Enzyme In Folate Metabolism Research Articles

Design, synthesis, in vitro and in silico studies of novel piperidine derived thiosemicarbazones as inhibitors of dihydrofolate reductase.

Dihydrofolate reductase (DHFR), an essential enzyme in folate metabolism, presents a promising target for drug development against various diseases, including cancer and tuberculosis. Herein, we present an integrated approach combining in vitro biochemical assays with in silico molecular docking analysis to evaluate the inhibitory potential of 4-piperidine-based thiosemicarbazones 5(a-s) against DHFR. In our in vitro study, a novel series of 4-piperidine-based thiosemicarbazones 5(a-s) were assessed for their inhibitory activity against DHFR enzyme. The synthesized compounds 5(a-s) exhibited potent inhibition with IC50 values in the range of 13.70 ± 0.25 µM to 47.30 ± 0.86 µM. Among all the derivatives 5p displayed highest inhibitory activity. Simultaneously, in silico analysis were performed and compared with standard drug (Methotrexate) to predict the binding affinity and interaction pattern of synthesized compounds with DHFR active site. SAR analysis was done to elucidate how structural modifications impact compound's biological activity, guiding the rational design of potent and selective drug candidates for targeted diseases. These findings may provide a comprehensive assessment of 4-piperdine-based thiosemicarbazones as DHFR inhibitors and contribute to the development of novel therapeutics targeting DHFR-associated diseases.

Standardization and application of ARMS TaqMan real-time PCR for screening of folate metabolism genes in Han Chinese.

Folate has antioxidant properties, and low concentration in seminal plasma may be associated with increased DNA damage in sperm. Mutations of the methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genes, including MTHFR C677T (rs1801133), MTHFR A1298C (rs1801131), and MTRR A66G (rs1801394), can lead to decreased activity of the encoded folate metabolic enzymes, thereby affecting male reproduction. The current SNP detection methods commonly used in clinical practice have some shortcomings, such as long time-consuming, complex detection steps, or high cost. The purpose of this study was to establish a simple, time-saving, sensitive, accurate, and easy to clinical popularization method for folate metabolism gene detection. We combined ARMS-PCR with TaqMan fluorescent probe to establish an ARMS TaqMan real-time PCR detection method. According to the variation of rs1801131, rs1801133, and rs1801394, two specific primers (one wild type and one mutant) were designed. Mismatched nucleotides were introduced at the penultimate or third position to improve the specificity of the primer. Specific TaqMan probe was introduced to detect PCR products to improve the sensitivity of the method. The results showed that the sensitivity of ARMS TaqMan real-time PCR in SNP genotyping was 1ng, and the accuracy was 100%. A total of 249 clinical samples were detected by the established method, and the correlation between three SNPs and semen quality was analyzed. We found that individuals carrying the AG+GG genotype of rs1801394 had a lower risk of abnormal semen quality. In conclusion, we developed a highly sensitive, accurate, rapid, and easy to be popularized method for detecting SNPs of rs1801394, rs1801131, and rs1801133. ARMS TaqMan real-time PCR is a reliable SNP genotyping method in folate metabolism genes.

Structural identification and comprehension of human ALDH1L1-Gossypol complex

The folate metabolism enzyme ALDH1L1 catalyzed 10-formyltetrahydrofolate to tetrahydrofolate and CO2. Non-small cell lung cancer cells (NSCLC) strongly express ALDH1L1. Gossypol binds to an allosteric site and disrupts the folate metabolism by preventing NADP+ binding. The Cryo-EM structures of tetrameric C-terminal aldehyde dehydrogenase human ALDH1L1 complex with gossypol were examined. Gossypol-bound ALDH1L1 interfered with NADP+ by shifting the allosteric site of the structural conformation, producing a closed-form NADP+ binding site. In addition, the inhibition activity of ALDH1L1 was targeted with gossypol in NSCLC. The gossypol treatment had anti-cancer effects on NSCLC by blocking NADPH and ATP production. These findings emphasize the structure characterizing ALDH1L1 with gossypol.

