Methionine Synthase Research Articles

Methyl transfer reactions catalyzed by cobalamin-dependent enzymes: Insight from molecular docking

Methyl transfer reactions, mediated by methyltransferases (MeTrs), such as methionine synthase (MetH) or monomethylamine: CoM (MtmBC), constitute one of the most important classes of vitamin B12-dependent reactions. The challenge in exploring the catalytic function of MeTrs is related to their modular structure. From the crystallographic point of view, the structure of each subunit has been determined, but there is a lack of understanding of how each subunit interacts with each other. So far, theoretical studies of methyl group transfer were carried out for the structural models of the active site of each subunit. However, those studies do not include the effect of the enzymatic environment, which is crucial for a comprehensive understanding of enzyme-mediated methyl transfer reactions. Herein, to explore how two subunits interact with each other and how the methyl transfer reaction is catalyzed by MeTrs, molecular docking of the functional units of MetH and MtmBC was carried out. Along with the interactions of the functional units, the reaction coordinates, including the Co–C bond distance for methylation of cob(I)alamin (CoICbl) and the C–S bond distance in demethylation reaction of cob(III)alamin (CoIIICbl), were considered. The functional groups should be arranged so that there is an appropriate distance to transfer a methyl group and present results indicate that steric interactions can limit the number of potential arrangements. This calls into question the possibility of SN2-type mechanism previously proposed for MeTrs. Further, it leads to the conclusion that the methyl transfer reaction involves some spatial changes of modules suggesting an alternate radical-based pathway for MeTrs-mediated methyl transfer reactions. The calculations also showed that changes in torsion angles induce a change in reaction coordinates, namely Co–C and C–S bond distances, for the methylation and demethylation reactions catalyzed both by MetH and MtmBC.

A novel gene-diet interaction promotes organismal lifespan and host protection during infection via the mitochondrial UPR.

Cells use a variety of mechanisms to maintain optimal mitochondrial function including the mitochondrial unfolded protein response (UPRmt). The UPRmt mitigates mitochondrial dysfunction by differentially regulating mitoprotective gene expression through the transcription factor ATFS-1. Since UPRmt activation is commensurate with organismal benefits such as extended lifespan and host protection during infection, we sought to identify pathways that promote its stimulation. Using unbiased forward genetics screening, we isolated novel mutant alleles that could activate the UPRmt. Interestingly, we identified one reduction of function mutant allele (osa3) in the mitochondrial ribosomal gene mrpl-2 that activated the UPRmt in a diet-dependent manner. We find that mrpl-2(osa3) mutants lived longer and survived better during pathogen infection depending on the diet they were fed. A diet containing low levels of vitamin B12 could activate the UPRmt in mrpl-2(osa3) animals. Also, we find that the vitamin B12-dependent enzyme methionine synthase intersects with mrpl-2(osa3) to activate the UPRmt and confer animal lifespan extension at the level of ATFS-1. Thus, we present a novel gene-diet pairing that promotes animal longevity that is mediated by the UPRmt.

Polymorphism of folate cycle genes as a risk factor of hyperhomocysteinemia

Hyperhomocysteinemia (HHc) is a new factor being considered at the moment that can cause damage to vessel walls. Its occurrence depends on genetic peculiarities of a body. Our research goal was to estimate frequency of genetic polymorphisms (SNP) in folate cycle genes among people living in Perm region and its influence on homocysteine (Hc) concentration in blood serum. We examined 189 women (32.2±5.25). Hc concentration in blood serum was determined with immune chemiluminescent procedure. We examined frequency of SNP in folate cycle genes with pyrosequencing. Homozygote state as per minor alleles in methylene tetrahydrofolate reductase (MTHFR) gene (rs 1801133 и rs 1801131) and MTR gene (rs 1805087) was registered 7.5, 5.4, and 13.75 times less frequently than homozygote state as per neutral alleles. Heterozygote state prevailed for genes of methionine synthase reductase and folate transport protein among examined SNP. Homozygotes as per minor allele SNP in MTHFR gene (Ala222Val; rs 1801133) had higher Hc concentration in blood serum that amounted to 8.476 ± 3.193 mmol/L and was 1.276 times higher than the same parameter in homozygotes as per neutral allele (р=0.0036). We didn’t establish any influence on Hc contents in blood serum for the remaining 4 SNP in folate cycle genes (р> 0.1). Examined SNP in MTHFR and MTR genes tended to have neutral alleles more frequently than minor ones. SNP in genes of other examined proteins belonging to folate cycle didn’t have any differences in frequency of examined alleles. We didn’t detect a combination of homozygote state as per two SNP in MTHFR gene or homozygote state as per one SNP and heterozygote state as per another one in a genome. Only SNP in MTHFR gene (Ala222Val, rs 1801133) authentically causes increase in homocysteine concentration out of all the examined SNP in genes of folate cycle enzymes and proteins

