Methionine Metabolism

Protein-protein interactions are an important mechanism for the regulation of enzyme function allowing metabolite channeling, crosstalk between pathways or the introduction of post-translational modifications. Therefore, a number of high-throughput studies have been carried out to shed light on the protein networks established under different pathophysiological settings. Surprisingly, this type of information is quite limited for enzymes of intermediary metabolism such as betaine homocysteine S-methyltransferase, despite its high hepatic abundancy and its role in homocysteine metabolism. Here, we have taken advantage of two approaches, affinity purification combined with mass spectrometry and yeast two-hybrid, to further uncover the array of interactions of betaine homocysteine S-methyltransferase in normal liver of Rattus norvegicus. A total of 131 non-redundant putative interaction targets were identified, out of which 20 were selected for further validation by coimmunoprecipitati...

Mitochondrial function affects many aspects of cellular physiology, and, most recently, its role in epigenetics has been reported. Mechanistically, how mitochondrial function alters DNA methylation patterns in the nucleus remains ill defined. Using a cell culture model of induced mitochondrial DNA (mtDNA) depletion, in this study we show that progressive mitochondrial dysfunction leads to an early transcriptional and metabolic program centered on the metabolism of various amino acids, including those involved in the methionine cycle. We find that this program also increases DNA methylation, which occurs primarily in the genes that are differentially expressed. Maintenance of mitochondrial nicotinamide adenine dinucleotide reduced (NADH) oxidation in the context of mtDNA loss rescues methionine salvage and polyamine synthesis and prevents changes in DNA methylation and gene expression but does not affect serine/folate metabolism or transsulfuration. This work provides a novel mechani...

Background: Oxidative aggression is a hallmark of varicocele and may depend on decreased reactive ability of the endogenous antioxidant system following heat stress. We aimed to investigate the underlying mechanisms. Therefore, the expression of the main enzyme proteins involved in the generation of endogenous antioxidant power, cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CSE), heme oxygenase (HO-1), and also, some of the metabolites (methionine, homocysteine, taurine and vitamin B6) reporting on their activity was investigated using a surgical varicocele model in rats. Materials and Methods: In this experimental study, thirty male Wistar rats (7-8 weeks old, weighing 180-220 g) were divided into three groups: control, sham, and varicocele. We evaluated sperm parameters and functional tests, as well as the expression of CBS, CSE, and HO-1 proteins using Western blot analysis. Serum levels of methionine, homocysteine, and taurine were analyzed through high-performance liquid chromatography, while vitamin B6 concentrations were measured using enzyme-linked immunosorbent assay. For group comparisons, we employed ANOVA and the Tukey test, considering a P<0.05 as significant. Results: We observed a significant reduction in both sperm quality and functional parameters. Additionally, there was a notable decrease in the expression of CBS, CSE, and HO-1 proteins, as well as circulating vitamin B6 levels, in the varicocele group compared to the control and sham groups (P<0.001). Conclusion: The results of this study suggest that one possible cause of increased oxidative stress in varicocele may be the reduced expression of testicular enzymes involved in the production of endogenous antioxidants, which are associated with the transsulfuration pathway. However, further studies are needed to confirm these findings.

Mitochondrial function affects many aspects of cellular physiology, and, most recently, its role in epigenetics has been reported. Mechanistically, how mitochondrial function alters DNA methylation patterns in the nucleus remains ill defined. Using a cell culture model of induced mitochondrial DNA (mtDNA) depletion, in this study we show that progressive mitochondrial dysfunction leads to an early transcriptional and metabolic program centered on the metabolism of various amino acids, including those involved in the methionine cycle. We find that this program also increases DNA methylation, which occurs primarily in the genes that are differentially expressed. Maintenance of mitochondrial nicotinamide adenine dinucleotide reduced (NADH) oxidation in the context of mtDNA loss rescues methionine salvage and polyamine synthesis and prevents changes in DNA methylation and gene expression but does not affect serine/folate metabolism or transsulfuration. This work provides a novel mechani...

Mitochondrial function affects many aspects of cellular physiology, and, most recently, its role in epigenetics has been reported. Mechanistically, how mitochondrial function alters DNA methylation patterns in the nucleus remains ill defined. Using a cell culture model of induced mitochondrial DNA (mtDNA) depletion, in this study we show that progressive mitochondrial dysfunction leads to an early transcriptional and metabolic program centered on the metabolism of various amino acids, including those involved in the methionine cycle. We find that this program also increases DNA methylation, which occurs primarily in the genes that are differentially expressed. Maintenance of mitochondrial nicotinamide adenine dinucleotide reduced (NADH) oxidation in the context of mtDNA loss rescues methionine salvage and polyamine synthesis and prevents changes in DNA methylation and gene expression but does not affect serine/folate metabolism or transsulfuration. This work provides a novel mechanistic link between mitochondrial function and epigenetic regulation of gene expression that involves polyamine and methionine metabolism responding to changes in the tricarboxylic acid (TCA) cycle. Given the implications of these findings, future studies across different physiological contexts and in vivo are warranted.

