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Enzymes of the methionine and homocysteine metabolism catalyze reactions belonging to the methionine and folate cycles and the transsulfuration pathway. The importance of the metabolites produced through these routes (e.g. S-adenosylmethionine, homocysteine) and their role in e.g. epigenetics or redox mechanisms makes their tight regulation essential for a correct cellular function. Pharmacological or pathophysiological insults induce, among others, changes in activity, oligomerization, protein levels, subcellular localization and expression of these enzymes. The abundance of these proteins in liver has made this organ the preferred system to study their regulation. Nevertheless, knowledge about their putative protein-protein interactions is limited in this and other tissues and cell types. High-throughput methods, including immunoprecipitation, affinity purification coupled to mass spectrometry and yeast two-hybrid have rendered the identification of a number of protein-protein interactions involving these enzymes in several systems. Validation by coimmunoprecipitation and/or pull-down has been made, mainly, after coexpression of bait and prey, but few of the interactions have been confirmed. Additionally, information concerning the role of these interactions in the regulation of this pathway and other cellular processes is scarce. Here, we review the current knowledge on mammalian protein-protein interactions involving methionine adenosyltransferases, S-adenosylhomocysteine hydrolase, betaine homocysteine S-methyltransferases, methionine synthase and cystathionine β-synthase, although references to data obtained in other organisms are also made. Moreover, the verified or putative implication of these interactions in the regulation of methionine and homocysteine metabolism, its interplay with other metabolic pathways and its putative link to pathophysiological processes, such as oncogenesis, is discussed., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Betaine-homocysteine S -methyltransferases (BHMTs) are methionine cycle enzymes that remethylate homocysteine; hence, their malfunction leads to hyperhomocysteinemia. Epidemiologic and experimental studies have revealed a correlation between hyperhomocysteinemia and hearing loss. Here, we have studied the expression of methionine cycle genes in the mouse cochlea and the impact of knocking out the Bhmt gene in the auditory receptor. We evaluated age-related changes in mouse hearing by recording auditory brainstem responses before and following exposure to noise. Also, we measured cochlear cytoarchitecture, gene expression by RNA-arrays and quantitative RT-PCR, and metabolite levels in liver and plasma by HPLC. Our results indicate that there is an age-dependent strain-specific expression of methionine cycle genes in the mouse cochlea and a further regulation during the response to noise damage. Loss of Bhmt did not cause an evident impact in the hearing acuity of young mice, but it produced higher threshold shifts and poorer recovery following noise challenge. Hearing loss was associated with increased cochlear injury, outer hair cell loss, altered expression of cochlear methionine cycle genes, and hyperhomocysteinemia. Our results suggest that BHMT plays a central role in the homeostasis of cochlear methionine metabolism and that Bhmt2 up-regulation could carry out a compensatory role in cochlear protection against noise injury in the absence of BHMT.-Partearroyo, T., Murillo-Cuesta, S., Vallecillo, N., Bermúdez-Muñoz, J. M., Rodríguez-de la Rosa, L., Mandruzzato, G., Celaya, A. M., Zeisel, S. H., Pajares, M. A., Varela-Moreiras, G., Varela-Nieto, I. Betaine-homocysteine S -methyltransferase deficiency causes increased susceptibility to noise-induced hearing loss associated with plasma hyperhomocysteinemia.

Nutritional imbalance is emerging as a causative factor of hearing loss. Epidemiologic studies have linked hearing loss to elevated plasma total homocysteine (tHcy) and folate deficiency, and have shown that folate supplementation lowers tHcy levels potentially ameliorating age-related hearing loss. The purpose of this study was to address the impact of folate deficiency on hearing loss and to examine the underlying mechanisms. For this purpose, 2-mo-old C57BL/6J mice (Animalia Chordata Mus musculus) were randomly divided into 2 groups (n = 65 each) that were fed folate-deficient (FD) or standard diets for 8 wk. HPLC analysis demonstrated a 7-fold decline in serum folate and a 3-fold increase in tHcy levels. FD mice exhibited severe hearing loss measured by auditory brainstem recordings and TUNEL-positive-apoptotic cochlear cells. RT-quantitative PCR and Western blotting showed reduced levels of enzymes catalyzing homocysteine (Hcy) production and recycling, together with a 30% increase in protein homocysteinylation. Redox stress was demonstrated by decreased expression of catalase, glutathione peroxidase 4, and glutathione synthetase genes, increased levels of manganese superoxide dismutase, and NADPH oxidase-complex adaptor cytochrome b-245, α-polypeptide (p22phox) proteins, and elevated concentrations of glutathione species. Altogether, our findings demonstrate, for the first time, that the relationship between hyperhomocysteinemia induced by folate deficiency and premature hearing loss involves impairment of cochlear Hcy metabolism and associated oxidative stress., (© FASEB.)