Полиморфизм генов фолатного цикла как фактор риска формирования гипергомоцистеинемии

Hyperhomocysteinemia (HHc) is a new factor being considered at the moment that can cause damage to vessel walls. Its occurrence depends on genetic peculiarities of a body. Our research goal was to estimate frequency of genetic polymorphisms (SNP) in folate cycle genes among people living in Perm region and its influence on homocysteine (Hc) concentration in blood serum. We examined 189 women (32.2±5.25). Hc concentration in blood serum was determined with immune chemiluminescent procedure. We examined frequency of SNP in folate cycle genes with pyrosequencing. Homozygote state as per minor alleles in methylene tetrahydrofolate reductase (MTHFR) gene (rs 1801133 и rs 1801131) and MTR gene (rs 1805087) was registered 7.5, 5.4, and 13.75 times less frequently than homozygote state as per neutral alleles. Heterozygote state prevailed for genes of methionine synthase reductase and folate transport protein among examined SNP. Homozygotes as per minor allele SNP in MTHFR gene (Ala222Val; rs 1801133) had higher Hc concentration in blood serum that amounted to 8.476 ± 3.193 mmol/L and was 1.276 times higher than the same parameter in homozygotes as per neutral allele (р=0.0036). We didn’t establish any influence on Hc contents in blood serum for the remaining 4 SNP in folate cycle genes (р> 0.1). Examined SNP in MTHFR and MTR genes tended to have neutral alleles more frequently than minor ones. SNP in genes of other examined proteins belonging to folate cycle didn’t have any differences in frequency of examined alleles. We didn’t detect a combination of homozygote state as per two SNP in MTHFR gene or homozygote state as per one SNP and heterozygote state as per another one in a genome. Only SNP in MTHFR gene (Ala222Val, rs 1801133) authentically causes increase in homocysteine concentration out of all the examined SNP in genes of folate cycle enzymes and proteins.

186 Young Scholar Presentation: Immunometabolism during periods of negative nutrient balance or heat stress is altered by dietary methyl donor supply in dairy cows

Abstract Our research examined the effects of enhancing methyl donor supply on immunometabolism during periods of negative nutrient balance (NNB) or heat stress (HS). The first experiment examined the effects of post-ruminal choline supply during NNB on production and pathways of 1-carbon metabolism. Ten primiparous rumen-cannulated cows (158±24 DIM) were used in a replicated 5×5 Latin square design with 4d treatment periods and 10d of recovery. Treatments were unrestricted intake with abomasal infusion of water, restricted intake (R; 60% of net energy for lactation requirements) with abomasal infusion of water or R plus abomasal infusion of 6.25, 12.5, or 25 g/d choline ion. Liver tissue was collected at the end of each treatment period. Enhancing choline supply increased milk yields, but decreased liver triacylglycerol. Activity of betaine homocysteine methyltransferase increased with choline, while methionine synthase tended to increase, and cystathionine β-synthase was decreased. These changes were associated with increased liver and plasma Met. Overall, enhanced supply of choline during NNB increases flux through the Met cycle to regenerate Met and reduce liver triacylglycerol. The second experiment examined the effects of rumen-protected Met (RPM) during HS on mTOR (mechanistic target of rapamycin)-related signaling proteins in the mammary gland. Thirty-two multiparous cows (184±59 DIM) were assigned to an environmental treatment, and a dietary treatment [TMR with RPM (0.105% DM) or without (CON)] in a crossover design. There were 2 periods with 2 phases per period. In phase 1 (9d), all cows were in thermoneutral conditions (TN) and fed ad libitum. During phase 2 (9d), group 1 (n=16) was exposed to HS using electric heat blankets while group 2 (n=16) remained in TN but were pair-fed to HS counterparts. After a washout period (21d), the study was repeated (period 2), with environmental treatments being inverted and dietary treatments remaining the same. Mammary tissue was collected at the end of phase 2. Abundance of phosphorylated mTOR was greater with RPM and tended to be greater with HS. Control cows had a greater decrease in milk protein (%) during phase 2 (difference from phase 1) compared with RPM cows, suggesting that RPM supplementation during HS may support greater milk protein synthesis via mTOR activation. The third experiment investigated the effects of RPM during HS on the response of mammary gland explants to lipopolysaccharide (LPS). Twenty-five mg of tissue obtained from cows in the second experiment was incubated with 0 or 3 μg/mL of LPS for 2h. Incubation with LPS increased abundance of genes associated with inflammation, while HS decreased genes associated with antioxidant responses. Expression of NFKB1was greater in LPS-treated explants from non-HS compared with HS cows. These data indicate that HS reduced immune and antioxidant responses while RPM did not attenuate the inflammatory response induced by LPS in vitro. Overall, data indicated a beneficial effect of choline during NNB and Met during HS on immunometabolism in dairy cows.

