Effect of methylation inhibitors on gene expression in HL-60 cells.

The methylation inhibitors Neplanocin A (Nep A), 3'-deazaadenosine (dzAdo), and 3-deaza(+/-)aristeromycin (Dari) were tested for their effect on the expression of histone H2B, actin, and the protooncogenes c-myc, and v-fos. Nep A and Dari bind to the S-adenosylhomocysteine hydrolase resulting in the accumulation of S-adenosylhomocysteine, while dzAdo served as a substrate for the enzyme. With dzAdo, inordinant amounts of 3-deazaadenosylhomocysteine (dzAdoHcy) accumulated in the cell, provided L-homocysteine (Hcy) was present. When added at sublethal concentrations, the methylation inhibitors had little or no effect on c-myc, v-fos, histone H2B, or actin expression, nor did any significant number of the drug-treated cells demonstrate myeloid characteristics. However, growth and gene expression were markedly inhibited upon the addition of Hcy and dzAdo. One of the earliest effects of dzAdoHcy on HL-60 cells was the disappearance of c-myc mRNA. Within 1 h of the addition of dzAdo and Hcy, only trace amounts of c-myc mRNA were detectable. After 4-5 h v-fos, histone H2B, and actin mRNAs also decreased to about 40% of control levels. Differences in the stability of preexisting mRNAs would appear to account for these results. Within 1 h following the addition of dzAdo and Hcy, the synthesis of rRNA and mRNA were completely blocked as measured by the incorporation of [3H]uridine.     

Inhibitory effect of Epimedium extract on S-adenosyl-L-homocysteine hydrolase and biomethylation

In the present paper, the inhibitory effect of Epimedium extract on the activity of S-adenosyl-L-homocysteine (AdoHcy) Hydrolase was studied. The results showed that Epimedium extract inhibited the activity of recombinant human AdoHcy hydrolase in a dose-dependent manner. This inhibitory effect was also observed in hepatic cell line 7701 and hepatoma HepG2, however, the effect in 7701 cells was more potent than in HepG2 cells. The extract could significantly reduce AdoMet/AdoHcy ratio in 7701 cells in a dose-dependent manner, suggesting reduced biomethylation level in 7701 cells. In contrast, it resulted in elevated AdoMet/AdoHcy ratio in the HepG2 cells. The result of MALDI-MS assay indicated that epimedin A and ikarisoside F from the extract could bind to AdoHcy hydrolase. The present data suggested that Epimedium extract could inhibit the activity of AdoHcy hydrolase, thus regulating the cellular biomethylation as well as reducing cellular Hcy level. These results will provide new clues to the mechanisms of Epimedium in curing of cardiovascular disease and regulating tumor cell growth.     

Altered methionine metabolism occurs in all members of a set of diverse human tumor cell lines

Methionine dependence is a metabolic defect found thus far only in transformed and malignant cells. The defect is manifested as the inability of cells to grow in media in which methionine (Met) is replaced by its immediate precursor homocysteine (Hcy). We have termed this Met ^? Hcy ⁺ media. We demonstrate here that methionine-dependent cells derived from human tumors, compared to normal methionine-independent cells, have low levels of free Met, low levels of S-adenosylmethionine (AdoMet) and elevated levels of S-adenosylhomocysteine (AdoHcy) when incubated in Met ^? Hcy ⁺ medium. Methionine-independent human tumor cells also have very low levels of free Met compared to normal cells but generally have levels of AdoMet and AdoHcy comparable to normal cells in Met ^? Hcy⁺ medium. All tumor cell types incorporate amounts of Met into protein similar to normal methionine-pindependent human fibroblasts when incubated in Met ^? Hcy⁺ medium, thereby indicating apparently normal levels of Met synthesis in the tumor cells. The methionine-independent tumor cell lines in Met ^? Hcy⁺ medium seem able to regulate their AdoMet/AdoHcy ratios normally despite this defect in having very low levels of free Met. Thus, in a diverse set of human tumor cell lines, all are defective in at least one aspect of Met metabolism, giving rise to the possibility of a general metabolic defect in cancer.     

