Protein methylation in animal cells. II. Inhibition of S-adenosyl-L-methionine:protein(arginine) N-methyltransferase by analogs of S-adenosyl-L-homocysteine

1. Protein methylase I (S-adenosyl-L-methionine: protein (arginine) N-methyltransferase, EC 2.1.1.23) has recently been purified in our laboratory from Krebs II ascites cells (Casellas, P. and Jeanteur, P. (1978) Biochim. Biophys. Acta 519, 243--254). In order to probe its binding site for S-adenosyl-L-methionine, three series of compounds deriving from the most potent competitive inhibitor, S-adenosyl-L-homocysteine, by specific alterations in each of the three regions of the molecule (amino acid side chain, ribose and adenine) have been tested for inhibitor activity. A competitive type of inhibition was assumed for all of them and demonstrated for five representative ones. The contribution of each of these regions to the binding could therefore be established as follows: (i) Any modification of the side chain results in a drop in affinity of about two orders of magnitude. Adenosine itself remained significantly inhibitory thereby demonstrating that the presence of a side chain was not critical, although important. (ii) The ribose moiety appears to be an essential part of the molecule as the loss of either 2'- or 3'-hydroxyls or their change to arabino configuration resulted in a nearly complete loss of activity. (iii) The amino group at position 6 and the nitrogen atom at position 7 of the adenine ring also play a crucial role although some substitutions can be tolerated. 2. S-Isobutyladenosine was shown to specifically inhibit the methylation of arginine residues as compared to lysine.     

Inhibitors of cyclic nucleotide phosphodiesterases inhibit protein carboxyl methylation in intact blood platelets

The cycle of protein-carboxyl methylation and demethylation was studied in intact blood platelets. Platelets rapidly incorporated L-[methyl-3H]methionine and after a delay of about 20 min, they evolved [3H]methanol. This evolution, and the amount of [3H] methanol liberated by treatment with base, was inhibited in a dose-dependent fashion by the cyclic nucleotide phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine, papaverine, dipyridamole, and RA233 (2,6-bis(diethanolamino)-4-piperidinopyrimido[5,4-d] pyrimidine). Each of these compounds increased the incorporation of [3H]methionine into platelets. The effects of RA233 were studied in more detail. Inhibition of [3H]methanol production was not potentiated by stimulators of the adenylate cyclase or the guanylate cyclase. The majority of the base-labile radioactivity was trichloroacetic acid precipitable. Thin layer chromatography of extracts of platelets incubated with L-[35S]methionine showed that RA233 did not induce a cellular accumulation of [35S]S-adenosylhomocysteine, and that it actually increased the amount of cellular [35S]S-adenosylmethionine. Discontinuous polyacrylamide gel electrophoresis at acid pH using the cationic detergent benzyldimethyl-n-hexadecylammonium chloride of platelets incubated with [3H]methionine showed incorporation of radioactivity into more than 30 protein bands, including one which co-migrates with calmodulin. The incorporation into the majority of these bands was inhibited by RA233 in a dose-dependent fashion. It is suggested that caution should be used in ascribing the pharmacological effects of known phosphodiesterase inhibitors to increases in cyclic nucleotides, because some of these effects could be due to inhibition of protein carboxyl methylation.     

Enhanced carboxyl methylation of membrane-associated hemoglobin in human erythrocytes

The alpha- and beta-chains of hemoglobin (Hb) are methylated in intact erythrocytes and in cellular extracts by a protein D-aspartate methyltransferase (EC 2.1.1.77) specific for D-aspartyl and L-isoaspartyl residues. During an 18-h incubation of intact erythrocytes with L-[methyl-3H]methionine, the subfraction of Hb molecules associated with the membrane becomes progressively enriched with methyl esters, reaching a specific activity 10-fold that of cytosolic Hb. The enhanced methylation of membrane Hb in intact cells appears not to result from its methylation at sites with inherently greater stability, since salt-extracted membrane Hb 3H-methyl esters and cytosolic Hb 3H-methyl esters are hydrolyzed at similar rates at pH 8.4 in vitro. Oxidative treatment of column-purified Hb with acetylphenylhydrazine produces an immediate 4-fold increase in its specific methyl-accepting activity coincident with the production of hemichrome forms known to possess a higher affinity for membrane binding sites. Together, the results suggest that the methyltransferase preferentially recognizes partially denatured Hb molecules which possess a higher affinity for membrane binding sites, similar to Hb forms observed in senescent erythrocytes.     

