Enzyme In Folate Metabolism Research Articles

Understanding the molecular mechanism of substrate channeling and domain communication in protozoal bifunctional TS-DHFR.

Most species, such as humans, have monofunctional forms of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) that are key folate metabolism enzymes making critical folate components required for DNA synthesis. In contrast, several parasitic protozoa, including Leishmania major (Lm), Plasmodium falciparum (Pf), Toxoplasma gondii (Tg) and Cryptosporidium hominis (Ch), contain a unique bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) having the two sequential catalytic activities contained on a single polypeptide chain. It has been suggested that the bifunctional nature of the two catalytic activities may enable substrate channeling. The 3D structures for each of these enzymes reveals distinct features for each species. While three of the four species (Pf, Tg and Ch) contain a junctional region linking the two domains, this is lacking in Lm. The Lm and Pf contain N-terminal amino acid extensions. A multidisciplinary approach using structural studies and transient kinetic analyses combined with mutational analysis has investigated the roles of these unique structural features for each enzyme. Additionally, the possibility of substrate channeling behavior was explored. These studies have identified unique, functional regions in both the TS and DHFR domains that govern efficient catalysis for each species. Surprisingly, even though there are structural similarities among the species, each is regulated in a distinct manner. This structural and mechanistic information was also used to exploit species-specific inhibitor design.

Metabolic Reprogramming by Folate Restriction Leads to a Less Aggressive Cancer Phenotype.

Metabolic reprogramming driven by folate restriction represents a therapeutic target for reducing the burden of breast cancer. Mol Cancer Res; 15(2); 189-200. ©2016 AACR.

Prognostic Relevance of Methylenetetrahydrofolate Reductase Polymorphisms for Prostate Cancer.

Folate metabolism has been associated with cancers via alterations in nucleotide synthesis, DNA methylation, and DNA repair. We hypothesized that genetic variants in methylenetetrahydrofolate reductase (MTHFR), a key enzyme of folate metabolism, would affect the prognosis of prostate cancer. Three haplotype-tagging single-nucleotide polymorphisms (SNPs) across the MTHFR gene region were genotyped in a cohort of 458 patients with clinically localized prostate cancer treated with radical prostatectomy. One SNP, rs9651118, was associated with disease recurrence, and the association persisted after multivariate analyses adjusting for known risk factors. Public dataset analyses suggested that rs9651118 affects MTHFR expression. Quantitative real-time polymerase chain reaction analysis revealed that MTHFR expression is significantly upregulated in prostate tumor tissues when compared with adjacent normal tissues. Furthermore, overexpression of MTHFR correlates with cancer recurrence and death in two independent publicly available prostate cancer datasets. In conclusion, our data provide rationale to further validate the clinical utility of MTHFR rs9651118 as a biomarker for prognosis in prostate cancer.

The importance of mitochondrial folate enzymes in human colorectal cancer.

Folate plays a pivotal role in the one-carbon metabolism needed for methylation reactions, nucleotide synthesis, and DNA repair. Although folate metabolism was recently shown to be associated with carcinogenesis in some solid tumors, the importance of folate metabolism in colorectal cancer remains unclear. In the present investigation we found that expression of three mitochondrial folate metabolic enzymes, serine hydroxymethyl transferase (SHMT2), methylenetetrahydrofolate dehydrogenase (MTHFD2) and aldehyde dehydrogenase1 family memberL2 (ALDH1L2), were upregulated in human colorectal tumor tissues compared to normal tissues. Colorectal cancer tissue samples were obtained from 117consecutive patients. We evaluated the expression of the enzymes with immunohistochemical analysis and determined their relevance to clinicopathological characteristics and prognosis. Rates of recurrence-free survival(RFS) and overall survival(OS) in patients with high expression of SHMT2, MTHFD2 and ALDH1L2 tended to be lower than in patients with low expression of SHMT2, MTHFD2 and ALDH1L2 (P=0.446 and P=0.337, P=0.099 and P=0.064, P=0.178 and P=0.257, respectively). Notably, the combined high expression of SHMT2, MTHFD2 and ALDH1L2 (triple high) was more highly associated with poor prognosis than the individual expression levels (RFS; P=0.004 and OS; P=0.037). A multivariate analysis showed that triple high expression was independently associated with RFS (P=0.017). These findings suggested that mitochondrial folate metabolic enzymes could provide a potential therapeutic strategy for treating colorectal cancer.