S-Adenosyl-l-homocysteine in brain

Administration of methionine sulfoximine (MSO) to rats and mice significantly decreased cerebral levels ofS-adenosyl-l-homocysteine (AdoHcy). Concurrent administration of methionine prevented this decrease and, when methionine was given alone, significantly elevated AdoHcy levels resulted in both species. Regionally, AdoHcy levels varied from 20 nmol/g in rat cerebellum and spinal cord to about 60 nmol/g in hypothalamus and midbrain. MSO decreased AdoHcy in all regions tested except striatum, midbrain, and spinal cord. AdoMet/AdoHcy ratios (methylation index) varied from 0.48 in hypothalamus to 2.4 in cerebellum, and MSO administration decreased these ratios in all regions except hypothalamus. AdoHcy hydrolase activity was lowest in hypothalamus, highest in brainstem and, generally, varied inversely with regional AdoHcy levels. MSO decreased AdoHcy hydrolase activity in all regions except hypothalamus and spinal cord. Cycloleucine administration resulted in significantly decreased levels of mouse brain AdoHcy, whereas the administration of dihydroxyphenylalanine (DOPA) failed to affect AdoHcy levels. It is concluded that (a) cerebral AdoHcy levels are more tightly regulated than are those of AdoMet after MSO administration, (b) slight fluctuations of AdoHcy levels may be important in regulating AdoHcy hydrolase activity and hence AdoHcy catabolism in vivo, (c) the AdoMet/AdoHcy ratio reflects the absolute AdoMet concentration rather than the transmethylation flux, (d) the decreased AdoMet levels in midbrain, cortex, and striatum after MSO with no corresponding decrease in AdoHcy suggest an enhanced AdoMet utilization, hence an increased transmethylation in the MSO preconvulsant state.Supported by USPHS, NINCDS grant NS-06294.     

Inhibition of macrophage phagocytosis by methylation inhibitors. Lack of correlation of protein carboxymethylation and phospholipid methylation with phagocytosis

Adenosine (Ado), deoxyadenosine (dAdo), and adenine arabinoside (AraA) inhibit the phagocytosis of IgG-coated erythrocytes and zymosan by resident and thioglycollate-elicited macrophages (thio-macrophages) in a dose-dependent and reversible manner. 3-Deazaadenosine (3cAdo) and adenine (Ade) also inhibit the phagocytosis by resident macrophages. Homocysteine thiolactonate (Hcy) potentiates the inhibition by Ado and 3cAdo while erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) potentiates the inhibition by Ado, dAdo and AraA. This inhibition has a very rapid onset and the drugs do not interfere with the binding of IgG-coated erythrocytes to macrophages. The combination of Ado, Hcy and EHNA does not appreciably affect the intracellular level of ATP and S-adenosyl-L-methionine (AdoMet) in thio-macrophages but causes accumulations of Ado and S-adenosyl-L-homocysteine (AdoHcy) up to 135 and 145 nmol/mg of protein, respectively. During phagocytosis reversal, Ado is metabolized within 15 min while AdoHcy decreases log-arithmically with a half-life of 50 min. Carboxymethylation and phospholipid methylation, however, resume about 60-90 min after phagocytosis has recovered, and thus cannot function as transmembrane signals for phagocytosis. Other evidence showing the lack of correlation between phagocytosis and carboxymethylation inhibition include 1) Ado + Hcy inhibit carboxymethylation much better than Ado + EHNA (91 versus 75%) in thio-macrophage, but the two combinations show comparable phagocytosis inhibition potency; 2) Ado + Hcy inhibit carboxymethylation almost as well as Ado + Hcy + EHNA, but the latter is a much more effective drug combination for phagocytosis inhibition; 3) Ade and 3cAdo, although inhibiting resident macrophage phagocytosis as well as Ado + EHNA + Hcy, are much weaker carboxymethylation inhibitors; 4) dAdo and AraA potently inhibit phagocytosis but not carboxymethylation. The difference in the apparent methylation levels is not due to changes in the specific activities of AdoMet, which decrease with a half-life of 88 min. Interestingly, after the initial lag phase of about 90 min after the initiation of inhibition reversal, carboxymethylation and phagocytosis increase in parallel. In a log-log plot of carboxymethylation, phospholipid methylation, or phagocytosis versus the intracellular AdoHcy accumulation, a linear relationship is obtained. It is possible that AdoHcy accumulation is responsible for phagocytosis inhibition but inhibits by a mechanism other than interfering with protein and lipid methylations.     

