Increased methylation of endogenous 20-kDa protein in HIT beta-cell during insulin secretion

Enzymatic methylation of endogenous proteins in clonal pancreatic beta-cell, HIT-T15, was investigated. When cell extract incubated with S-adenosyl-L-[methyl-3H]methionine was subjected to SDS-PAGE followed by fluorography, endogenous 20-kDa protein was highly [methyl-3H]-labeled. The increase of methylation was correlated with insulin secretion, when the cells were treated with secretagogue; at 5.5mM glucose, insulin secretion increased by 2.5-fold, while the 20-kDa methylation to about 3.2-fold. In the case of forskolin, another secretagogue, at 0.1mM, the methylation increased by approximately 4.5-fold. This increase of 20-kDa methylation was inhibited when the cells were treated with 3mM EGTA to inhibit insulin secretion by depleting extracellular calcium ion, indicating intercausal relation between methylation and insulin secretion. The [methyl-3H]-labeled amino acids were identified by thin layer chromatography as N(G)-methylated arginines. While arginyl residues in Gly-Arg-Gly sequence are known to be posttranslationally methylated, a synthetic nonapeptide, GGRGRGRGG, competed with the 20-kDa methylation; at 1 and 10 micro M nonapeptides, 62% and 78% of 20-kDa methylation were inhibited, respectively. Furthermore, Western immunoblot analysis of HIT cell extract against GGRGRGRGG antibodies strongly immunoreacted with the 20-kDa protein. These results suggested that methylation of the endogenous 20-kDa protein might play some role in insulin secretion.     

Synthesis of tritiated rat insulin with high specific activity: A method for radiolabeling the active site of insulin

Insulin was synthesized by rat islets from tritiated amino acids under conditions designed to achieve high specific activity. Islets were isolated by the collagenase method. Stores of unlabeled insulin were depleted by culturing them for 40 hours in the presence of 3-isobutyl-1-methylxanthine. The islets were then incubated for 22 hours in the presence of [³H]Isoleucine or [³H]Phenylalanine. These amino acids were chosen because they are specific markers from the A and B chains of insulin respectively. Labeled insulin was extracted from the islets and purified by gel filtration. Its biological activity was indistinguishable from monoiodinated insulin as assessed by binding to receptors on cultured human lymphocytes and by precipitation by anti-insulin antibodies. The specific activity was (18 Ci/mmole) and (37 Ci/mmole) for [³H]Ile and [³H]Phe insulin respectively.     

Methylation of phospholipids in microsomes of the rat aorta

The methylation of phospholipids by S-adenosyl-L-methionine was characterized in microsomes prepared from strips of rat aorta. In the presence of 0.5 microM S-adenosyl-L-methionine, endogenous phosphatidylethanolamine was methylated to form three products: phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine and phosphatidylcholine. In the presence of 150 microM S-adenosyl-L-methionine the methylation activity increased more than 50-fold and the principal radioactive product was phosphatidylcholine. Optimal activity was at pH 9 and no magnesium requirement was detected. Exogenous phosphatidylethanolamine, phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine served as substrates for the enzyme. The methylation of exogenous phosphatidyl-N,N-dimethylethanolamine proceeded at a slower rate. Incubation of trypsin with the aorta microsomes reduced the enzymatic activity and reduced the relative yield of phosphatidyl-N-monomethylethanolamine. Phospholipase C degraded the methylated phospholipids, but phosphatidyl-N,N-dimethylethanolamine appeared to be less accessible to the phospholipase. The phospholipid methylation activity was inhibited by the addition of S-adenosyl-L-homocysteine or by L-homocysteinethiolactone. When intact strips of rat aorta were incubated with L-[methyl-3H]methionine, [3H]methyl groups were incorporated into phospholipids. This incorporation was inhibited when L-homocysteinethiolactone was added to the incubation. Polarized fluorescence of diphenylhexatriene in aorta microsomes was measured to determine the apparent membrane fluidity. When intact strips of aorta were incubated with methionine or with L-homocysteinethiolactone, methionine enhanced and L-homocysteinethiolactone decreased apparent fluidity of the microsomal membranes. Phospholipid methylation activity was examined in aorta microsomes prepared from genetically spontaneous hypertensive SHR strain rats. Phospholipid methylation activity was substantially greater in the SHR aorta microsomes than in microsomes prepared from Wistar-Kyoto WKY control strain aorta. Membrane fluidity was greater in the SHR aorta microsomes than in the WKY aorta microsomes. The hypothesis that phospholipid methylation activity influences fluidity of membranes and the possible involvement of methylated phospholipids in aorta membrane functions are discussed.     

