. However, Mthfd1 deficiency increased tumor incidence 2.5-fold, tumor number 3.5-fold and tumor load 2-fold in AOM-treated mice. DNA uracil content in the colon was lower in Mthfd1(gt/+) mice, indicating that thymidylate biosynthesis capacity does not play a significant role in AOM-induced colon tumorigenesis. Mthfd1 deficiency-modified cellular methylation potential, as indicated by the AdoMet: S-adenosylhomocysteine ratio and gene expression profiles, suggesting that changes in the transcriptome and/or decreased de novo purine biosynthesis and associated mutability cause cellular transformation in the AOM CRC model. This study emphasizes the impact and complexity of gene-nutrient interactions with respect to the relationships among folate metabolism and colon cancer initiation and progression.
Assuntos
Aminoidrolases/fisiologia , Neoplasias do Colo/genética , DNA de Neoplasias/metabolismo , Formiato-Tetra-Hidrofolato Ligase/fisiologia , Meteniltetra-Hidrofolato Cicloidrolase/fisiologia , Metilenotetra-Hidrofolato Desidrogenase (NADP)/fisiologia , Complexos Multienzimáticos/fisiologia , Enzimas Multifuncionais/fisiologia , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Aminoidrolases/genética , Animais , Apoptose , Azoximetano/toxicidade , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Carcinógenos/toxicidade , Proliferação de Células , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Feminino , Formiato-Tetra-Hidrofolato Ligase/genética , Perfilação da Expressão Gênica , Técnicas Imunoenzimáticas , Masculino , Meteniltetra-Hidrofolato Cicloidrolase/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multienzimáticos/genética , Enzimas Multifuncionais/genética , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Uracila/metabolismoRESUMO
Folate co-enzymes play a pivotal role in one-carbon transfer cellular processes. Many eukaryotes encode the tri-functional tetrahydrofolate dehydrogenase/cyclohydrolase/synthetase (deh/cyc/syn) enzyme, which consists of a N-terminal bifunctional domain (deh/cyc) and a C-terminal monofunctional domain (syn). Here, we report the first analogous archeal enzyme structures, for the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase from Thermoplasma acidophilum (TaMTHFDC) as the native protein and also as its NADP complex. The TaMTHFDC structure is a dimer with a polar interface, as well as a NADP binding site that shows minor conformational change. The orientations of the residues in the NADP binding site do not change on ligand binding, incorporating three water molecules which are hydrogen bonded with phosphate groups of NADP in the structure of the complex. Our structural information will contribute to an improved understanding of the basis of THF and one-carbon metabolism.
Assuntos
Meteniltetra-Hidrofolato Cicloidrolase/química , Thermoplasma/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Cristalização , Cristalografia por Raios X , Meteniltetra-Hidrofolato Cicloidrolase/genética , Dados de Sequência Molecular , NADP/química , Multimerização Proteica , Estrutura Secundária de Proteína , Tetra-Hidrofolatos/químicaRESUMO
Folate and choline are interconnected metabolically. The MTHFD1 R653Q SNP is a risk factor for birth defects and there are concerns that choline deficiency may interact with this SNP and exacerbate health risks. 80-90% of women do not meet the Adequate Intake (AI) for choline. The objective of this study was to assess the effects of choline deficiency on maternal one-carbon metabolism and reproductive outcomes in the MTHFD1-synthetase deficient mouse (Mthfd1S), a model for MTHFD1 R653Q. Mthfd1S+/+ and Mthfd1S+/- females were fed control (CD) or choline-deficient diets (ChDD; 1/3 the amount of choline) before mating and during pregnancy. Embryos were evaluated for delays and defects at 10.5 days gestation. Choline metabolites were measured in the maternal liver, and total folate measured in maternal plasma and liver. ChDD significantly decreased choline, betaine, phosphocholine, and dimethylglycine in maternal liver (p < 0.05, ANOVA), and altered phosphatidylcholine metabolism. Maternal and embryonic genotype, and diet-genotype interactions had significant effects on defect incidence. Mild choline deficiency and Mthfd1S+/- genotype alter maternal one-carbon metabolism and increase incidence of developmental defects. Further study is required to determine if low choline intakes contribute to developmental defects in humans, particularly in 653QQ women.