Altered levels of S-adenosylmethionine and S-adenosylhomocysteine in the brains of L-isoaspartyl (D-Aspartyl) O-methyltransferase-deficient mice

L-Isoaspartyl (D-aspartyl) O-methyltransferase (PCMT1) is a protein repair enzyme that initiates the conversion of abnormal D-aspartyl and L-isoaspartyl residues to the normal L-aspartyl form. In the course of this reaction, PCMT1 converts the methyl donor S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy). Due to the high level of activity of this enzyme, particularly in the brain, it seemed of interest to investigate whether the lack of PCMT1 activity might alter the concentrations of these small molecules. AdoMet and AdoHcy were measured in mice lacking PCMT1 (Pcmt1-/-), as well as in their heterozygous (Pcmt1+/-) and wild type (Pcmt1+/+) littermates. Higher levels of AdoMet and lower levels of AdoHcy were found in the brains of Pcmt1-/- mice, and to a lesser extent in Pcmt1+/- mice, when compared with Pcmt1+/+ mice. In addition, these levels appear to be most significantly altered in the hippocampus of the Pcmt1-/- mice. The changes in the AdoMet/AdoHcy ratio could not be attributed to increases in the activities of methionine adenosyltransferase II or S-adenosylhomocysteine hydrolase in the brain tissue of these mice. Because changes in the AdoMet/AdoHcy ratio could potentially alter the overall excitatory state of the brain, this effect may play a role in the progressive epilepsy seen in the Pcmt1-/- mice.     

Biotin synthase exhibits burst kinetics and multiple turnovers in the absence of inhibition by products and product-related biomolecules

Biotin synthase (BS) is a member of the "SAM radical" superfamily of enzymes, which catalyze reactions in which the reversible or irreversible oxidation of various substrates is coupled to the reduction of the S-adenosyl-l-methionine (AdoMet) sulfonium to generate methionine and 5'-deoxyadenosine (dAH). Prior studies have demonstrated that these products are modest inhibitors of BS and other members of this enzyme family. In addition, the in vivo catalytic activity of Escherichia coli BS requires expression of 5'-methylthioadenosine/S-adenosyl-l-homocysteine nucleosidase, which hydrolyzes 5'-methylthioadenosine (MTA), S-adenosyl-l-homocysteine (AdoHcy), and dAH. In the present work, we confirm that dAH is a modest inhibitor of BS (K(i) = 20 μM) and show that cooperative binding of dAH with excess methionine results in a 3-fold enhancement of this inhibition. However, with regard to the other substrates of MTA/AdoHcy nucleosidase, we demonstrate that AdoHcy is a potent inhibitor of BS (K(i) ≤ 650 nM) while MTA is not an inhibitor. Inhibition by both dAH and AdoHcy likely accounts for the in vivo requirement for MTA/AdoHcy nucleosidase and may help to explain some of the experimental disparities between various laboratories studying BS. In addition, we examine possible inhibition by other AdoMet-related biomolecules present as common contaminants in commercial AdoMet preparations and/or generated during an assay, as well as by sinefungin, a natural product that is a known inhibitor of several AdoMet-dependent enzymes. Finally, we examine the catalytic activity of BS with highly purified AdoMet in the presence of MTAN to relieve product inhibition and present evidence suggesting that the enzyme is half-site active and capable of undergoing multiple turnovers in vitro.     

Adenosine dialdehyde and neplanocin A: Potent inhibitors of S-adenosylhomocysteine hydrolase in neuroblastoma N2a cells

S-Adenosylhomocysteine hydrolase (AdoHcy hydrolase, E.C. 3.3.1.1) catalyzes the metabolism of S-adenosylhomocysteine (AdoHcy) to adenosine (Ado) and homocysteine (Hcy) in mouse neuroblastoma N2a cells. AdoHcy hydrolase in N2a cells can be inhibited completely by adenosine dialdehyde (Ado dialdehyde) or neplanocin A. The inhibitory effects of Ado dialdehyde (2.5 μM) and neplanocin A (1 μM) on cellular AdoHcy hydrolase were time-dependent, with total enzyme inhibition occurring after 30 min and 15 min of incubation, respectively. The inhibition of AdoHcy hydrolase produced by Ado dialdehyde and neplanocin A persisted for up to 72 h of incubation, and was paralleled by a time-dependent increase in endogenous AdoHcy levels reaching a maximum 4-fold elevation after 8 h of incubation with Ado dialdehyde and an 11-fold increase in the neplanocin A-treated cells. This increase in AdoHcy levels produced a subsequent inhibition of S-adenosylmethionine (AdoMet)-dependent cellular methylations (e.g. protein carboxylmethylation (PCM), lipid methylation). In addition, neplanocin A was metabolically converted to the corresponding AdoMet analog, S-neplanocylmethionine (NepMet), in neuroblastoma N2a cells. NepMet reached maximum levels after 8 h of incubation of the cells with neplanocin A.     