Mitochondrial function affects many aspects of cellular physiology, and, most recently, its role in epigenetics has been reported. Mechanistically, how mitochondrial function alters DNA methylation patterns in the nucleus remains ill defined. Using a cell culture model of induced mitochondrial DNA (mtDNA) depletion, in this study we show that progressive mitochondrial dysfunction leads to an early transcriptional and metabolic program centered on the metabolism of various amino acids, including those involved in the methionine cycle. We find that this program also increases DNA methylation, which occurs primarily in the genes that are differentially expressed. Maintenance of mitochondrial nicotinamide adenine dinucleotide reduced (NADH) oxidation in the context of mtDNA loss rescues methionine salvage and polyamine synthesis and prevents changes in DNA methylation and gene expression but does not affect serine/folate metabolism or transsulfuration. This work provides a novel mechani...

Ethanol is well known as a teratogenic factor that is capable of inducing a wide range of developmental abnormalities if the developing fetus is exposed to it. Duration and dose are the critical parameters of exposure that affect teratogenic variation to the developing fetus. It is suggested that ethanol interferes with epigenetic processes especially DNA methylation. We aimed to organize all of the available information on the alteration of DNA methylation by ethanol in utero. Thus, we have summarized all published information regarding alcohol-mediated alterations in DNA methylation during gestation. We tried to arrange information in a way that anyone can easily find the alcohol exposure time, doses, sampling time, and major changes in genomic level. Manuscript texts will also represent the correlation between ethanol metabolites and subsequent changes in methylome patterns. We hope that this review will help future researchers to further examine the issues associated with ethanol exposure.

The increased life expectancy, urbanization, and unhealthy lifestyle characterized by a shift towards a sedentary lifestyle and decreased energy expenditure are considered the main drivers of epidemiological transition. In particular, developing countries are facing a double burden caused by coexisting under- and over-nutrition, which causes a change in the disease profile from infectious diseases to a chronic degenerative pattern. This review discusses the under- and over-nutrition context in Mauritania and India, two countries that are experiencing a nutritional transition, and where we began a collaboration with local medical staff to integrate interventional and diagnostic guidelines. If many studies about diet and its relationship to non-communicable diseases are available for India, very few nutrition and cardiovascular risk studies have been conducted in Mauritania. Presently, with the exponential increase of nutrition-related diseases, targeted approaches are needed to provi...

Protein-protein interactions are an important mechanism for the regulation of enzyme function allowing metabolite channeling, crosstalk between pathways or the introduction of post-translational modifications. Therefore, a number of high-throughput studies have been carried out to shed light on the protein networks established under different pathophysiological settings. Surprisingly, this type of information is quite limited for enzymes of intermediary metabolism such as betaine homocysteine S-methyltransferase, despite its high hepatic abundancy and its role in homocysteine metabolism. Here, we have taken advantage of two approaches, affinity purification combined with mass spectrometry and yeast two-hybrid, to further uncover the array of interactions of betaine homocysteine S-methyltransferase in normal liver of Rattus norvegicus. A total of 131 non-redundant putative interaction targets were identified, out of which 20 were selected for further validation by coimmunoprecipitati...

One of the main issues in kidney transplantation is the optimal functional preservation of the organ until its transplantation into the appropriate recipient. Despite intensive efforts, the functional preservation period remains limited to hours. During this time, as a result of cellular injury, various proteins, peptides, and other molecules are released by the organ into the preservation medium. In this study, we used proteomic techniques to analyze the protein profiles of preservation solutions in which organs had been preserved prior to their transplantation. Samples were obtained from the preservation solutions of 25 deceased donor kidneys scheduled for transplantation. The protein profiles of the solutions were analyzed using 2D gel electrophoresis/MALDI-TOF and LC-MS/MS. We identified and quantified 206 proteins and peptides belonging to 139 different groups. Of these, 111 proteins groups were belonging to kidney tissues. This study used proteomic techniques to analyze the protein profiles of organ preservation solutions. These findings will contribute to the development of improved preservation solutions to effectively protect organs for transplantation.

Age related hearing loss (ARHL) affects about one third of the elderly population. It is suggested that the senescence of the hair cells could be modulated by inflammation. Thus, intake of anti- and pro-inflammatory foods is of high interest. Methods: From the MICOL study population, 734 participants were selected that participated in the 2013 to 2018 examination including hearing ability and from which past data collected in 2005/2008 was available. ARHL status was determined and compared cross-sectionally and retrospectively according to clinical and lifestyle data including food and micronutrient intake. Results: ARHL status was associated with higher age but not with education, smoking, relative weight (BMI), and clinical-chemical blood markers in the crossectional and retrospective analyses. Higher intake of fruit juices among ARHL-participants was seen cross-sectionally, and of sugary foods, high-caloric drinks, beer, and spirits retrospectively. No difference was found for th...

5-(N-Ethyl-N-isopropyl)amiloride (EIPA), a potent inhibitor of Na+/H+ antiport, reduced [35S]methionine incorporation in proteins and induced the phosphorylation of a Mr 95,000 protein in bovine aortic endothelial cells. This protein was previously shown to become phosphorylated in response to ATP, bradykinin, and A23187 (1) and was identified as elongation factor-2 (2). The action of EIPA was independent of changes in cytosolic pH, because it was neither mimicked by sodium acetate nor inhibited by ammonium chloride, and it was reproduced by 2&#39;,4&#39;-dimethylbenzamil, an analog of amiloride that is inactive on the Na+/H+ antiport. Furthermore, EIPA enhanced the Ca2(+)-dependent phosphorylation of a similar Mr 95,000 protein in a cell-free system, rabbit reticulocyte lysate, where an inhibitory effect of amiloride on protein synthesis has already been described (3). Because phosphorylation decreases the activity of elongation factor-2, our observation might explain why amiloride...

