Dysregulation of hepatic one-carbon metabolism in classical homocystinuria: Implications of redox-sensitive DHFR repression and tetrahydrofolate depletion for pathogenesis and treatment.

Cystathionine beta-synthase-deficient homocystinuria (HCU) is a life-threatening disorder of sulfur metabolism. HCU can be treated by using betaine to lower tissue and plasma levels of homocysteine (Hcy). Here, we show that mice with severely elevated Hcy and potentially deficient in the folate species tetrahydrofolate (THF) exhibit a very limited response to betaine indicating that THF plays a critical role in treatment efficacy. Analysis of a mouse model of HCU revealed a 10-fold increase in hepatic levels of 5-methyl -THF and a 30-fold accumulation of formiminoglutamic acid, consistent with a paucity of THF. Neither of these metabolite accumulations were reversed or ameliorated by betaine treatment. Hepatic expression of the THF-generating enzyme dihydrofolate reductase (DHFR) was significantly repressed in HCU mice and expression was not increased by betaine treatment but appears to be sensitive to cellular redox status. Expression of the DHFR reaction partner thymidylate synthase was also repressed and metabolomic analysis detected widespread alteration of hepatic histidine and glutamine metabolism. Many individuals with HCU exhibit endothelial dysfunction. DHFR plays a key role in nitric oxide (NO) generation due to its role in regenerating oxidized tetrahydrobiopterin, and we observed a significant decrease in plasma NOx (NO2 + NO3) levels in HCU mice. Additional impairment of NO generation may also come from the HCU-mediated induction of the 20-hydroxyeicosatetraenoic acid generating cytochrome CYP4A. Collectively, our data shows that HCU induces dysfunctional one-carbon metabolism with the potential to both impair betaine treatment and contribute to multiple aspects of pathogenesis in this disease.

Is there a multidisciplinary role for 5-methyltetrahydrofolate? The obstetric evidence in perspective.

5-methyltetrahydrofolate (5-MTHF), or its synthetic precursor, folic acid, is traditionally used as a supplement for improving fertility and for the prevention of embryonal neural tube defects. However, in the last decade, starting from the effectiveness of this preventive treatment in the gynecological setting, the use of 5-MTHF was extended to other medical and pathological areas. Thus, there might be a rationale for the use of 5-MTHF for purposes other than the protection of the growing embryo linked to the possible effect of MTHFR variants in different pathological conditions. A narrative review was conducted to provide an overview of the available evidence on the use of 5-MTHF in the obstetric field and to critically discuss the available data regarding the use of 5-MTHF across other different therapeutic areas. Results showed that the use of 5-MTHF in pregnancy presents some advantages if compared with folic acid, such as immediate action, the non-necessity of metabolic activation, and the immediate bioavailability of the mother and fetus. Otherwise, the role of 5-MTHF in the management of cardiovascular risk is still debated due to the multiple confounding factors that characterize this patient setting. A link between folate deficiency in pregnancy and postpartum depression has been proposed, as well as between folate levels and the onset of depression. In conclusion, evidence from the literature supports the additional role of 5-MTHF as a pleiotropic drug with a transversal effect in different therapeutic contexts. With regard to the prevention of cardiovascular disorders, available evidence is not conclusive.

Effect of Methylfolate, Pyridoxal-5'-Phosphate, and Methylcobalamin (SolowaysTM) Supplementation on Homocysteine and Low-Density Lipoprotein Cholesterol Levels in Patients with Methylenetetrahydrofolate Reductase, Methionine Synthase, and Methionine Synthase Reductase Polymorphisms: A Randomized Controlled Trial.