Assuntos
Aminoidrolases/genética , Deficiência de Colina/genética , Deficiências do Desenvolvimento/genética , Formiato-Tetra-Hidrofolato Ligase/deficiência , Formiato-Tetra-Hidrofolato Ligase/genética , Fenômenos Fisiológicos da Nutrição Materna/genética , Meteniltetra-Hidrofolato Cicloidrolase/deficiência , Metilenotetra-Hidrofolato Desidrogenase (NADP)/deficiência , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Complexos Multienzimáticos/genética , Enzimas Multifuncionais/deficiência , Animais , Colina/análise , Deficiências do Desenvolvimento/epidemiologia , Modelos Animais de Doenças , Desenvolvimento Embrionário/genética , Feminino , Ácido Fólico/metabolismo , Genótipo , Incidência , Fígado/metabolismo , Camundongos , Polimorfismo de Nucleotídeo Único , GravidezRESUMO
Obesity has been followed with interest as a risk factor for COVID-19, with triglycerides as one of four common criteria used to define obesity, which have been used to study the mechanism of obesity. In this study, we showed that angiotensin-converting enzyme-2 (ACE2) is widely expressed in the mouse body, including the kidney, spleen, brain, heart, lung, liver, and testis, and that ACE2 levels increased after a high-fat diet. The ACE2 levels were recorded at 0 days, 3 days, 7 days, and 14 days after a high-fat diet, and they increased at 14 days after high-fat diet initiation. In addition, triglyceride levels were also significantly increased at 14 days after high-fat diet initiation, but body weight was not changed. Furthermore, we examined the ACE2 levels in Calu3 cells (a lung cancer cell line) after triglyceride treatment, and the results indicated that ACE2 levels were increased at 25 µM and reached their peak at 200 µM. Finally, we found that the mRNA level of mthfd1 was significantly increased in the high-fat diet group. Given these findings, we hypothesize that triglycerides can regulate the expression of ACE2 and Mthfd1.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Dieta Hiperlipídica/efeitos adversos , Formiato-Tetra-Hidrofolato Ligase/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Meteniltetra-Hidrofolato Cicloidrolase/metabolismo , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Enzimas Multifuncionais/metabolismo , Triglicerídeos/farmacologia , Enzima de Conversão de Angiotensina 2/genética , Animais , Biomarcadores/sangue , COVID-19/etiologia , COVID-19/virologia , Linhagem Celular , Sobrevivência Celular , Formiato-Tetra-Hidrofolato Ligase/genética , Humanos , Masculino , Meteniltetra-Hidrofolato Cicloidrolase/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Camundongos , Camundongos Endogâmicos C57BL , Enzimas Multifuncionais/genética , Obesidade/complicações , Obesidade/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Risco , SARS-CoV-2 , Triglicerídeos/administração & dosagemRESUMO
10-Formyl tetrahydrofolate (10-CHO-THF) is a key metabolite in C1 carbon metabolism, arising through the action of formate-tetrahydrofolate ligase (FTL) and/or 5,10-methenyltetrahydrofolate cyclohydrolase/5,10-methylene tetrahydrofolate dehydrogenase (DHCH). Leishmania major possesses single DHCH1 and FTL genes encoding exclusively cytosolic proteins, unlike other organisms where isoforms occur in the mitochondrion as well. Recombinant DHCH1 showed typical NADP(+)-dependent methylene tetrahydrofolate DH and 5,10-methenyltetrahydrofolate CH activities, and the DH activity was potently inhibited by a substrate analogue 5,10-CO-THF (K(i) 105 nM), as was Leishmania growth (EC(50) 1.1 microM). Previous studies showed null ftl(-) mutants were normal, raising the possibility that loss of the purine synthetic pathway had rendered 10-CHO-THF dispensable in evolution. We were unable to generate dhch1(-) null mutants by gene replacement, despite using a wide spectrum of nutritional supplements expected to bypass DHCH function. We applied an improved method for testing essential genes in Leishmania, based on segregational loss of episomal complementing genes rather than transfection; analysis of approximately 1400 events without successful loss of DHCH1 again established its requirement. Lastly, we employed 'genetic metabolite complementation' using ectopically expressed FTL as an alternative source of 10-CHO-THF; now dhch1(-) null parasites were readily obtained. These data establish a requirement for 10-CHO-THF metabolism in L. major, and provide genetic and pharmacological validation of DHCH as a target for chemotherapy, in this and potentially other protozoan parasites.