Betaine-homocysteine S-methyltransferase (BHMT) and BHMT2 convert homocysteine to methionine using betaine and S-methylmethionine, respectively, as methyl donor substrates. Increased levels of homocysteine in blood are associated with cardiovascular disease. Given their role in human health and nutrition, we identified BHMT and BHMT2 genes and proteins from 38 species of deuterostomes including human and non-human primates. We aligned the genes to look for signatures of selection, to infer evolutionary rates and events across lineages, and to identify the evolutionary timing of a gene duplication event that gave rise to two genes, BHMT and BHMT2. We found that BHMT was present in the genomes of the sea urchin, amphibians, reptiles, birds and mammals; BHMT2 was present only across mammals. BHMT and BHMT2 were present in tandem in the genomes of all monotreme, marsupial and placental species examined. Evolutionary rates were accelerated for BHMT2 relative to BHMT. Selective pressure varied across lineages, with the highest dN/dS ratios for BHMT and BHMT2 occurring immediately following the gene duplication event, as determined using GA Branch analysis. Nine codons were found to display signatures suggestive of positive selection; these contribute to the enzymatic or oligomerization domains, suggesting involvement in enzyme function. Gene duplication likely occurred after the divergence of mammals from other vertebrates but prior to the divergence of extant mammalian subclasses, followed by two deletions in BHMT2 that affect oligomerization and methyl donor specificity. The faster evolutionary rate of BHMT2 overall suggests that selective constraints were reduced relative to BHMT. The dN/dS ratios in both BHMT and BHMT2 was highest following the gene duplication, suggesting that purifying selection played a lesser role as the two paralogs diverged in function.

5-(N-Ethyl-N-isopropyl)amiloride (EIPA), a potent inhibitor of Na+/H+ antiport, reduced [35S]methionine incorporation in proteins and induced the phosphorylation of a Mr 95,000 protein in bovine aortic endothelial cells. This protein was previously shown to become phosphorylated in response to ATP, bradykinin, and A23187 (1) and was identified as elongation factor-2 (2). The action of EIPA was independent of changes in cytosolic pH, because it was neither mimicked by sodium acetate nor inhibited by ammonium chloride, and it was reproduced by 2&#39;,4&#39;-dimethylbenzamil, an analog of amiloride that is inactive on the Na+/H+ antiport. Furthermore, EIPA enhanced the Ca2(+)-dependent phosphorylation of a similar Mr 95,000 protein in a cell-free system, rabbit reticulocyte lysate, where an inhibitory effect of amiloride on protein synthesis has already been described (3). Because phosphorylation decreases the activity of elongation factor-2, our observation might explain why amiloride...

Ethanol is well known as a teratogenic factor that is capable of inducing a wide range of developmental abnormalities if the developing fetus is exposed to it. Duration and dose are the critical parameters of exposure that affect teratogenic variation to the developing fetus. It is suggested that ethanol interferes with epigenetic processes especially DNA methylation. We aimed to organize all of the available information on the alteration of DNA methylation by ethanol in utero. Thus, we have summarized all published information regarding alcohol-mediated alterations in DNA methylation during gestation. We tried to arrange information in a way that anyone can easily find the alcohol exposure time, doses, sampling time, and major changes in genomic level. Manuscript texts will also represent the correlation between ethanol metabolites and subsequent changes in methylome patterns. We hope that this review will help future researchers to further examine the issues associated with ethanol exposure.

The relationship between metabolism and methylation is considered to be an important aspect of cancer development and drug efficacy. However, it remains poorly defined how to apply this aspect to improve preclinical disease characterization and clinical treatment outcome. Using available molecular information from Kyoto Encyclopedia of Genes and Genomes (KEGG) and literature, we constructed a large-scale knowledge-based metabolic model. For the purpose of model validation, we applied data from the Cancer Cell Line Encyclopedia (CCLE) to investigate computationally the impact of metabolism on chemotherapy efficacy. In our model, different metabolic components such as MAT2A, ATP6V0E1, NNMT involved in methionine cycle correlate with biologically measured chemotherapy outcome (IC50) that are in agreement with findings of independent studies. These proteins are potentially also involved in cellular methylation processes. In addition, several components such as 3,4-dihydoxymandelate, PAP...

Betaine homocysteine S-methyltransferase (BHMT) is one of the two enzymes known to methylate homocysteine to generate methionine in the liver. It presents a Zn(2+) atom linked to three essential Cys residues. The crystal structure of rat liver BHMT has been solved at 2.5A resolution, using crystals with P2(1) symmetry and 45% solvent content in the cell. The asymmetric unit contains the whole functional tetramer showing point symmetry 222. The overall fold of the subunit consists mostly of a (alpha/beta)(8) barrel, as for human BHMT. From the end of the barrel, the polypeptide chain extends away and makes many interactions with a different subunit, forming tight dimers. The most remarkable structural feature of rat liver BHMT is the presence of a helix including residues 381-407, at the C terminus of the chain, which bind together the dimers AB to CD. A strong ion-pair and more than 60 hydrophobic interactions keep this helix stacked to the segment 316-349 from the opposite subunit....