Effect of guanidinoacetic acid and betaine supplementation in soybean meal-based diets on growth performance, muscle energy metabolism and methionine utilization in the bullfrog Lithobates catesbeianus

This study was carried out to evaluate the impacts and interactions of guanidinoacetic acid (GAA) and betaine (BT) supplementation of soybean-meal-based diets on the growth, meat quality, and methionine utilization in the bullfrog Lithobates catesbeianus. A basal diet was supplemented with 0 or 4 g/kg of BT and/or 0, 0.4, or 4 g/kg GAA to formulate six experimental diets, designated Control (BT/GAA: 0/0), B0G0.4 (0/0.4), B0G4 (0/4), B4G0 (4/0), B4G0.4 (4/0.4) and B4G4 (4/4). Each diet was fed to three individual bullfrogs (initially weighing 51.70 ± 0.33 g) for 8 weeks. As a result, bullfrogs fed diets with GAA showed greater weight gain than the control group (P < 0.05). The addition of GAA or BT to the diets increased whole-body protein deposition and the hind leg index (P < 0.05). Diets supplemented with GAA led to reduced muscle glycolysis, which increased the muscle pH value 24 h after euthanasia (P < 0.05). However, a combination of BT and GAA had an insignificant effect on the expression of betaine homocysteine methyltransferase mRNA compared with the diets supplemented with GAA-only (P > 0.05). The findings of this study suggest that dietary GAA supplementation improves bullfrog growth performance and muscle energy metabolism after slaughter, but GAA combined BT supplementation does not improve methionine utilization for bullfrog.

Peripheral blood mononuclear cells are hypomethylated in active rheumatoid arthritis and methylation correlates with disease activity.

Epigenetic modifications are dynamic and influence cellular disease activity. The aim of this study was to investigate global DNA methylation in peripheral blood mononuclear cells (PBMCs) of RA patients to clarify whether global DNA methylation pattern testing might be useful in monitoring disease activity as well as the response to therapeutics. Flow cytometric measurement of 5-methyl-cytosine (5'-mC) was established using the cell line U937. In the subsequent prospective study, 62 blood samples were investigated, including 17 healthy donors and 45 RA patients at baseline and after 3 months of treatment with methotrexate, the IL-6 receptor inhibitor sarilumab, and Janus kinase inhibitors. Methylation status was assessed with an anti-5'-mC antibody and analysed in PBMCs and CD4+, CD8+, CD14+ and CD19+ subsets. Signal intensities of 5'-mC were correlated with 28-joint DASs with ESR and CRP (DAS28-ESR and DAS28-CRP). Compared with healthy individuals, PBMCs of RA patients showed a significant global DNA hypomethylation. Signal intensities of 5'-mC correlated with transcription levels of DNMT1, DNMT3B and MTR genes involved in methylation processes. Using flow cytometry, significant good correlations and linear regression values were achieved in RA patients between global methylation levels and DAS28-ESR values for PBMCs (r = -0.55, P = 0.002), lymphocytes (r = -0.57, P = 0.001), CD4+ (r = -0.57, P = 0.001), CD8+ (r = -0.54, P = 0.001), CD14+ (r = -0.49, P = 0.008) and CD19+ (r = -0.52, P = 0.004) cells. The degree of global DNA methylation was found to be associated with disease activity. Based on this novel approach, the degree of global methylation is a promising biomarker for therapy monitoring and the prediction of therapy outcome in inflammatory diseases.