Exploring the link between genetic polymorphisms in folate metabolism genes (MTHFR, MTR, and MTRR) and cardiovascular disease (CVD), this study evaluates the effect of B vitamin supplements (methylfolate, pyridoxal-5'-phosphate, and methylcobalamin) on homocysteine and lipid levels, potentially guiding personalized CVD risk management. In a randomized, double-blind, placebo-controlled trial, 54 patients aged 40-75 with elevated homocysteine and moderate LDL-C levels were divided based on MTHFR, MTR, and MTRR genetic polymorphisms. Over six months, they received either a combination of methylfolate, P5P, and methylcobalamin, or a placebo. At the 6 months follow-up, the treatment group demonstrated a significant reduction in homocysteine levels by 30.0% (95% CI: -39.7% to -20.3%) and LDL-C by 7.5% (95% CI: -10.3% to -4.7%), compared to the placebo (p < 0.01 for all). In the subgroup analysis, Homozygous Minor Allele Carriers showed a more significant reduction in homocysteine levels (48.3%, 95% CI: -62.3% to -34.3%, p < 0.01) compared to mixed allele carriers (18.6%, 95% CI: -25.6% to -11.6%, p < 0.01), with a notable intergroup difference (29.7%, 95% CI: -50.7% to -8.7%, p < 0.01). LDL-C levels decreased by 11.8% in homozygous carriers (95% CI: -15.8% to -7.8%, p < 0.01) and 4.8% in mixed allele carriers (95% CI: -6.8% to -2.8%, p < 0.01), with a significant between-group difference (7.0%, 95% CI: -13.0% to -1.0%, p < 0.01). Methylfolate, P5P, and methylcobalamin supplementation tailored to genetic profiles effectively reduced homocysteine and LDL-C levels in patients with specific MTHFR, MTR, and MTRR polymorphisms, particularly with homozygous minor allele polymorphisms.

Tetrahydrofolate Attenuates Cognitive Impairment after Hemorrhagic Stroke by Promoting Hippocampal Neurogenesis via PTEN Signaling.

Intracerebral hemorrhage (ICH), the most common subtype of hemorrhagic stroke, leads to cognitive impairment and imposes significant psychological burdens on patients. Hippocampal neurogenesis has been shown to play an essential role in cognitive function. Our previous study has shown that tetrahydrofolate (THF) promotes the proliferation of neural stem cells (NSCs). However, the effect of THF on cognition after ICH and the underlying mechanisms remain unclear. Here, we demonstrated that administration of THF could restore cognition after ICH. Using Nestin-GFP mice, we further revealed that THF enhanced the proliferation of hippocampal NSCs and neurogenesis after ICH. Mechanistically, we found that THF could prevent ICH-induced elevated level of PTEN and decreased expressions of phosphorylated AKT and mTOR. Furthermore, conditional deletion of PTEN in NSCs of the hippocampus attenuated the inhibitory effect of ICH on the proliferation of NSCs and abnormal neurogenesis. Taken together, these results provide molecular insights into ICH-induced cognitive impairment and suggest translational clinical therapeutic strategy for hemorrhagic stroke.

Tetrahydrofolate levels influence 2-aminoacrylate stress in Salmonella enterica.

In Salmonella enterica, the absence of the RidA deaminase results in the accumulation of the reactive enamine 2-aminoacrylate (2AA). The resulting 2AA stress impacts metabolism and prevents growth in some conditions by inactivating a specific target pyridoxal 5'-phosphate (PLP)-dependent enzyme(s). The detrimental effects of 2AA stress can be overcome by changing the sensitivity of a critical target enzyme or modifying flux in one or more nodes in the metabolic network. The catabolic L-alanine racemase DadX is a target of 2AA, which explains the inability of an alr ridA strain to use L-alanine as the sole nitrogen source. Spontaneous mutations that suppressed the growth defect of the alr ridA strain were identified as lesions in folE, which encodes GTP cyclohydrolase and catalyzes the first step of tetrahydrofolate (THF) synthesis. The data here show that THF limitation resulting from a folE lesion, or inhibition of dihydrofolate reductase (FolA) by trimethoprim, decreases the 2AA generated from endogenous serine. The data are consistent with an increased level of threonine, resulting from low folate levels, decreasing 2AA stress.IMPORTANCERidA is an enamine deaminase that has been characterized as preventing the 2-aminoacrylate (2AA) stress. In the absence of RidA, 2AA accumulates and damages various cellular enzymes. Much of the work describing the 2AA stress system has depended on the exogenous addition of serine to increase the production of the enamine stressor. The work herein focuses on understanding the effect of 2AA stress generated from endogenous serine pools. As such, this work describes the consequences of a subtle level of stress that nonetheless compromises growth in at least two conditions. Describing mechanisms that alter the physiological consequences of 2AA stress increases our understanding of endogenous metabolic stress and how the robustness of the metabolic network allows perturbations to be modulated.