Murine MTHFD1-synthetase deficiency, a model for the human MTHFD1 R653Q polymorphism, decreases growth of colorectal tumors.

The common R653Q variant (∼20% 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.

Heterogenous Distribution of MTHFR Gene Variants among Mestizos and Diverse Amerindian Groups from Mexico.

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in folate metabolism. Folate deficiency has been related to several conditions, including neural tube defects (NTDs) and cardiovascular diseases. Hence, MTHFR genetic variants have been studied worldwide, particularly the C677T and A1298C. We genotyped the C677T and A1298C MTHFR polymorphisms in Mexican Amerindians (MAs), from the largest sample included in a genetic study (n = 2026, from 62 ethnic groups), and in a geographically-matched Mexican Mestizo population (MEZ, n = 638). The 677T allele was most frequent in Mexican individuals, particularly in MAs. The frequency of this allele in both MAs and MEZs was clearly enriched in the South region of the country, followed by the Central East and South East regions. In contrast, the frequency of the 1298C risk allele in Mexicans was one of the lowest in the world. Both in MAs and MEZs the variants 677T and 1298C displayed opposite allele frequency gradients from southern to northern Mexico. Our findings suggest that in Mestizos the 677T allele was derived from Amerindians while the 1298C allele was a European contribution. Some subgroups showed an allele frequency distribution that highlighted their genetic diversity. Notably, the distribution of the frequency of the 677T allele was consistent with that of the high incidence of NTDs reported in MEZ.

Converging Evolutionary, Environmental and Clinical Ideas on Folate Metabolism

Background & objectivesFolate is a UV-labile vitamin, critical to maintenance of DNA synthesis and repair mechanisms. Evidence supports a number of folate-dependent enzymes as being UV-responsive, suggesting that the activity of pathways operating through such enzymes may differ between geographic areas of varied UV exposure. Therefore, we explored the possible association of global UV levels with the prevalence of common polymorphisms in folate-metabolism enzymes.

Association of the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism with primary glaucoma in Saudi population.

BackgroundMethylenetetrahydrofolate reductase (MTHFR), a critical enzyme in folate metabolism is involved in DNA synthesis, DNA repair and DNA methylation. The functional polymorphism of MTHFR gene, C677T has been shown to impact various diseases and implicated as a risk factor for the development of various neurodegenerative disorders including glaucoma.MethodsWe investigated MTHFR C677T genotypes and alleles frequencies in primary glaucoma [primary open angle glaucoma (POAG) and primary angle closure glaucoma (PACG)] patients and matched healthy controls in a case-control study. Two hundred ten primary glaucoma cases were studied for MTHFR C677T polymorphism and compared with 280 controls taken from the healthy population, employing the polymerase chain reaction-restriction fragment length polymorphism technique (PCR-RFLP). The MTHFR gene was amplified using specific primers. The PCR products (294 bp) was subsequently digested with HinfI (New England Biolabs) at 37 °C for 12 h, separated by electrophoresis on 2 % agarose gels, and visualized with ethidium bromide staining. The restriction digestion yielded 168 and 126 bp fragments for TT, 294, 168 and 126 bp fragments for CT and undigested PCR product 294 bp indicating CC genotype.ResultsWe found the frequency of the genotypes and alleles of MTHFR C677T differ significantly between cases and controls. The frequencies of allele T and genotype CT were significantly higher while the frequencies of allele C and genotype CC were lower in primary glaucoma patients as compared to controls (p <0.05). Upon stratification of our results into POAG and PACG, significantly higher frequencies of allele T (19.44 %) and genotype CT (38.89 %) were found in POAG patients compared to controls (12.5 % and 25 % respectively). The frequencies of alleles and genotypes were almost similar in PACG and controls (p = 0.8).ConclusionThis study indicates that the allele T and genotype CT of MTHFR C677T polymorphism are significantly associated with POAG while allele C and CC genotype may be protective for it. We conclude that the MTHFR C677T polymorphism increases the risk for POAG development in Saudi population and can be a genetic marker however, further studies are needed with multiple-ethnic populations affected with POAG to strengthen these findings.