Associations of methylene tetrahydrofolate reductase (MTHFR) polymorphism with hepatocellular carcinoma in Egyptian population

Liver serves as a hub for key metabolic pathways such as folate cycle that provides one-carbon units for a network of metabolic reactions. Methylenetetrahydrofolate reductase (MTHFR) is a rate limiting enzyme in folate metabolism and thus it is vital for DNA methylation, synthesis and repair [1]. The objective of this study was to evaluate an eventual association between MTHFR polymorphisms C677T (rs1801133) and A1298C (rs1801131) and the susceptibility to hepatocellular carcinoma (HCC) in Egyptian population.Blood samples from patients and controls from Mansoura university hospital were used after signed consent and approval from Medical ethical committee. The two genetic loci were designed for amplification and genotyped by using PCR–RFLP.Our results clarify that, the most important predictors for HCC are T/T genotype of variant C677T and C/C genotype of variant (A1298C) with odds ratio 3.28 and 2.99 respectively. Also, MTHFR variant C677T genotype C/C or T/T combined with MTHFR variant A1298C genotype C/C were associated with an increased risk of HCC, with the OR, 2.6 and 7 respectively. CT genotype of MTHFR variant C677T showed significant difference between HCC grades and C allele of variant C677T showed significant difference in BCLC stages of HCC.Our data indicates that, the two variants (C677T and A1298C) constitute a risk factor for the development of HCC and this could be attributed to the low activities of the enzyme MTHFR that disturb one carbon metabolism and subsequently, DNA synthesis, repair and methylation, thus cellular redox state, growth, and proliferation.

Folate gene polymorphisms CBS 844ins68 and RFC1 A80G and risk of Down syndrome offspring in young Iranian women: A cross-sectional study

Background: Cytogenetics and association studies showed that folate gene polymorphisms can increase the risk of chromosomal nondisjunction and aneuploidies. The folate-metabolizing gene polymorphisms in Down syndrome mothers (DSM) have been assessed in a variety of populations. Reduced folate carrier 1 (RFC1) and cystathionine beta-synthase (CBS) are key enzymes in folate metabolism.
 Objective: 2 common polymorphisms, CBS 844ins68 and RFC1 A80G, were analyzed to determine their probable risk for having Down syndrome (DS) babies in young mothers of Khuzestan province, Iran.
 Materials and Methods: This study was conducted on 100 mothers who had trisomy 21 DS children. 100 age- and ethnic-matched mothers with at least 2 healthy children and no history of abnormal pregnancies were considered as control. The samples were collected from all the mothers from June 2019 to April 2021. Genomic DNA was extracted from peripheral blood. The CBS-844ins68 and RFC1-A80G were genotyped using polymerase chain reaction-electrophoresis and restriction fragment length polymorphism, respectively.
 Results: The frequency of RFC1 AG and GG genotypes in DSM was significantly higher than the control mothers (odds ratio [OR] of 2.38 and 3.07, respectively). The heterozygote genotype of CBS 844ins68 was significantly more prevalent among DSM than the control (OR: 2.419). The OR was significantly increased to 6.667 when the homozygote of both variants was found together.
 Conclusion: Studying polymorphisms possibly increases the susceptibility of having a DS child. However, ethnicity, nutrition, and epistatic interactions are considerable factors to be evaluated in future studies.
 Key words: Down syndrome, Folic acid, Polymorphism, CBS, RFC1.