Inhibition of protein synthesis by polypeptide antibiotics. I. Inhibition in intact bacteria

Ennis, Herbert L. (St. Jude Children's Research Hospital, Memphis, Tenn.). Inhibition of protein synthesis by polypeptide antibiotics. I. Inhibition in intact bacteria. J. Bacteriol. 90:1102-1108. 1965.-The mechanism of inhibition of growth of cells by the polypeptide antibiotics of the PA 114, vernamycin, and streptogramin complexes was studied. This inhibition apparently was due to the selective inhibition of protein synthesis by these antibiotics. Ribonucleic acid synthesis was unaffected by concentrations of the antibiotics which completely inhibited protein synthesis. Deoxyribonucleic acid synthesis was slightly inhibited. These antibiotics are composed of a number of components. Mixtures of equal amounts of PA 114 A and PA 114 B or vernamycin A and Balpha were more active in stopping protein synthesis in intact cells than each of the components of the antibiotic complex alone. Mutants resistant to one of the antibiotics were resistant to all of the group and, in addition, were resistant to erythromycin and oleandomycin.     

Enzymatic methylation of band 3 anion transporter in intact human erythrocytes

Band 3, the anion transport protein of erythrocyte membranes, is a major methyl-accepting substrate of the intracellular erythrocyte protein carboxyl methyltransferase (S-adenosyl-L-methionine: protein-D-aspartate O-methyltransferase; EC 2.1.1.77) [Freitag, C., & Clarke, S. (1981) J. Biol. Chem. 256, 6102-6108]. The localization of methylation sites in intact cells by analysis of proteolytic fragments indicated that sites were present in the cytoplasmic N-terminal domain as well as the membranous C-terminal portion of the polypeptide. The amino acid residues that serve as carboxyl methylation sites of the erythrocyte anion transporter were also investigated. 3H-Methylated band 3 was purified from intact erythrocytes incubated with L-[methyl-3H]methionine and from trypsinized and lysed erythrocytes incubated with S-adenosyl-L-[methyl-3H]methionine. After proteolytic digestion with carboxypeptidase Y, D-aspartic acid beta-[3H]methyl ester was isolated in low yields (9% and 1%, respectively) from each preparation. The bulk of the radioactivity was recovered as [3H]methanol, and the amino acid residue(s) originally associated with these methyl groups could not be determined. No L-aspartic acid beta-[3H]methyl ester or glutamyl gamma-[3H]methyl ester was detected. The formation of D-aspartic acid beta-[3H]methyl esters in this protein in intact cells resulted from protein carboxyl methyltransferase activity since it was inhibited by adenosine and homocysteine thiolactone, which increases the intracellular concentration of the potent product inhibitor S-adenosylhomocysteine, and cycloleucine, which prevents the formation of the substrate S-adenosyl-L-[methyl-3H]methionine.     

Inhibition of Na,K-dependent adenosine triphosphatase by adenosine in intact pigeon erythrocytes

In intact pigeon erythrocytes, adenosine is a potent inhibitor of Na,K-dependent adenosine triphosphatase. In purified cell-membrane preparations, adenosine is only a weak competitive inhibitor of Na,K-ATPase, with respect to ATP. This indicates that adenosine must not be a direct inhibitor of the sodium pump in intact red cells per se; instead, adenosine exerts its inhibitory effect via endogenous cell factors.     

Protein carboxyl methylation in kidney brush-border membranes.

Protein carboxyl methylation activity was detected in the cytosol and in purified brush-border membranes (BBM) from the kidney cortex. The protein carboxyl methyltransferase (PCMT) activity associated with the BBM was specific for endogenous membrane-bound protein substrates, while the cytosolic PCMT methylated exogenous substrates (ovalbumin and gelatin) as well as endogenous proteins. The apparent Km for S-adenosyl-L-methionine with endogenous proteins as substrates were 30 microM and 4 microM for the cytosolic and BBM enzymes, respectively. These activities were sensitive to S-adenosyl-L-homocysteine, a well known competitor of methyltransferase-catalyzed reactions, but were not affected by the presence of chymostatin and E-64, two protein methylesterase inhibitors. The activity of both cytosolic and BBM PCMT was maximal at pH 7.5, while BBM-phospholipid methylation was predominant at pH 10.0. Separation of the = methylated proteins by acidic gel electrophoresis in the presence of the cationic detergent benzyldimethyl-n-hexadecylammonium chloride revealed distinct methyl accepting proteins in the cytosol (14, 17, 21, 27, 31, 48, 61 and 168 kDa) and in the BBM (14, 60, 66, 82, and 105 kDa). Most of the labelling was lost following electrophoresis under moderately alkaline conditions, except for a 21 kDa protein in the cytosol and a 23 kDa protein in the BBM fraction. These results suggest the existence of two distinct PCMT in the kidney cortex: a cytosolic enzyme with low selectivity and affinity, methylating endogenous and exogenous protein substrates, and a high-affinity BBM-associated methylating activity.     