The Relationship Between Methylation Defects and Different Genetic Disorders: Two Case Reports

5, 10-methylenetetrahydrofolate reductase (MTHFR) is a coding gene, for a key enzyme in methionine-homocysteine and folate metabolism. This pathway has been associated with gene specific DNA hypo and hypermethylation as a result of gene switching on or off.Methylenetetrahydrofolate reductase C677T and A1298C gene polymorphisms are associated with folate metabolism disorders; such as, it results in an impaired DNA methylation and chromosomal abnormalities, gene deficiencies and structural anomalies. Here, we reported two cases of compound heterozygote and homozygote MTHFR gene mutation association with genetical disorders during the pregnancies.

46 MTHFR gene polymorphism A1298C and development of pre-eclampsia

Introduction Pre-eclampsia (PE) is a pregnancy-specific disease diagnosed by hypertension and proteinuria after 20th week of pregnancy. The methylenetetrahydrofolate reductase (MTHFR) is a crucial enzyme for folate and homocysteine metabolism. The A1298C (rs1801131) is a common polymorphism of MTHFR gene and has also been suspected as a contributor of altered serum homocysteine levels. Hyperhomocysteinemia has been associated with pregnancy complications such as pre-eclampsia. Objective Determine wheter MTHFR A1298C polymorphism is associated with PE susceptibility. Methods In this case-control study, a total of 53 preeclamptic pregnant women were recruited from Departament of Obstetrics and Gynecology of Clinical Hospital, of Triangulo Mineiro Federal University, Uberaba, Brazil. PE was defined according clinical findings, including increased blood pressure ( ⩾ 140 mmHg systolic or ⩾ 90 mmHg diastolic on 2 or more measurements at least 6 h apart) and proteinuria ⩾ 0.3 g/24 h or ⩾ +1 on a urine dipstick after 20 weeks of gestation. The control group included 179 normal pregnant women. None of PE patients and healthy controls had hypertension history. Genomic DNA was extracted from EDTA-treated whole blood by standard procedure of phenol–chloroform. Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) was performed for MTHFR A1298C (rs1801131) genotyping and subsequent digestion of PCR products with MboII restriction enzyme. Differences in the distribution of genotypes and alleles between groups and deviations from the Hardy-Weinberg equilibrium were tested using the chi-square test. Results No deviation from the Hardy–Weinberg equilibrium was observed for MTHFR A1298C polymorphism either in PE patients or in healthy controls (p = 0.81 and p = 0.21 respectively). The frequency of 1298C allele in PE patients and controls was 28% and 20% and was not statistically different. The frequency of AA genotype was 54.2% and 62.3%, AC genotype was 36.3 and 33.9% and CC genotype was 9.5 and 3.8% in PE patients and controls, respectively, which was not significantly different (p = 0.337). Conclusion The MTHFR A1298C (rs1801131) polymorphism is not associated with risk of developing pre-eclampsia. Conflict of interest The authors declare that they have no conflicts of interest.

Riboflavin status, MTHFR genotype and blood pressure: current evidence and implications for personalised nutrition

Clinical deficiency of the B-vitamin riboflavin (vitamin B2) is largely confined to developing countries; however accumulating evidence indicates that suboptimal riboflavin status is a widespread problem across the developed world. Few international data are available on riboflavin status as measured by the functional biomarker, erythrocyte glutathione reductase activation coefficient, considered to be the gold standard index. One important role of riboflavin in the form of flavin dinucleotide is as a co-factor for the folate-metabolising enzyme methylenetetrahydrofolate reductase (MTHFR). Homozygosity for the common C677T polymorphism in MTHFR, affecting over 10 % of the UK and Irish populations and up to 32 % of other populations worldwide, has been associated with an increased risk of CVD, and more recently with hypertension. This review will explore available studies reporting riboflavin status worldwide, the interaction of riboflavin with the MTHFR C677T polymorphism and the potential role of riboflavin in personalised nutrition. Evidence is accumulating for a novel role of riboflavin as an important modulator of blood pressure (BP) specifically in individuals with the MTHFR 677TT genotype, with results from a number of recent randomised controlled trials demonstrating that riboflavin supplementation can significantly reduce systolic BP by 5-13 mmHg in these genetically at risk adults. Studies are however required to investigate the BP-lowering effect of riboflavin in different populations and in response to doses higher than 1·6 mg/d. Furthermore, work focusing on the translation of this research to health professionals and patients is also required.