Monoamine oxidase A from human placenta and monoamine oxidase B from bovine liver both have one FAD per subunit.

Choline and C1 metabolism pathways intersect at the formation of methionine from homocysteine. Hepatic S-adenosylmethionine (AdoMet) concentrations are decreased in animals ingesting diets deficient in choline, and it has been suggested that this occurs because the availability of methionine limits AdoMet synthesis. If the above hypothesis is correct, changes in hepatic AdoMet concentrations should relate in some consistent manner to changes in hepatic methionine concentrations. Rats were fed on a choline-deficient or control diet for 1-42 days. Hepatic choline concentrations in control animals were 105 nmol/g, and decreased to 50% of control after the first 7 days on the choline-deficient diet. Hepatic methionine concentrations decreased by less than 20%, with most of this decrease occurring between days 3 and 7 of choline deficiency. Hepatic AdoMet concentrations decreased by 25% during the first week, and continued to decrease (in total, by over 60%) during each subsequent week during which animals consumed a choline-deficient diet. Hepatic S-adenosylhomocysteine (AdoHcy) concentrations increased by 50% when animals consumed a choline-deficient diet. AdoHcy is formed when AdoMet is utilized as a methyl donor. In summary, choline deficiency can deplete hepatic stores of AdoMet under dietary conditions that only minimally decrease the availability of methionine within liver. Thus decreased availability of methionine may not have been the only mechanism whereby choline deficiency lowers hepatic AdoMet concentrations. We suggest that increased utilization of AdoMet might also have occurred.     

Decreased Transmethylation of Biogenic Amines After In Vivo Elevation of Brain S-Adenosyl-l-Homocysteine

Abstract: The ability of S -adenosyl- l -homocysteine (AdoHcy) to inhibit biologic transmethylation reactions in vitro has led us to explore the possibility of pharmacologically manipulating AdoHcy levels in vivo and examining the consequences of these alterations on the transmethylation of some biogenic amines. Swiss-Webster mice were injected intraperitoneally with different doses of adenosine (Ado) and d,l -homocysteine thiolactone (Hcy) and were killed at various times thereafter. S -Adenosyl- l -methionine (AdoMet) and AdoHcy concentrations were determined by using a modified isotope dilution-ion exchange chromatography-high pressure liquid chromatography technique sensitive to less than 10 pmol. Increasing doses of Ado + Hcy (50-1000 mg/kg of each) produced a dose-related increase in blood, liver, and brain AdoHcy levels. At a dose level of 200 mg/kg Ado + Hcy, AdoHcy levels were markedly elevated, with minimal concomitant perturbations of AdoMet. This elevation was maximal 40 min after giving Ado + Hcy, returning to control values within 6 h. Ado + Hcy treatment resulted in decreased activities of catechol- O -methyltransferase, histamine- N -methyltransferase, and AdoHcy hydrolase in vitro. The cerebral catabolism of intraventricularly administered [³H]histamine (HA) was decreased in a dose-related manner by Ado + Hcy treatment as evidenced by higher amounts of nonutilized [³H]HA in brain, concurrent decreases in [³H]methylhistamine formation, and decreases in the transmethylation conversion index. Steady state levels of HA also showed dose-related increases after Ado + Hcy treatment. It is concluded that injections of Ado + Hcy can markedly elevate AdoHcy levels in vivo , which can, in turn, decrease the rate of transmethylation reactions.     