Alteration of intramolecular disulfides in insulin receptor/kinase by insulin and dithiothreitol: insulin potentiates the apparent dithiothreitol-dependent subunit reduction of insulin receptor

Dithiothreitol (DTT) was observed to increase both beta-subunit autophosphorylation and exogenous substrate phosphorylation of the insulin receptor in the absence of insulin. The natural protein reducing agent thioredoxin was also observed to increase the insulin receptor beta-subunit autophosphorylation. The activation of the insulin receptor/kinase by both DTT and thioredoxin was found to be additive with that of insulin. Further, the increase in the insulin receptor beta-subunit autophosphorylation in the presence of DTT and insulin was demonstrated to be due to an increase in the initial rate of autophosphorylation without alteration in the extent of phosphorylation. Similarly, the increase in the exogenous substrate phosphorylation was due to an increase in the Vmax of phosphorylation without significant effect on the apparent Km of substrate binding. In the presence of relatively low concentrations of DTT, insulin was found to potentiate the apparent insulin receptor subunit reduction of the native alpha 2 beta 2 heterotetrameric complex into alpha beta heterodimers, when observed by silver staining of sodium dodecyl sulfate-polyacrylamide gels. N-[3H]Ethylmaleimide ([3H]NEM) labeling in the absence of DTT pretreatment demonstrated that only the beta subunit had accessible sulfhydryl group(s). However, treatment of insulin receptors with DTT increased the amount of [3H]NEM labeling in the beta subunit as well as exposing sites on the alpha subunit. Further, incubation of the insulin receptors with the combination of DTT and insulin also demonstrated the apparent insulin-potentiated subunit reduction without any increase in the total amount of [3H]NEM labeling.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of hormones on protein and amino acid metabolism in mammary-gland explants of mice.

The effects of insulin, cortisol and prolactin on amino acid uptake and protein biosynthesis were determined in mammary-gland explants from mid-pregnant mice. Insulin stimulated [3H]leucine incorporation into protein within 15 min of adding insulin to the incubation medium. Insulin also had a rapid stimulatory effect on the rate of aminoiso[14C]butyric acid uptake, but it had no effect on the intracellular accumulation of [3H]leucine. Cortisol inhibited the rate of [3H]leucine incorporation into protein during the initial 4h of incubation, but it had no effect at subsequent times. [3H]Leucine uptake was unaffected by cortisol, but amino[14C]isobutyric acid uptake was inhibited after a 4h exposure period to this hormone. Prolactin stimulated the rate of [3H]leucine incorporation into protein when tissues were exposed to this hormone for 4h or more; up to 4h, however, no effect of prolactin was detected. At all times tested, prolactin had no effect on the uptake of either amino[14C]isobutyric acid or [3H]leucine. Incubation with actinomycin D abolished the prolactin stimulation of protein biosynthesis, but this antibiotic did not affect the insulin response. A distinct difference in the mechanism of action of these hormones on protein biosynthesis in the mammary gland is thus apparent.     

Fluorometric determination of phosphatidylcholine as a measure of phospholipid methylation.

The successive methylation of phosphatidylethanolamine to phosphatidylcholine (phospholipid methylation) has been measured by the incorporation of S-[methyl-3H]adenosylmethionine or colorimetric assay of phosphatidylcholine extracted from adipocyte plasma membranes. A fluorometric assay for phosphatidylcholine was developed to measure phospholipid methylation. This assay is 10 times more sensitive than the colorimetric assay and demonstrates no significant interference with other methylated phospholipids. The fluorometric assay was used to determine a biphasic insulin dose response in adipocyte plasma membranes. This fluorometric assay for phosphatidylcholine represents an alternative method for monitoring phospholipid methylation, especially when increased sensitivity is required.     

Methyl esterification of glutamic acid residues of methyl-accepting chemotaxis proteins in Bacillus subtilis.