We have overexpressed recombinant human liver betaine-homocysteine methyltransferase (BHMT; EC 2.1.1.5) in Escherichia coli and have purified the enzyme to homogeneity. The Michaelis constants for betaine and L-homocysteine are 2.2 mM and 4 M, respectively. Analysis of the pure protein for metals by inductively coupled plasma emission spectrometry indicate that the recombinant enzyme contains zinc. Extensive dialysis in buffer containing high levels of EDTA could not strip the protein of zinc. However, dialysis against buffer containing EDTA and methyl methanethiosulfonate, followed by buffer containing EDTA and dithiothreitol, could remove zinc from the enzyme with concomitant loss of activity. Dialyzing the zinc-depleted enzyme against buffer containing 1 M urea and 2 mM zinc, followed by dialysis with buffer alone, completely restored BHMT activity and zinc content. BHMT was also partially purified from human liver. The purest BHMT-containing fractions also contained zinc and the enzyme was kinetically indistinguishable from the recombinant enzyme. As with the recombinant enzyme, the partially purified human liver enzyme could be inactivated by treatment with methyl methanethiosulfonate, EDTA, and dithiothreitol. Reconstitution of the zinc-depleted enzyme completely restored activity. We conclude that BHMT is a major zinc metalloenzyme in liver and that cysteine residues are likely involved in zinc binding.

Age related hearing loss (ARHL) affects about one third of the elderly population. It is suggested that the senescence of the hair cells could be modulated by inflammation. Thus, intake of anti- and pro-inflammatory foods is of high interest. Methods: From the MICOL study population, 734 participants were selected that participated in the 2013 to 2018 examination including hearing ability and from which past data collected in 2005/2008 was available. ARHL status was determined and compared cross-sectionally and retrospectively according to clinical and lifestyle data including food and micronutrient intake. Results: ARHL status was associated with higher age but not with education, smoking, relative weight (BMI), and clinical-chemical blood markers in the crossectional and retrospective analyses. Higher intake of fruit juices among ARHL-participants was seen cross-sectionally, and of sugary foods, high-caloric drinks, beer, and spirits retrospectively. No difference was found for th...

1Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str.13, 13092 Berlin, Germany; 2Max Planck Institute of Colloids and Interfaces, Department of Theory and Bio-Systems, 14424 Potsdam, Germany; 3MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh EH4, UK; 4Max-Planck-Institut für Biochemie, Am Klopferspitz 18, 82152 Martinsried, Germany; 5TU Kaiserslautern, Paul-Ehrlich Str. 24, 67663 Kaiserslautern, Germany; 6Charité Universitätsmedizin Berlin, Germany

In mammals, methionine adenosyltransferase (MAT), the enzyme responsible for S-adenosylmethionine (AdoMet) synthesis, is encoded by two genes, MAT1A and MAT2A. In liver, MAT1A expression is associated with high AdoMet levels and a differentiated phenotype, whereas MAT2A expression is associated with lower AdoMet levels and a dedifferentiated phenotype. In the current study, we examined regulation of MAT2A gene expression by L-methionine availability using HepG2 cells. In L-methionine-deficient cells, MAT2A gene expression is rapidly induced, and methionine adenosyltransferase activity is increased. Restoration of L-methionine rapidly down-regulates MAT2A mRNA levels; for this effect, L-methionine needs to be converted into AdoMet. This novel action of AdoMet is not mediated through a methyl transfer reaction. MAT2A gene expression was also regulated by 5-methylthioadenosine, but this was dependent on 5-methylthioadenosine conversion to methionine through the salvage pathway. The transcription rate of the MAT2A gene remained unchanged during L-methionine starvation; however, its mRNA half-life was significantly increased (from 100 min to more than 3 h). The effect of L-methionine withdrawal on MAT2A mRNA stabilization requires both gene transcription and protein synthesis. We conclude that MAT2A gene expression is modulated as an adaptive response of the cell to L-methionine availability through its conversion to AdoMet.

Dietary folic acid deficiency induced early hearing loss in C57BL/6J mice after 2-months, corroborates the epidemiological association previously described between vitamin deficiency and this sensory impairment. However, this strain is prone to early hearing loss, and hence we decided to analyze whether the effects exerted by folate deprivation follow the same pattern in a mouse strain such as CBA/Ca, which is resistant to hearing impairment. Here, we show results of a long-term study on hearing carried out on CBA/Ca mice subjected to dietary folate deprivation. Systemic changes included decreased serum folate levels, hyperhomocysteinemia and signs of anemia in the group fed with folate-deficient (FD) diet. Initial signs of hearing loss were detected in this strain after 8-months of vitamin deficiency, and correlated with histological damage in the cochleae. In conclusion, the data presented reinforce the importance of adequate folic acid levels for the auditory system and suggest that the impact of dietary deficiencies may depend on the genetic background.

Betaine homocysteine S-methyltransferase (BHMT) is one of the two enzymes known to methylate homocysteine to generate methionine in the liver. It presents a Zn(2+) atom linked to three essential Cys residues. The crystal structure of rat liver BHMT has been solved at 2.5A resolution, using crystals with P2(1) symmetry and 45% solvent content in the cell. The asymmetric unit contains the whole functional tetramer showing point symmetry 222. The overall fold of the subunit consists mostly of a (alpha/beta)(8) barrel, as for human BHMT. From the end of the barrel, the polypeptide chain extends away and makes many interactions with a different subunit, forming tight dimers. The most remarkable structural feature of rat liver BHMT is the presence of a helix including residues 381-407, at the C terminus of the chain, which bind together the dimers AB to CD. A strong ion-pair and more than 60 hydrophobic interactions keep this helix stacked to the segment 316-349 from the opposite subunit....