Study on the characterization of 6S-5-methyltetrahydrofolate calcium salt crystal form C and its stability improvement by ascorbic acid and cysteine

6S-5-methyltetrahydrofolate calcium salt crystal form C (MTHF-CAC) is a stable form of active folate. Compared with folic acid, MTHF-CAC is unaffected by the polymorphism of genes for folate-metabolizing enzymes and can be directly absorbed by the human body. In this study, MTHF-CAC was characterized by XRD, FTIR, DSC, and TGA. The effects of temperature, light, pH, and freeze–thaw cycles on the stability of MTHF-CAC solution were studied. The protective effects of ascorbic acid (AA) and cysteine (Cys) on MTHF-CAC were investigated by HPLC. The retention rate of MTHF-CAC solution was 82.6% after 6 days of storage at 4 °C but only 0.8% after heating at 75 °C for 330 min. Under UHT conditions, AA and Cys exerted protective effects on MTHF-CAC. 120 μg/mL of AA and Cys increased the retention of MTHF-CAC by 89.9% and 85.3%, respectively. The protective effect of AA was better than that of Cys. The research on the characterization and stability of MTHF-CAC provided the basic information for its application in the food industry.

Abstract A110: Targeting methionine metabolism inhibits ovarian cancer stem cells

Abstract Ovarian cancer (OC) is the leading cause of gynecologic cancer death. Platinum (Pt)- based chemotherapy can reprogram cancer cells to ovarian cancer stem cells (OCSCs) and contribute to disease recurrence. In the current study, we demonstrated that platinum drugs can enrich for OCSCs. In high grade serous ovarian cancer (HGSOC) cell lines, cisplatin treatment (IC50 for 16hrs) increased the percentage of cells with high aldehyde dehydrogenase (ALDH+) activity, a known OCSCs marker, and altered methionine (Met) metabolism. Extracellular Met uptake is converted to S-adenosyl-methionine (SAM) by methionine adenosyltransferase 2A (MAT2A), the rate-limiting enzyme of Met utilization. Short-term cisplatin treatment decreased SAM level and increased expression of MAT2A. It has been reported that CSCs have a high-flux metabolic state and are dependent on Met metabolism. We observed higher MAT2A expression in ALDH+ cells compared to the whole cell population, and Met depletion blocked the cisplatin-induced increase in %ALDH+ cells and spheroid formation ability. Furthermore, both MAT2A siRNA knockdown and the MAT2A inhibitor FIDAS-5 prevented cisplatin-induced increase in %ALDH+ cells, suggesting that blocking Met metabolism phenotypically inhibited the platinum enrichment of OCSCs. MAT2A inhibition also downregulated Cyclin E and arrested HGSOC cells in G1/S phase, indicating that a lower level of Met utilization re-established G1/S checkpoint in p53-mutant OC. As SAM is the universal methyl-group donor, it was of interest to examine whether the observed cisplatin-induced alterations in SAM level resulted in changes in DNA methylation. Methylation specific-PCR analysis demonstrated that siMAT2A or FIDAS-5 treatment of HGSOC cells inhibited cisplatin-induced hypermethylation of CpG islands of BRCA1 and ALDH1L1, a folate metabolic enzyme of which promoter hypermethylation is associated to tumor progression. Collectively, these results demonstrated a crucial role of Met metabolism in cisplatin-induced OCSCs enrichment. MAT2A has been recognized as a therapeutic target for the treatment of solid tumors and Met inhibition by targeting MAT2A could represent a therapeutic approach for inhibiting CSC in ovarian cancer. Citation Format: Shu Zhang, Tara X. Metcalfe, Christiane A. Hassel, Heather M. O'Hagan, Kenneth P. Nephew. Targeting methionine metabolism inhibits ovarian cancer stem cells [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A110.

Computational investigation of turmeric phytochemicals targeting PTR1 enzyme of Leishmania species