Assuntos
Leishmania major/enzimologia , Leucovorina/análogos & derivados , Meteniltetra-Hidrofolato Cicloidrolase/metabolismo , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Animais , Clonagem Molecular , Antagonistas do Ácido Fólico/farmacologia , Técnicas de Inativação de Genes , Genes Essenciais , Genes de Protozoários , Leishmania major/efeitos dos fármacos , Leishmania major/genética , Leucovorina/metabolismo , Meteniltetra-Hidrofolato Cicloidrolase/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Mutação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
One-carbon metabolism produces methionine and N10-formyl-tetrahydrofolate (N10-fTHF) required for aminoacylation and formylation of initiator tRNA (i-tRNA), respectively. In Escherichia coli, N10-fTHF is made from 5, 10-methylene-THF by a two-step reaction using 5,10-methylene-THF dehydrogenase/cyclohydrolase (FolD). The i-tRNAs from all domains of life possess a highly conserved sequence of three consecutive G-C base pairs (3GC pairs) in their anticodon stem. A 3GC mutant i-tRNA (wherein the 3GC pairs are mutated to those found in elongator tRNAMet) is incompetent in initiation in E. coli (even though it is efficiently aminoacylated and formylated). Here, we show that E. coli strains having mutations in FolD (G122D or C58Y or P140L) allow a plasmid encoded 3GC mutant i-tRNA to participate in initiation. In vitro, the FolD mutants are highly compromised in their dehydrogenase/cyclohydrolase activities leading to reduced production of N10-fTHF and decreased rates of i-tRNA formylation. The perturbation of one-carbon metabolism by trimethoprim (inhibitor of dihydrofolate reductase) phenocopies FolD deficiency and allows initiation with the 3GC mutant i-tRNA. This study reveals an important crosstalk between one-carbon metabolism and the fidelity of translation initiation via formylation of i-tRNA, and suggests that augmentation of the age old sulfa drugs with FolD inhibitors could be an important antibacterial strategy.
Assuntos
Escherichia coli/metabolismo , Formiltetra-Hidrofolatos/metabolismo , Iniciação Traducional da Cadeia Peptídica , RNA de Transferência de Metionina/metabolismo , Anticódon/metabolismo , Proteínas de Escherichia coli/metabolismo , Análise do Fluxo Metabólico , Meteniltetra-Hidrofolato Cicloidrolase/metabolismo , Modelos MolecularesRESUMO
The pluripotency of stem cells determines their developmental potential. While the pluripotency states of pluripotent stem cells are variable and interconvertible, the mechanisms underlying the acquisition and maintenance of pluripotency remain largely elusive. Here, we identified that methylenetetrahydrofolate dehydrogenase (NAD+-dependent), methenyltetrahydrofolate cyclohydrolase (Mthfd2) plays an essential role in maintaining embryonic stem cell pluripotency and promoting complete reprogramming of induced pluripotent stem cells. Mechanistically, in mitochondria, Mthfd2 maintains the integrity of the mitochondrial respiratory chain and prevents mitochondrial dysfunction. In the nucleus, Mthfd2 stabilizes the phosphorylation of EXO1 to support DNA end resection and promote homologous recombination repair. Our results revealed that Mthfd2 is a dual-function factor in determining the pluripotency of pluripotent stem cells through both mitochondrial and nuclear pathways, ultimately ensuring safe application of pluripotent stem cells.