The amino acid residue modified in the reversible methylation of Bacillus subtilis methyl-accepting chemotaxis proteins was identified as glutamic acid; methylation results in the formation of glutamate 5-methyl ester. Identification was made by comparing the behaviour of a 3H-labelled compound isolated from proteolytically hydrolysed methyl-accepting chemotaxis proteins labelled in vivo with that of authentic methylated amino acids by chromatographic and electrophoretic techniques. Also, the isolated compound on mild alkaline hydrolysis shows behaviour identical with that of authentic glutamate 5-methyl ester. [3H]Methanol released by mild alkaline hydrolysis was made to react with 3,5-dinitrobenzyl chloride to form [3H]methyl 3,5-dinitrobenzoate, which was identified by reverse-phase high-pressure liquid chromatography.     

Transmembrane signalling by insulin via an insulin receptor mutated at tyrosines 1158, 1162, and 1163

In order to study the role of tyrosine autophosphorylation in insulin receptor signalling, we investigated a mutant human insulin receptor whereby the three major tyrosine autophosphorylation sites at positions 1158, 1162, and 1163 in the receptor beta-subunit were mutated to phenylalanines. When these mutant receptors were expressed in HTC rat hepatoma cells, there was no enhanced beta-subunit autophosphorylation and tyrosine kinase activity. In these cells there was enhanced insulin stimulation of [3H]AIB uptake and [3H]thymidine incorporation when compared to wild type HTC cells. The present study suggests therefore that the presence of the major insulin autophosphorylation sites is not a requirement for insulin stimulation of amino acid transport and mitogenesis.     

Amino acid dependent and independent insulin stimulation of cartilage metabolism

The effects of insulin on embryonic chicken cartilage in organ culture and the dependence of these effects on essential amino acids have been studied. In the presence of all essential amino acids, insulin: (1) increases 2-deoxy-D-glucose and alpha-aminoisobutyric acid uptake; (2) increases [5(-3H] uridine flux into uridine metabolites and the intracellular UTP pool; (3) expands the size of the intracellular UTP pool; (4) does not change the specific activity of the UTP pool; and (5) stimulates RNA, proteoglycan, and total protein synthesis. In lysine (or other essential amino acid)-deficient medium, the effects of insulin are different. While insulin stimulates incorporation of [5(-3)H] uridine into RNA, it does so by increasing the specific activity of the UTP pool without increasing RNA synthesis. Insulin stimulates 2-deoxy-D-glucose and alpha-aminoisobutyric acid uptake but no longer stimulates proteoglycan, total protein, or RNA synthesis or expands the size of the UTP pool. These data indicate that there are amino acid dependent and independent effects of insulin on cartilage. Transport processes are amino acid independent, while synthetic processes are amino acid dependent.     

The glycophospholipid anchor of Thy-1. Biosynthetic labeling experiments with wild-type and class E Thy-1 negative lymphomas

The Thy-1 antigen of the surface of lymphocytes and neurons is anchored to the plasma membrane via a glycophospholipid moiety. In contrast, the Thy-1 synthesized by the class E Thy-1 negative mutant lymphoma is secreted as a hydrophilic species. The present investigation uses the approach of biosynthetic labeling to investigate further the structure of the intracellular Thy-1 of wild-type cells and the secreted Thy-1 of these mutant cells. In the wild-type cells, Thy-1 can be labeled with [3H] mannose, [3H]galactose, [3H]fucose, [3H]ethanolamine, and [3H]palmitic acid. In the latter two cases the label is recovered almost exclusively in a detergent-binding Pronase fragment of the protein. The incorporated label is in the form of [3H]ethanolamine, or [3H]palmitate and stearate, respectively. Reductive methylation of biosynthetically labeled Thy-1 and a nonradioactive sample of Thy-1 shows that [3H]ethanolamine is incorporated equally into two residues of ethanolamine, only one of which has a free amino group. A single residue of glucosamine with a free amino group is also detected. Each of the sugar precursors is incorporated with extensive conservation of chemical identity. In the class E cells, each of the labeled sugars but neither [3H]ethanolamine nor [3H]palmitate is incorporated into Thy-1. The anchor moiety therefore appears to be entirely missing, although N-linked oligosaccharide processing is essentially normal. We postulate that the anchor deficiency in the mutant cells results from a biosynthetic lesion.     