In this study, we used in silico techniques to identify available parasite treatments, representing a promising therapeutic avenue. Building upon our computational initiatives aimed at discovering natural inhibitors for various target enzymes from parasites causing neglected tropical diseases (NTDs), we present novel findings on three turmeric-derived phytochemicals as inhibitors of Leishmania pteridine reductase I (PTR1) through in silico methodologies. PTR1, a crucial enzyme in the unique folate metabolism of trypanosomatid parasites, holds established therapeutic significance. Employing MOE software, a molecular docking analysis assesses the efficacy of turmeric phytochemicals against Leishmania PTR1. Validation of the docking protocol is confirmed with an RMSD value of 2. Post-docking, compounds displaying notable interactions with critical residues and binding affinities ranging between −6 and −8 kcal/mol are selected for interaction pattern exploration. Testing twelve turmeric phytochemicals, including curcumin, zingiberene, curcumol, curcumenol, eugenol, bisdemethoxycurcumin, tetrahydrocurcumin, tryethylcurcumin, turmerones, turmerin, demethoxycurcumin, and turmeronols, revealed binding affinities ranging from −5.5 to −8 kcal/mol. Notably, curcumin, demethoxycurcumin, and bisdemethoxycurcumin exhibit binding affinities within −6.5 to −8 kcal/mol and establish substantial interactions with catalytic residues. These phytochemicals hold promise as lead structures for rational drug design targeting Leishmania spp. PTR in future applications. This work underscores the potential of these identified phytochemicals in the development of more effective inhibitors, demonstrating their relevance in addressing neglected tropical diseases caused by parasites.

The effects of genetic polymorphism on toxicity and pharmacokinetics of methotrexate in Egyptian adult patients with leukaemia or lymphoma

Polymorphisms in genes coding folate-metabolising enzymes might alter the pharmacokinetics and sensitivity for methotrexate “MTX”. The aim of the study aimed to investigate the influence of MTHFR C677T, DHFR19 Ins/del, GGH −401 C > T, and MTR A2756G polymorphisms on MTX toxicity and pharmacokinetics in Egyptian patients with Acute lymphoblastic leukaemia (ALL) or Non-Hodgkin lymphoma (NHL). Fifty adult Egyptian patients with ALL and NHL, treated with high dose MTX, were prospectively enrolled in the study. Clinical and biochemical data was collected objectively from medical records after each cycle of MTX. Plasma concentrations of MTX were measured after 72 h of initiation of infusion. Genotyping was done with a PCR-ARMS and PCR-RFLP assays. The MTHFR C677T T variants significantly increased the risk of leukopoenia, whereas the genotype MTHFR 677 C > T TT significantly associated with lymphocytopenia, thrombocytopenia, and anaemia. The genotype GGH-401 TT was significantly correlated with anaemia. Plasma MTX level was significantly higher in patients with MTR A2756G G variants. MTHFR polymorphism played the main role in MTX toxicities. The pharmacokinetics of MTX was affected by MTR polymorphism. GGH mutation was mainly concerned with anaemia. Pharmacogenetic testing are recommended to optimise MTX therapy.

Abstract LB_B03: Lentivirus-induced ALDH1L1 knock-in inhibits proliferation and migration and activates apoptosis of human A549 lung adenocarcinoma cells

