AdoMet Synthetase Activity Research Articles

Glucocorticoid-induced S-Adenosylmethionine Enhances the Interferon Signaling Pathway by Restoring STAT1 Protein Methylation in Hepatitis B Virus-infected Cells

Patients with chronic hepatitis B usually exhibit a low response to treatment with interferon α (IFN-α). An alternative approach to increase the response rate of IFN-α might be to immunologically stimulate the host with glucocorticoids (GCs) before treatment with IFN-α, but the underlying mechanism remains unclear. We hypothesized that the GCs enhance IFN signaling by inducing S-adenosylmethionine (AdoMet) when hepatitis B virus (HBV) replication was effectively suppressed by IFN-α. Here, we investigated the effect of GCs and IFN-α on AdoMet production and methionine adenosyltransferase 1A (MAT1A) expression in vitro. Furthermore, we determined whether post-transcriptional regulation is involved in HBV-repressed MAT1A expression and AdoMet production induced by dexamethasone (Dex). We found that AdoMet homeostasis was disrupted by Dex and that Dex directly regulated MAT1A expression by enhancing the binding of the glucocorticoid receptor (GR) to the glucocorticoid-response element (GRE) of the MAT1A promoter. HBV reduced AdoMet production by increasing methylation at GRE sites within the MAT1A promoter. The X protein of hepatitis B virus led to hypermethylation in the MAT1A promoter by recruiting DNA methyltransferase 1, and it inhibited GR binding to the GRE in the MAT1A promoter. Dex could increase an antiviral effect by inducing AdoMet production via a positive feedback loop when HBV is effectively suppressed by IFN-α, and the mechanism that involves Dex-induced AdoMet could increase STAT1 methylation rather than STAT1 phosphorylation. These findings provide a possible mechanism by which GC-induced AdoMet enhances the antiviral activity of IFN-α by restoring STAT1 methylation in HBV-infected cells.

Acetaminophen-induced suppression of hepatic AdoMet synthetase activity is attenuated by prodrugs of l-cysteine

Administration of acetaminophen (ACP, 400 mg/kg, i.p.) to fasted, male Swiss-Webster mice caused a rapid 90% decrease in total hepatic glutathione (GSH) and a 58% decrease in mitochondrial GSH by 2 h post ACP. This was followed by a time-dependent decrease (72%) in hepatic AdoMet synthetase activity and rise in plasma ALT levels (>10 000 U/l) at 24 h post ACP treatment. AdoMet synthetase activity was maintained at 82, 78 and 60% of controls, respectively, by the cysteine prodrugs PTCA, CySSME and NAC. Total hepatic and mitochondrial GSH levels were also protected from severe ACP-induced depletion by CySSME and MTCA. These results suggest that the maintenance of GSH homeostasis by cysteine prodrugs can protect mouse hepatic AdoMet synthetase, a sulfhydryl enzyme whose integrity is dependent on GSH, as well as the liver itself from the consequences of oxidative stress elicited by toxic metabolites of xenobiotics.

Pulsatile growth hormone secretion decreases S-adenosylmethionine synthetase in rat liver.

S-adenosylmethionine synthetase (AdoMet synthetase) is responsible for the synthesis of the major methyl donor S-adenosylmethionine. The AdoMet synthetase gene was identified by subtractive suppressive hybridization as being expressed at higher levels in the liver of rats continuously exposed to growth hormone (GH) than in rats intermittently exposed to the hormone. Further studies on the regulation of AdoMet synthetase showed that the activity and mRNA levels were higher in female than in male rats. Hypophysectomy increased AdoMet synthetase mRNA in both male and female rats. Combined thyroxine and cortisol treatment of hypophysectomized rats had no effect on AdoMet synthetase mRNA levels. Two daily injections of GH for 7 days, mimicking the male secretory pattern of GH, decreased AdoMet synthetase activity and mRNA levels. A continuous infusion of GH, mimicking the female secretory pattern of GH, had small or no effects on AdoMet synthetase activity and decreased the mRNA levels to a lesser degree than two daily injections. It is concluded that the lower AdoMet synthetase activity in male rats is due to an inhibitory effect of the male characteristic pulsatile secretory pattern of GH on AdoMet synthetase mRNA expression.

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.

S-Adenosylmethionine and Pneumocystis carinii

We previously reported that S-adenosylmethionine (AdoMet), a key molecule in methylation reactions and polyamine biosynthesis, enhances axenic culture of the AIDS-associated opportunistic fungal pathogen Pneumocystis carinii. Here we report that AdoMet is absolutely required for continuous growth. Two transporters are present, one high affinity, K(m) = 4.5 microm, and one low affinity, K(m) = 333 microm. The physiologically relevant high affinity transporter has a pH optimum of 7.5 and no related natural compounds compete for uptake. Transport is 98% inhibited at 4 degrees C, 24% inhibited by 20 mm sodium azide, and 95% inhibited by the combination of 20 mm sodium azide and 1 mm salicylhydroxamic acid; thus transport is active and dependent on both a cytochrome chain and an alternative oxidase. In vitro, AdoMet is used at a rate of 1. 40 x 10(7) molecules cell(-1) min(-1). AdoMet synthetase activity was not detected by a sensitive radiolabel incorporation assay capable of detecting 0.1% of the activity in rat liver. In addition, the AdoMet plasma concentration of rats is inversely correlated with the number of P. carinii in the lungs. These findings demonstrate that P. carinii is an AdoMet auxotroph. The uptake and metabolism of this compound are rational chemotherapeutic targets.

