In vivo regulation by glutathione of methionine adenosyltransferase S-nitrosylation in rat liver

BACKGROUND/AIMS: Ethanol consumption and pathological conditions such as cirrhosis lead to a reduction of hepatic glutathione. Hepatic methionine adenosyltransferase, the enzyme that synthesizes S-adenosylmethionine, the major methylating agent, is regulated in vivo by glutathione levels. We have previously shown that nitric oxide inactivates methionine adenosyltransferase in vivo by S-nitrosylation. In this study, we aimed to investigate the regulation by glutathione of methionine adenosyltransferase S-nitrosylation in rat liver. METHODS: Rat hepatocytes and whole animals were treated with buthionine sulfoximine, an inhibitor of glutathione synthesis, and methionine adenosyltransferase S-nitrosylation and activity were determined. RESULTS: In hepatocytes, buthionine sulfoximine led to the S-nitrosylation and inactivation of methionine adenosyltransferase. Restoring glutathione levels in hepatocytes treated with buthionine sulfoximine, by the addition of glutathione monoethyl ester, a permeable derivative of glutathione, led to the denitrosylation and reactivation of methionine adenosyltransferase. In whole animals, buthionine sulfoximine led also to methionine adenosyltransferase S-nitrosylation and inactivation. S-Nitrosylation and inactivation of methionine adenosyltransferase induced by buthionine sulfoximine in whole animals was prevented by glutathione monoethyl ester. CONCLUSIONS: These results indicate that in vivo hepatic methionine adenosyltransferase exists in two forms in equilibrium, nitrosylated (inactive) and denitrosylated (active), which are regulated by both the cellular levels of nitric oxide and glutathione

Hysteretic behavior of methionine adenosyltransferase III. Methionine switches between two conformations of the enzyme with different specific activity

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

Nitric oxide inactivates rat hepatic methionine adenosyltransferase In vivo by S-nitrosylation

We investigated the mechanism of nitric oxide (NO) action on hepatic methionine adenosyltransferase (MAT) activity using S-nitrosoglutathione (GSNO) as NO donor. Hepatic MAT plays an essential role in the metabolism of methionine, converting this amino acid into S-adenosylmethionine. Hepatic MAT exists in two oligomeric states: as a tetramer (MAT I) and as a dimer (MAT III) of the same subunit. This subunit contains 10 cysteine residues. In MAT I, S-nitrosylation of 1 thiol residue per subunit was associated with a marked inactivation of the enzyme (about 70%) that was reversed by glutathione (GSH). In MAT III, S-nitrosylation of 3 thiol residues per subunit led to a similar inactivation of the enzyme, which was also reversed by GSH. Incubation of isolated rat hepatocytes with S-nitrosoglutathione monoethyl ester (EGSNO), a NO donor permeable through the cellular membrane, induced a dose-dependent inactivation of MAT that was reversed by removing the NO donor from the cell suspension. MAT, purified from isolated rat hepatocytes, contained S-nitrosothiol groups and the addition of increasing concentrations of EGSNO to the hepatocyte suspension led to a progressive S-nitrosylation of the enzyme. Removal of the NO donor from the incubation media resulted in loss of most NO groups associated to the enzyme. Finally, induction in rats of the production of NO, by the administration of bacterial lipopolysaccharide (LPS), induced a fivefold increase in the S-nitrosylation of hepatic MAT, which led to a marked inactivation of the enzyme. Thus, the activity of liver MAT appears to be regulated in vivo by S-nitrosylation

S-Adenosylmethionine: a control switch that regulates liver function

Genome sequence analysis reveals that all organisms synthesize S-adenosylmethionine (AdoMet) and that a large fraction of all genes is AdoMet-dependent methyltransferases. AdoMet-dependent methylation has been shown to be central to many biological processes. Up to 85% of all methylation reactions and as much as 48% of methionine metabolism occur in the liver, which indicates the crucial importance of this organ in the regulation of blood methionine. Of the two mammalian genes (MAT1A, MAT2A) that encode methionine adenosyltransferase (MAT, the enzyme that makes AdoMet), MAT1A is specifically expressed in adult liver. It now appears that growth factors, cytokines, and hormones regulate liver MAT mRNA levels and enzyme activity and that AdoMet should not be viewed only as an intermediate metabolite in methionine catabolism, but also as an intracellular control switch that regulates essential hepatic functions such as regeneration, differentiation, and the sensitivity of this organ to injury. The aim of this review is to integrate these recent findings linking AdoMet with liver growth, differentiation, and injury into a comprehensive model. With the availability of AdoMet as a nutritional supplement and evidence of its beneficial role in various liver diseases, this review offers insight into its mechanism of action

Methionine adenosyltransferase S-nitrosylation is regulated by the basic and acidic amino acids surrounding the target thiol