Betaine-homocysteine S-methyltransferase (BHMT) is one of the most abundant proteins in the liver and regulates homocysteine metabolism. However, the molecular mechanisms underlying Bhmt transcription have not yet been elucidated. This study aimed to assess the molecular mechanisms underlying Bhmt transcription and the effect of BHMT deficiency on metabolic functions in the liver mediated by liver receptor homolog-1 (LRH-1). During fasting, both Bhmt and Lrh-1 expression increased in the liver of Lrh-1f/f mice; however, Bhmt expression was decreased in LRH-1 liver specific knockout mice. Promoter activity analysis confirmed that LRH-1 binds to a specific site in the Bhmt promoter region. LRH-1 deficiency was associated with elevated production of reactive oxygen species (ROS), lipid peroxidation, and mitochondrial stress in hepatocytes, contributing to hepatic triglyceride (TG) accumulation. In conclusion, this study suggests that the absence of an LRH-1-mediated decrease in Bhmt expression promotes TG accumulation by increasing ROS levels and inducing mitochondrial stress. Therefore, LRH-1 deficiency not only leads to excess ROS production and mitochondrial stress in hepatocytes, but also disrupts the methionine cycle. Understanding these regulatory pathways may pave the way for novel therapeutic interventions against metabolic disorders associated with hepatic lipid accumulation. [ABSTRACT FROM AUTHOR]

Cystathionine beta-synthase deficient homocystinuria (HCU) is a life-threatening disorder of sulfur metabolism. Our knowledge of the metabolic changes induced in HCU are based almost exclusively on data derived from plasma. In the present study, we present a comprehensive analysis on the effects of HCU upon the hepatic metabolites and enzyme expression levels of the methionine-folate cycles in a mouse model of HCU. HCU induced a 10-fold increase in hepatic total homocysteine and in contrast to plasma, this metabolite was only lowered by approximately 20% by betaine treatment indicating that this toxic metabolite remains unacceptably elevated. Hepatic methionine, S -adenosylmethionine, S -adenosylhomocysteine, N -acetlymethionine, N -formylmethionine, methionine sulfoxide, S -methylcysteine, serine, N -acetylserine, taurocyamine and N -acetyltaurine levels were also significantly increased by HCU while cysteine, N -acetylcysteine and hypotaurine were all significantly decreased. In terms of polyamine metabolism, HCU significantly decreased spermine and spermidine levels while increasing 5′-methylthioadenosine. Betaine treatment restored normal spermine and spermidine levels but further increased 5′-methylthioadenosine. HCU induced a 2-fold induction in expression of both S -adenosylhomocysteine hydrolase and methylenetetrahydrofolate reductase. Induction of this latter enzyme was accompanied by a 10-fold accumulation of its product, 5-methyl-tetrahydrofolate, with the potential to significantly perturb one‑carbon metabolism. Expression of the cytoplasmic isoform of serine hydroxymethyltransferase was unaffected by HCU but the mitochondrial isoform was repressed indicating differential regulation of one‑carbon metabolism in different sub-cellular compartments. All HCU-induced changes in enzyme expression were completely reversed by either betaine or taurine treatment. Collectively, our data show significant alterations of polyamine, folate and methionine cycle metabolism in HCU hepatic tissues that in some cases, differ significantly from those observed in plasma, and have the potential to contribute to multiple aspects of pathogenesis. [ABSTRACT FROM AUTHOR]

Potyviral helper component proteinase (HCPro) is a well-characterized suppressor of antiviral RNA silencing, but its mechanism of action is not yet fully understood. In this study, we used affinity purification coupled with mass spectrometry to identify binding partners of HCPro in potyvirus-infected plant cells. This approach led to identification of various HCPro interactors, including two key enzymes of the methionine cycle, S-adenosyl-L-methionine synthase and S-adenosyl-L-homocysteine hydrolase. This finding, together with the results of enzymatic activity and gene knockdown experiments, suggests a mechanism in which HCPro complexes containing viral and host proteins act to suppress antiviral RNA silencing through local disruption of the methionine cycle. Another group of HCPro interactors identified in this study comprised ribosomal proteins. Immunoaffinity purification of ribosomes demonstrated that HCPro is associated with ribosomes in virus-infected cells. Furthermore, we show that HCPro and ARGONAUTE1 (AGO1), the core component of the RNA-induced silencing complex (RISC), interact with each other and are both associated with ribosomes in planta. These results, together with the fact that AGO1 association with ribosomes is a hallmark of RISC-mediated translational repression, suggest a second mechanism of HCPro action, whereby ribosome-associated multiprotein complexes containing HCPro relieve viral RNA translational repression through interaction with AGO1. [ABSTRACT FROM AUTHOR]