Assuntos
Aminoidrolases/metabolismo , Reparo do DNA , Células-Tronco Pluripotentes Induzidas/metabolismo , Meteniltetra-Hidrofolato Cicloidrolase/metabolismo , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Mitocôndrias/metabolismo , Complexos Multienzimáticos/metabolismo , Animais , Proteína Quinase CDC2/metabolismo , Núcleo Celular/metabolismo , Autorrenovação Celular/genética , Dano ao DNA , Enzimas Reparadoras do DNA/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Exodesoxirribonucleases/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Glicólise , Meteniltetra-Hidrofolato Cicloidrolase/deficiência , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Fosforilação Oxidativa , Fosforilação , Ligação ProteicaRESUMO
Methylenetetrahydrofolate dehydrogenase)methenyltetrahydrofolate cyclohydrolase)formyltetrahydrofolate synthetase (MTHFD1) is a trifunctional enzyme that interconverts tetrahydrofolate (THF) derivatives for nucleotide synthesis. A common variant in MTHFD1, p.Arg653Gln (c.1958G>A), may increase the risk for neural tube defects (NTD). To examine the biological impact of this variant on MTHFD1 function, we measured enzyme activity and stability in vitro and assessed substrate flux in transfected mammalian cells. The purified Arg653Gln enzyme has normal substrate affinity but a 36% reduction in half)life at 42 degrees C. Thermolability is reduced by magnesium adenosine triphosphate and eliminated by the substrate analog folate pentaglutamate, suggesting that folate status may modulate impact of the variant. The mutation reduces the metabolic activity of MTHFD1 within cells: formate incorporation into DNA in murine Mthfd1 knockout cells transfected with Arg653Gln is reduced by 26%+/-7.7% (P<0.05), compared to cells transfected with wild)type protein, indicating a disruption of de novo purine synthesis. We assessed the impact of the variant on risk for congenital heart defects (CHD) in a cohort of Quebec children (158 cases, 110 controls) and mothers of children with heart defects (199 cases, 105 controls). The 653QQ genotype in children is associated with increased risk for heart defects (odds ratio [OR], 2.11; 95% confidence interval [CI], 1.01-4.42), particularly Tetralogy of Fallot (OR, 3.60; 95% CI, 1.38-9.42) and aortic stenosis (OR, 3.13; 95% CI, 1.13-8.66). There was no effect of maternal genotype. Our results indicate that the Arg653Gln polymorphism decreases enzyme stability and increases risk for CHD. Further evaluation of this polymorphism in folate)related disorders and its potential interaction with folate status is warranted.
Assuntos
Substituição de Aminoácidos , Predisposição Genética para Doença , Cardiopatias Congênitas/enzimologia , Cardiopatias Congênitas/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Mutação/genética , Adolescente , Animais , Estudos de Casos e Controles , Coenzimas , Inibidores Enzimáticos , Estabilidade Enzimática , Feminino , Formiato-Tetra-Hidrofolato Ligase/genética , Formiato-Tetra-Hidrofolato Ligase/isolamento & purificação , Formiato-Tetra-Hidrofolato Ligase/metabolismo , Frequência do Gene , Humanos , Cinética , Meteniltetra-Hidrofolato Cicloidrolase/genética , Meteniltetra-Hidrofolato Cicloidrolase/isolamento & purificação , Meteniltetra-Hidrofolato Cicloidrolase/metabolismo , Metilenotetra-Hidrofolato Desidrogenase (NADP)/isolamento & purificação , Camundongos , Antígenos de Histocompatibilidade Menor , Polimorfismo Genético , Homologia Estrutural de Proteína , Especificidade por Substrato , TemperaturaRESUMO
Bacillus subtilis mutants with resistance against peptide deformylase inhibitors were isolated. All showed a bypass of the pathway through mutations in three genes required for formylation of Met-tRNA(fMet), fmt, folD, and glyA. glyA corresponds to a yet uncharacterized locus inducing resistance. The bypass of formylation caused robust fitness reduction but was not accompanied by alterations of the transcription profile. A subtle adaptation of the enzymes of the intermediary metabolism was observed.