Variants c.677 C>T, c.1298 A>C in MTHFR, and c.66 A>G in MTRR Affect the Occurrence of Recurrent Pregnancy Loss in Chinese Women.

Objective: Although genetic variants of key enzymes in the folic acid-methionine metabolic circulation, including methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) were thought to be related to the risk of recurrent pregnancy loss (RPL), the results of recent studies have been inconsistent. Therefore, the present retrospective case–control study was designed to explore whether the variants c.66A>G in MTRR and c.677C>T and c.1298A>C in MTHFR are associated with the susceptibility of RPL in Southeast Chinese women.Materials and Methods: In total, samples from 237 RPL patients and 618 healthy controls were collected and genotyped by fluorescent quantitative polymerase chain reaction. The frequencies of the variants were calculated and compared between the two groups. The relative risk of the various genotypes was further determined by calculating the odds ratio (OR) at a 95% confidence interval (CI).Results: A significant positive correlation was observed between the variants MTHFR c.677C>T, MTHFR c.1298A>C, MTRR c.66A>G, and RPL susceptibility (MTHFR c.677C>T, OR = 0.74, 95% CI = 0.58–0.95, p = 0.02; MTHFR c.1298A>C, OR = 1.39, 95% CI = 1.09–1.77, p = 0.008; MTRR c.66A>G, OR = 1.38, 95% CI = 1.10–1.73, p = 0.006). Further analysis of the genotypic distributions of the three variants between the two groups showed that the MTHFR c.677C>T heterozygote was associated with lower RPL risk, while the MTHFR c.1298A>C variant and MTRR c.66A>G heterozygote were correlated with higher RPL risk (dominant model, MTHFR c.677C>T, OR = 0.70, 95% CI = 0.52–0.95, p = 0.02; MTHFR c.1298A>C, OR = 1.39, 95% CI = 1.03–1.88, p = 0.032; MTRR c.66A>G, OR = 1.62, 95% CI = 1.20–2.19, p = 0.002).Conclusion: MTHFR c.677C>T and c.1298A>C and MTRR c.66A>G were associated with RPL in Southeast Chinese women.

Association study of polymorphisms at A66G (rs1801394) of MTRR gene and autism spectrum disorders in a Kurdish population

Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders that are distinguished by the inability in social interaction, communication, and repetitive behavior pattern. One of the etiologic factors of the disease is believed to be methionine synthase reductase (MTRR) A66G polymorphism, which participates in homocysteine (Hcy)/folate metabolism. The aim of this study was to explore the link between polymorphism A66G (rs1801394) of MTRR and susceptibility of Kurdish autistic children to develop ASD in Iraq. In this study, 200 samples were included and divided into two equal groups: 100 autistic and 100 control children. After extraction of genomic DNA, the allele-specific polymerase chain reaction (AS-PCR) was performed. Our results showed that a significant association between heterozygote and homozygote variants (AG vs. AA: OR = 3.333, 95%CI: 1.723 to 6.449; P = 0.0004) and (GG vs. AA: OR = 2.500, 95%CI: 1.362 to 18.35; P = 0.021) respectively, when compared with wild homozygote. Also, this study demonstrated a statistical difference in the frequencies of the two alleles in MTRR A66G (G vs. A: OR = 1.857, 95%CI: 1.243 to 2.775; P = 0.003). Our study revealed that the polymorphism of MTRR A66G genotypes and alleles could influence the ASD susceptibility among Kurdish children in Erbil, Iraq.