Abstract Lung Adenocarcinoma (LUAD), one of the most common life-threatening malignancies worldwide, represents about 40% of all lung cancers. Human Protein Atlas and OncoLnc database indicate that LUAD tumors have significantly lower mRNA levels of ALDH1L1 (10-formyltetrahydrofolate dehydrogenase, a regulatory enzyme in folate metabolism) than in non-tumor lung tissues. Moreover, higher intratumoral ALDH1L1 expression in LUAD patients correlates with better clinical outcome. These findings raise the question of whether ALDH1L1 knock-in can have a therapeutic effect. As the first step in addressing this question, we knocked-in ALDH1L1 in A549 lung adenocarcinoma cells using the lentiviral delivery. Of note, as most cancer cell lines, A549 cells do not express ALDH1L1. We have constructed the lentiviral vector carrying ALDH1L1 and green fluorescent protein (EGFP) cDNA under the control of CMV and SV40 promoters, respectively. Recombinant lentivirus (Lv-ALDH1L1), generated and packed in HEK293 cells, was used to transduce A549 cells. The efficiency of the transduction was evaluated by monitoring the GFP expression using the fluorescence microscopy of live cells. The expression of ALDH1L1 was determined at the mRNA and protein levels by RT-PCR and western blot assays, accordingly. Our results showed a significant expression of ALDH1L1 mRNA and protein expression in A549 cells carrying the Lv-ALDH1L1 vector. The lentivirus-induced ALDH1L1 elevation produced strong inhibitory effects on cellular proliferation and colony formation under nutrient-rich conditions. Of note, it also induced alterations of cell morphology. To explore the underlying mechanisms of these effects, apoptosis-related markers were examined. Elevated levels of the signal transduction pathways involved in initiating apoptosis; BID, BAX, and BAK in A549 cells transduced with Lv-ALDH1L1 were observed. Furthermore, using the wound healing assay, we showed that ALDH1L1 strongly suppresses the migratory ability of A549 cells. Interestingly, this effect was associated with a robust down-regulation of the actin depolymerizing factor cofilin and its phosphorylated form. Overall, this study demonstrated that lentiviral ALDH1L1 knock-in produces strong anti-tumorigenesis effects in A549 cells. This further suggests that ALDH1L1 should be investigated as a therapeutic drug target in cancer treatment. Citation Format: Amira Abdellatef, Sergey Krupenko. Lentivirus-induced ALDH1L1 knock-in inhibits proliferation and migration and activates apoptosis of human A549 lung adenocarcinoma cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_B03.

Abstract LB_B09: Loss of folate enzyme ALDH1L1 promotes cancer cell proliferation and xenograft tumor growth

Abstract Background: ALDH1L1 (aldehyde dehydrogenase 1 family member L1, or cytosolic 10-formyltetrahydrofolate dehydrogenase), an enzyme in folate metabolism, is abundantly expressed in several tissues. This enzyme converts 10-formyltetrahydrofolate to tetrahydrofolate (THF) and CO2 while reducing NADP+ to NADPH. ALDH1L1 is ubiquitously downregulated human cancers through DNA promoter methylation, while one cancer cell line, RT4 (derived from papillary bladder cancer), maintains high expression of ALDH1L1. Aims: We tested the hypothesis that loss of ALDH1L1 expression could provide a proliferative and survival advantage for cancer cells and tumor growth. Methods: The ALDH1L1 gene in RT4 cells was knocked down using the shRNA technique. Proliferation was evaluated using MTT assays and xCELLigence real-time cellular analysis (RTCA). Cell migration was assessed by wound healing assays. NADP+/NADPH levels and reduced folate pools were measured by ELISA and the ternary complex assays, respectively. RT4 tumors were generated in nude mice from RT4 lines by subcutaneous injections into both flanks of athymic nude mice. Tumor growth was monitored every week for 12-weeks, after which the tumors were harvested. ALDH1L1 levels in tumors were measured by Western Blot assays, while tumor proliferation was assessed by the Ki-67/H&E staining of the tumor tissue sections. Results: We found that doubling time did not differ between the wild-type (RT4) and shControl (shC) cell lines. Doubling time for the sh506 and sh572 clones was shorter than the controls by 68% and 79%, respectively. Proliferation and migration were significantly increased in sh506 and sh572 cells compared to the control lines. NADP+/NADPH levels were significantly increased for only the sh572 clone. Interestingly, tumor volume and weight were increased in both sh506 and sh572 derived tumors compared to shC tumors. The sh506 and sh572 tumors also showed a slightly increased Ki-67 proliferation index. The ALDH1L1 protein expression was completely lost in sh572 tumors but only partially decreased in sh506 tumors. Conclusions: Our study indicates that RT4 clones lacking ALDH1L1 have increased cell proliferation and tumor growth compared to wild-type RT4 cells, supporting the role of ALDH1L1 as a metabolic regulator of proliferation. Citation Format: Halle M Fogle, Mikyoung You, Jaspreet Sharma, Jonathan McCormac, Natalia I Krupenko, Sergey A Krupenko. Loss of folate enzyme ALDH1L1 promotes cancer cell proliferation and xenograft tumor growth [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_B09.

