RESUMO
BACKGROUND: One-carbon metabolism, which includes the folate and methionine cycles, involves the transfer of methyl groups which are then utilised as a part of multiple physiological processes including redox defence. During the methionine cycle, the vitamin B12-dependent enzyme methionine synthetase converts homocysteine to methionine. The enzyme S-adenosylmethionine (SAM) synthetase then uses methionine in the production of the reactive methyl carrier SAM. SAM-binding methyltransferases then utilise SAM as a cofactor to methylate proteins, small molecules, lipids, and nucleic acids. RESULTS: We describe a novel SAM methyltransferase, RIPS-1, which was the single gene identified from forward genetic screens in Caenorhabditis elegans looking for resistance to lethal concentrations of the thiol-reducing agent dithiothreitol (DTT). As well as RIPS-1 mutation, we show that in wild-type worms, DTT toxicity can be overcome by modulating vitamin B12 levels, either by using growth media and/or bacterial food that provide higher levels of vitamin B12 or by vitamin B12 supplementation. We show that active methionine synthetase is required for vitamin B12-mediated DTT resistance in wild types but is not required for resistance resulting from RIPS-1 mutation and that susceptibility to DTT is partially suppressed by methionine supplementation. A targeted RNAi modifier screen identified the mitochondrial enzyme methylmalonyl-CoA epimerase as a strong genetic enhancer of DTT resistance in a RIPS-1 mutant. We show that RIPS-1 is expressed in the intestinal and hypodermal tissues of the nematode and that treating with DTT, ß-mercaptoethanol, or hydrogen sulfide induces RIPS-1 expression. We demonstrate that RIPS-1 expression is controlled by the hypoxia-inducible factor pathway and that homologues of RIPS-1 are found in a small subset of eukaryotes and bacteria, many of which can adapt to fluctuations in environmental oxygen levels. CONCLUSIONS: This work highlights the central importance of dietary vitamin B12 in normal metabolic processes in C. elegans, defines a new role for this vitamin in countering reductive stress, and identifies RIPS-1 as a novel methyltransferase in the methionine cycle.
Assuntos
Sulfeto de Hidrogênio , Ácidos Nucleicos , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Carbono/metabolismo , Ditiotreitol/metabolismo , Ácido Fólico/metabolismo , Homocisteína/metabolismo , Sulfeto de Hidrogênio/metabolismo , Ligases/metabolismo , Lipídeos , Mercaptoetanol/metabolismo , Metionina/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Oxigênio/metabolismo , Substâncias Redutoras/metabolismo , S-Adenosilmetionina/metabolismo , Compostos de Sulfidrila/metabolismo , Vitamina B 12/metabolismo , Vitamina B 12/farmacologia , Vitaminas/metabolismoRESUMO
Methyl-Cobalamin (Cbl) derives from dietary vitamin B12 and acts as a cofactor of methionine synthase (MS) in mammals. MS encoded by MTR catalyzes the remethylation of homocysteine to generate methionine and tetrahydrofolate, which fuel methionine and cytoplasmic folate cycles, respectively. Methionine is the precursor of S-adenosyl methionine (SAM), the universal methyl donor of transmethylation reactions. Impaired MS activity results from inadequate dietary intake or malabsorption of B12 and inborn errors of Cbl metabolism (IECM). The mechanisms at the origin of the high variability of clinical presentation of impaired MS activity are classically considered as the consequence of the disruption of the folate cycle and related synthesis of purines and pyrimidines and the decreased synthesis of endogenous methionine and SAM. For one decade, data on cellular and animal models of B12 deficiency and IECM have highlighted other key pathomechanisms, including altered interactome of MS with methionine synthase reductase, MMACHC, and MMADHC, endoplasmic reticulum stress, altered cell signaling, and genomic/epigenomic dysregulations. Decreased MS activity increases catalytic protein phosphatase 2A (PP2A) and produces imbalanced phosphorylation/methylation of nucleocytoplasmic RNA binding proteins, including ELAVL1/HuR protein, with subsequent nuclear sequestration of mRNAs and dramatic alteration of gene expression, including SIRT1. Decreased SAM and SIRT1 activity induce ER stress through impaired SIRT1-deacetylation of HSF1 and hypomethylation/hyperacetylation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α), which deactivate nuclear receptors and lead to impaired energy metabolism and neuroplasticity. The reversibility of these pathomechanisms by SIRT1 agonists opens promising perspectives in the treatment of IECM outcomes resistant to conventional supplementation therapies.
Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase , Sirtuína 1 , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Animais , Ácido Fólico , Mamíferos/metabolismo , Metionina , Sirtuína 1/genética , Sirtuína 1/metabolismo , Vitamina B 12/genética , Vitamina B 12/metabolismo , VitaminasRESUMO
Mammalian cells require activated folates to generate nucleotides for growth and division. The most abundant circulating folate species is 5-methyl tetrahydrofolate (5-methyl-THF), which is used to synthesize methionine from homocysteine via the cobalamin-dependent enzyme methionine synthase (MTR). Cobalamin deficiency traps folates as 5-methyl-THF. Here, we show using isotope tracing that MTR is only a minor source of methionine in cell culture, tissues or xenografted tumours. Instead, MTR is required for cells to avoid folate trapping and assimilate 5-methyl-THF into other folate species. Under conditions of physiological extracellular folates, genetic MTR knockout in tumour cells leads to folate trapping, purine synthesis stalling, nucleotide depletion and impaired growth in cell culture and as xenografts. These defects are rescued by free folate but not one-carbon unit supplementation. Thus, MTR plays a crucial role in liberating THF for use in one-carbon metabolism.
Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Neoplasias/metabolismo , Tetra-Hidrofolatos/metabolismo , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , Linhagem Celular Tumoral , Proliferação de Células , Ácido Fólico/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Redes e Vias Metabólicas , Metionina/metabolismo , Metilação , Mutação , Neoplasias/etiologia , Purinas/biossíntese , Deficiência de Vitamina B 12/metabolismoRESUMO
BACKGROUND: Periconceptional folic acid (FA) supplementation is recommended to prevent the occurrence of neural tube defects. Currently, most over-the-counter FA supplements in Canada and the United States contain 1 mg FA and some women are prescribed 5 mg FA/d. High-dose FA is hypothesized to impair 1-carbon metabolism. We aimed to determine folate and 1-carbon metabolism biomarkers in pregnant women exposed to 1 mg or 5 mg FA. OBJECTIVES: This was an ancillary study within the Folic Acid Clinical Trial (FACT), a randomized, double-blinded, placebo-controlled, phase III trial designed to assess the efficacy of high-dose FA to prevent preeclampsia. METHODS: For FACT, women were randomized at 8-16 gestational weeks to receive daily 4.0 mg FA (high dose) or placebo (low dose) plus their usual supplementation (≤1.1 mg). Women were recruited from 3 Canadian FACT centers and provided nonfasting blood samples at 24-26 gestational weeks for measurement of RBC and serum total folate, serum unmetabolized FA (UMFA), tetrahydrofolate (THF), 5-methylTHF, 5-formylTHF, 5,10-methenylTHF, and MeFox (pyrazino-s-triazine derivative of 4α-hydroxy-5-methylTHF, a 5-methylTHF oxidation product); total vitamins B-12 and B-6; and plasma total homocysteine. Group differences were determined using χ2, Fisher exact, and Wilcoxon rank-sum tests. RESULTS: Nineteen (38%) women received high-dose FA and 31 (62%) received low-dose FA. The median RBC folate concentration was 2701 (IQR: 2243-3032) nmol/L and did not differ between groups. The high-dose group had higher serum total folate (median: 148.4 nmol/L, IQR: 110.4-181.2; P = 0.007), UMFA (median: 4.6 nmol/L, IQR: 2.5-33.8; P = 0.008), and 5-methylTHF (median: 126.6 nmol/L, IQR: 98.8-158.6; P = 0.03) compared with the low-dose group (median: 122.8 nmol/L, IQR: 99.5-136.0; median: 1.9 nmol/L, IQR: 0.9-4.1; median: 108.6 nmol/L, IQR: 96.4-123.2, respectively). Other biomarkers of 1-carbon metabolism did not differ. CONCLUSIONS: High-dose FA supplementation in early pregnancy increases maternal serum folate but not RBC folate concentrations, suggesting tissue saturation. Higher UMFA concentrations in women receiving high-dose FA supplements suggest that these doses are supraphysiologic but with no evidence of altered 1-carbon metabolism.
Assuntos
Suplementos Nutricionais , Ácido Fólico/administração & dosagem , Ácido Fólico/farmacologia , Complexo Vitamínico B/administração & dosagem , Complexo Vitamínico B/farmacologia , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Biomarcadores/sangue , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Polimorfismo de Nucleotídeo Único , GravidezRESUMO
Small-for-gestational-age (SGA) is associated with significant perinatal morbidity and mortality. Our aim was to investigate gene-nutrient interactions between maternal one-carbon single nucleotide polymorphisms (SNPs) and folic acid supplement (FAS) use, and their association with SGA. Nulliparous New Zealand women with singleton pregnancy were recruited as part of the Screening for Pregnancy Endpoints prospective cohort study. Data on FAS use was collected via face-to-face interview at 15 weeks' gestation; participants were followed prospectively and birth outcome data collected within 72 h of delivery. Participants were genotyped for MTHFR 677, MTHFR 1298, MTHFD1 1958, MTR 2756, MTRR 66 and TCN2 776 SNPs. Genotype data for at least one SNP was available for 1873 (93%) of eligible participants. Analysis showed a significant SNP-FAS interaction for MTHFR 1298 (p = 0.020), MTHFR 677 (p = 0.019) and TCN2 776 (p = 0.017) in relation to SGA: MTHFR 1298 CC variant non-FAS users had an increased likelihood [Odds Ratio (OR) = 2.91 (95% Confidence Interval (CI) = 1.52, 5.60] compared with wild-type (MTHFR 1298 AA) FAS users. MTHFR 677 variant allele carrier (MTHFR 677 CT + MTHFR 677 TT) non-FAS users had an increased likelihood [OR = 1.87 (95% CI = 1.21, 2.88)] compared to wild-type (MTHFR 677 CC) FAS users. TCN2 776 variant (TCN2 776 GG) non-FAS users had an increased likelihood [OR = 2.16 (95% CI = 1.26, 3.71)] compared with wild type homozygote + heterozygote (TCN2 776 CC + TCN2 776 CG) FAS users. No significant interactions were observed for MTHFD1 1958, MTR 2756 or MTRR 66 (p > 0.05). We observed an overall pattern of FAS attenuating differences in the likelihood of SGA seen between genotype groups in FAS non-users. Future research should focus on how intake of other one-carbon nutrients might mediate these gene-nutrient interactions.