Assuntos
Amidoidrolases/antagonistas & inibidores , Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Mutação , Bacillus subtilis/genética , Farmacorresistência Bacteriana , Glicina Hidroximetiltransferase/genética , Ácidos Hidroxâmicos/farmacologia , Meteniltetra-Hidrofolato Cicloidrolase/genética , Reação em Cadeia da Polimerase , RNA de Transferência de Metionina/genéticaRESUMO
BACKGROUND: Periconceptional intake of supplemental folic acid can reduce the incidence of neural tube defects by as much as 70%, but the mechanisms by which folic acid supports cellular processes during neural tube closure are unknown. The mitochondrial 10-formyl-tetrahydrofolate synthetase MTHFD1L catalyzes production of formate, thus generating one-carbon units for cytoplasmic processes. Deletion of Mthfd1l causes embryonic lethality, developmental delay, and neural tube defects in mice. METHODS: To investigate the role of mitochondrial one-carbon metabolism during cranial neural tube closure, we have analyzed cellular morphology and function in neural tissues in Mthfd1l knockout embryos. RESULTS: The head mesenchyme showed significantly lower cellular density in Mthfd1l nullizygous embryos compared to wildtype embryos during the process of neural tube closure. Apoptosis and neural crest cell specification were not affected by deletion of Mthfd1l. Sections from the cranial region of Mthfd1l knockout embryos exhibited decreased cellular proliferation, but only after completion of neural tube closure. Supplementation of pregnant dams with formate improved mesenchymal density and corrected cell proliferation in the nullizygous embryos. CONCLUSIONS: Deletion of Mthfd1l causes decreased density in the cranial mesenchyme and this defect is improved with formate supplementation. This study reveals a mechanistic link between folate-dependent mitochondrially produced formate, head mesenchyme formation and neural tube defects.
Assuntos
Formiato-Tetra-Hidrofolato Ligase/genética , Meteniltetra-Hidrofolato Cicloidrolase/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Enzimas Multifuncionais/genética , Defeitos do Tubo Neural/genética , Animais , Embrião de Mamíferos/metabolismo , Feminino , Ácido Fólico/genética , Ácido Fólico/metabolismo , Formiato-Tetra-Hidrofolato Ligase/metabolismo , Formiatos/metabolismo , Masculino , Mesoderma/metabolismo , Meteniltetra-Hidrofolato Cicloidrolase/metabolismo , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Enzimas Multifuncionais/metabolismo , Crista Neural/metabolismo , Defeitos do Tubo Neural/metabolismo , Neurulação , Deleção de SequênciaRESUMO
The MTHFD1 gene encodes for methylenetetrahydrofolate dehydrogenase 1, an enzyme that has an important role in folate-mediated one-carbon metabolism. In people, a single nucleotide polymorphism of this gene (1958G>A; rs2236225) is associated with increased risk for bipolar disorder and schizophrenia, neural tube and other birth defects. Mice homozygous for a loss of Mthfd1 via a gene-trap mutation are not viable, and heterozygotes, though they appear healthy, have metabolic imbalances in the folate- and choline-mediated 1-carbon metabolic pathways. In this study, we evaluated cognitive function in Mthfd1gt/+ male and female mice using a behavioral battery composed of eight different tests. We found that these mice display impaired cue-conditioned learning, while other behaviors remain intact.