The paradigm of a cytoplasmic methionine cycle synthesizing/eliminating metabolites that are transported into/out of the nucleus as required has been challenged by detection of significant nuclear levels of several enzymes of this pathway. Here, we show betaine homocysteine S-methyltransferase (BHMT), an enzyme that exerts a dual function in maintenance of methionine levels and osmoregulation, as a new component of the nuclear branch of the cycle. In most tissues, low expression of Bhmt coincides with a preferential nuclear localization of the protein. Conversely, the liver, with very high Bhmt expression levels, presents a main cytoplasmic localization. Nuclear BHMT is an active homotetramer in normal liver, although the total enzyme activity in this fraction is markedly lower than in the cytosol. N-terminal basic residues play a role in cytoplasmic retention and the ratio of glutathione species regulates nucleocytoplasmic distribution. The oxidative stress associated with D-galact...

Methionine adenosyltransferase III (MATIII) catalyzes S-adenosylmethionine (AdoMet) synthesis and, as part of its reaction mechanism, it also hydrolyzes tripolyphosphate. Tripolyphosphatase activity was linear over time and had a slightly sigmoidal behavior with an affinity in the low micromolar range. On the contrary, AdoMet synthetase activity showed a lag phase that was independent of protein concentration but decreased at increasing substrate concentrations. Tripolyphosphatase activity, which appeared to be slower than AdoMet synthesis, was stimulated by preincubation with ATP and methionine so that it matched AdoMet synthetase activity. This stimulation process, which is probably the origin of the lag phase, represents the slow transition between two conformations of the enzyme that could be distinguished by their different tripolyphosphatase activity and sensitivity to S-nitrosylation. Tripolyphosphatase activity appeared to be the rate-determining reaction in AdoMet synthesis and the one inhibited by S-nitrosylation. The methionine concentration necessary to obtain half-maximal stimulation was in the range of physiological methionine fluctuations. Moreover, stimulation of MAT activity by methionine was demonstrated in vivo. We propose that the hysteretic behavior of MATIII, in which methionine induces the transition to a higher specific activity conformation, can be considered as an adaptation to the specific functional requirements of the liver.

Aims. The discovery of methionine metabolism enzymes in the cell nucleus, together with their association with key nuclear processes, suggested a putative relationship between alterations in their subcellular distribution and disease. Results. Using the rat model of D-galactosamine intoxication severe changes in hepatic steady-state mRNA levels were found; the largest decreases corresponded to enzymes exhibiting the highest expression in normal tissue. Cytoplasmic protein levels, activities and metabolite concentrations suffered more moderate changes following a similar trend. Interestingly, galactosamine-treatment induced hepatic nuclear accumulation of MATα1 and Sadenosylhomocysteine hydrolase tetramers, their active assemblies. In fact, galactosamine-treated livers showed enhanced nuclear methionine adenosyltransferase activity. Acetaminophen intoxication mimicked most galactosamine effects on hepatic MATα1, including accumulation of nuclear tetramers. H35 cells overexpressing tagged-MATα1 reproduced the subcellular distribution observed in liver, and the changes induced by galactosamine and acetaminophen that were also observed upon glutathione depletion by buthionine sulfoximine. The H35 nuclear accumulation of tagged-MATα1 induced by these agents correlated with decreased GSH/GSSG ratios and was prevented by N-acetylcysteine and glutathione ethyl ester. However, the changes in epigenetic modifications associated with tagged-MATα1 nuclear accumulation were only prevented by N-acetylcysteine in galactosamine-treated cells. Innovation. Cytoplasmic and nuclear changes in proteins regulating the methylation index follow opposite trends in acute liver injury, their nuclear accumulation showing potential as disease marker. Conclusion. Altogether these results demonstrate galactosamine-and acetaminopheninduced nuclear accumulation of methionine metabolism enzymes as active oligomers Delgado et al. 3 and unveil the implication of redox-dependent mechanisms in the control of MATα1 subcellular distribution.

Betaine homocysteine S-methyltransferase (BHMT) is one of the two enzymes known to methylate homocysteine to generate methionine in the liver. It presents a Zn(2+) atom linked to three essential Cys residues. The crystal structure of rat liver BHMT has been solved at 2.5A resolution, using crystals with P2(1) symmetry and 45% solvent content in the cell. The asymmetric unit contains the whole functional tetramer showing point symmetry 222. The overall fold of the subunit consists mostly of a (alpha/beta)(8) barrel, as for human BHMT. From the end of the barrel, the polypeptide chain extends away and makes many interactions with a different subunit, forming tight dimers. The most remarkable structural feature of rat liver BHMT is the presence of a helix including residues 381-407, at the C terminus of the chain, which bind together the dimers AB to CD. A strong ion-pair and more than 60 hydrophobic interactions keep this helix stacked to the segment 316-349 from the opposite subunit....

Transsulfuration allows conversion of methionine into cysteine using homocysteine (Hcy) as an intermediate. This pathway produces S-adenosylmethionine (AdoMet), a key metabolite for cell function, and provides 50% of the cysteine needed for hepatic glutathione synthesis. The route requires the intake of essential nutrients (e.g., methionine and vitamins) and is regulated by their availability. Transsulfuration presents multiple interconnections with epigenetics, adenosine triphosphate (ATP), and glutathione synthesis, polyol and pentose phosphate pathways, and detoxification that rely mostly in the exchange of substrates or products. Major hepatic diseases, rare diseases, and sensorineural disorders, among others that concur with oxidative stress, present impaired transsulfuration. Recent Advances: In contrast to the classical view, a nuclear branch of the pathway, potentiated under oxidative stress, is emerging. Several transsulfuration proteins regulate gene expression, suggesting...