Interleukin-2 induces gamma-S-adenosyl-L-methionine synthetase gene expression during T-lymphocyte activation.

The regulation of expression of the gamma-S-adenosyl-L-methionine (AdoMet) synthetase gene was investigated in T-cells during G0/G1 transition, as well as throughout the G1 phase. Stimulation of G0 T-lymphocytes with concanavalin A induces AdoMet synthetase gene expression, starting 8 h after stimulation. Interleukin-2 (IL-2) stimulates the induction of this gene expression and AdoMet synthetase activity in G1 lymphoblasts, in part by an increase in the transcription rate of the gene. Phorbol esters, which also stimulate the proliferation of G1 lymphoblasts, show a similar kinetics of AdoMet synthetase mRNA induction. In contrast, the mRNA levels of the S-adenosyl-L-homocysteine hydrolase, another enzyme of the methionine cycle, remain unchanged upon IL-2 or phorbol 12,13-dibutyrate treatment. Dexamethasone and 8Br-cAMP, both inhibitors of lymphocyte proliferation, are able to block the expression of the AdoMet synthetase gene and, consequently, AdoMet synthetase activity. Together these findings indicate that the AdoMet synthetase gene is subject to cell-cycle regulation in T-lymphocytes.

TheSAM2 gene product catalyzes the formation of S-adenosyl-ethionine from ethionine inSaccharomyces cerevisiae

Ethionine is the toxic S-ethyl analog of the essential amino acid methionine. Whereas in prokaryotes the ethionine just competes with the methionine, in eukaryotes it can also be transformed into S-adenosyl-ethionine (Ado-Eth), competing with the S-adenosyl-methionine (Ado-Met). When the Ado-Met synthetase activity was studied in strains defective in either of the two isoenzymes, the one coded by theSAM1 gene was totally unable to convert ethionine into Ado-Eth and was inhibited by the analog, whereas the enzyme coded by theSAM2 gene was able to bind ethionine and was not inhibited by it. This has allowed the development of a procedure to measure Ado-Met synthetase and differentiate between the two isoenzymes present inSaccharomyces cerevisiae.

Differential accumulation of S-adenosylmethionine synthetase transcripts in response to salt stress.

NaCl stress causes the accumulation of several mRNAs in tomato seedlings. An upregulated cDNA clone, SAM1, was found to encode a S-adenosyl-L-methionine synthetase enzyme (AdoMet synthetase). Expression of the cDNA SAM1 in a yeast mutant lacking functional SAM genes resulted in high AdoMet synthetase activity and AdoMet accumulation. We show that tomato plants contain at least four SAM isogenes. Clones corresponding to isogenes SAM2 and SAM3 have also been isolated and sequenced. They encode predicted polypeptides 95% and 92% identical, respectively, to the SAM1-encoded AdoMet Synthetase. RNA hybridization analysis showed a differential response of SAM genes to salt and other stress treatments. SAM1 and SAM3 mRNAs accumulated in the root in response to NaCl, mannitol or ABA treatments. SAM1 mRNA accumulated also in leaf tissue. These increases of mRNA level were apparent as soon as 8 h after the initiation of the salt treatment and were maintained for at least 3 days. A possible role for AdoMet synthetases in the adaptation to salt stress is discussed.

Modulation of rat liver S-adenosylmethionine synthetase activity by glutathione.

Rat liver S-adenosylmethionine (AdoMet) synthetase appears as high-M(r) (tetramer) and low-M(r) (dimer) forms. Both are inhibited in the presence of GSSG at pH 8. The calculated Ki values are 2.14 and 4.03 mM for the high- and low-M(r) forms, respectively. No effect on enzyme activity was observed in the presence of GSH, but modulation of inhibition by GSSG can be obtained by addition of GSH. At a total glutathione concentration (GSH + GSSG) of 10 mM, a KOX of 1.74 was calculated for the high-M(r) form, whereas this constant was 2.85 for the low-M(r) AdoMet synthetase. No incorporation of [35S]GSSG was observed in either of the enzyme forms, and inhibition of enzyme activity was correlated with dissociation of both AdoMet synthetases to a monomer. The data obtained in the presence of GSSG seem to suggest that oxidation leads to the formation of an intrasubunit disulfide. The possible regulation of AdoMet synthetase activity by the GSH/GSSG ratio is discussed, as well as its in vivo significance.