Biological function of cobalamin: causes and effects of hypocobalaminemia at the molecular, cellular, tissue and organism level

Cobalamin (vitamin B12) is a complex compound, which is classified as a water-soluble vitamin. Absorption of cobalamin in the gut and its transport to cells is a unique process, in which many proteins are involved. The loss of function of these proteins causes serious cell homeostasis disturbance, which may result in the dysfunction of many tissues and organs. Vitamin B12, a cofactor of methionine synthase, provides methylation process and nucleic acid synthesis. Cobalamin is also necessary for methylmalonyl-CoA mutase activity. The enzyme synthesizes succinyl-CoA, an intermediate in tricarboxylic acid cycle. Vitamin B12 deficiency is an important and current health problem. It may be caused by insufficient dietary intake, age, or disease-related malabsorption and genetic defects of mechanisms involved in the absorption, transport and metabolism of cobalamin. Hypocobalaminemia can also result from long-term pharmacotherapy with medicines: metformin, proton pump inhibitors (e.g. omeprazole) and H2-receptor antagonists (e.g. ranitidine). Significant clinical symptoms of cobalamin deficiency include hematological abnormalities, mainly megaloblastic anemia, as well as neurological disorders resulting from degeneration within the nervous system. Early diagnosis and starting treatment with vitamin B12 increase chances for a complete cure. Therefore, the diagnostically important symptom of hypocobalaminemia may be skin manifestations, mainly hyperpigmentations, but also premature graying of hair. The aim of this review article was to summarize the current state of knowledge on the biological function of cobalamin, as well as the causes and consequences of its deficiency at the molecular, cellular, tissue and organism level.

Optimization of protein extraction for proteomic analyses of fresh and frozen “Musang King” durian pulps

Four different methods were evaluated to extract proteins from “Musang King” durian pulps and subsequently proteins with different abundance between fresh and long term frozen storage were identified using two-dimensional polyacrylamide gel electrophoresis coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analyses. The acetone-phenol method was found to produce good protein yields and gave the highest gel resolution and reproducibility. Differential protein analyses of the durian pulp revealed that 15 proteins were down-regulated and three other proteins were up-regulated after a year of frozen storage. Isoflavone reductase-like protein, S-adenosyl methionine synthase, and cysteine synthase isoform were up-regulated during frozen storage. The down-regulation of proteins in frozen durian pulps indicated that frozen storage has affected proteins in many ways, especially in their functions related to carbohydrate and energy metabolisms, cellular components, and transport processes. This study will enable future detailed investigations of proteins associated with quality attributes of durians to be studied.

Cobamide remodeling in the freshwater microalga Chlamydomonas reinhardtii.

Microalgae are not able to produce cobamides (Cbas, B12 vitamers) de novo. Hence, the production of catalytically active Cba-containing methionine synthase (MetH), which is present in selected representatives, is dependent on the availability of exogenous B12 vitamers. Preferences in the utilization of exogenous Cbas equipped with either adenine or 5,6-dimethylbenzimidazole as lower base have been reported for some microalgae. Here, we investigated the utilization of norcobamides (NorCbas) for growth by the Cba-dependent Chlamydomonas reinhardtii mutant strain (ΔmetE). The growth yields in the presence of NorCbas were lower in comparison to those achieved with Cbas. NorCbas lack a methyl group in the linker moiety of the nucleotide loop. C. reinhardtii was also tested for the remodeling of NorCbas (e.g. adeninyl-norcobamide) in the presence of different benzimidazoles. Extraction of the NorCbas from C. reinhardtii, their purification, and identification confirmed the exchange of the lower base of the vitamers. However, the linker moiety of the NorCbas nucleotide loop was not exchanged. This observation strongly indicates the presence of an alternative mode of Cba deconstruction in C. reinhardtii that differs from the amidohydrolase (CbiZ)-dependent pathway described in Cba-remodeling bacteria and archaea.

Targeting Methionine Synthase in a Fungal Pathogen Causes a Metabolic Imbalance That Impacts Cell Energetics, Growth, and Virulence.