Engineering Redox Cofactor Balance for Improved 5-Methyltetrahydrofolate Production in Escherichia coli.

5-Methyltetrahydrofolate (5-MTHF) is the sole active form of folate functioning in the human body and is widely used as a nutraceutical. Unlike the pollution from chemical synthesis, microbial synthesis enables green production of 5-MTHF. In this study, Escherichia coli BL21 (DE3) was selected as the host. Initially, by deleting 6-phosphofructokinase 1 and overexpressing glucose-6-phosphate 1-dehydrogenase and 6-phosphogluconate dehydrogenase, the glycolysis pathway flux decreased, while the pentose phosphate pathway flux enhanced. The ratios of NADH/NAD+ and NADPH/NADP+ increased, indicating elevated NAD(P)H supply. This led to more folate being reduced and the successful accumulation of 5-MTHF to 44.57 µg/L. Subsequently, formate dehydrogenases from Candida boidinii and Candida dubliniensis were expressed, which were capable of catalyzing the reaction of sodium formate oxidation for NAD(P)H regeneration. This further increased the NAD(P)H supply, leading to a rise in 5-MTHF production to 247.36 µg/L. Moreover, to maintain the balance between NADH and NADPH, pntAB and sthA, encoding transhydrogenase, were overexpressed. Finally, by overexpressing six key enzymes in the folate to 5-MTHF pathway and employing fed-batch cultivation in a 3 L fermenter, strain Z13 attained a peak 5-MTHF titer of 3009.03 µg/L, the highest level reported in E. coli so far. This research is a significant step toward industrial-scale microbial 5-MTHF production.

Human milk oligosaccharide composition following supplementation with folic acid vs (6S)-5-methyltetrahydrofolic acid during pregnancy and mediation by human milk folate forms.

Supplementation with folic acid versus (6S)-5-methyltetrahydrofolic acid (5-MTHF) results in different folate forms in human milk, with folic acid increasing unmetabolized folic acid (UMFA) at the expense of reduced folate forms. It is unknown whether folate forms present in human milk have further effects on human milk composition, such as human milk oligosaccharide (HMO) concentrations. We randomized 60 pregnant women in Canada to 0.6 mg/day folic acid or (6S)-5-MTHF. Human milk folate forms (LC-MS/MS) and nineteen HMOs (HPLC) were quantified at 1 week postpartum. Linear regression and causal mediation analysis were used to evaluate the effect of folate supplementation on HMO concentrations, and possible mediation by concentrations of UMFA and reduced folate forms in human milk (controlling for secretor status and parity). HMO concentrations were not different between groups, with no evidence of mediation by reduced folate forms; however, increased UMFA was associated with reduced concentrations of total HMOs and 3'-sialyllactose.

Novel tetrahydrofolate-dependent d-serine dehydratase activity of serine hydroxymethyltransferases.