Homocysteine, a sulfur-containing amino acid derived from the methionine metabolism, is located at the branch point of two pathways of the methionine cycle, i.e. remethylation and transsulfuration. Gene abnormalities in the enzymes catalyzing reactions in both pathways lead to hyperhomocysteinemia. Hyperhomocysteinemia is associated with increased risk for congenital disorders, including neural tube closure defects, heart defects, cleft lip/palate, Down syndrome, and multi-system abnormalities in adults. Since hyperhomocysteinemia is known to affect the extent of DNA methylation, it is likely that abnormal DNA methylation during embryogenesis, may be a pathogenic factor for these congenital disorders. In this review we highlight the importance of homocysteinemia by describing the genes encoding for enzymes of homocysteine metabolism relevant to the clinical practice, especially cystathionine-β-synthase and methylenetetrahydrofolate reductase mutations , and the impairment of related metabolites levels. Moreover, a possible correlation between hyperhomocysteine and congenital disorders through the involvement of abnormal DNA methylation during embryogenesis is discussed. Finally, the relevance of present and future diagnostic tools such as tandem mass spectrometry and next generation sequencing in newborn screening is highlighted. [ABSTRACT FROM AUTHOR]

It is well established that homocysteine (Hcy) is an independent risk factor for atherosclerosis (AS), which is characterized by vascular smooth muscle cell (VSMC) proliferation. However, the molecular mechanism underlying AS in VSMCs is yet to be elucidated. The aim of this study was to investigate the potential involvement of aberrant DNA methylation of the platelet-derived growth factor (PDGF) gene in Hcy-mediated VSMC proliferation and its underlying mechanism. Cultured human VSMCs were treated with varying concentrations of Hcy. VSMC proliferation, PDGF mRNA and protein expression and PDGF promoter demethylation showed a dose-dependent increase with Hcy concentration, suggesting an association among them. Cell cycle analysis revealed a decreased proportion of VSMCs in G0/G1 and an increased proportion in S phase, indicating that VSMC proliferation was increased under Hcy treatment. Furthermore, S-adenosylhomocysteine (SAH) levels were observed to increase and those of S-adenosylmethionine (SAM) were observed to decrease. The consequent decrease in the ratio of SAM/SAH may partially explain the hypomethylation of PDGF with Hcy treatment. Folate treatment exhibited an antagonistic effect against Hcy-induced VSMC proliferation, aberrant PDGF methylation and PDGF expression. These data suggest that Hcy may stimulate VSMC proliferation through the PDGF signaling pathway by affecting the epigenetic regulation of PDGF through the demethylation of its promoter region. These findings may provide novel insight into the molecular association between aberrant PDGF gene demethylation and the proliferation of VSMCs in Hcy-associated AS. [ABSTRACT FROM AUTHOR]

Background/Aims: Folic acid (FA) deficiency/supplementation effects seem to be dependent on age group and/or physiological status. The aim was to evaluate changes associated with rapid growth in relation to methionine metabolism in rats. Methods: Four groups (n = 10 each) of male Sprague Dawley rats (5 weeks old) were on diets that varied in their FA content: 0 mg FA/kg diet (deficient), 2 mg FA/kg diet (control), 8 mg FA/kg diet (moderate supplementation), 40 mg FA/kg diet (supranormal supplementation). Animals were fed ad libitum for 30 days. Biomarkers of methionine metabolism and antioxidant status were evaluated. Results: Serum total homocysteine concentration increased (p < 0.01) in FA deficient animals, with no differences between the supplemented groups. The hepatic ‘methylation ratio’ (S-adenosylmethionine/S-adenosylhomocysteine) of the FA content groups reached similar values, which were significantly higher compared to the deficient group. The brain ‘methylation ratio’, however, remained unmodified independently of FA content in the diet. FA deficiency induced hepatic DNA hypomethylation, and supranormal FA supplementation exerted the most protective effect (p < 0.01). Serum folate levels increased according to FA dietary level, whereas no differences were seen for vitamin B12 and vitamin B6. Conclusions: FA deficiency compromises methionine metabolism whereas supplementation does not show an additional positive effect compared to the control diet in growing animals. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