There is an urgent need to develop novel antifungals to tackle the threat fungal pathogens pose to human health. Here, we have performed a comprehensive characterization and validation of the promising target methionine synthase (MetH). We show that in Aspergillus fumigatus the absence of this enzymatic activity triggers a metabolic imbalance that causes a reduction in intracellular ATP, which prevents fungal growth even in the presence of methionine. Interestingly, growth can be recovered in the presence of certain metabolites, which shows that metH is a conditionally essential gene and consequently should be targeted in established infections for a more comprehensive validation. Accordingly, we have validated the use of the tetOFF genetic model in fungal research and improved its performance in vivo to achieve initial validation of targets in models of established infection. We show that repression of metH in growing hyphae halts growth in vitro, which translates into a beneficial effect when targeting established infections using this model in vivo Finally, a structure-based virtual screening of methionine synthases reveals key differences between the human and fungal structures and unravels features in the fungal enzyme that can guide the design of novel specific inhibitors. Therefore, methionine synthase is a valuable target for the development of new antifungals.IMPORTANCE Fungal pathogens are responsible for millions of life-threatening infections on an annual basis worldwide. The current repertoire of antifungal drugs is very limited and, worryingly, resistance has emerged and already become a serious threat to our capacity to treat fungal diseases. The first step to develop new drugs is often to identify molecular targets in the pathogen whose inhibition during infection can prevent its growth. However, the current models are not suitable to validate targets in established infections. Here, we have characterized the promising antifungal target methionine synthase in great detail, using the prominent fungal pathogen Aspergillus fumigatus as a model. We have uncovered the underlying reason for its essentiality and confirmed its druggability. Furthermore, we have optimized the use of a genetic system to show a beneficial effect of targeting methionine synthase in established infections. Therefore, we believe that antifungal drugs to target methionine synthase should be pursued and additionally, we provide a model that permits gaining information about the validity of antifungal targets in established infections.

When to measure plasma homocysteine and how to place it in context: The homocystinurias.

This review presents clinical patterns that should trigger homocysteine measurement in blood, as well as the further diagnostic work-up focused on inborn errors of metabolism and disorders of vitamin B12 (cobalamin) absorption and supply. The numerous conditions (e.g. cardiovascular disease, Alzheimer’s disease) for which mild-to-moderate hyperhomocysteinaemia caused by genetic polymorphisms or acquired reasons is considered a risk factor are beyond the scope of this review.Homocysteine is a sulphur-containing amino acid, which is derived from the amino acid methionine. Homocysteine is either trans-sulphurated to form cystathionine and then cysteine, or re-methylated to methionine. The trans-sulphuration reaction depends on the enzyme cystathionine beta synthase and its cofactor vitamin B6. The re-methylation reaction not only involves the enzymes methionine synthase and methionine synthase reductase but also depends on the cofactor cobalamin and on the provision of methyl groups from the folate cycle. Because the homocysteine–methionine cycle provides for the vast majority of methyl groups in the body, it is central to numerous pathways that depend on methyl group supply, such as creatine synthesis or DNA methylation. Based on this premise, the severity of clinical presentations of inborn errors of metabolism, such as classical homocystinuria or the cobalamin C (cblC) defect, affecting this pathway is unsurprising.

Alterations in Sulfur Amino Acids as Biomarkers of Disease

Homocysteine (Hcy) is methylated by methionine synthase to form methionine with methyl-cobalamin as a cofactor. The reaction demethylates 5-methyltetrahydrofolate to tetrahydrofolate, which is required for DNA and RNA synthesis. Deficiency of either of the cobalamin (Cbl) and/or folate cofactors results in elevated Hcy and megaloblastic anemia. Elevated Hcy is a sensitive biomarker of Cbl and/or folate status and more specific than serum vitamin assays. Elevated Hcy normalizes when the correct vitamin is given. Elevated Hcy is associated with alcohol use disorder and drugs that target folate or Cbl metabolism, and is a risk factor for thrombotic vascular disease. Elevated methionine and cystathionine are associated with liver disease. Elevated Hcy, cystathionine, and cysteine, but not methionine, are common in patients with chronic renal failure. Higher cysteine predicts obesity and future weight gain. Serum S-adenosylhomocysteine (AdoHcy) is elevated in Cbl deficiency and chronic renal failure. Drugs that require methylation for catabolism may deplete liver S-adenosylmethionine and raise AdoHcy and Hcy. Deficiency of Cbl or folate or perturbations of their metabolism cause major changes in sulfur amino acids.