Malic enzyme tracers reveal hypoxia-induced switch in adipocyte NADPH pathway usage.

The critical cellular hydride donor NADPH is produced through various means, including the oxidative pentose phosphate pathway (oxPPP), folate metabolism and malic enzyme. In growing cells, it is efficient to produce NADPH via the oxPPP and folate metabolism, which also make nucleotide precursors. In nonproliferating adipocytes, a metabolic cycle involving malic enzyme holds the potential to make both NADPH and two-carbon units for fat synthesis. Recently developed deuterium ((2)H) tracer methods have enabled direct measurement of NADPH production by the oxPPP and folate metabolism. Here we enable tracking of NADPH production by malic enzyme with [2,2,3,3-(2)H]dimethyl-succinate and [4-(2)H]glucose. Using these tracers, we show that most NADPH in differentiating 3T3-L1 mouse adipocytes is made by malic enzyme. The associated metabolic cycle is disrupted by hypoxia, which switches the main adipocyte NADPH source to the oxPPP. Thus, (2)H-labeled tracers enable dissection of NADPH production routes across cell types and environmental conditions.

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.

Polymorphisms of 5,10-methylenetetrahydrofolate reductase and thymidylate synthase, dietary folate intake, and the risk of leukemia in adults.

The 5,10-methylenetetrahydrofolate reductase (MTHFR) and thymidylate synthase (TS) are critical enzymes in folate metabolism. Previous studies have reported conflicting results on the associations between MTHFR/TS polymorphisms and adult leukemia risk, which may due to the lack of information on folate intake. We investigated the risks of adult leukemia with genetic polymorphisms of folate metabolic enzymes (MTHFR C677T, A1298C, and TS) and evaluated if the associations varied by dietary folate intake from a multicenter case-control study conducted in Chinese. This study comprised 442 incident adult leukemia cases and 442 outpatient controls, individually matched to cases by gender, birth quinquennium, and study site. Genotypes were determined by a polymerase chain reaction (PCR) or PCR-based restriction fragment length polymorphism assay. Dietary folate intake was assessed by face-to-face interviews using a validated food-frequency questionnaire. The MTHFR 677TT genotype conferred a significant higher risk of leukemia in males than in females and exhibited an increased risk of acute myeloid leukemia (AML) but a decreased risk of acute lymphoblastic leukemia (ALL). The MTHFR 1298AC genotype appeared to decrease the risks of leukemia in both genders, in AML and ALL. Stratified analysis by dietary folate intake showed the increased risks of leukemia with the MTHFR 677TT and TS 2R3R/2R2R genotypes were only significant in individuals with low folate intake. A significant interaction between TS polymorphism and dietary folate intake was observed (P = 0.03). This study suggests that dietary folate intake and gender may modify the associations between MTHFR/TS polymorphisms and adult leukemia risk.

Folate-Dependent Purine Nucleotide Biosynthesis in Humans.

Purine nucleotide biosynthesis de novo (PNB) requires 2 folate-dependent transformylases-5'-phosphoribosyl-glycinamide (GAR) and 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR) transformylases-to introduce carbon 8 (C8) and carbon 2 (C2) into the purine ring. Both transformylases utilize 10-formyltetrahydrofolate (10-formyl-H4folate), where the formyl-carbon sources include ring-2-C of histidine, 3-C of serine, 2-C of glycine, and formate. Our findings in human studies indicate that glycine provides the carbon for GAR transformylase (exclusively C8), whereas histidine and formate are the predominant carbon sources for AICAR transformylase (C2). Contrary to the previous notion, these carbon sources may not supply a general 10-formyl-H4folate pool, which was believed to equally provide carbons to C8 and C2. To explain these phenomena, we postulate that GAR transformylase is in a complex with the trifunctional folate-metabolizing enzyme (TFM) and serine hydroxymethyltransferase to channel carbons of glycine and serine to C8. There is no evidence for channeling carbons of histidine and formate to AICAR transformylase (C2). GAR transformylase may require the TFM to furnish 10-formyl-H4folate immediately after its production from serine to protect its oxidation to 10-formyldihydrofolate (10-formyl-H2folate), whereas AICAR transformylase can utilize both 10-formyl-H2folate and 10-formyl-H4folate. Human liver may supply AICAR to AICAR transformylase in erythrocytes/erythroblasts. Incorporation of ring-2-C of histidine and formate into C2 of urinary uric acid presented a circadian rhythm with a peak in the morning, which corresponds to the maximum DNA synthesis in the bone marrow, and it may be useful in the timing of the administration of drugs that block PNB for the treatment of cancer and autoimmune disease.