Assuntos
Suplementos Nutricionais , Desenvolvimento Fetal/genética , Desenvolvimento Fetal/fisiologia , Ácido Fólico/administração & dosagem , Genótipo , Recém-Nascido Pequeno para a Idade Gestacional , Fenômenos Fisiológicos da Nutrição Materna/genética , Fenômenos Fisiológicos da Nutrição Materna/fisiologia , Nutrigenômica , Polimorfismo de Nucleotídeo Único , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , Adulto , Feminino , Ferredoxina-NADP Redutase/genética , Humanos , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Antígenos de Histocompatibilidade Menor/genética , Nova Zelândia , Gravidez , Estudos Prospectivos , Adulto JovemRESUMO
Vitamin B12 or cobalamin (Cbl) metabolism can be affected by genetic defects leading to defective activity of either methylmalonyl-CoA mutase or methionine synthase or both enzymes. Patients usually present with a wide spectrum of pathologies suggesting that various cellular processes could be affected by modifications in gene expression. We have previously demonstrated that these genetic defects are associated with subcellular mislocalization of RNA-binding proteins (RBP) and subsequent altered nucleo-cytoplasmic shuttling of mRNAs. In order to characterize the possible changes of gene expression in these diseases, we have investigated global gene expression in fibroblasts from patients with cblC and cblG inherited disorders by RNA-seq. The most differentially expressed genes are strongly associated with developmental processes, neurological, ophthalmologic and cardiovascular diseases. These associations are consistent with the clinical presentation of cblC and cblG disorders. Multivariate analysis of transcript processing revaled splicing alterations that led to dramatic changes in cytoskeleton organization, response to stress, methylation of macromolecules and RNA binding. The RNA motifs associated with this differential splicing reflected a potential role of RBP such as HuR and HNRNPL. Proteomic analysis confirmed that mRNA processing was significantly disturbed. This study reports a dramatic alteration of gene expression in fibroblasts of patients with cblC and cblG disorders, which resulted partly from disturbed function of RBP. These data suggest to evaluate the rescue of the mislocalization of RBP as a potential strategy in the treatment of severe cases who are resistant to classical treatments with co-enzyme supplements.
Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , Oxirredutases/genética , Deficiência de Vitamina B 12/genética , Vitamina B 12/genética , Processamento Alternativo/genética , Linhagem Celular , Proteína Semelhante a ELAV 1/genética , Fibroblastos/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Proteômica , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genética , Vitamina B 12/metabolismo , Deficiência de Vitamina B 12/patologiaRESUMO
BACKGROUND: The risk of neural tube defects (NTDs) is influenced by nutritional factors and genetic determinants of one-carbon metabolism. A key pathway of this metabolism is the vitamin B-12- and folate-dependent remethylation of homocysteine, which depends on methionine synthase (MS, encoded by MTR), methionine synthase reductase, and methylenetetrahydrofolate reductase. Methionine, the product of this pathway, is the direct precursor of S-adenosylmethionine (SAM), the universal methyl donor needed for epigenetic mechanisms. OBJECTIVES: This study aimed to evaluate whether the availability of vitamin B-12 and folate and the expression or activity of the target enzymes of the remethylation pathway are involved in NTD risk. METHODS: We studied folate and vitamin B-12 concentrations and activity, expression, and gene variants of the 3 enzymes in liver from 14 NTD and 16 non-NTD fetuses. We replicated the main findings in cord blood from pregnancies of 41 NTD fetuses compared with 21 fetuses with polymalformations (metabolic and genetic findings) and 375 control pregnancies (genetic findings). RESULTS: The tissue concentration of vitamin B-12 (P = 0.003), but not folate, and the activity (P = 0.001), transcriptional level (P = 0.016), and protein expression (P = 0.003) of MS were decreased and the truncated inactive isoforms of MS were increased in NTD livers. SAM was significantly correlated with MS activity and vitamin B-12. A gene variant in exon 1 of GIF (Gastric Intrinsic Factor gene) was associated with a dramatic decrease of liver vitamin B-12 in 2 cases. We confirmed the decreased vitamin B-12 in cord blood from NTD pregnancies. A gene variant of GIF exon 3 was associated with NTD risk. CONCLUSIONS: The decreased vitamin B-12 in liver and cord blood and decreased expression and activity of MS in liver point out the impaired remethylation pathway as hallmarks associated with NTD risk. We suggest evaluating vitamin B-12 in the nutritional recommendations for prevention of NTD risk beside folate fortification or supplementation.
Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , Doenças Fetais/enzimologia , Fígado/metabolismo , Defeitos do Tubo Neural/enzimologia , Vitamina B 12/metabolismo , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Estudos de Casos e Controles , Feminino , Ferredoxina-NADP Redutase/genética , Ferredoxina-NADP Redutase/metabolismo , Doenças Fetais/genética , Doenças Fetais/metabolismo , Ácido Fólico/análise , Ácido Fólico/metabolismo , Idade Gestacional , Humanos , Fígado/química , Fígado/embriologia , Fígado/enzimologia , Masculino , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Defeitos do Tubo Neural/embriologia , Defeitos do Tubo Neural/genética , Defeitos do Tubo Neural/metabolismo , Gravidez , Vitamina B 12/análiseRESUMO
Cobalamin G (cblG) and cobalamin J (cblJ) defects are rare disorders of cbl metabolism caused by MTR and ABCD4 mutations, respectively. Patients with atypical biochemical features can be missed by current newborn screening using tandem mass spectrometry (MS/MS), in which total homocysteine (tHCY) in dried blood spots (DBS) is not a primary biomarker. Two Chinese patients suspected of cbl defect but missed by newborn screening were studied. Using comprehensive metabolic analyses including MS/MS assay for tHCY in DBS, slightly low methionine in Patient 1, methymalonic aciduria in Patient 2, and homocysteinemia in both patients were detected, and DBS tHCY of two patients were obviously elevated (59.22 µmol/L, 17.75 µmol/L) compared to 140 healthy controls (2.5th-97.5th percentile, 1.05-8.22 µmol/L). Utilizing whole-exome sequencing, we found two novel MTR variants c.871C>T (p.Pro291Ser) and c.1771C>T (p.Arg591*) in Patient 1, and a ABCD4 homozygous variant c.423C>G (p.Asn141Lys) in Patient 2. Our study identified the first cblG patient and cblJ patient in mainland China, and highlighted comprehensive metabolic analyses and genetic tests in patients suspected of cbl defects. It also indicated that supplementary MS/MS assay for tHCY in DBS may be practical for early diagnosis of homocysteinemia, without repeated blood sampling.
Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , Transportadores de Cassetes de Ligação de ATP/genética , Erros Inatos do Metabolismo dos Aminoácidos/sangue , Triagem Neonatal , Vitamina B 12/genética , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/fisiopatologia , Criança , Pré-Escolar , China , Feminino , Homocisteína/sangue , Humanos , Lactente , Recém-Nascido , Masculino , Mutação , Espectrometria de Massas em Tandem , Vitamina B 12/sangue , Sequenciamento do ExomaRESUMO
OBJECTIVE: To assess the association between maternal gene polymorphisms of the enzymes involved in folate metabolism and the risk of having a Down syndrome (DS) offspring in southern China mothers. METHODS: Gene polymorphisms in folate metabolizing and the levels of homocysteine (HCY) were analyzed in 84 southern China mothers with DS babies (the case group) and 120 healthy mothers (the control group). Methylenetetrahydrofolate reductase (MTHFR) C677T (rs1801133) and A1298C (rs1801131), methionine synthase (MTR) A2756G (rs1805087), and methionine synthase reductase (MTRR) A66G (rs1801394) were studied. RESULTS: We found no significant differences (p > .05) in the frequencies of four genetic polymorphisms between the two groups. We found gene-gene interactions had a 1.997-fold increased risk in MTHFR 677 CT with MTR AA (OR: 1.997, 95% CI: 1.038-3.841, p = .038) and a 2.588-fold increased risk in MTHFR 677 CT with MTRR AG (OR: 2.588, 95% CI: 1.111-6.031, p = .028) in the case group than control. The levels of HCY were significantly higher in MTHFR 677 TT than MTHFR 677 CC in the case group (TT 17.2167±5.1051, CC 12.1969±5.0299, F = 2.194, p < .05), and it was significantly higher in MTHFR 677 TT in the case group than control (TT 17.2167±5.1051 in the case group, TT 10.2286±1.4373 in the control group, F = 2.546, p < .05). CONCLUSION: These results suggest that genetic polymorphisms involved in folate metabolism may have population specificity in determining the susceptibility of having DS offsprings. The gene-nutrition, gene-gene interactions and ethnicity are important variables to be considered in periconceptional nutritional supplementation and antenatal care for reducing the risk of DS babies.
Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , Síndrome de Down/genética , Ferredoxina-NADP Redutase/genética , Ácido Fólico/metabolismo , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Adulto , Povo Asiático/genética , Estudos de Casos e Controles , Síndrome de Down/metabolismo , Feminino , Frequência do Gene , Homocisteína/sangue , Humanos , Polimorfismo Genético , Gravidez , Fatores de Risco , Adulto JovemRESUMO
BACKGROUND: Colorectal cancer (CRC) is one of the major health issues worldwide. 5-Fluorouracil (5-FU) is a cornerstone of chemotherapy for CRC and the major targets of 5-FU are folate-metabolizing enzymes. METHODS: A total of 103 CRC patients with complete clinical data were included in this prospective cohort study. Genotyping was performed using polymerase chain reaction (PCR) followed by sequencing. Using Kaplan-Meier curves, log-rank tests, and Cox proportional hazard models, we evaluated associations between functional polymorphisms in four genes MTHFR (1298A>C and 677C>T), DPYD (496A>G and 85T>C), DHFR 19 bp del, and MTR (2756 A>G) with disease-free survival (DFS). RESULTS: The minor allele frequencies of MTHFR 1298A>C, MTHFR 677C>T, DPYD 496A>G, DPYD 85T>C, DHFR 19 bp del, and MTR 2756 A>G were 0.364, 0.214, 0.116, 0.209, 0.383, and 0.097, respectively. CRC patients carrying the homozygous GG genotype in DPYD 496A>G had 4.36 times shorter DFS than wild-type AA carriers, (DFSGG vs AA: 8.0 ± 4 vs 69.0 ± 10 months; HR 4.36, 95% CI 1.04-18; p = 0.04). Moreover, female carriers of homozygous CC genotype of DPYD 85T>C had shorter DFS compared to either heterozygous or wild-type genotypes, and were 12.7 times shorter than wild-type TT carriers (DFSCC vs TT: 5.0 ± 1.5 vs 42.0 ± 7.6 months; HR 12.7, 95% CI 2.2-71.4; p = 0.004). However, there were no significant associations with the other studied polymorphisms. CONCLUSION: Genetic polymorphism in DPYD seems to be associated with DFS in CRC patients receiving an adjuvant regimen of 5-FU/capecitabine-based chemotherapy. Further studies are needed to verify these findings.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Ácido Fólico/metabolismo , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Adulto , Idoso , Capecitabina/administração & dosagem , Quimioterapia Adjuvante , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/metabolismo , Di-Hidrouracila Desidrogenase (NADP)/genética , Di-Hidrouracila Desidrogenase (NADP)/metabolismo , Feminino , Fluoruracila/administração & dosagem , Humanos , Masculino , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Adulto JovemRESUMO
Folate and other B vitamins are essential co-factors of one-carbon metabolism, and genetic variants, such as polymorphisms, can alter the metabolism. Furthermore, the adoption of food fortification with folic acid showed a decrease of homocysteine concentration. The aim of this study was to investigate the frequencies of the polymorphisms of enzymes and carrier proteins involved in one-carbon metabolism, and to evaluate homocysteine concentrations in the presence of these genetic variants in a population exposed to mandatory food fortification with folic acid. Using data from a population-based cross-sectional study in São Paulo, Brazil, the study population comprised 750 participants above 12 years of age of both genders. A linear regression model was used to evaluate the homocysteine concentrations according to genetic variants and folate level. The results showed that the minor allelic frequencies were 0.33 for MTHFR (rs1801133), 0.24 for MTHFR (rs1801131), 0.19 for MTR (rs1805087), 0.42 for MTRR (rs1801394), 0.46 for RFC1 (rs1051266), and 0.47 for DHFR (19-bp deletion). The genetic variants of MTHFR 677C>T, MTRR 66A>G and RFC-1 80G>A were different according to race. The homocysteine concentrations increased in the CT and TT compared to CC genotypes of polymorphism MTHFR 677C>T in all populations, and differences between the homocysteine concentrations according to the genotypes of MTHFR 677C>T were observed regardless of folate level.
Assuntos
Ferredoxina-NADP Redutase/metabolismo , Ácido Fólico/farmacologia , Homocisteína/sangue , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Polimorfismo Genético , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Estudos Transversais , Inquéritos sobre Dietas , Feminino , Ferredoxina-NADP Redutase/genética , Ácido Fólico/administração & dosagem , Alimentos Fortificados , Genótipo , Humanos , Masculino , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Pessoa de Meia-Idade , Proteína de Replicação C/genética , Proteína de Replicação C/metabolismoRESUMO
BACKGROUND: Compared with choline, Met enhances milk yield and feed intake, and elicits a better immuno-metabolic status in periparturient cows. It is unknown whether hepatic activity and transcription of betaine-homocysteine methyltransferase (BHMT), 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), and cystathionine ß-synthase (CBS) are responsive to Met and choline supply. OBJECTIVE: This study sought to characterize hepatic BHMT, MTR, and CBS transcription and activity in response to Met and choline supplementation. METHODS: Forty multiparous cows were used in a 2 × 2 factorial design from -21 d through 30 d around parturition to assess effects of dietary rumen-protected Met (0% or 0.08% dry matter basis) or rumen-protected choline (0 or 60 g · cow-1 · d-1). Liver tissue obtained on days -10, 7, 20, and 30 was used for analyses. RESULTS: Met-supplemented cows had greater methionine adenosyltransferase 1A (MAT1A) (0.38 compared with 0.27; SEM = 0.05; P = 0.02) and phosphatidylethanolamine methyltransferase (PEMT) (0.74 compared with 0.58; SEM = 0.08; P = 0.05) expression. Greater S-adenosylhomocysteine hydrolase (SAHH) (0.93 compared with 0.74; SEM = 0.05; P = 0.01) and CBS (1.16 compared with 1.02; SEM = 0.07; P = 0.04), as well as lower MTR activity (23.4 compared with 29.7 nmol product · h-1 · mg protein-1; SEM = 2.9; P = 0.04), also were detected in Met- but not choline-supplemented cows. Although BHMT and MTR expression and BHMT enzyme activity did not change (P > 0.05), MTR enzyme activity was lower in choline-supplemented cows (23.5 compared with 29.6 nmol product · h-1 · mg protein-1; SEM = 2.9; P = 0.05). CONCLUSIONS: These findings indicate that greater synthesis of phosphatidylcholine and antioxidants contribute to the better performance and immuno-metabolic status in Met-supplemented cows. Failure to generate a comparable amount of endogenous Met from choline could be one reason that choline-fed cows fail to achieve comparable performance and health benefits during the periparturient period.
Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Betaína-Homocisteína S-Metiltransferase/metabolismo , Bovinos/fisiologia , Colina/administração & dosagem , Cistationina beta-Sintase/metabolismo , Metionina/administração & dosagem , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal , Animais , Betaína-Homocisteína S-Metiltransferase/genética , Cistationina beta-Sintase/genética , Dieta/veterinária , Feminino , Regulação Enzimológica da Expressão Gênica , Fígado/enzimologia , Fígado/metabolismo , Período PeripartoRESUMO
Metabolizable methionine (Met) concentrations can be increased by feeding rumen-protected dl-Met or the isopropyl ester of 2-hydroxy-4-(methylthio) butanoic acid (HMBi). Hepatic responses to increasing concentrations of metabolizable Met as a result of supplementation of different Met sources have not been comparatively examined. The objective of this experiment was to examine the regulation of key genes for Met metabolism, gluconeogenesis, and fatty acid oxidation in response to increasing concentrations of dl-Met or 2-hydroxy-4-(methylthio) butanoic acid (HMB) in bovine primary hepatocytes. Hepatocytes isolated from 4 Holstein calves less than 7d old were maintained as monolayer cultures for 24h before addition of treatments. Cells were then exposed to treatments of dl-Met or HMB (0, 10, 20, 40, or 60 µM) in Met-free medium for 24h and collected for RNA isolation and quantification of gene expression by quantitative PCR. Expression of betaine-homocysteine methyltransferase (BHMT), 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), and 5,10 methylenetetrahydrofolate reductase (MTHFR) genes, which catalyze regeneration of Met from betaine and homocysteine, decreased linearly with increasing dl-Met concentration. We observed similar effects with increasing HMB concentration, except expression of MTHFR, which was not altered. Expression of Met adenosyltransferase 1A (MAT1A), which catalyzes the first step of Met metabolism to generate S-adenosylmethionine (SAM), a primary methyl donor, was decreased with increasing dl-Met or HMB concentration. Expression of S-adenosylhomocysteine hydrolase (SAHH) was decreased linearly with increasing HMB concentration, but not altered by dl-Met. Increasing concentrations of dl-Met and HMB decreased cytosolic phosphoenolpyruvate carboxykinase (PCK1) expression, but did not alter the expression of mitochondrial phosphoenolpyruvate carboxykinase (PCK2) or pyruvate carboxylase (PC). Expression of glucose-6-phosphatase(G6PC) decreased linearly with increasing HMB concentration, but not altered by dl-Met. Neither dl-Met nor HMB altered the expression of carnitine palmitoyltransferase 1A(CPT1a). These findings demonstrate reduced necessity for Met regeneration with increased Met concentrations in the medium, regardless of the Met source. The lack of upregulation of gluconeogenesis indicates that increased dl-Met or HMB is not prioritized for glucose synthesis in primary bovine hepatocytes.
Assuntos
Fígado/efeitos dos fármacos , Metionina/análogos & derivados , Metionina/farmacologia , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , Adenosil-Homocisteinase/genética , Animais , Animais Recém-Nascidos , Betaína/metabolismo , Betaína-Homocisteína S-Metiltransferase/genética , Carnitina O-Palmitoiltransferase/genética , Bovinos , Regulação para Baixo , Gluconeogênese/genética , Glucose-6-Fosfatase/genética , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Homocisteína/metabolismo , Fígado/metabolismo , Metionina Adenosiltransferase/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , S-Adenosilmetionina/metabolismo , Regulação para CimaRESUMO
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.
Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Ácido Fólico/metabolismo , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Placenta/metabolismo , Placentação , Progesterona/metabolismo , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/antagonistas & inibidores , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Coriocarcinoma/metabolismo , Coriocarcinoma/patologia , Gonadotropina Coriônica/metabolismo , Feminino , Humanos , Metilenotetra-Hidrofolato Desidrogenase (NADP)/antagonistas & inibidores , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Antígenos de Histocompatibilidade Menor , Invasividade Neoplásica , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Concentração Osmolar , Placenta/citologia , Placenta/patologia , Gravidez , Interferência de RNA , RNA Interferente Pequeno , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologiaRESUMO
BACKGROUND: Recent research suggests that maternal folic acid supplementation is associated with a reduced risk of childhood brain tumors (CBT); polymorphisms in folate pathway genes could modify this association or directly influence CBT risk. METHODS: Associations between risk of CBT and folate pathway polymorphisms were investigated in a population-based case-control study in Australia (2005-2010). Cases were recruited through all Australian pediatric oncology centers and controls by national random digit dialing. Data were available from 321 cases and 552 controls. Six polymorphisms were genotyped in children and parents (MTHFR 677C>T, MTHFR 1298A>C, MTRR 66A>G, MTR 2756A>G, MTR 5049C>A, and CBS 2199 T>C). Maternal folic acid use was ascertained via questionnaire. ORs were estimated using unconditional logistic regression. Case-parent trio