Requirements of drug-induced endocytosis by intact human erythrocytes.

Study of drug-induced endocytosis in intact human erythrocytes continues to provide an opportunity for correlating membrane functions such as invagination and fusion with erythrocytic energetics and other determinants of plasma membrane function like Ca++. The studies reported indicate that high concentrations of vinblastine and chlorpromazine can produce endocytic vacuoles, albeit in reduced amounts, even in severely ATP depleted erythrocytes. In contrast, primaquine-induced endocytosis seems definitely dependent upon persistence of erythrocytic ATP stores. The ionophore mediated entry of Ca++ into erythrocytes potentiates primaquine endocytosis, inhibits vinblastine endocytosis, and has no regular effect on chlorpromazine endocytosis. Sodium lactate enhances primaquine endocytosis, probably by causing an increase in the entry of primaquine into erythrocytes. Cytochalasin B neither enhances nor inhibits erythrocytic endocytosis, thereby suggesting that microfibrils or analogues of microfibrils in erythrocytes are not involved in endocytosis. Cyclic nucleotide inhibition of endocytosis is confined to a very high concentration range of nucleotides in the medium. Primaquine and chlorpromazine endocytosis are inhibited by cyclic nucleotides as is vinblastine endocytosis.     

Methylation of microinjected isoaspartyl peptides in Xenopus oocytes. Competition with protein carboxyl methylation reactions

Xenopus oocytes possess a highly conserved protein carboxyl methyltransferase postulated to function in the repair or metabolism of age-damaged protein aspartyl residues (O'Connor, C. M. (1987) J. Biol. Chem. 262, 10398-10403). Three hexapeptides of the general sequence Val-Tyr-Pro-isoAsp-X-Ala, in which isoAsp represents an L-isoaspartyl residue and X represents Gly, Ser, or Ala, are methylated with the same order of preference following their microinjection into oocytes as in a purified system containing bovine brain protein carboxyl methyltransferase and S-adenosyl-L-[methyl-3H]methionine. The affinities of the enzyme for the glycyl, seryl, and alanyl variants of the peptides in vitro are 4.25, 3.04, and 1.67 microM, respectively. A nonapeptide of the sequence Lys-Ala-Ser-Ala-isoAsp-Leu-Ala-Lys-Tyr is a higher affinity substrate for the methyltransferase in vitro, characterized by a Km of 0.88 microM, but it is modified to a lesser extent in oocytes, partially because of its reduced stability in cytoplasm. The hexapeptide Val-Tyr-Pro-Asp-Gly-Ala, which contains an aspartyl residue in the usual stereoconfiguration, is not methylated either in vitro or in intact oocytes. Microinjection of any of the four isoaspartyl-containing peptides greatly stimulates total carboxyl methylation in oocytes, with rate increases ranging from 19- to 51-fold after the injection of 30 pmol of peptide. The protein ovalbumin is also modified following its microinjection into oocytes to near its calculated methyl-accepting capacity. Each of the isoaspartyl peptides can act as a competitive inhibitor of ovalbumin methylation both in vitro and in microinjected oocytes. The inhibitory potencies of the peptides parallel their specific methyl-accepting activities. The results demonstrate that the oocyte may be a useful model for studying the significance of protein carboxyl methylation because of the large functional excess of methylation capacity and the fidelity of the reactions compared to those observed in purified systems. This excess capability may have physiological significance when structurally abnormal proteins accumulate as a result of cellular stress and or aging.     