Effects of DL-methionine and a methionine hydroxy analogue (MHA-Ca) on growth, amino acid profiles and the expression of genes related to taurine and protein synthesis in common carp (Cyprinus carpio)

In this trial, nine diets were formulated to investigate the effects of DL-methionine (DL-Met) and a methionine hydroxy analogue (MHA-Ca) on growth, amino acid profiles and the expression of genes related to taurine and protein synthesis in common carp. The basal diet was prepared without methionine supplementation and the other diets were formulated with 0.1%, 0.2%, 0.3% and 0.4% of DL-Met or MHA-Ca, respectively. After the eight-week feeding trial, fish fed with DL-Met or MHA-Ca supplemented diets had higher growth performance and feed utilization when compared with basal diet and two-way ANOVA showed the growth performance and feed utilization were significantly increased in fish fed DL-Met supplemented diets (P < 0.05). The concentrations of free amino acids in serum (Met, Leu, His, Lys, Val, Arg, Ile, Phe and Tau) and muscle (Tau) significantly increased as dietary DL-Met and MHA-Ca increased. Moreover, two-way ANOVA showed that taurine was higher in common carp fed with DL-Met diets compared with MHA-Ca diets. To find out how DL-Met and MHA-Ca affect taurine synthesis, genes involved in methionine degradation and taurine synthesis were further investigated. DL-Met or MHA-Ca supplementation decreased the expression of methionine adenosyltransferase II, alpha (MAT2A) and methionine synthase (MS) and increased the expression of the cysteine dioxygenase (CDO). Moreover, DL-Met supplementation significantly increased the expression of CDO and cysteine sulfinate acid decarboxylase (CSD) when compared with MHA-Ca supplementation. As for protein synthesis, the mRNA expression of mechanistic target of rapamycin (mTOR) in liver was not significantly different among all diet groups. But the expression of eukaryotic translation initiation factor 4E-Binding protein 1 (4EBP1), general control nonderepressible2 (GCN2) and asparagine synthetase (ASNS) in liver were significantly down-regulated as dietary DL-Met or MHA-Ca increased. Compared with MHA-Ca diets, the expression of S6 (ribosomal protein s6) and eIF4E (eukaryotic translation initiation factor 4E binding protein 2) were markedly elevated in DL-Met groups by the analysis of two-way ANOVA. This study indicated that dietary DL-Met or MHA-Ca supplement could increase free essential amino acids in serum, as well as improve taurine synthesis and protein synthesis in common carp. Multi-exponential regression analysis showed that MHA-Ca was less utilized by common carp than DL-Met with bioavailability values of 41% to 50% on weight-for-weight basis.

High-dose folic acid supplementation results in significant accumulation of unmetabolized homocysteine, leading to severe oxidative stress in Caenorhabditis elegans

Using Caenorhabditis elegans as a model animal, we evaluated the effects of chronical supplementation with high-dose folic acid on physiological events such as life cycle and egg-laying capacity and folate metabolism. Supplementation of high-dose folic acid significantly reduced egg-laying capacity. The treated worms contained a substantial amount of unmetabolized folic acid and exhibited a significant downregulation of the mRNAs of cobalamin-dependent methionine synthase reductase and 5,10-methylenetetrahydrofolate reductase. In vitro experiments showed that folic acid significantly inhibited the activity of cobalamin-dependent methionine synthase involved in the metabolism of both folate and methionine. In turn, these metabolic disorders induced the accumulation of unmetabolized homocysteine, leading to severe oxidative stress in worms. These results were similar to the phenomena observed in mammals during folate deficiency.

Structural insight into the effect of polymorphic variation on the functional dynamics of methionine synthase reductase: Implications in neural tube defects

Neural tube defects (NTDs), one of the most common birth defects, are strongly associated with the variations of several single nucleotide polymorphisms (SNPs) in the MTRR gene. The gene codes a key enzyme that is involved in the rejuvenation of methionine synthase activity. An allelic variant of the protein leads to missense mutation at 49th position from isoleucine to methionine (I49M) is associated with higher disease prevalence in different populations. Here, extensive molecular dynamics simulations and interaction network analysis reveal that the 49th isoleucine is a crucial residue that allosterically regulates the dynamics between the flavin mononucleotide (FMN) and NADP(H) binding domains. I49M variation alters the functional dynamics in a way that might impede the electron transport chain along the NADP(H)→flavin adenine dinucleotide→FMN pathway. The present study provides functional insights into the effect of the genetic variations of the MTRR gene on the NTDs disease pathogenesis.