MTHFR-Ala222Val and male infertility: a study in Iranian men, an updated meta-analysis and an in silico-analysis

Methylenetetrahydrofolate reductase (MTHFR) functions as a main regulatory enzyme in folate metabolism. The association of MTHFR gene Ala222Val polymorphism with male infertility in an Iranian population was investigated by undertaking a meta-analysis and in-silico approach. A genetic association study included 497 men; 242 had unexplained infertility and 255 were healthy controls. Polymerase chain reaction restriction fragment length polymorphism was used for genotyping MTHFR-Ala222Val. OpenMeta[Analyst] software was used to conduct the analysis; 22 studies were identified by searching PubMed and the currently reported genetic association study. A novel in-silico approach was used to analyse the effects of Ala222Val substitution on the structure of mRNA and protein. Genetic association study revealed a significant association of MTHFR-222Val/Val genotype with oligozoospermia (OR 2.32; 95% CI, 1.12 to 4.78; P = 0.0451) and azoospermia (OR 2.59; 95% CI 1.09 to 6.17; P = 0.0314). Meta-analysis for allelic, dominant and codominant models showed a significant association between Ala222Val polymorphism and the risk of male infertility (P < 0.001). In silico-analysis showed MTHFR-Ala222Val affects enzyme structure and could also change the mRNA properties (P = 0.1641; P < 0.2 is significant). The meta-analysis suggested significant association of MTHFR-Ala222Val with risk of male infertility, especially in Asian populations.

Abstract 4709: Compartmental flexibility in mammalian folate metabolism

Abstract Background: Folate metabolism occurs in parallel cytosolic and mitochondrial pathways. Increased mitochondrial, but not cytosolic, folate dependent serine catabolism is among the most consistent metabolic alterations in human tumors. This study employs genetics coupled with quantitative metabolic analysis to determine the respective requirements for compartmentalized folate metabolism in proliferating human cells. Methods: Using CRISPR-Cas9 technology, we created a library of deletion mutant cell lines for enzymes in folate metabolism. Cellular metabolites from these cells were quantified by liquid chromatography mass spectrometry (LC-MS). Compartmental specific folate metabolic fluxes were assayed by labeling experiments using isotopic tracers of serine, glycine and formate. Results: We show that in HEK 293T cells, as well as in many but not all human cancer cell lines, folate dependent serine catabolism occurs predominantly in the mitochondria. In mutant cell lines deficient for mitochondrial folate genes, the reversal of the direction of cytosolic one-carbon flux enables cell growth, albeit at a reduced rate and with metabolic hallmarks of 10-formyltetrahydrofolate deficiency. Addition of exogenous formate rescued the 10-formyltetrahydrofolate deficiency in all mutants, but the growth defect in only a subset. The growth defect persisted in cell lines mutant for enzymes required for the generation of mitochondrial one-carbon units and folate dependent generation of NAD(P)H but not for the production of formate. Conclusions: This study finds that folate metabolism is flexible with respect to compartmentalization. Cytosolic folate metabolism can provide sufficient one-carbon flux to fuel cell growth even in the complete absence of mitochondrial activity. High expression of mitochondrial folate genes in cancer may function to meet biosynthetic requirements under the stressful conditions of the tumor or to provide necessary compartmentalized one-carbon units and/or reducing equivalents. Citation Format: Gregory S. Ducker, Li Chen, Xin Teng, Joshua D. Rabinowitz. Compartmental flexibility in mammalian folate metabolism. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4709. doi:10.1158/1538-7445.AM2015-4709

Ubiquitin-like protein MNSFβ covalently binds to cytosolic 10-formyltetrahydrofolate dehydrogenase and regulates thymocyte function