The paradigm of a cytoplasmic methionine cycle synthesizing/eliminating metabolites that are transported into/out of the nucleus as required has been challenged by detection of significant nuclear levels of several enzymes of this pathway. Here, we show betaine homocysteine S-methyltransferase (BHMT), an enzyme that exerts a dual function in maintenance of methionine levels and osmoregulation, as a new component of the nuclear branch of the cycle. In most tissues, low expression of Bhmt coincides with a preferential nuclear localization of the protein. Conversely, the liver, with very high Bhmt expression levels, presents a main cytoplasmic localization. Nuclear BHMT is an active homotetramer in normal liver, although the total enzyme activity in this fraction is markedly lower than in the cytosol. N-terminal basic residues play a role in cytoplasmic retention and the ratio of glutathione species regulates nucleocytoplasmic distribution. The oxidative stress associated with D-galact...

Dietary folic acid deficiency induced early hearing loss in C57BL/6J mice after 2-months, corroborates the epidemiological association previously described between vitamin deficiency and this sensory impairment. However, this strain is prone to early hearing loss, and hence we decided to analyze whether the effects exerted by folate deprivation follow the same pattern in a mouse strain such as CBA/Ca, which is resistant to hearing impairment. Here, we show results of a long-term study on hearing carried out on CBA/Ca mice subjected to dietary folate deprivation. Systemic changes included decreased serum folate levels, hyperhomocysteinemia and signs of anemia in the group fed with folate-deficient (FD) diet. Initial signs of hearing loss were detected in this strain after 8-months of vitamin deficiency, and correlated with histological damage in the cochleae. In conclusion, the data presented reinforce the importance of adequate folic acid levels for the auditory system and suggest that the impact of dietary deficiencies may depend on the genetic background.

Aims. The discovery of methionine metabolism enzymes in the cell nucleus, together with their association with key nuclear processes, suggested a putative relationship between alterations in their subcellular distribution and disease. Results. Using the rat model of D-galactosamine intoxication severe changes in hepatic steady-state mRNA levels were found; the largest decreases corresponded to enzymes exhibiting the highest expression in normal tissue. Cytoplasmic protein levels, activities and metabolite concentrations suffered more moderate changes following a similar trend. Interestingly, galactosamine-treatment induced hepatic nuclear accumulation of MATα1 and Sadenosylhomocysteine hydrolase tetramers, their active assemblies. In fact, galactosamine-treated livers showed enhanced nuclear methionine adenosyltransferase activity. Acetaminophen intoxication mimicked most galactosamine effects on hepatic MATα1, including accumulation of nuclear tetramers. H35 cells overexpressing tagged-MATα1 reproduced the subcellular distribution observed in liver, and the changes induced by galactosamine and acetaminophen that were also observed upon glutathione depletion by buthionine sulfoximine. The H35 nuclear accumulation of tagged-MATα1 induced by these agents correlated with decreased GSH/GSSG ratios and was prevented by N-acetylcysteine and glutathione ethyl ester. However, the changes in epigenetic modifications associated with tagged-MATα1 nuclear accumulation were only prevented by N-acetylcysteine in galactosamine-treated cells. Innovation. Cytoplasmic and nuclear changes in proteins regulating the methylation index follow opposite trends in acute liver injury, their nuclear accumulation showing potential as disease marker. Conclusion. Altogether these results demonstrate galactosamine-and acetaminopheninduced nuclear accumulation of methionine metabolism enzymes as active oligomers Delgado et al. 3 and unveil the implication of redox-dependent mechanisms in the control of MATα1 subcellular distribution.

Hearing loss has been recently ranked as the fifth leading cause of years lived with disability, ahead of many other chronic diseases such as diabetes, dementia, or chronic obstructive pulmonary disease. Moreover, according to the World Health Organization, moderate-to-profound hearing loss affects about 466 million people worldwide. Its incidence varies in each population segment, affecting approximately 10% of children and increasing to 30% of the population over 65 years. However, hearing loss receives still very limited research funding and public awareness. This sensory impairment is caused by genetic and environmental factors, and among the latter, the nutritional status has acquired relevance due its association to hearing loss detected in recent epidemiological studies. Several experimental models have proved that the onset and progression of hearing loss are closely linked to the availability of nutrients and their metabolism. Here, we have reviewed studies focused on nutri...

Dietary folic acid deficiency induced early hearing loss in C57BL/6J mice after 2-months, corroborates the epidemiological association previously described between vitamin deficiency and this sensory impairment. However, this strain is prone to early hearing loss, and hence we decided to analyze whether the effects exerted by folate deprivation follow the same pattern in a mouse strain such as CBA/Ca, which is resistant to hearing impairment. Here, we show results of a long-term study on hearing carried out on CBA/Ca mice subjected to dietary folate deprivation. Systemic changes included decreased serum folate levels, hyperhomocysteinemia and signs of anemia in the group fed with folate-deficient (FD) diet. Initial signs of hearing loss were detected in this strain after 8-months of vitamin deficiency, and correlated with histological damage in the cochleae. In conclusion, the data presented reinforce the importance of adequate folic acid levels for the auditory system and suggest that the impact of dietary deficiencies may depend on the genetic background.