Update analysis on the association between Methionine synthase rs1805087 A/G variant and risk of prostate cancer

Previous studies have investigated the association of the rs1805087 A/G variant of Methionine synthase gene with the susceptibility to prostate cancer (PCa). Nevertheless, the conclusions remain divergent. We performed a systemic analysis with odds ratios (ORs) and 95% confidence intervals (95% CIs) to assess Methionine synthase rs1805087 A/G variant and PCa risk. Furthermore, we utilized in silico analysis to investigate the relationship between Methionine synthase expression and the overall survival (OS) time. Totally, 10,666 PCa patients and 40,750 controls were included. We observed that Methionine synthase rs1805087 A/G variant is associated with an elevated risk of PCa (G-allele vs. A-allele: OR = 1.06, 95% CI = 1.01–1.11, P = 0.013; heterozygous model: OR = 1.08, 95% CI = 1.02–1.14, P = 0.009; dominant model: OR = 1.08, 95% CI = 1.02–1.14, P = 0.007). During stratified analysis, similar results were obtained in Asian populations, hospital-based, high quality studies and that with large sample size. Moreover, in silico analysis indicated the Methionine synthase expression is down-regulated in both young and old PCa subjects (P < 0.05). Compared with the normal subjects, the down-regulated expression of Methionine synthase was found in PCa cases with Gleason score 6 to 9. Our study showed that Methionine synthase rs1805087 A/G variant may be associated with susceptibility of PCa, especially in Asian populations, hospital-based studies and that with high quality and large sample size. Furthermore, Methionine synthase rs1805087 A/G variant may be related to the prognosis of PCa.

Genetic predictors of vascular complications in adolescents involved in sports

Introduction. The urgency of the problem is because the carriage of genetic predictors of thrombogenic risk in adolescents can be a significant factor in the formation of arterial and venous thromboses, leading to disability and even death, especially after significant sports overloads. Materials and methods. DNA genotyping was carried out in 259 boys aged 14 to 17 years who were professionally involved in sports for a long time. The reference group consisted of 228 students who were not additionally involved in sports sections at the age of 14-17 years of I-II health groups. Genetic testing was performed using the Real-Time PCR method using competing TagMan probes. 12 genetic allelic DNA polymorphisms were studied. The analysis of laboratory and instrumental methods of research. The results were statistically processed using the STATISTICA 10.0 software package. Results. Among adolescent athletes, a group of children with a high risk of vascular complications was identified on the basis of the studied thrombogenic DNA polymorphisms and folate metabolism genes. The frequency of the minor C1565 allele of the GPIIIa gene was significantly more often determined in adolescent athletes, while the variant of the minor 4G allele (-675) of the PAI-I gene was significantly more often detected in relatively healthy schoolchildren. The heterozygous genotype 1565 TC of the GPIIIa gene, the heterozygous genotype 1298 of the AC MTHFR gene, and the heterozygous genotype 66 AG of the methionine synthase reductase gene (MTRR) were found to be more pronounced in adolescent athletes. The frequency of associations of 4 (17.4%), 5 (28.6%), 6 (25.8%), 7 (10.8%) and 8 (5.4%) genetic polymorphisms was significantly increased in adolescent athletes, which may have pathogenetic significance. Moreover, in athletes, combinations of C677T polymorphisms of the MTHFR gene, 4G (-675) 5G of the PAI-I gene, G (-455) A of the FGB gene were unfavorable for the implementation of vascular lesions. Conclusion. For the first time, criteria have been formulated for the selection of teenage athletes in the high-risk group for vascular complications, taking into account genetic factors. It has been established that the most informative predictors of the risk of vascular complications are the variants of the GPIIIa T1565C, MTRR A66G genes, and gene gene associations of polymorphisms.