Age-related decline in S-adenosylmethionine and protein methyl esterification levels in bovine lenses

The enzymatic methyl esterification of proteins, catalyzed by the enzyme S-adenosylmethionine: protein carboxyl-O-methyl transferase (PCMT), has been investigated in two age-related fractions of bovine lenses. The eukaryotic PCMT methyl esterifies peptides and proteins containing altered aspartyl residues, such as D-aspartyls and L-isoaspartyls, which can arise from the age-dependent deamidation of labile Asn residues. When intact bovine lenses were incubated in a cell culture medium under physiological conditions (pH 7.4, 37 degrees C), in the presence of L-(methyl-(14)C)methionine, the in vivo precursor of the methyl donor substrate S-adenosylmethionine (AdoMet), a significant decrease in the level of protein methyl esterification was observed in the oldest cell fraction compared to the youngest one; in contrast, the in vitro activity of PCMT does not change during cell aging. It has also been demonstrated that the cell aging in the lens is accompanied by a reduction of the AdoMet synthetase activity and, consequently, of the endogenous level of the methyl donor AdoMet. Furthermore, the activity of the enzyme S-adenosylhomocysteine (AdoHcy) hydrolase, which cleaves the methylation inhibitor AdoHcy, also appears dramatically reduced in the oldest cells, probably leading to an increase in the AdoHcy cellular level. Finally, the possible correlation between the decrease in the efficiency of the overall methyl esterification process during aging and the accumulation of aberrant forms of proteins observed in aged tissues is also discussed.

Phytohormonal regulation of S-adenosylmethionine synthetase and S-adenosylmethionine levels in dwarf pea epicotyls

A significant stimulation (2- to 2.5-fold) of AdoMet synthetase was witnessed in glibberellicd acid (GA 3, 1μM)-treated epicotyls of the dwarf pea ( Pisum sativum). This was accompanied by a 2.4-fold increase in the endogenous pool of S-adenosylmethionine. Both abscisic acid (10 μM) and cycloheximide (20 μg ml ) inhibited the GA3-mediated enhancement of AdoMet synthetase activity. Three isozymes of AdoMet synthetase were detected in GA 3-treated epicotyls, whereas a single activity peak was observed in controls. Thus, GA 3 seems to control the induction of two new isozymes of AdoMet synthetase in the dwarf pea. By contrast, the tall pea exhibited three isozymes of AdoMet synthetase even in the absence of GA 3 treatment. High concentration of L-methionine (2 mM) mimicked the GA 3-elicited induction of two new isozymes of AdoMet synthetase in dwarf pea epicotyls.

Relative effects of S-adenosylmethionine depletion on nucleic acid methylation and polyamine biosynthesis.

Treatment of cultured L1210 cells with 1 mM-L-2-amino-4-methoxy-cis-but-3-enoic acid (L-cisAMB), a methionine-analogue inhibitor of S-adenosylmethionine (AdoMet) synthetase (EC 2.5.1.6), produced a rapid and near-total depletion of AdoMet by 4 h. After this, the pools recovered to 60% of control by 48 h, apparently because of an increase in AdoMet synthetase activity. Both AdoMet depletion and the accompanying increase in synthetase activity were substantially enhanced by lowering methionine concentrations in the media from 100 microM to 30 microM, the minimal concentration that supports cell growth at control values. During a 4 h incubation in media containing 30 microM-methionine, 1-5 mM-L-cisAMB depleted cellular AdoMet to undetectable values, and inhibited nucleic acid methylation by 44-72% and RNA methylation by 60-87%. Under these same treatment conditions, putrescine pools increased by about 3-fold, whereas spermidine pools decreased by only 20% and spermine pools remained the same. Pool changes were accompanied by a 2-4-fold increase in ornithine decarboxylase activities and AdoMet activities. Thus the rapid depletion of AdoMet pools by L-cisAMB results immediately in a decrease in methyl-transfer reactions involving nucleic acids, whereas, by contrast, biosynthesis of higher polyamines appears to be minimally affected, owing to compensatory increases in key enzyme activities.

Stimulation of S-adenosylmethionine synthetase in human lymphocytes by streptococcal M protein.

The effects of the specific antigen M5 protein of group A streptococci on AdoMet synthetase activity and AdoMet levels in peripheral blood (PB) lymphocytes were studied and were compared with the effects of the nonspecific polyclonal T cell mitogen PHA. M5 protein stimulated AdoMet synthetase activity, whereas PHA had a biphasic effect with an early inhibitory effect and a later stimulatory effect on AdoMet synthetase activity. S-Carbamyl-L-cysteine (SCC), an inhibitor of human lymphocyte AdoMet synthetase, reduced AdoMet levels and inhibited the blastogenic response of PB lymphocytes to both M5 protein and PHA. Inhibition of the response to M5 protein was stronger than that to PHA. However, the inhibitory effects of SCC were totally reversible by washing the cells. It is our hypothesis that such differences in the biochemical events triggered by specific antigen as opposed to a polyclonal mitogen may determine the direction of the functional differentiation of T lymphocytes.

Differential effects of dimethylsulfoxide on S-adenosylmethionine synthetase from rat liver and hepatoma

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