Assuntos
Formiato-Tetra-Hidrofolato Ligase/deficiência , Deficiências da Aprendizagem/enzimologia , Meteniltetra-Hidrofolato Cicloidrolase/deficiência , Metilenotetra-Hidrofolato Desidrogenase (NADP)/deficiência , Enzimas Multifuncionais/deficiência , Animais , Peso Corporal , Cognição/fisiologia , Feminino , Formiato-Tetra-Hidrofolato Ligase/genética , Masculino , Meteniltetra-Hidrofolato Cicloidrolase/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Camundongos Transgênicos , Enzimas Multifuncionais/genética , Testes Neuropsicológicos , FenótipoRESUMO
To sustain their proliferation, cancer cells become dependent on one-carbon metabolism to support purine and thymidylate synthesis. Indeed, one of the most highly upregulated enzymes during neoplastic transformation is MTHFD2, a mitochondrial methylenetetrahydrofolate dehydrogenase and cyclohydrolase involved in one-carbon metabolism. Because MTHFD2 is expressed normally only during embryonic development, it offers a disease-selective therapeutic target for eradicating cancer cells while sparing healthy cells. Here we report the synthesis and preclinical characterization of the first inhibitor of human MTHFD2. We also disclose the first crystal structure of MTHFD2 in complex with a substrate-based inhibitor and the enzyme cofactors NAD+ and inorganic phosphate. Our work provides a rationale for continued development of a structural framework for the generation of potent and selective MTHFD2 inhibitors for cancer treatment. Cancer Res; 77(4); 937-48. ©2017 AACR.
Assuntos
Inibidores Enzimáticos/química , Meteniltetra-Hidrofolato Cicloidrolase/química , Metilenotetra-Hidrofolato Desidrogenase (NADP)/química , Mitocôndrias/enzimologia , Sítios de Ligação , Cristalização , Ácido Fólico/análogos & derivados , Ácido Fólico/metabolismo , Humanos , Leucovorina/análogos & derivados , Leucovorina/metabolismo , Meteniltetra-Hidrofolato Cicloidrolase/antagonistas & inibidores , Metilenotetra-Hidrofolato Desidrogenase (NADP)/antagonistas & inibidores , Antígenos de Histocompatibilidade Menor , NAD/metabolismo , Multimerização ProteicaRESUMO
Drugs targeting metabolism have formed the backbone of therapy for some cancers. We sought to identify new such targets in acute myeloid leukemia (AML). The one-carbon folate pathway, specifically methylenetetrahydrofolate dehydrogenase-cyclohydrolase 2 (MTHFD2), emerged as a top candidate in our analyses. MTHFD2 is the most differentially expressed metabolic enzyme in cancer versus normal cells. Knockdown of MTHFD2 in AML cells decreased growth, induced differentiation, and impaired colony formation in primary AML blasts. In human xenograft and MLL-AF9 mouse leukemia models, MTHFD2 suppression decreased leukemia burden and prolonged survival. Based upon primary patient AML data and functional genomic screening, we determined that FLT3-ITD is a biomarker of response to MTHFD2 suppression. Mechanistically, MYC regulates the expression of MTHFD2, and MTHFD2 knockdown suppresses the TCA cycle. This study supports the therapeutic targeting of MTHFD2 in AML.
Assuntos
Ciclo do Ácido Cítrico , Regulação Enzimológica da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/epidemiologia , Meteniltetra-Hidrofolato Cicloidrolase/biossíntese , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Técnicas de Silenciamento de Genes , Células HL-60 , Xenoenxertos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Meteniltetra-Hidrofolato Cicloidrolase/genética , Camundongos , Transplante de Neoplasias , Proteínas Proto-Oncogênicas c-myc/genética , Células U937RESUMO
In response to growth signals, mechanistic target of rapamycin complex 1 (mTORC1) stimulates anabolic processes underlying cell growth. We found that mTORC1 increases metabolic flux through the de novo purine synthesis pathway in various mouse and human cells, thereby influencing the nucleotide pool available for nucleic acid synthesis. mTORC1 had transcriptional effects on multiple enzymes contributing to purine synthesis, with expression of the mitochondrial tetrahydrofolate (mTHF) cycle enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) being closely associated with mTORC1 signaling in both normal and cancer cells. MTHFD2 expression and purine synthesis were stimulated by activating transcription factor 4 (ATF4), which was activated by mTORC1 independent of its canonical induction downstream of eukaryotic initiation factor 2α eIF2α phosphorylation. Thus, mTORC1 stimulates the mTHF cycle, which contributes one-carbon units to enhance production of purine nucleotides in response to growth signals.