The small heat shock protein (sHsp) Hsp22 from Drosophila melanogaster (DmHsp22) is part of the family of sHsps in this diptera. This sHsp is characterized by its presence in the mitochondrial matrix as well as by its preferential expression during ageing. Although DmHsp22 has been demonstrated to be an efficient in vitro chaperone, its function within mitochondria in vivo remains largely unknown. Thus, determining its protein-interaction network (interactome) in the mitochondrial matrix would help to shed light on its function(s). In the present study we combined immunoaffinity conjugation (IAC) with mass spectroscopy analysis of mitochondria from HeLa cells transfected with DmHsp22 in non-heat shock condition and after heat shock (HS). 60 common DmHsp22-binding mitochondrial partners were detected in two independent IACs. Immunoblotting was used to validate interaction between DmHsp22 and two members of the mitochondrial chaperone machinery; Hsp60 and Hsp70. Among the partners of DmHsp22, several ATP synthase subunits were found. Moreover, we showed that expression of DmHsp22 in transiently transfected HeLa cells increased maximal mitochondrial oxygen consumption capacity and ATP contents, providing a mechanistic link between DmHsp22 and mitochondrial functions.

The first corresponded to methionine synthase that catalyses the last step in the plant methionine biosynthetic pathway. This protein was present at low level in dry mature seeds, and its level was increased strongly at 1-day imbibition, prior to radicle emergence. Its level was not increased further at 2day imbibition, coincident with radicle emergence. However, its level in 1-day imbibed seeds strongly decreased upon subsequent drying of the imbibed seeds back to the original water content of the dry mature seeds. The second enzyme corresponded to S-adenosylmethionine synthetase that catalyses the synthesis of S-adenosylmethionine from methionine and ATP. In this case, this enzyme was detected in the form of two isozymes with different pI and Mr. Both proteins were

Protein-protein interactions are an important mechanism for the regulation of enzyme function allowing metabolite channeling, crosstalk between pathways or the introduction of post-translational modifications. Therefore, a number of high-throughput studies have been carried out to shed light on the protein networks established under different pathophysiological settings. Surprisingly, this type of information is quite limited for enzymes of intermediary metabolism such as betaine homocysteine S-methyltransferase, despite its high hepatic abundancy and its role in homocysteine metabolism. Here, we have taken advantage of two approaches, affinity purification combined with mass spectrometry and yeast two-hybrid, to further uncover the array of interactions of betaine homocysteine S-methyltransferase in normal liver of Rattus norvegicus. A total of 131 non-redundant putative interaction targets were identified, out of which 20 were selected for further validation by coimmunoprecipitati...

Transsulfuration allows conversion of methionine into cysteine using homocysteine (Hcy) as an intermediate. This pathway produces S-adenosylmethionine (AdoMet), a key metabolite for cell function, and provides 50% of the cysteine needed for hepatic glutathione synthesis. The route requires the intake of essential nutrients (e.g., methionine and vitamins) and is regulated by their availability. Transsulfuration presents multiple interconnections with epigenetics, adenosine triphosphate (ATP), and glutathione synthesis, polyol and pentose phosphate pathways, and detoxification that rely mostly in the exchange of substrates or products. Major hepatic diseases, rare diseases, and sensorineural disorders, among others that concur with oxidative stress, present impaired transsulfuration. Recent Advances: In contrast to the classical view, a nuclear branch of the pathway, potentiated under oxidative stress, is emerging. Several transsulfuration proteins regulate gene expression, suggesting...

The paradigm of a cytoplasmic methionine cycle synthesizing/eliminating metabolites that are transported into/out of the nucleus as required has been challenged by detection of significant nuclear levels of several enzymes of this pathway. Here, we show betaine homocysteine S-methyltransferase (BHMT), an enzyme that exerts a dual function in maintenance of methionine levels and osmoregulation, as a new component of the nuclear branch of the cycle. In most tissues, low expression of Bhmt coincides with a preferential nuclear localization of the protein. Conversely, the liver, with very high Bhmt expression levels, presents a main cytoplasmic localization. Nuclear BHMT is an active homotetramer in normal liver, although the total enzyme activity in this fraction is markedly lower than in the cytosol. N-terminal basic residues play a role in cytoplasmic retention and the ratio of glutathione species regulates nucleocytoplasmic distribution. The oxidative stress associated with D-galact...

Dietary folic acid deficiency induced early hearing loss in C57BL/6J mice after 2-months, corroborates the epidemiological association previously described between vitamin deficiency and this sensory impairment. However, this strain is prone to early hearing loss, and hence we decided to analyze whether the effects exerted by folate deprivation follow the same pattern in a mouse strain such as CBA/Ca, which is resistant to hearing impairment. Here, we show results of a long-term study on hearing carried out on CBA/Ca mice subjected to dietary folate deprivation. Systemic changes included decreased serum folate levels, hyperhomocysteinemia and signs of anemia in the group fed with folate-deficient (FD) diet. Initial signs of hearing loss were detected in this strain after 8-months of vitamin deficiency, and correlated with histological damage in the cochleae. In conclusion, the data presented reinforce the importance of adequate folic acid levels for the auditory system and suggest that the impact of dietary deficiencies may depend on the genetic background.