Protein methylation in pea chloroplasts

The methylation of chloroplast proteins has been investigated by incubating intact pea (Pisum sativum) chloroplasts with [³H-methyl]-S-adenosylmethionine. Incubation in the light increases the amount of methylation in both the thylakoid and stromal fractions. Numerous thylakoid proteins serve as substrates for the methyltransfer reactions. Three of these thylakoid proteins are methylated to a significantly greater extent in the light than in the dark. One is a polypeptide with a molecular mass of 64 kD, a second has an M_r of 48 kD, and the third has a molecular mass of less than 10 kD. The primary stromal polypeptide methylated is the large subunit of ribulose bisphosphate carboxylase/oxygenase. One other stromal polypeptide, having a molecular mass of 24 kD, is also methylated much more in the light than in the dark. Two distinct types of protein methylation occur. One methyl-linkage is stable to basic conditions whereas a second type is base labile. The base-stable linkage is indicative of N-methylation of amino acid residues while base-lability is suggestive of carboxymethylation of amino acid residues. Labeling in the light increases the percentage of methylation that is base labile in the thylakoid fraction while no difference is observed in the amount of base-labile methylations in light-labeled and dark-labeled stromal proteins. Also suggestive of carboxymethylation is the detection of volatile [³H]methyl radioactivity which increases during the labeling period and is greater in chloroplasts labeled in the light as opposed to being labeled in the dark; this implies in vivo turnover of the [³H]methyl group.     

Secretory Proteins from Adrenal Medullary Cells Are Carboxyl-Methylated In Vivo and Released Under Their Methylated Form by Acetylcholine

The carboxyl methylation of secretory proteins in vivo was investigated in bovine adrenal medullary cells in culture. Chromogranin A, the major intragranular secretory protein in adrenal medullary cells, and other secretory proteins were found to be carboxyl-methylated within secretory vesicles. The in vivo labeling pattern using [methyl-3H]methionine and the in vitro labeling pattern using S-adenosyl-[methyl-14C]methionine of intravesicular secretory proteins were similar. The detection of methylated chromogranin A in mature secretory vesicles required 3-6 h, a time consistent with the synthesis and storage of secretory proteins in this tissue. Carboxyl-methylated chromogranin A was secreted from medullary cells by exocytosis via activation of nicotinic cholinergic receptor and recovered still under the methylated form in the incubation medium. Since protein-carboxyl-methylase is cytosolic, these results suggest that methylation of secretory proteins is a cotranslational phenomenon.     

Relationship among methylation,isoprenylation, and GTP binding in 21-to 23-kDa proteins of neuroblastoma

1. Dimethylsulfoxide-induced differentiated neuroblastoma express high levels of membrane 21 to 23-kDa carboxyl methylated proteins. Relationships among methylation, isoprenylation, and GTP binding in these proteins were investigated. Protein carboxyl methylation, protein isoprenylation, and [alpha-32P]GTP binding were determined in the electrophoretically separated proteins of cells labeled with the methylation precursor [methyl-3H]methionine or with an isoprenoid precursor [3H]mevalonate. 2. A broad band of GTP-binding proteins, which overlaps with the methylated 21 to 23-kDa proteins, was detected in [alpha-32P]GTP blot overlay assays. This band of proteins was separated in two-dimensional gels into nine methylated proteins, of which four bound GTP. 3. The carboxyl-methylated 21 to 23-kDa proteins incorporated [3H]mevalonate metabolites with characteristics of protein isoprenylation. The label was not removed by organic solvents or destroyed by hydroxylamine. Incorporation of radioactivity from [3H]mevalonate was enhanced when endogenous levels of mevalonate were reduced by lovastatin, an inhibitor of mevalonate synthesis. Lovastatin blocked methylation of the 21 to 23-kDa proteins as well (greater than 70%). 4. Methylthioadenosine, a methylation inhibitor, inhibited methylation of these proteins (greater than 80%) but did not affect their labeling by [3H]mevalonate. The results suggest that methylation of the 21 to 23-kDa proteins depends on, and is subsequent to, isoprenylation. The sequence of events may be similar to that known in ras proteins, i.e., carboxyl methylation of a C-terminal cysteine that is isoprenylated. 5. Lovastatin reduced the level of small GTP-binding proteins in the membranes and increased GTP binding in the cytosol. Methylthioadensoine blocked methylation without affecting GTP binding. 6. Thus, isoprenylation appears to precede methylation and to be important for membrane association, while methylation is not required for GTP binding or membrane association. The role of methylation remains to be determined but might be related to specific interactions of the small GTP-binding proteins with other proteins.     