analyses were also undertaken. RESULTS: There was weak evidence of a reduced risk of CBT for the MTRR 66GG genotype in the child or father: ORs 0.71 [95% confidence interval (CI), 0.48-1.07]; 0.54 (95% CI, 0.34-0.87), respectively. Maternal prepregnancy folic acid supplementation showed a stronger negative association with CBT risk where the child, mother, or father had the MTRR 66GG genotype (Pinteraction = 0.07, 0.10, and 0.18, respectively). CONCLUSIONS: Evidence for an association between folate pathway genotypes and CBT is limited in this study. There was possible protection by the MTRR 66GG genotype, particularly when combined with maternal prepregnancy folic acid supplementation; these results are novel and require replication. IMPACT: The possible interaction between folic acid supplementation and MTRR 66A>G, if confirmed, would strengthen evidence for prepregnancy folate protection against CBT.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Encefálicas/epidemiologia , Neoplasias Encefálicas/genética , Suplementos Nutricionais , Ácido Fólico/genética , Polimorfismo de Nucleotídeo Único/genética , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , Adolescente , Adulto , Austrália/epidemiologia , Neoplasias Encefálicas/dietoterapia , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Ferredoxina-NADP Redutase/genética , Ácido Fólico/administração & dosagem , Seguimentos , Predisposição Genética para Doença , Genótipo , Humanos , Incidência , Lactente , Recém-Nascido , Masculino , Metionina Sulfóxido Redutases/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Proteínas dos Microfilamentos , Prognóstico , Fatores de Risco , Fatores de Transcrição/genéticaRESUMO
Methionine synthase (MS) and betaine-homocysteine methyltransferase (BHMT) are both zinc (Zn)-dependent methyltransferases and involved in the methylation of homocysteine. The objective of this study was to investigate the effects of dietary Zn supply on homocysteine levels and expression of the two enzymes in growing rats. Male weanling Sprague-Dawley rats were assigned randomly to four dietary groups (n=8/group) for 3 weeks: Zn deficient (ZD; <1mg Zn/kg); Zn control (ZC; 30mg Zn/kg); Zn supplemented (ZS; 300mg Zn/kg); pair fed (PF; 30mg Zn/kg) to the ZD group. Serum and femur Zn concentrations were 83% and 58% lower in ZD, and 49% and 62% higher in ZS compared to ZC (P<0.001), respectively. The ZD rats had lower feed intake (37%), body weight gains (45%), liver (43%) and kidney (31%) weights than those of ZC (P<0.001), but these parameters in ZD were not significantly different from the PF controls. Serum homocysteine concentrations were 65% higher in ZD compared to PF (P<0.05), and there was no significant difference in serum folate levels between ZD and PF groups. The mRNA expression of liver and kidney MS was 57% and 38% lower in ZD than PF (P<0.001), respectively. Hepatic and renal BHMT mRNA levels were not altered in ZD compared to controls. The aforementioned measurements were not significantly different between ZS and ZC groups, except Zn levels. These results demonstrated that homocysteine homeostasis appeared to be disturbed by Zn deficiency but not Zn supplementation, and elevated serum homocysteine might be due to reduced expression of MS during Zn deficiency.
Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Betaína-Homocisteína S-Metiltransferase/metabolismo , Suplementos Nutricionais , Homocisteína/sangue , Zinco/deficiência , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , Animais , Betaína-Homocisteína S-Metiltransferase/genética , Peso Corporal , Dieta , Comportamento Alimentar , Ácido Fólico , Regulação Enzimológica da Expressão Gênica , Rim/enzimologia , Fígado/enzimologia , Masculino , Tamanho do Órgão , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Zinco/sangueRESUMO
We investigated the association between dietary intake of folate, vitamin B6, and the 5,10-methylenetetrahydrofolate reductase (MTHFR) genotype with breast cancer. A matched case-control study was conducted, and 413 patients with newly diagnosed and histologically confirmed breast cancer and 436 controls were recruited. Folate intake, vitamin B6, and vitamin B12 levels were calculated, and the MTHFR C677T and A1298C and MTR A2756G polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism. Breast cancer cases were generally older, older at first live birth, and younger at menarche, had a higher body mass index, were smokers, had higher energy intake, and more first-degree relatives with breast cancer as well as more live births compared to controls. With respect to energy intake, we found that higher energy intake were more likely to increase the risk of breast cancer. The MTHFR 667TT genotype was associated with a moderately increased risk of breast cancer when compared with the CC genotype, and a significant odds ratio (OR; 95% confidence interval, CI) was found (OR = 1.70, 95%CI = 1.06-2.73). Individuals carrying T allele were associated with higher risk of breast cancer when compared with C allele (OR = 1.34, 95%CI = 1.06-1.70). We did not find a significant effect of the MTHFR A1298C and MTR A2756G on the risk of breast cancer. We did not find any association between folate intake and MTHFR C677T polymorphisms. In conclusion, we found that the MTHFR C667T polymorphism is associated with the risk of breast cancer, indicating that this genotype plays a role in breast cancer development.
Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , Neoplasias da Mama/genética , Ácido Fólico/administração & dosagem , Predisposição Genética para Doença/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Polimorfismo de Nucleotídeo Único , Adulto , Alelos , Neoplasias da Mama/metabolismo , Suplementos Nutricionais , Feminino , Ácido Fólico/metabolismo , Frequência do Gene , Genótipo , Humanos , Pessoa de Meia-Idade , Razão de Chances , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Fatores de Risco , Vitamina B 12/administração & dosagem , Vitamina B 12/metabolismo , Vitamina B 6/administração & dosagem , Vitamina B 6/metabolismo , Vitaminas/administração & dosagem , Vitaminas/metabolismoRESUMO
In Brassicaceae, a self-incompatibility (SI) system mediates pollen-pistil interactions. Self-pollen could be recognized and rejected by incompatible pistils. Several components involved in the SI response have been determined, including S-locus receptor kinase (SRK), S-locus cysteine-rich protein/S-locus protein 11, and arm repeat-containing protein 1 (ARC1). However, the components involved in the SI system of Brassicaceae are not fully understood. Here, we detected expression patterns of 24 SI-related genes in non-heading Chinese cabbage (Brassica campestris ssp chinensis Makino) after compatible and incompatible pollination, and potential interaction relationships of these genes were predicted. SRK and ARC1 transcripts increased initially 0.25 h after incompatible pollination, while kinase-associated protein phosphatase had an expression pattern that was opposite that of SRK transcripts during self-pollination. Plant U-box 8 was not required in the SI response of non-heading Chinese cabbage. Our results showed that the SI signal of non-heading Chinese cabbage could occur within 0.25 h after self-pollination. The hypothetical interaction relationships indicated that plastid-lipid-associated protein and malate dehydrogenase could be negatively regulated by chaperonin 10, glutathione transferase, cytidylate kinase/uridylate kinase, and methionine synthase by indirect interactions. Our findings could be helpful to better understand potential roles of these components in the SI system of non-heading Chinese cabbage.
Assuntos
Brassica/genética , Regulação da Expressão Gênica de Plantas , Glicoproteínas/genética , Proteínas de Plantas/genética , Polinização/genética , Proteínas Quinases/genética , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Brassica/classificação , Brassica/metabolismo , Chaperonina 10/genética , Chaperonina 10/metabolismo , Flores/genética , Redes Reguladoras de Genes , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Glicoproteínas/metabolismo , Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Núcleosídeo-Fosfato Quinase/genética , Núcleosídeo-Fosfato Quinase/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Filogenia , Proteínas de Plantas/metabolismo , Pólen/genética , Proteínas Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Photosynthetic microalgae play a vital role in primary productivity and biogeochemical cycling in both marine and freshwater systems across the globe. However, the growth of these cosmopolitan organisms depends on the bioavailability of nutrients such as vitamins. Approximately one-half of all microalgal species requires vitamin B12 as a growth supplement. The major determinant of algal B12 requirements is defined by the isoform of methionine synthase possessed by an alga, such that the presence of the B12-independent methionine synthase (METE) enables growth without this vitamin. Moreover, the widespread but phylogenetically unrelated distribution of B12 auxotrophy across the algal lineages suggests that the METE gene has been lost multiple times in evolution. Given that METE expression is repressed by the presence of B12, prolonged repression by a reliable source of the vitamin could lead to the accumulation of mutations and eventually gene loss. Here, we probe METE gene regulation by B12 and methionine/folate cycle metabolites in both marine and freshwater microalgal species. In addition, we identify a B12-responsive element of Chlamydomonas reinhardtii METE using a reporter gene approach. We show that complete repression of the reporter occurs via a region spanning -574 to -90 bp upstream of the METE start codon. A proteomics study reveals that two other genes (S-Adenosylhomocysteine hydrolase and Serine hydroxymethyltransferase2) involved in the methionine-folate cycle are also repressed by B12 in C. reinhardtii. The strong repressible nature and high sensitivity of the B12-responsive element has promising biotechnological applications as a cost-effective regulatory gene expression tool.
Assuntos
Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Microalgas/genética , Vitamina B 12/farmacologia , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/química , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Sequência de Aminoácidos , Chlamydomonas/efeitos dos fármacos , Chlamydomonas/genética , Genes Reporter , Microalgas/efeitos dos fármacos , Microalgas/enzimologia , Dados de Sequência Molecular , Proteômica , Elementos de Resposta/genéticaRESUMO
Methylenetetrahydrofolate reductase (MTHFR) is necessary for the synthesis of methionine and S-adenosylmethionine, which is necessary for CNS (re-)myelination. The MTHFR variant c.1298A>C was associated with the development of relapsing remitting multiple sclerosis (RRMS) in a German population. This study aimed at analyzing whether further genetic variants of methionine metabolism are associated with the development or the clinical course of RRMS. Therefore, genomic DNA of 147 serial German RRMS patients and 147 matched healthy controls was genotyped for five polymorphic variants of methionine metabolism. Statistical analyses were performed using multivariate binary and linear regression analyses. We show that the insertion allele of cystathionine beta-synthase (CBS) c.844_855ins68bp and the G-allele of reduced folate carrier 1 (RFC1) c.80G>A were associated with an earlier age of onset of MS, suggesting gene-dose effects (median age of onset in years: 25-26-32; standardized regression coefficient beta: 0.216; p=0.030, and 29-31-35 years; beta: 0.282; p=0.005, respectively). Conclusively, mutant variants of CBS and RFC1 may be associated with the age of RRMS onset. Since methionine metabolism can be manipulated by supplementation of vitamins and amino acids, our data provide a rationale for novel ideas of preventive and therapeutic strategies in RRMS.