d-Serine plays vital physiological roles in the functional regulation of the mammalian brain, where it is produced from l-serine by serine racemase and degraded by d-amino acid oxidase. In the present study, we identified a new d-serine metabolizing activity of serine hydroxymethyltransferase (SHMT) in bacteria as well as mammals. SHMT is known to catalyze the conversion of l-serine and tetrahydrofolate (THF) to glycine and 5,10-methylenetetrahydrofolate, respectively. In addition, we found that human and Escherichia coli SHMTs have d-serine dehydratase activity, which degrades d-serine to pyruvate and ammonia. We characterized this enzymatic activity along with canonical SHMT activity. Intriguingly, SHMT required THF to catalyze d-serine dehydration and did not exhibit dehydratase activity toward l-serine. Furthermore, SHMT did not use d-serine as a substrate in the canonical hydroxymethyltransferase reaction. The d-serine dehydratase activities of two isozymes of human SHMT were inhibited in the presence of a high concentration of THF, whereas that of E. coli SHMT was increased. The pH and temperature profiles of d-serine dehydratase and serine hydroxymethyltransferase activities of these three SHMTs were partially distinct. The catalytic efficiency (kcat /Km ) of dehydratase activity was lower than that of hydroxymethyltransferase activity. Nevertheless, the d-serine dehydratase activity of SHMT was physiologically important because d-serine inhibited the growth of an SHMT deletion mutant of E. coli, ∆glyA, more than that of the wild-type strain. Collectively, these results suggest that SHMT is involved not only in l- but also in d-serine metabolism through the degradation of d-serine.

Efficacy and Mechanism Study of 6S-5-Methyltetrahydrofolate-Calcium Against Telencephalon Infarction Injury in Zebrafish Model of Ischemic Stroke.

Ischemic stroke is a heterogeneous brain injury with complex pathophysiology and it is also a time sensitive neurological injury disease. At present, the treatment options for ischemic stroke are still limited. 6S-5-methyltetrahydrofolate-calcium (MTHF-Ca) is the calcium salt of the predominant form of dietary folate in circulation. MTHF-Ca has potential neuroprotective effect on neurocytes, but whether it can be used for ischemic stroke treatment remains unknown. We established zebrafish ischemic stroke model through photothrombotic method to evaluate the protective effect of MTHF-Ca on the ischemic brain injury of zebrafish. We demonstrated that MTHF-Ca reduced the brain damage by reducing motor dysfunction and neurobehavioral defects of zebrafish with telencephalon infarction injury. MTHF-Ca counteracted oxidative damages after Tel injury by increasing the activities of GSH-Px and SOD and decreasing the content of MDA. RNA-seq and RT-qPCR results showed that MTHF-Ca played a neuroprotective role by alleviating neuroinflammation, inhibiting blood coagulation, and neuronal apoptosis processes. Overall, we have demonstrated that MTHF-Ca has neuroprotective effect in ischemic stroke and can be used as a potential treatment for ischemic stroke.

Analysis of the mediating role of BMI in associations of different folate forms with hepatic steatosis and liver fibrosis in adolescents in the USA: results from the NHANES 2017-2018.

Background: Most studies have explored the relationship between serum total folate and nonalcoholic fatty liver disease (NAFLD) in adults, but there has been no study on the relationship between different folate forms and hepatic steatosis or liver stiffness in adolescents. Objective: To investigate the association of different folate forms with hepatic steatosis or liver stiffness in adolescents, and further explore the intermediary role of BMI in this relationship. Methods: The cross-sectional study included 549 participants from the 2017-2018 National Health and Nutrition Inspection Survey (NHANES) survey cycle who had complete data. Four folate data (red blood cell folate, serum total folate, 5-methyl-tetrahydrofolate and folic acid) were included in our study. Controlled attenuation parameters (CAP) and liver stiffness came from the results of liver ultrasound transient elastography. We used linear regression to analyze the relationship between different forms of folate and CAP or liver stiffness, and logistic regression to analyze the relationship between different forms of folate and NAFLD or significant fibrosis. We also used restricted cubic splines to analyze the nonlinear relationship between different forms of folate and NAFLD or significant fibrosis. Finally, we used regression-based intermediary analysis to distinguish the direct and BMI-mediated effects of folate on CAP or liver stiffness. All the analyses adjusted the relevant covariates. Results: The means of CAP and liver hardness in this study were 223.02dB/m and 5.03kPa, respectively. We found that in model 2, there was a negative correlation between serum total folate (ß: -18.53; 95%CI: -29.32 to -7.73) or 5-methyltetrahydrofolate (ß: -14.13; 95%CI: -28.98 to -7.86) and CAP. However, when the BMI was further adjusted in model 3, this negative correlation no longer existed (serum total folate: ß: -8.36; 95%CI: -17.69 to 0.97; 5-methyltetrahydrofolate: ß: -8.05; 95%CI: -17.19 to 1.09). Similarly, we found a negative correlation between serum total folate or 5-Methyl-tetrahydrofolate and liver stiffness in model 2. There was no significant correlation between red blood cell folate or folic acid and CAP or liver stiffness in either model 2 or model 3. The nonlinear relationship between different folate forms and NAFLD or significant fibrosis was not significant. It is estimated that 76% of the total association between serum total folate and CAP is mediated by BMI. The mediating proportion of BMI in the total correlation between serum total folate and liver stiffness was 50%. Similarly, we found that BMI significantly mediated the relationship between 5-Methyl-tetrahydrofolate and CAP or liver stiffness, with a mediating ratio of 77% and 49%, respectively. Conclusion: Our results show that serum total folate or 5-Methyl-tetrahydrofolate are negatively correlated with hepatic steatosis or liver stiffness in adolescents, and BMI plays major mediating role in this relationship. Our findings emphasize the importance of monitoring the concentration of serum folate, not just the serum total folate concentration.