Methylation of erythrocyte membrane proteins at extracellular and intracellular D-aspartyl sites in vitro. Saturation of intracellular sites in vivo

A cytosolic protein carboxyl methyltransferase (S-adenosyl-L-methionine:protein O-methyltransferase, E.C. 2.1.1.24) purified from human erythrocytes catalyzes the methylation of erythrocyte membrane proteins in vitro using S-adenosyl-L-[methyl-3H]methionine as the methyl group donor. The principal methyl-accepting proteins have been identified by sodium dodecyl sulfate-gel electrophoresis at pH 2.4 and fluorography as the anion transport protein (band 3), ankyrin (band 2.1), and integral membrane proteins with molecular weights of 45,000, 28,000, and 21,000. Many of the methylation sites associated with intrinsic membrane proteins may reside in their extracellular portions, since these same proteins are methylated when intact cells are used as the substrate. The maximal number of methyl groups transferred in these experiments is approximately 30 pmol/mg of membrane protein, a value which represents less than one methyl group/50 polypeptide chains of any methyl-accepting species. The number of methylation sites associated with the membranes is increased, but not to stoichiometric levels, by prior demethylation of the membranes. The additional sites are associated primarily with bands 2.1 and 4.1, the principal methyl acceptors in vivo, suggesting that most methylation sites are fully modified in vivo. Extracellular methylation sites are not increased by demethylation of membranes. The aspartic acid beta-methyl ester which can be isolated from carboxypeptidase Y digests of [3H]methylated membranes is in the unusual D-stereoconfiguration. Similar results have been obtained with [3H]methylated membranes isolated from intact cells (McFadden, P.N., and Clarke, S. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2460-2464). It is proposed that the methyltransferase recognizes D-aspartyl residues in proteins and is involved with the metabolism of damaged proteins in vivo.     

Increased methyl esterification of membrane proteins in aged red-blood cells. Preferential esterification of ankyrin and band-4.1 cytoskeletal proteins

The enzymatic carboxyl methyl esterification of erythrocyte membrane proteins has been investigated in three different age-related fractions of human erythrocytes. When erythrocytes of different mean age, separated by density gradient centrifugation, were incubated under physiological conditions (pH 7.4, 37 degrees C) in the presence of L-[methyl-3H]methionine, the precursor in vivo of the methyl donor S-adenosylmethionine, a fourfold increase in membrane-protein carboxyl methylation was observed in the oldest cells compared with the youngest ones. The identification of methylated species, based on comigration of radioactivity with proteins stained with Coomassie blue, analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, shows, in all cell fractions, a pattern similar to that reported for unfractionated erythrocytes. However in the membrane of the oldest erythrocytes the increase in methylation of the cytoskeletal proteins, bands 2.1 and 4.1, appears to be significantly more marked compared with that observed in the other methylated polypeptides. Furthermore the turnover rate of incorporated [3H]methyl groups in the membrane proteins of the oldest cells markedly increases during cell ageing. Particularly in band 4.1 the age-related increase in methyl esterification is accompanied by a significant reduction of the half-life of methyl esters. The activity of cytoplasmic protein methylase II does not change during cell ageing, while the isolated ghosts from erythrocytes of different age show an age-related increased ability to act as methyl-accepting substrates, when incubated in presence of purified protein methylase II and methyl-labelled S-adenosylmethionine, therefore the relevance of membrane structure in determining membrane protein methylation levels can be postulated. Finally the possible correlation of this posttranslational protein modification with erythrocyte ageing is discussed.     

Effect of hexoses and mannoheptulose on cyclic AMP accumulation and insulin secretion in rat pancreatic islets.