S-Adenosylmethionine serves as the methyl donor for many biological methylation reactions and provides the propylamine group for the synthesis of polyamines. S-Adenosylmethionine is synthesized from methionine and ATP by the enzyme methionine adenosyltransferase. The cellular factors regulating S-adenosylmethionine synthesis have not been well defined. Here we show that in rat hepatocytes S-nitrosoglutathione monoethyl ester, a cell-permeable nitric oxide donor, markedly reduces cellular S-adenosylmethionine content via inactivation of methionine adenosyltransferase by S-nitrosylation. Removal of the nitric oxide donor from the incubation medium leads to the denitrosylation and reactivation of methionine adenosyltransferase and to the rapid recovery of cellular S-adenosylmethionine levels. Nitric oxide inactivates methionine adenosyltransferase via S-nitrosylation of cysteine 121. Replacement of the acidic (aspartate 355) or basic (arginine 357 and arginine 363) amino acids located in the vicinity of cysteine 121 by serine leads to a marked reduction in the ability of nitric oxide to S-nitrosylate and inactivate hepatic methionine adenosyltransferase. These results indicate that protein S-nitrosylation is regulated by the basic and acidic amino acids surrounding the target cysteine

Methylthioadenosine

5'-Methylthioadenosine (MTA) is a naturally occurring sulfur-containing nucleoside present in all mammalian tissues. MTA is produced from S-adenosylmethionine mainly through the polyamine biosynthetic pathway, where it behaves as a powerful inhibitory product. This compound is metabolized solely by MTA-phosphorylase, to yield 5-methylthioribose-1-phosphate and adenine, a crucial step in the methionine and purine salvage pathways, respectively. Abundant evidence has accumulated over time suggesting that MTA can affect cellular processes in many ways. MTA has been shown to influence numerous critical responses of the cell including regulation of gene expression, proliferation, differentiation and apoptosis. Although most of these responses have been observed at the pharmacological level, their specificity makes it tempting to speculate that endogenous MTA could play a regulatory role in the cell. Finally, observations carried out in models of liver damage and cancer demonstrate a therapeutic potential for MTA that deserves further consideration

Folding of dimeric methionine adenosyltransferase III: identification of two folding intermediates

Methionine adenosyl transferase (MAT) is an essential enzyme that synthesizes AdoMet. The liver-specific MAT isoform, MAT III, is a homodimer of a 43.7-kDa subunit that organizes in three nonsequential alpha-beta domains. Although MAT III structure has been recently resolved, little is known about its folding mechanism. Equilibrium unfolding and refolding of MAT III, and the monomeric mutant R265H, have been monitored using different physical parameters. Tryptophanyl fluorescence showed a three-state folding mechanism. The first unfolding step was a folding/association process as indicated by its dependence on protein concentration. The monomeric folding intermediate produced was the predominant species between 1.5 and 3 m urea. It had a relatively compact conformation with tryptophan residues and hydrophobic surfaces occluded from the solvent, although its N-terminal region may be very unstructured. The second unfolding step monitored the denaturation of the intermediate. Refolding of the intermediate showed first order kinetics, indicating the presence of a kinetic intermediate within the folding/association transition. Its presence was confirmed by measuring the 1,8-anilinonaphtalene-8-sulfonic acid binding in the presence of tripolyphosphate. We propose that the folding rate-limiting step is the formation of an intermediate, probably a structured monomer with exposed hydrophobic surfaces, that rapidly associates to form dimeric MAT III

Interaction between an adcy3 genetic variant and two weight-lowering diets affecting body fatness and body composition outcomes depending on macronutrient distribution: a randomized trial

The adenylate cyclase 3 (ADCY3) gene is involved in the regulation of several metabolic processes including the development and function of adipose tissue. The effects of the ADCY3 rs10182181 genetic variant on changes in body composition depending on the macronutrient distribution intake after 16 weeks of the dietary intervention were tested. The ADCY3 genetic variant was genotyped in 147 overweight or obese subjects, who were randomly assigned to one of the two diets varying in macronutrient content: a moderately-high-protein diet and a low-fat diet. Anthropometric and body composition measurements (DEXA scan) were recorded. Significant interactions between the ADCY3 genotype and dietary intervention on changes in weight, waist circumference, and body composition were found after adjustment for covariates. Thus, in the moderately-high-protein diet group, the G allele was associated with a lower decrease of fat mass, trunk and android fat, and a greater decrease in lean mass. Conversely, in the low-fat diet group carrying the G allele was associated with a greater decrease in trunk, android, gynoid, and visceral fat. Subjects carrying the G allele of the rs10182181 polymorphism may benefit more in terms of weight loss and improvement of body composition measurements when undertaking a hypocaloric low-fat diet as compared to a moderately-high-protein diet