MNSFβ is a ubiquitously expressed member of the ubiquitin-like family that has been involved in various biological functions. Previous studies have demonstrated that MNSFβ covalently binds to various target proteins including Bcl-G, a proapoptotic protein. In this study, we purified a 115 kDa MNSFβ adduct from murine liver lysates by sequential chromatography on DEAE and anti-MNSFβ IgG-conjugated Sepharose in the presence of ATP. MALDI-TOF MS fingerprinting revealed that this MNSFβ adduct consists of an 8.5 kDa MNSFβ and 10-formyltetrahydrofolate dehydrogenase (FDH), an abundant enzyme of folate metabolism. Interestingly, MNSFβ preferably binds to cytosolic but not mitochondrial FDH. Fingerprinting analysis of the MNSFβ adduct demonstrate that MNSFβ conjugates to cytosolic FDH with a linkage between the C-terminal Gly74 and Lys72. The 115 kDa MNSFβ/FDH complex was not expressed in any of the tissues examined, indicating that this adduct formation is not ubiquitous. We found that MNSFβ/FDH complex formation was induced by dexamethasone in thymocytes. Double knockdown of MNSFβ and FDH strongly reduced dexamethasone-induced apoptosis. Collectively, MNSFβ/FDH complex formation may positively regulate apoptosis in thymocytes.

The Recessive model of 677C&gt;T Polymorphism in MTHFR gene increase moderately the risk of Colorectal Cancer in Moroccan patients

Methylenetetrahydrofolate reductase (MTHFR) is a critical folate-metabolising enzyme and a polymorphism at position 677 (C677T), is associated with reduced enzyme activity. We investigated whether this functional polymorphism modulates the risk of developing Colorectal Cancer (CRC). We conducted a hospital-based case-control study to assess the association of MTHFR gene polymorphism C677T with risk for colorectal cancer in a Moroccan population. Odds ratios [ORs] with corresponding 95 % confidence intervals (Cis) were used to assess the association. The analysis had shown the significant elevated risk of cancer was associated with theMTHFR C677T polymorphism in recessive model (OR = 2.81, 95 % CI; (1.13-7.06), P=0.027) compared the other genetic models. Simultaneously, the T-allele genotype versus C-allele genotype was not associated with CRC risk (ORa = 1.355; 95% CI = 0.94-1.96and p = 0.10). Thusrecessive model are significantly associated in the risk of colorectal cancer. Further larger-scale studies are necessary to confirm our finding.

MTHFR deficiency or reduced intake of folate or choline in pregnant mice results in impaired short-term memory and increased apoptosis in the hippocampus of wild-type offspring

Genetic or nutritional disturbances in one-carbon metabolism, with associated hyperhomocysteinemia, can result in complex disorders including pregnancy complications and neuropsychiatric diseases. In earlier work, we showed that mice with a complete deficiency of methylenetetrahydrofolate reductase (MTHFR), a critical enzyme in folate and homocysteine metabolism, had cognitive impairment with disturbances in choline metabolism. Maternal demands for folate and choline are increased during pregnancy and deficiencies of these nutrients result in several negative outcomes including increased resorption and delayed development. The goal of this study was to investigate the behavioral and neurobiological impact of a maternal genetic deficiency in MTHFR or maternal nutritional deficiency of folate or choline during pregnancy on 3-week-old Mthfr+/+ offspring. Mthfr+/+ and Mthfr+/− females were placed on control diets (CD); and Mthfr+/+ females were placed on folate-deficient diets (FD) or choline-deficient diets (ChDD) throughout pregnancy and lactation until their offspring were 3weeks of age. Short-term memory was assessed in offspring, and hippocampal tissue was evaluated for morphological changes, apoptosis, proliferation and choline metabolism. Maternal MTHFR deficiency resulted in short-term memory impairment in offspring. These dams had elevated levels of plasma homocysteine when compared with wild-type dams. There were no differences in plasma homocysteine in offspring. Increased apoptosis and proliferation was observed in the hippocampus of offspring from Mthfr+/− mothers. In the maternal FD and ChDD study, offspring also showed short-term memory impairment with increased apoptosis in the hippocampus; increased neurogenesis was observed in ChDD offspring. Choline acetyltransferase protein was increased in the offspring hippocampus of both dietary groups and betaine was decreased in the hippocampus of FD offspring. Our results reveal short-term memory deficits in the offspring of dams with MTHFR deficiency or dietary deficiencies of critical methyl donors. We suggest that deficiencies in maternal one-carbon metabolism during pregnancy can contribute to hippocampal dysfunction in offspring through apoptosis or altered choline metabolism.