The role of methionine synthases in fungal metabolism and virulence.

Methionine synthases (MetH) catalyse the methylation of homocysteine (Hcy) with 5-methyl-tetrahydrofolate (5, methyl-THF) acting as methyl donor, to form methionine (Met) and tetrahydrofolate (THF). This function is performed by two unrelated classes of enzymes that differ significantly in both their structures and mechanisms of action. The genomes of plants and many fungi exclusively encode cobalamin-independent enzymes (EC.2.1.1.14), while some fungi also possess proteins from the cobalamin-dependent (EC.2.1.1.13) family utilised by humans. Methionine synthase's function connects the methionine and folate cycles, making it a crucial node in primary metabolism, with impacts on important cellular processes such as anabolism, growth and synthesis of proteins, polyamines, nucleotides and lipids. As a result, MetHs are vital for the viability or virulence of numerous prominent human and plant pathogenic fungi and have been proposed as promising broad-spectrum antifungal drug targets. This review provides a summary of the relevance of methionine synthases to fungal metabolism, their potential as antifungal drug targets and insights into the structures of both classes of MetH.

Tailored extraction and ion mobility-mass spectrometry enables isotopologue analysis of tetrahydrofolate vitamers.

Climate change directs the focus in biotechnology increasingly on one-carbon metabolism for fixation of CO2 and CO2-derived chemicals (e.g. methanol, formate) to reduce our reliance on both fossil and food-competing carbon sources. The tetrahydrofolate pathway is involved in several one-carbon fixation pathways. To study such pathways, stable isotope-labelled tracer analysis performed with mass spectrometry is state of the art. However, no such method is currently available for tetrahydrofolate vitamers. In the present work, we established a fit-for-purpose extraction method for the methylotrophic yeast Komagataella phaffii that allows access to intracellular methyl- and methenyl-tetrahydrofolate (THF) with demonstrated stability over several hours. To determine isotopologue distributions of methyl-THF, LC-QTOFMS provides a selective fragment ion with suitable intensity of at least two isotopologues in all samples, but not for methenyl-THF. However, the addition of ion mobility separation provided a critical selectivity improvement allowing accurate isotopologue distribution analysis of methenyl-THF with LC-IM-TOFMS. Application of these new methods for 13C-tracer experiments revealed a decrease from 83 ± 4 to 64 ± 5% in the M + 0 carbon isotopologue fraction in methyl-THF after 1 h of labelling with formate, and to 54 ± 5% with methanol. The M + 0 carbon isotopologue fraction of methenyl-THF was reduced from 83 ± 2 to 78 ± 1% over the same time when using 13C-methanol labelling. The labelling results of multiple strains evidenced the involvement of the THF pathway in the oxygen-tolerant reductive glycine pathway, the presence of the in vivo reduction of formate to formaldehyde, and the activity of the spontaneous condensation reaction of formaldehyde with THF in K. phaffii.