The Methylation Cycle and its Possible Functions in Barley Endosperm Development

Barley endosperm development can be subdivided into the pre-storage, intermediate, storage and desiccation phase. Nothing is known about DNA methylation events involved in different endosperm-specific developmental programmes. A complete set of methylation cycle enzyme genes was identified and investigated by mRNA expression analysis. During the pre-storage phase, methionine synthase and S-adenosylmethionine (AdoMet) synthase genes are expressed at high levels, mainly to produce AdoMet, which might be used for methylation processes as indicated by high expression of methyltransferases HvMET1, HvCMT1 and HvDnmt3-1 as well as AdoHcy hydrolase genes. The methyltransferases, core histones and DNA-unwinding ATPases are co-expressed at the mRNA level. On the contrary, storage protein (prolamin) gene expression is repressed due to CpG methylation. Expression of genes responsible for starch biosynthesis is also developmentally regulated but not methylation-dependent. Thus, during pre-storage phase, activity of HvMET1 and HvCMT1 possibly maintains DNA replication and suppresses specific pathways of maturation. Besides, HvDnmt3-1 might be responsible for differentiation-specific de novo methylation. Expression of methyltransferases HvDnmt3-2 and HvCMT2 peaks during the onset of massive starch accumulation. The enzymes are likely responsible for DNA methylation involved in determining plastid division and amyloplast differentiation as concluded from the patterns of co-expressed genes. Levels of AdoMet decarboxylase mRNA, but not methyltransferase- and AdoHcy mRNA, increase at the beginning of desiccation together with methionine synthase and AdoMet synthase levels. This increase may be indicative for utilization of AdoMet in polyamine production protecting aleuron and embryo cell membranes during desiccation.     

Purification and characterization of a novel NADPH(NADH)-dependent hydroxypyruvate reductase from spinach leaves. Comparison of immunological properties of leaf hydroxypyruvate reductases.

5'-Deoxy-5'-methylthioadenosine, a by-product of polyamine synthesis, can support the growth of Raji cells in a methionine-free medium, but not the growth of CCL39 cells, although these cells are also able to incorporate radiolabelled 5'-deoxy-5'-methylthioadenosine (MeSAdo) into methionine, S-adenosyl-L-methionine (AdoMet) and proteins [Christa, Kersual, Augé & Pérignon (1986) Biochem. Biophys. Res. Commun. 135, 131-138]. We first tested the hypothesis of a toxic effect of MeSAdo in the conditions of growth experiments: we could not demonstrate any toxic effect of MeSAdo on the synthesis of macromolecules, nor any toxicity mediated by polyamines or pyrimidine starvation, and we found that the growth of CCL39 cells was strictly dependent on the supply of exogenous methionine. We then tried to determine whether the ability of CCL39 cells to metabolize MeSAdo to methionine and AdoMet was modulated by the proliferation state of CCL39 cells, which is dependent on the supply of exogenous methionine. Studies of the incorporation of radiolabelled MeSAdo show that: (i) the total synthesis of methionine from MeSAdo is twice as high in subconfluent cells (grown in 100 microM-methionine) as in resting cells (cultured in 0 microM-methionine); (ii) the incorporation into proteins does not parallel the total protein synthesis, and the methionine derived from MeSAdo mostly flows out of the cell; (iii) addition of methionine to resting cells immediately leads to a transient and marked increase in metabolism of MeSAdo to AdoMet, presumably reflecting the rapid replenishment of the AdoMet pool of the cells. Taken together, these results suggest that the methionine derived from MeSAdo is preferentially used to synthesize AdoMet rather than proteins, and that this synthesis of AdoMet depends on the ability of the CCL39 cells to grow, and hence on the supply of exogenous methionine. It is proposed that, in CCL39 cells, the metabolic pathway leading from MeSAdo (a by-product of polyamine synthesis) to methionine and to AdoMet (a precursor of polyamine synthesis) is part of a metabolic cycle the activity of which depends, like polyamine synthesis itself, on cell proliferation.     

S-adenosylhomocysteine metabolism in different cell lines: effect of hypoxia and cell density.

BACKGROUND/AIMS: The methylation potential (MP) is defined as the ratio of S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy). It was shown recently that hypoxia increases AdoMet/AdoHcy ratio in HepG2 cells (Hermes et al., Exp Cell Res 294: 325-334, 2004). In the present study, we compared AdoMet/AdoHcy ratio and energy metabolism in HepG2, HEK-293, HeLa, MCF-7 and SK-HEP-1 cell lines under normoxia and hypoxia. METHODS: Metabolite concentrations were measured by HPLC. In addition, AdoHcy hydrolase (AdoHcyase) activity was determined photometrically. RESULTS: Under normoxia HepG2 cells show the highest AdoMet/AdoHcy ratio of 53.4 +/- 3.3 followed by MCF-7 and SK-HEP-1 cells with a AdoMet/AdoHcy ratio of 14.4 +/- 1.1 and 21.1 +/- 1.3, respectively. The lowest AdoMet/AdoHcy ratios are exhibited by HeLa and HEK-293 cells (6.6 +/- 0.7 and 7.1 +/- 0.3). Hypoxia does not significantly change the MP in MCF-7 and HeLa cells, but alters the MP in HepG2, HEK-293 and SK-HEP-1 cells. These alterations are dependent on the cell density. Under normoxia HepG2 cells exhibit AdoHcyase activity of 2.5 +/- 0.2 nmol min(-1) mg(-1) protein. All other cell lines show 3-5 times lower enzyme activity. Interestingly, hypoxia affects AdoHcyase activity only in HepG2 cells. CONCLUSIONS: Our data clearly show that the cell lines are characterized by different MP and different behavior under hypoxia. That implies that a lower MP is not necessarily associated with impaired transmethylation activity and cellular function.     