There are nationwide folic acid (FA) fortification programmes of staple foods established or under consideration in order to prevent neural tube defects. Universal FA fortification still remains controversial because of the concern that additional FA in the diets of population groups (e.g. children and elderly) not initially targeted for fortification may suffer adverse effects. However, dietary surveys regarding folate generally deal with adults and little is known about the consumption and long-term effects of fortified food and supplements in growing individuals. Recent reports from our laboratory show several effects of high-dose folate supplementation in rats. In the present work, we studied the effect of FA on the methionine cycle in weanling (3-week-old) male rats after 4 weeks of supplementation with 40??mg FA/kg diet v. control (1??mg FA/kg diet). FA supplementation resulted in a reduction of homocysteine and creatinine concentrations v. control group. FA supplementation did not alter S-adenosylmethionine/S-adenosylhomocysteine ratio, DNA methylation, enzymatic activities or concentrations of vitamins involved in the nutritional regulation of the methionine cycle, except for folate. FA supplementation of 40??mg/kg did not lead to hepatic or renal damage. In conclusion, there were no apparent adverse effects on one-carbon metabolism after FA supplementation in the studied conditions. [ABSTRACT FROM PUBLISHER]

Background: The effects of vitamin B-6 status on steady-state kinetics of homocysteine metabolism in humans are unclear. Objective: The objective was to determine the effects of dietary vitamin B-6 restriction on the rates of homocysteine remethylation and synthesis in healthy humans. Design: Primed, constant infusions of [13C5]methionine, [3-13C]serine, and [2H3]leucine were conducted in healthy female (n=5) and male (n=4) volunteers (20-30 y) before and after 4 wk of dietary vitamin B-6 restriction (<0.5 mg vitamin B-6/d) to establish whether vitamin B-6 status affects steady-state kinetics of homocysteine metabolism in the absence of concurrent methionine intake. Effects of dietary vitamin B-6 restriction on vitamin B-6 status, plasma amino acid concentrations, and the rates of reactions of homocysteine metabolism were assessed. Results: Dietary vitamin B-6 restriction significantly reduced plasma pyridoxal 5'-phosphate (PLP) concentrations (55.1 ± 8.3 compared with 22.6 ± 1.3 nmol/L; P = 0.004), significantly increased plasma glycine concentrations (230 ± 14 compared with 296 ± 15; P = 0.008), and significantly reduced basal (43%; P < 0.001) and PLP-stimulated (35%; P = 0.004) lymphocyte serine hydroxymethyltransferase activities in vitro. However, the in vivo fluxes of leucine, methionine, and serine; the rates of homocysteine synthesis and remethylation (total and vitamin B-6-dependent); and the concentrations of homocysteine, methionine, and serine in plasma were not significantly affected by dietary vitamin B-6 restriction. Conclusions: Moderate vitamin B-6 deficiency does not significantly alter the rates of homocysteine remethylation or synthesis in healthy young adults in the absence of dietary methionine intake. [ABSTRACT FROM AUTHOR]

Background: Since homocysteine (Hcy) is a risk factor for cardiovascular and other diseases, it is important to know how exercise can modify it. Previous studies have suggested that endurance training influences Hcy. However, little is known about the effect of training intensity on Hcy. Materials and Methods: We investigated Hcy, vitamin B[sub12] , vitamin B[sub6] , folate and methylmalonic acid (MMA) before and after 3 weeks of volume-oriented training (VOL) (30 km/week) and high-intensity interval training (HIT) (20 km/week) in 20 young swimmers (16±2 years). Afterward, the athletes completed 5 days of recovery training. Results: The training induced a Hcy increase in HIT and VOL (6.47±0.95 µmol/l vs. 7.44±1.17 µmol/l and 7.33± 1.92 µmol/l vs. 8.28±1.42 µmol/l, respectively) that persisted during the recovery period (8.02±1.69 µmol/l and 8.00±1.81 µmol/l, respectively). Vitamin B[sub12] was unchanged after the training (539±166 ng/l vs. 556±192 ng/l and 480±144 ng/l vs. 491±124 ng/l, respectively) but decreased during the recovery period (459±134 ng/l and 451±116 ng/l, respectively). Folate showed an increase during the training (9.07±2.01 µg/l vs. 11.71±4.08 µg/l and 10.34±2.32 µg/l vs. 11.13 ± 4.64 µg/l, respectively), which was reversible by the end of the recovery training (8.57±1.98 µg/l and 9.60±2.38 µg/l, respectively). Vitamin B[sub6] and MMA did not change. For none of the measured parameters were there significant differences between HIT and VOL. Conclusion: Three weeks of strenuous swimming caused a prolonged Hcy increase, which was accompanied by changes in vitamin B[sub12] and folate. The magnitude of these effects was not influenced by the training intensity. [ABSTRACT FROM AUTHOR]