Inhibition by S-adenosyl-L-homocysteine of dopamine dependent adenylate cyclase in rat retina

S-adenosyl-L-homocysteine (S-AH), a potent inhibitor of biological transmethylation, decreased the response of rat retina adenylate cyclase to dopamine and to 2-amino-6, 7-dihydroxytetrahydronaphtalene (ADTN). This effect appeared for 10^?7M of S-adenosyl-L-homocysteine and was linear for concentration ranging to 10^?4M. S-adenosyl-L-homocysteine did not decrease the cyclic AMP accumulation with sodium fluoride, a non specific adenylate cyclase activator. On the other hand, the incorporation of methyl group was reduced in rat retina homogenates by S-adenosyl-L-homocysteine. These findings suggest that the activity of the dopamine dependent adenylate cyclase is linked to a methylation process.     

Effects of farnesylcysteine analogs on protein carboxyl methylation and signal transduction

Several proteins associated with signal transduction in eukaryotes are carboxyl methylated at COOH-terminal S-farnesylcysteine residues. These include members of the Ras superfamily and gamma-subunits of heterotrimeric G-proteins. The enzymes that catalyze the carboxyl methylation reaction also methylate small molecules such as N-acetyl-S-trans, trans-farnesyl-L-cysteine (AFC). AFC inhibits carboxyl methylation of p21ras and related proteins both in vitro and in vivo. Saturating concentrations of AFC cause a greater than 80% inhibition of chemotactic responses of mouse peritoneal macrophages. Our results suggest that carboxyl methylation may play a role in the regulation of receptor-mediated signal transduction processes in eukaryotic cells.     

Hypoxanthine-guanine exchange by intact human erythrocytes

The uptake and release of [14C]hypoxanthine by human erythrocytes, suspended in a tris(hydroxymethyl)aminomethane (Tris)-glucose-NaCl isotonic medium (pH 7.4), have been studied at 37 degrees C. The uptake of hypoxanthine, mediated by its incorporation into inosine 5'-monophosphate (IMP), was markedly stimulated by preincubating the cells in phosphate-buffered saline. After a lag time, [14C]IMP-enriched erythrocytes released [14C]hypoxanthine in the medium. Formycin B, at concentrations known to inhibit purine nucleoside phosphorylase in intact erythrocytes, affected hypoxanthine uptake and release and led to an increase in the intracellular concentration of inosine, suggesting that the main catabolic path of IMP is the sequential degradation of the nucleotide to inosine and hypoxanthine. The addition of guanine to a suspension of [14C]IMP-enriched erythrocytes led to an increase in the rate of [14C]hypoxanthine release, which was unaffected by the presence of formycin B. During the guanine-induced hypoxanthine release, guanine was taken up by the cells as GMP. These results suggest that the presence of guanine in the incubation medium activates a catabolic path in human erythrocytes leading to IMP degradation without formation of inosine.     

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.     

Inhibition of GTPgammaS-dependent L-isoaspartyl protein methylation by tyrosine kinase inhibitors in kidney

Protein carboxyl methylation in rat kidney cytosol is increased by the addition of guanosine 5'-O-[gamma-thio]triphosphate (GTPgammaS), a non-hydrolysable analogue of GTP. GTPgammaS-stimulated methyl ester group incorporation takes place on isoaspartyl residues, as attested by the alkaline sensitivity of the labelling and its competitive inhibition by L-isoaspartyl-containing peptides. GTPgammaS was the most potent nucleotide tested, whereas GDPbetaS and ATPgammaS also stimulated methylation but to a lesser extent. Maximal stimulation (5-fold) of protein L-isoaspartyl methytransferase (PIMT) activity by GTPgammaS was reached at a physiological pH in the presence of 10 mM MgCl2. Other divalent cations, such as Cu2+, Zn2+ and Co2+ (100 microM), totally inhibited GTPgammaS-dependent carboxyl methylation. The phosphotyrosine phosphatase inhibitor vanadate potentiated the GTPgammaS stimulation of PIMT activity in the kidney cytosol at a concentration lower than 40 microM, but increasing the vanadate concentration to more than 40 microM resulted in a dose-dependent inhibition of the GTPgammaS effect. The tyrosine kinase inhibitors genistein (IC50 = 4 microM) and tyrphostin (IC50 = 1 microM) abolished GTPgammaS-dependent PIMT activity by different mechanisms, as was revealed by acidic gel analysis of methylated proteins. Whereas tyrphostin stabilised the methyl ester groups, genistein acted by blocking a crucial step required for the activation of PIMT activity by GTPgammaS. The results obtained with vanadate and genistein suggest that tyrosine phosphorylation regulates GTPgammaS-stimulated PIMT activity in the kidney cytosol.