Quantitative evaluation and regulation of S-adenosylmethionine-dependent transmethylation in sheep tissues

The overall rates of S-adenosylmethionine (AdoMet)-dependent transmethylation were estimated in various tissues from the initial rate of S-adenosylhomocysteine (AdoHcy) plus AdoMet accumulation after blocking hydrolysis of AdoHcy. The rates were found to differ widely among the tissues of sheep and the highest rate was in the pancreas, being 600 times higher than that in the muscle. Sheep liver possessed approximately 75% of total-body capacity for transmethylation although the transmethylation rate was approximately half that in rat liver. The minimum estimate of daily requirement of AdoMet for transmethylation for adult sheep was approximately 18 mmol, far in excess of methionine intake. Methionine loading elevated AdoMet levels only in the tissues with a high or moderate rate of transmethylation. The kinetic properties of major methyltransferases in sheep liver along with tissue distribution of AdoMet and AdoHcy suggest that transmethylation rate is subject to physiological regulation by tissue levels of AdoMet and AdoHcy.     

Increase in S-adenosylhomocysteine concentration in interferon-treated HeLa cells and inhibition of methylation of vesicular stomatitis virus mRNA

A fraction of the viral mRNA synthesized in interferon-treated HeLa cells infected with vesicular stomatitis virus (VSV) lacks the 7-methyl group in the 5'-terminal guanosine of the cap; this mRNA is not associated with polyribosomes and does not bind to ribosomes in an assay for initiation of protein synthesis (de Ferra, F., and Baglioni, C. (1981) Virology 112, 426-435). To establish whether this defect in methylation is due to changes in the level of the methyl donor S-adenosylmethionine (AdoMet) and of its competitive inhibitor S-adenosylhomocysteine (AdoHcy), we measured the concentration of these compounds in HeLa cells treated with interferon. An increase in both AdoMet and AdoHcy was detected 3 to 6 h after addition of interferon. The level of these compounds increased gradually and in proportion to the interferon concentration used. With 125 reference units/ml of beta interferon, for example, the AdoHcy concentration increased more than 3-fold and that of AdoMet about 1.5-fold with a consequent change in the AdoHcy/AdoMet ratio. An increased AdoHcy/AdoMet ratio was also found in HeLa cells treated with pure alpha 2 interferon produced in Escherichia coli by recombinant DNA techniques. When the methylation of VSV mRNA was measured in assays carried out with permeabilized virions at the AdoHcy and AdoMet concentrations found in interferon-treated cells, a preferential inhibition of the viral (guanine-7-)methyltransferase activity was observed. Such an inhibition may account for the synthesis of VSV mRNA lacking the 7-methyl group of guanosine in the cap.     

Protein Arginine Methylation Is More Prone to Inhibition by S-Adenosylhomocysteine than DNA Methylation in Vascular Endothelial Cells

Methyltransferases use S-adenosylmethionine (AdoMet) as methyl group donor, forming S-adenosylhomocysteine (AdoHcy) and methylated substrates, including DNA and proteins. AdoHcy inhibits most methyltransferases. Accumulation of intracellular AdoHcy secondary to Hcy elevation elicits global DNA hypomethylation. We aimed at determining the extent at which protein arginine methylation status is affected by accumulation of intracellular AdoHcy. AdoHcy accumulation in human umbilical vein endothelial cells was induced by inhibition of AdoHcy hydrolase by adenosine-2,3-dialdehyde (AdOx). As a measure of protein arginine methylation status, the levels of monomethylarginine (MMA) and asymmetric and symmetric dimethylated arginine residues (ADMA and SDMA, respectively) in cell protein hydrolysates were measured by HPLC. A 10% decrease was observed at a 2.5-fold increase of intracellular AdoHcy. Western blotting revealed that the translational levels of the main enzymes catalyzing protein arginine methylation, protein arginine methyl transferases (PRMTs) 1 and 5, were not affected by AdoHcy accumulation. Global DNA methylation status was evaluated by measuring 5-methylcytosine and total cytosine concentrations in DNA hydrolysates by LC-MS/MS. DNA methylation decreased by 10% only when intracellular AdoHcy concentration accumulated to 6-fold of its basal value. In conclusion, our results indicate that protein arginine methylation is more sensitive to AdoHcy accumulation than DNA methylation, pinpointing a possible new player in methylation-related pathology.     