The effects of various sugars on the simultaneous release of insulin and accumulation of cyclic AMP were studied in collagenase isolated rat pancreatic islets. D-Glucose stimulated the formation of cyclic AMP at 3 and 60 min of incubation, whether measured by a label incorporation technique, or by the protein kinase binding assay of Gilman. Only D-glucose and D-mannose were able to stimulate insulin release and cyclic [3H]AMP accumulation in the absence of other substrate. D-fructose had a stimulatory effect in the presence of 3.3 mM D-glucose only at a high concentration (33.8 mM), and enhanced the effects of 8.3 mM glucose when added at the concentration of 8.3 mM. D-Galactose was effective only together with 8.3 mM D-glucose. The order of potency of these hexoses, both regarding insulin secretion and cyclic [3H]AMP accumulation, was glucose-mannose-fructose-galactose. L-Glucose and 3-O-methylglucose had no effects at 60 min when incubated together with 8.3 mM D-glucose, whereas at 3 min, 3-O-methylglucose induced a small stimulation of the cyclic [3H]AMP response. D-mannoheptulose and D-glucosamine inhibited the insulin and cyclic [3H]AMP responses to 27.7 mM glucose. Mannoheptulose suppressed completely the glucose effect on cyclic nucleotide accumulation within 90 s. Although under all incubation conditions, the threshold stimulatory or inhibitory concentration of a given agent was identical for insulin release and cyclic [3H]AMP accumulation, these two variables showed quantitative differences in incubations of 60 min, the magnitude of the changes in insulin secretion being larger than that for the cyclic nucleotide. It is suggested that modulation of islet cyclic AMP level is an important step in the transmission of the effect of various sugars on insulin release; however, glucose and possibly other sugars may also enhance insulin release by additional mechanisms not involving the adenylate cyclase-cyclic AMP system of the beta-cell.     

Regulation of pyruvate kinase in Reuber H35 hepatoma cells by insulin and fructose

1. Kinetic and immunological studies as well as electrophoretic behaviour indicated that pyruvate kinase in Reuber H35 hepatoma cells is of the M2-type. 2. Addition of 0.1 microM insulin or 2 mM fructose to the incubation medium for 72 hr increased the activity of the M2-type pyruvate kinase in Reuber H35 hepatoma cells by 103 and 25% respectively. 3. Incorporation studies with [3H]leucine followed by immunoprecipitation showed that the apparent rate of synthesis of the M2-type pyruvate kinase was increased by both insulin and fructose. 4. Degradation studies indicated that the addition of insulin and fructose to the incubation medium increased the half-life of the M2-type pyruvate kinase from 4.8 to 8.6 and 6.8 hr respectively.     

In vivo studies on yeast cytochrome c methylation in relation to protein synthesis

Methylation of cytochrome c was studied in vivo using double label with L-[methyl-3H]methionine and DL-[2-14C]methionine. In pulse-chase experiments the cytochrome c associated with the mitochondrial fraction possessed a higher ratio of 3H/14C label, suggesting the presence of methylated cytochrome c. The appearance of methylated cytochrome c in mitochondria showed no lag phase. The inhibition of cytochrome c methylation in presence of cycloheximide indicated that both the methylation and protein synthesis were tightly coupled and cycloheximide selectively inhibited cytochrome c methylation. There was also an indication of selective turnover of incorporation methyl groups in preformed cytochrome c.     

Methylation of Drosophila histones at proline, lysine, and arginine residues during heat shock

Heat shock or arsenite treatment alter the pattern of histone methylation in Drosophila cells. Both types of stress induce a rapid increase in the methylation level of histone H2B. The methylated amino acid residue of H2B has been identified by thin layer chromatography and electrophoresis as methylproline and is located at the N-terminal end of H2B. Heat shock also induces a decrease in the level of methylation of histone H3. Under normal growth temperature conditions, histone H3 is shown to be methylated on lysine residues. However under heat shock conditions, there is a decrease in the extent of methylation of lysine residues and the appearance of new methylation on arginine residues in H3. These new heat shock-induced methylated residues have been identified as the symmetrical and asymmetrical forms of dimethylarginine. The methylated amino acid residue of histone H4 is lysine with mono-, di-, and trimethyl forms found in both control and heat or chemically stressed cells. These stress-induced changes in the methylation level of the N-terminal proline residue of histone H2B and shift in the methylation sites of histone H3 may be involved in the restructuration of chromatin accompanying the inactivation of normal genes in response to stress. Moreover, we suggest that the hypermethylation of H2B may also be involved in its protection from increased ubiquitin-mediated proteolytic activity under these conditions of cellular stress.