Hearing loss has been recently ranked as the fifth leading cause of years lived with disability, ahead of many other chronic diseases such as diabetes, dementia, or chronic obstructive pulmonary disease. Moreover, according to the World Health Organization, moderate-to-profound hearing loss affects about 466 million people worldwide. Its incidence varies in each population segment, affecting approximately 10% of children and increasing to 30% of the population over 65 years. However, hearing loss receives still very limited research funding and public awareness. This sensory impairment is caused by genetic and environmental factors, and among the latter, the nutritional status has acquired relevance due its association to hearing loss detected in recent epidemiological studies. Several experimental models have proved that the onset and progression of hearing loss are closely linked to the availability of nutrients and their metabolism. Here, we have reviewed studies focused on nutri...

Aging is associated with a drastic decline in fertility/fecundity and with an increased risk of pregnancy complications. Resveratrol (RES), a natural polyphenolic compound, has shown anti-oxidant and anti-inflammatory activities in both human and animal models, thus representing a potential therapeutic and prophylactic anti-aging supplement. Here, we investigated whether preconceptional resveratrol supplementation improved reproductive outcomes in mid-aged (8-month-old) and old (12-month-old) C57BL/6J female mice. Female siblings were cohoused and assigned to either RES or vehicle supplementation to drinking water for 10 consecutive weeks. Subsequently, females were mated with non-supplemented males and their pregnancy outcomes were monitored. RES improved mating success in old, but not in mid-aged females, and prevented the occurrence of delivery complications in the latter. These results indicate that preconceptional RES supplementation could partially improve age-related reproduc...

Age related hearing loss (ARHL) affects about one third of the elderly population. It is suggested that the senescence of the hair cells could be modulated by inflammation. Thus, intake of anti- and pro-inflammatory foods is of high interest. Methods: From the MICOL study population, 734 participants were selected that participated in the 2013 to 2018 examination including hearing ability and from which past data collected in 2005/2008 was available. ARHL status was determined and compared cross-sectionally and retrospectively according to clinical and lifestyle data including food and micronutrient intake. Results: ARHL status was associated with higher age but not with education, smoking, relative weight (BMI), and clinical-chemical blood markers in the crossectional and retrospective analyses. Higher intake of fruit juices among ARHL-participants was seen cross-sectionally, and of sugary foods, high-caloric drinks, beer, and spirits retrospectively. No difference was found for th...

In recent years, there has been increasing research interest in the correlation between hearing impairment and cognitive decline, two conditions that have demonstrated a strong association. Hearing loss appears as a risk factor for cognitive impairment, especially among certain populations, notably nursing home residents. Furthermore, hearing loss has been identified as a modifiable age-related condition linked to dementia, and it has been estimated that midlife hearing loss, if eliminated, might decrease the risk of dementia in the general population. Several mechanisms have been suggested to explain the pathologic connections between hearing loss and dementia; however, clear evidence is missing, and the common pathophysiological basis is still unclear. In this review, we discussed current knowledge about the relationship between hearing loss and dementia, and future perspectives in terms of the effects of hearing rehabilitation for early prevention of cognitive decline.

Objective Evidence shows that anemic individuals are at a higher risk of hearing loss. However, there is no systematic review and meta-analysis study. Thus, we aimed to meta-analyze the existing evidence on the association of iron deficiency anemia (IDA) with sensorineural hearing loss (SNHL). We searched PubMed, MEDLINE, Embase, Scopus, and Google Scholar from inception through October 30, 2017, for studies done on the association of the IDA with SNHL. Pooled odds ratio (OR) was calculated by random effect meta-analysis method. Heterogeneity was assessed by I2 metrics. Result Four studies, covering a total of 344,080 adults and children, were included. The odds of SNHL was higher by 55% in individuals with IDA, compared with individuals without IDA (OR = 1.55, 95% CI 1.17–2.06; P = 0.03). The age-specific ORs were 1.36 (95% CI 1.15–1.61; P = 0.27) and 3.67 (95% CI 1.72–7.84) for adults and children, respectively. IDA may be a contributing factor to hearing loss. Further studies are...

The Nogo-B receptor (NgBR) is involved in oncogenic Ras signaling through directly binding to farnesylated Ras. It recruits farnesylated Ras to the non-lipid-raft membrane for interaction with downstream effectors. However, the cytosolic domain of NgBR itself is only partially folded. The lack of several conserved secondary structural elements makes this domain unlikely to form a complete farnesyl binding pocket. We find that inclusion of the extracellular and transmembrane domains that contain additional conserved residues to the cytosolic region results in a well folded protein with a similar size and shape to the E.coli cis-isoprenyl transferase (UPPs). Small Angle X-ray Scattering (SAXS) analysis reveals the radius of gyration (Rg) of our NgBR construct to be 18.2 Å with a maximum particle dimension (Dmax) of 61.0 Å. Ab initio shape modeling returns a globular molecular envelope with an estimated molecular weight of 23.0 kD closely correlated with the calculated molecular weight...

Hearing loss has been recently ranked as the fifth leading cause of years lived with disability, ahead of many other chronic diseases such as diabetes, dementia, or chronic obstructive pulmonary disease. Moreover, according to the World Health Organization, moderate-to-profound hearing loss affects about 466 million people worldwide. Its incidence varies in each population segment, affecting approximately 10% of children and increasing to 30% of the population over 65 years. However, hearing loss receives still very limited research funding and public awareness. This sensory impairment is caused by genetic and environmental factors, and among the latter, the nutritional status has acquired relevance due its association to hearing loss detected in recent epidemiological studies. Several experimental models have proved that the onset and progression of hearing loss are closely linked to the availability of nutrients and their metabolism. Here, we have reviewed studies focused on nutri...