The effect of S-adenosyl methionine on the TSH receptor function in human thyroid tissue: increase in binding of TSH and decrease in adenylate cyclase coupling

To determine the effects of S-Adenosyl methionine (AdoMet) on TSH receptor function and adenylate cyclase coupling, human thyroidal crude membrane fraction was pretreated with AdoMet and with S-Adenosyl homocysteine (AdoHcy), separately or in combination. 125I-TSH binding to the pretreated membrane and adenylate cyclase activity of the membrane were examined. In contrast to the reported effect of AdoMet on the decrease in GH binding to lactogenic receptor, AdoMet 0.5 mumoles/ml significantly increased the binding of TSH to the receptor by increasing the affinity of the binding, whereas it decreased the coupling of adenylate cyclase significantly. The effect of AdoMet was partially counteracted by the pretreatment of the membrane with AdoHcy. This effect of AdoMet is very similar to that of diamide previously reported. The result implies that the effect is due to an alteration in the tertiary structure of receptor protein triggered by methylation.     

Tissue-specific effects of testosterone on S-adenosylmethionine formation and utilization in the mouse.

Exogenous administration of testosterone produced several metabolic tissue-specific changes in female mouse kidneys, but not in the liver. The hormone induced ornithine decarboxylase (ODC) activity, and also profoundly influenced metabolism of S-adenosylmethionine (AdoMet). Therefore, the activity of the AdoMet-synthesizing enzyme (AdoMet synthetase) and of cystathionine synthase, which commits homocysteine irreversibly to the transsulfuration pathway, were significantly increased. In contrast to the level of AdoMet in the liver the renal level of this metabolite was augmented, whereas the level of S-adenosylhomocysteine (AdoHcy) did not change. This resulted in an increase of the AdoMet/AdoHcy ratio. In testosterone-treated mice, pulse-labelled with [methyl-14C]methionine, the radioactivity recovered in the kidneys doubled, but in the liver remained the same. The rise in radioactivity recovered occurred mainly in TCA-soluble compounds and lipids, and to a smaller extent, in proteins and nucleic acids.     

The role of hyperhomocysteinemia in nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilatation.

Hyperhomocysteinemia (HHcy) is associated with impaired endothelial-dependent vasodilatation and increased risk of atherosclerosis and thrombosis. Here, we summarize some of our previous work on the effect of HHcy on pathways involved in endothelium-dependent vasodilatation, and present new data concerning the endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilatation. We showed that the 894 G>T single-nucleotide polymorphism in the human endothelial nitric oxide synthase gene (eNOS) increased the risk of recurrent venous thrombosis in individuals with elevated homocysteine levels, indicating that the pathophysiological mechanism in HHcy involves impaired NO-mediated vasodilatation. In addition, the EDHF-mediated vasodilatation of the renal artery was disturbed in diet-induced hyperhomocysteinemic rats. Interestingly, we demonstrated that pretreatment of rats with periodate-oxidized adenosine (Adox), which is an inhibitor of S-adenosylhomocysteine hydrolase, prevented the methionine-induced rise in plasma total Hcy (tHcy) levels but not the inhibition of the EDHF pathway. Furthermore, we demonstrated that S-adenosylhomocysteine (AdoHcy) and S-adenosylmethionine (AdoMet) levels were increased in the kidneys of diet-induced HHcy rats, resulting in a decreased AdoMet:AdoHcy ratio. In addition, we demonstrated that mRNA expression of Connexin 40, which is one of the structural subunits of gap-junctions, was down-regulated in endothelial cells of HHcy rats, and correlated with elevated AdoHcy levels in kidney of these rats. These finding suggest a key role for AdoHcy in relation to decreased Cx40 mRNA expression and impaired EDHF-mediated vasodilatation of HHcy rats.