Association of methylene tetrahydrofolate reductase polymorphisms with cycle day two hormonal characteristics

The 5,10-methylenetetrahydrofolate reductase (MTHFR) enzyme is a key methionine and folate metabolism enzyme that is found in oocytes and embryos in preimplantation stages and plays a vital role in female reproduction.To assess the impact of MTHFR genetic polymorphisms (C677T and A1298C) on follicular stimulating hormone (FSH), luteinizing hormone(LH), Estradiol (E2), progesterone and antimullerian hormone (AMH) in female receiving intracytoplasmic sperm injection (ICSI), 85 infertile women undergoing ICSI treatment at the High Institute of Infertility Diagnosis and Assisted Reproductive Technologies in Baghdad, Iraq, were recruited. These patients had their MTHFR polymorphism genotypes examined, FSH, LH, E2, progesterone, and AMH levels were compared between groups. Furthermore, all demographic characteristics as well as ICSI outcomes were compared between pregnant and non-pregnant women. The research revealed no discernible variation between the means of FSH, LH, E2 and AMH with respect to the 219A>C(rs1801131) (A1298C) genotypes (p > 0.05). Progesterone level was significantly differed with high level found in CC followed by AA and AC. There was no statistically significant variation in hormonal features between CC, CT, and TT for A1298C, 219A>C(rs1801133). The demographic features (age, duration, type of infertility, cause of infertility and previous trial) were not notably different between women who were pregnant and those who were not. In women undergoing ICSI, the maternal MTHFR polymorphisms is not related to FSH, LH, E2 and AMH. MTHFR polymorphisms therefore don't offer useful information regarding the hormonal levels in our groups

Structure of full-length cobalamin-dependent methionine synthase and cofactor loading captured in crystallo

Cobalamin-dependent methionine synthase (MS) is a key enzyme in methionine and folate one-carbon metabolism. MS is a large multi-domain protein capable of binding and activating three substrates: homocysteine, folate, and S-adenosylmethionine for methylation. Achieving three chemically distinct methylations necessitates significant domain rearrangements to facilitate substrate access to the cobalamin cofactor at the right time. The distinct conformations required for each reaction have eluded structural characterization as its inherently dynamic nature renders structural studies difficult. Here, we use a thermophilic MS homolog (tMS) as a functional MS model. Its exceptional stability enabled characterization of MS in the absence of cobalamin, marking the only studies of a cobalamin-binding protein in its apoenzyme state. More importantly, we report the high-resolution full-length MS structure, ending a multi-decade quest. We also capture cobalamin loading in crystallo, providing structural insights into holoenzyme formation. Our work paves the way for unraveling how MS orchestrates large-scale domain rearrangements crucial for achieving challenging chemistries.

Use of recombinant microRNAs as antimetabolites to inhibit human non-small cell lung cancer.

During the development of therapeutic microRNAs (miRNAs or miRs), it is essential to define their pharmacological actions. Rather, miRNA research and therapy mainly use miRNA mimics synthesized invitro. After experimental screening of unique recombinant miRNAs produced invivo, three lead antiproliferative miRNAs against human NSCLC cells, miR-22-3p, miR-9-5p, and miR-218-5p, were revealed to target folate metabolism by bioinformatic analyses. Recombinant miR-22-3p, miR-9-5p, and miR-218-5p were shown to regulate key folate metabolic enzymes to inhibit folate metabolism and subsequently alter amino acid metabolome in NSCLC A549 and H1975cells. Isotope tracing studies further confirmed the disruption of one-carbon transfer from serine to folate metabolites by all three miRNAs, inhibition of glucose uptake by miR-22-3p, and reduction of serine biosynthesis from glucose by miR-9-5p and -218-5p in NSCLC cells. With greater activities to interrupt NSCLC cell respiration, glycolysis, and colony formation than miR-9-5p and -218-5p, recombinant miR-22-3p was effective to reduce tumor growth in two NSCLC patient-derived xenograft mouse models without causing any toxicity. These results establish a common antifolate mechanism and differential actions on glucose uptake and metabolism for three lead anticancer miRNAs as well as antitumor efficacy for miR-22-3p nanomedicine, which shall provide insight into developing antimetabolite RNA therapies.