Dual effect of methylene-tetrahydrofolate reductase and angiotensinconverting enzyme gene polymorphisms on the risk of acute ischemic stroke.

Ischemic stroke, a prevalent neurological disease, is the major reason of serious disability and death worldwide. Methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms increase homocysteine levels which also raise the risk of vascular diseases. Angiotensin-converting enzyme (ACE) gene polymorphisms can cause vascular reorganization and disrupt arterial wall stability. The aim of this study was to explore how the MTHFR and ACE gene polymorphisms are related to acute ischemic stroke. A total of 200 individuals (102 acute ischemic stroke patients and 98 healthy controls) were included in this case-control research. MTHFR gene C677T (rs1801133) and A1298C (rs1801131) polymorphisms were studied through polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) assays, ACE gene I/D polymorphism (rs1799752) was studied by PCR. The MTHFR C667T and ACE I/D polymorphisms did not show any statistically meaningful differences between healthy controls and acute ischemic stroke patients (P>0.05). However, compared to healthy controls, acute ischemic stroke patients had almost nine-fold higher prevalence of the CC genotype based on the MTHFR A1298C polymorphism (P=0.024, OR=8.8, 95%Cl=1.27-208.2). Additionally, individuals with acute ischemic stroke had greater frequencies of the combined genotypes of MTHFR and ACE gene polymorphisms in the forms of CC/CC (C667T/A1298C), CC/ DD (A1298C/ACE I/D) and CC/CC/DD (C677T/A1298C/ACE I/D) (P = 0.027, P = 0.015 and P = 0.037, respectively). A statistically significant correlation was assessed between MTHFR gene A1298C polymorphism and acute ischemic stroke. Additionally, it was discovered that the genotype combinations of CC/CC (C667T/A1298C), CC/DD (A1298C/ACE I/D) and CC/CC/DD (C677T/A1298C/ ACE I/D) have risk-increasing effects on acute ischemic stroke. To employ these genetic variations as alternative treatments for ischemic stroke, these findings should be validated by more research.

A new metabolic trait in an acetogen: Mixed acid fermentation of fructose in a methylene-tetrahydrofolate reductase mutant of Acetobacterium woodii.

To inactivate the Wood-Ljungdahl pathway in the acetogenic model bacterium Acetobacterium woodii, the genes metVF encoding two of the subunits of the methylene-tetrahydrofolate reductase were deleted. As expected, the mutant did not grow on C1 compounds and also not on lactate, ethanol or butanediol. In contrast to a mutant in which the first enzyme of the pathway (hydrogen-dependent CO2 reductase) had been genetically deleted, cells were able to grow on fructose, albeit with lower rates and yields than the wild-type. Growth was restored by addition of an external electron sink, glycine betaine + CO2 or caffeate. Resting cells pre-grown on fructose plus an external electron acceptor fermented fructose to two acetate and four hydrogen, that is, performed hydrogenogenesis. Cells pre-grown under fermentative conditions on fructose alone redirected carbon and electrons to form lactate, formate, ethanol as well as hydrogen. Apparently, growth on fructose alone induced enzymes for mixed acid fermentation (MAF). Transcriptome analyses revealed enzymes potentially involved in MAF and a quantitative model for MAF from fructose in A. woodii is presented.

Formate overflow drives toxic folate trapping in MTHFD1 inhibited cancer cells.

Cancer cells fuel their increased need for nucleotide supply by upregulating one-carbon (1C) metabolism, including the enzymes methylenetetrahydrofolate dehydrogenase-cyclohydrolase 1 and 2 (MTHFD1 and MTHFD2). TH9619 is a potent inhibitor of dehydrogenase and cyclohydrolase activities in both MTHFD1 and MTHFD2, and selectively kills cancer cells. Here, we reveal that, in cells, TH9619 targets nuclear MTHFD2 but does not inhibit mitochondrial MTHFD2. Hence, overflow of formate from mitochondria continues in the presence of TH9619. TH9619 inhibits the activity of MTHFD1 occurring downstream of mitochondrial formate release, leading to the accumulation of 10-formyl-tetrahydrofolate, which we term a 'folate trap'. This results in thymidylate depletion and death of MTHFD2-expressing cancer cells. This previously uncharacterized folate trapping mechanism is exacerbated by physiological hypoxanthine levels that block the de novo purine synthesis pathway, and additionally prevent 10-formyl-tetrahydrofolate consumption for purine synthesis. The folate trapping mechanism described here for TH9619 differs from other MTHFD1/2 inhibitors and antifolates. Thus, our findings uncover an approach to attack cancer and reveal a regulatory mechanism in 1C metabolism.

In silico screening of inhibitors against human dihydrofolate reductase to identify potential anticancer compounds.

In all species, dihydrofolate reductase (DHFR) is an essential enzyme that regulates the cellular amount of tetrahydrofolate. Human DHFR (hDHFR) activity inhibition results in tetrahydrofolate depletion and cell death. This property has made hDHFR a therapeutic target for cancer. Methotrexate is a well-known hDHFR inhibitor, but its administration has shown some light to severe adverse effects. Therefore, we aimed to find new potential hDHFR inhibitors using structure-based virtual screening, ADMET prediction, molecular docking, and molecular dynamics simulations. Here, we used the PubChem database to find all compounds with at least 90% structural similarity to known natural DHFR inhibitors. To explore their interaction pattern and estimate their binding affinities, the screened compounds (2023) were subjected to structure-based molecular docking against hDHFR. The fifteen compounds that showed higher binding affinity to the hDHFR than the reference compound (methotrexate) displayed important molecular orientation and interactions with key residues in the enzyme's active site. These compounds were subjected to Lipinski and ADMET prediction. PubChem CIDs: 46886812 and 638190 were identified as putative inhibitors. In addition, molecular dynamics simulations revealed that the binding of compounds (CIDs: 46886812 and 63819) stabilized the hDHFR structure and caused minor conformational changes. Our findings suggest that two compounds (CIDs: 46886812 and 63819) could be promising potential inhibitors of hDHFR in cancer therapy.Communicated by Ramaswamy H. Sarma.

Impact of methylene-tetrahydrofolate reductase gene C677T and A1298C polymorphisms as a risk factor for hepatitis B virus infection.

Chronic hepatitis B infection caused by Hepatitis B virus (HBV), influences over two billion people worldwide despite having an effective vaccine. With a total prevalence of 4.57%, there are 3.3 million estimated HBV carriers in Türkiye. Methylene-tetrahydrofolate reductase (MTHFR) arrange folate metabolism through nucleic acid synthesis and DNA methylation. C677T (rs1801133, p.Ala222Val) and A1298C (rs1801131, p.Glu429Ala) polymorphisms of MTHFR gene have effect of reducing the activity of enzyme. We purposed to investigate the correlation between C677T and A1298C polymorphisms of MTHFR gene with HBV infection in a Turkish population. One hundred eighteen HBV-infected participants and ninety healthy controls were incorporated in this research. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was applied to discover the genotypes of MTHFR polymorphisms. We demonstrated that T allele and CT + TT genotype frequencies of C677T polymorphism were significantly increased in HBV-infected participants than healthy controls [p = 0.015, OR (95% Cl) = 1.7 (1.11-2.79) and p = 0.020, OR (95% Cl) = 1.9 (1.10-3.42), respectively). No significant associations were noted concerning the A1298C polymorphism (p > 0.05). CC-AA composite genotype was observed to be significantly elevated in healthy controls than HBV-infected participants (32.2% vs. 13.6%, p = 0.001). In addition, the frequency of T-C haplotype was found to be considerably higher in the patient group than control group (15.8% vs 11.8%, p = 0.018). In conclusion, we found that T allele of C677T polymorphism poses a risk factor for HBV infection. We also discovered a protective impact of the CC-AA composite genotype against HBV infection and a risk effect of the T-A haplotype for HBV-infection.

Structural insights into translation regulation by the THF-II riboswitch.