Exploration and Biological Evaluation of 1,3-Diamino-7H-pyrrol[3,2-f]quinazoline Derivatives as Dihydrofolate Reductase Inhibitors.

Dihydrofolate reductase (DHFR), a core enzyme of folate metabolism, plays a crucial role in the biosynthesis of purines and thymidylate for cell proliferation and growth in both prokaryotic and eukaryotic cells. However, the development of new DHFR inhibitors is challenging due to the limited number of scaffolds available for drug development. Hence, we designed and synthesized a new class of DHFR inhibitors with a 1,3-diamino-7H-pyrrol[3,2-f]quinazoline derivative (PQD) structure bearing condensed rings. Compound 6r exhibited therapeutic effects on mouse models of systemic infection and thigh infection caused by methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300. Moreover, methyl-modified PQD compound 8a showed a strong efficacy in a murine model of breast cancer, which was better than the effects of taxol. The findings showcased in this study highlight the promising capabilities of novel DHFR inhibitors in addressing bacterial infections as well as breast cancer.

Structural and Functional Characterization of a Mycobacterial Methylenetetrahydrofolate Reductase Utilizing NADH as the Exclusive Cofactor.

5,10-Methylenetetraydrofolate reductase (MTHFR) is a key enzyme in folate metabolism. MSMEG_6649, a non-canonical MTHFR from Mycobacterium smegmatis, was previously reported as a monomeric protein lacking the flavin coenzyme. However, the structural basis for its unique flavin-independent catalytic mechanism remains poorly understood. Here, we determined the crystal structures of apo MTHFR MSMEG_6649 and its complex with NADH from M. smegmatis. Structural analysis revealed that the groove formed by the loops 4 and 5 of non-canonical MSMEG_6649 interacting with FAD was significantly larger than that of canonical MTHFR. Meanwhile, the NADH binding site in MSMEG_6649 is highly similar to the FAD binding site in canonical MTHFR, suggesting that NADH plays the same role (immediate hydride donor for methylenetetraydrofolate) as FAD in the catalytic reaction. Using biochemical analysis, molecular modeling, and site-directed mutagenesis, the critical residues participating in the binding of NADH and the substrate 5,10-methylenetetrahydrofolate as well as the product 5-methyltetrahydrofolate were identified and validated. Taken together, this work not only provides a good starting point for understanding the potential catalytic mechanism for MSMEG_6649, but also identifies an exploitable target for the development of anti-mycobacterial drugs.

Genetic impairment of folate metabolism regulates cortical interneurons and social behavior.

The implications of folate deficiency in neuropsychiatric disorders were demonstrated in numerous studies. Genetic deficiency in a key folate metabolism enzyme, MTHFR, is an example of the interaction between genetic and environmental risk factors: the maternal MTHFR deficiency governs in-utero nutrient availability, and the embryo's Mthfr genotype influences its ability to metabolize folates. Here, we explore how the maternal and offspring Mthfr genotypes affect cortical interneuron densities and distributions, mouse social outcome, and the relation of the different interneuron patterns to cortical excitability. Two experiments were conducted to examine the effects of maternal and offspring Mthfr-KO heterozygosity. Mice were tested for direct social interactions (DSIs), repetitive behavior and cortical laminar distribution of interneuron populations expressing glutamate-decarboxylase-65, parvalbumin and somatostatin. Susceptibility to seizure was tested by exposure to pentylenetetrazole (PTZ). Maternal Mthfr+/- genotype was associated with suppressed social activities and reduced interneuron densities in all layers of the retrosplenial cortex (RSC). Somatostatin density and the somatostatin/parvalbumin ratio in the RSC and frontal cortex positively correlated with social behavior in the mice. An interaction between maternal and offspring Mthfr genotypes resulted in higher susceptibility of wild-type offspring to PTZ induced seizure. Maternal folate metabolism was shown to be critical to interneuron ontogenesis. Our results demonstrate that interneurons have a specific susceptibility to folate deficiency that may mediate folate's involvement in neuropsychiatric disease. The relations between cortical somatostatin interneuron patterns and social behavior highlight this subpopulation of interneurons as a target for further research.