In bacteria, expression of folate-related genes is controlled by the tetrahydrofolate (THF) riboswitch in response to specific binding of THF and its derivatives. Recently, a second class of THF riboswitches, named THF-II, was identified in Gram-negative bacteria, which exhibit distinct architecture from the previously characterized THF-I riboswitches found in Gram-positive bacteria. Here, we present the crystal structures of the ligand-bound THF-II riboswitch from Mesorhizobium loti. These structures exhibit a long rod-like fold stabilized by continuous base pair and base triplet stacking across two helices of P1 and P2 and their interconnecting ligand-bound binding pocket. The pterin moiety of the ligand docks into the binding pocket by forming hydrogen bonds with two highly conserved pyrimidines in J12 and J21, which resembles the hydrogen-bonding pattern at the ligand-binding site FAPK in the THF-I riboswitch. Using small-angle X-ray scattering and isothermal titration calorimetry, we further characterized the riboswitch in solution and reveal that Mg2+ is essential for pre-organization of the binding pocket for efficient ligand binding. RNase H cleavage assay indicates that ligand binding reduces accessibility of the ribosome binding site in the right arm of P1, thus down-regulating the expression of downstream genes. Together, these results provide mechanistic insights into translation regulation by the THF-II riboswitch.

Assaying plant formate-tetrahydrofolate ligase with monoglutamylated and polyglutamylated substrates using a fluorescence-HPLC based method.

Formate-tetrahydrofolate ligase catalyzes reversible, ATP-dependent conversion of tetrahydrofolate and formate to 10-formyltetrahydrofolate, simultaneously releasing ADP and inorganic phosphate. This enzyme has traditionally been assayed in the direction of 10-CHO-tetrahydrofolate formation by lowering pH of the reaction post-incubation, thus converting the product of the reaction to 5,10-methenyltetrahydrofolate, which is then quantified spectrophotometrically. To increase sensitivity of the product detection, which is particularly useful when determining the kinetic parameters of the enzyme with polyglutamylated substrates, we have replaced the spectrophotometric detection with HPLC separation and fluorescence detection. In addition to the modified enzyme assay protocol, we are also providing protocols for producing recombinant formate-tetrahydrofolate ligase from Arabidopsis in Escherichia coli cells, producing crude Arabidopsis leaf and root extracts suitable for assaying this enzyme, and for synthesis of polyglutamylated tetrahydrofolate substrates.

Association between Methylene-Tetrahydrofolate Reductase C677T Polymorphism and Human Immunodeficiency Virus Type 1 Infection in Morocco.

Human immunodeficiency virus type 1 (HIV-1) infection varies substantially among individuals. One of the factors influencing viral infection is genetic variability. Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism is a genetic factor that has been correlated with different types of pathologies, including HIV-1. The MTHFR gene encodes the MTHFR enzyme, an essential factor in the folate metabolic pathway and in maintaining circulating folate and methionine at constant levels, thus preventing the homocysteine accumulation. Several studies have shown the role of folate on CD4+ T lymphocyte count among HIV-1 subjects. In this case-control study we aimed to determine the association between the MTHFR C677T polymorphism and HIV-1 infection susceptibility, AIDS development, and therapeutic outcome among Moroccans. The C677T polymorphism was genotyped by polymerase chain reaction followed by fragment length polymorphism digestion in 214 participants living with HIV-1 and 318 healthy controls. The results of the study revealed no statistically significant association between MTHFR C677T polymorphism and HIV-1 infection (P > .05). After dividing HIV-1 subjects according to their AIDS status, no significant difference was observed between C677T polymorphism and AIDS development (P > .05). Furthermore, regarding the treatment response outcome, as measured by HIV-1 RNA viral load and CD4+ T cell counts, no statistically significant association was found with MTHFR C677T polymorphism. We conclude that, in the genetic context of the Moroccan population, MTHFR C677T polymorphism does not affect HIV-1 infection susceptibility, AIDS development, or response to treatment. However, more studies should be done to investigate both genetic and nutritional aspects for more conclusive results.