The role of folate metabolism enzymes snps in non-obstructive azoospermia: A systematic review and meta-analysis

The role of folate metabolism enzymes snps in non-obstructive azoospermia: A systematic review and meta-analysis

MTHFD1L confers a poor prognosis and malignant phenotype in esophageal squamous cell carcinoma by activating the ERK5 signaling pathway

MTHFD1L, a key enzyme of folate metabolism, is seldom reported in cancer. In this study, we investigate the role of MTHFD1L in the tumorigenicity of esophageal squamous cell carcinoma (ESCC). ESCC tissue microarrays (TMAs) containing 177 samples from 109 patients were utilized to evaluate whether MTHFD1L expression, determined using immunohistochemical analysis, is a prognostic indicator for ESCC patients. The function of MTHFD1L in the migration and invasion of ESCC cells was studied with wound healing, Transwell, and three-dimensional spheroid invasion assays in vitro and a lung metastasis mouse model in vivo. The mRNA microarrays and Ingenuity pathway analysis (IPA) were used to explore the downstream of MTHFD1L. Elevated expression of MTHFD1L in ESCC tissues was significantly associated with poor differentiation and prognosis. These phenotypic assays revealed that MTHFD1L significantly promote the viability and metastasis of ESCC cell in vivo and in vitro. Further detailed analyses of the molecular mechanism demonstrated that the ESCC progression driven by MTHFD1L was through up-regulation ERK5 signaling pathways. These findings reveal that MTHFD1L is positively associated with the aggressive phenotype of ESCC by activating ERK5 signaling pathways, suggesting that MTHFD1L is a new biomarker and a potential molecular therapeutic target for ESCC.

Association of MTHFR C677T polymorphism with risk of preterm birth in Indian mothers: a case–control study

BackgroundSince MTHFR is the key enzyme in folate metabolism, its reduction can lead to hyperhomocysteinemia, which can have a negative impact on pregnancy outcome. Moreover, MTHFR polymorphism has also been linked with oxidative stress and genotoxicity. Identifying its ethnicity-specific association can help to reduce the incidence of preterm birth (PTB). Material and methods: Age-matched preterm birth mothers (< 37 weeks) and full-term mothers (> 37 weeks) were carefully selected for the study. The polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method was adopted to analyse MTHFR C677T polymorphism. Oxidative stress (OS) analysis was performed by measuring the levels of antioxidants (superoxide dismutase (SOD) and catalase (CAT)) and OS damage markers (lipid peroxidation (LPO), total protein). Genotoxicity was confirmed by the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay. The statistical analysis was performed by Student’s t test, chi-square test, and one-way ANOVA. The relevant risk of premature delivery was represented by odds ratios (ORs) with 95% confidence intervals (95% CIs).ResultsThe MTHFR polymorphism showed statistical significance for PTB outcome with CT and TT genotype frequencies at p < 0.01 and p < 0.05, respectively, between cases and controls. Within the PTB categories (extreme-, very-, moderate-PTB), TT genotype showed statistical significance at (p < 0.05), while CT genotype remained insignificant. Also, statistically high oxidative stress and DNA damage were observed in cases compared to controls for all genotypes. Furthermore, the T allele of the MTHFR gene was found to be linked with significantly increased OS and DNA damage on comparison within the groups.ConclusionsThis study confirms the MTHFR C677T polymorphism, oxidative stress, and genotoxicity biomarkers are associated with the PTB outcome. Analysis of these biomarkers during pregnancy can be of clinical significance.