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Spontaneous protein deamidation of labile Asn residues, generating L-isoaspartates and D-aspartates, is associated with cell aging and is enhanced by an oxidative microenvironment; to minimize the damage, the isoaspartate residues can be 'repaired' by a specific L-isoaspartate (D-aspartate) protein O-methyltransferase (PIMT). As both premature aging and chronic oxidative stress are typical features of Down's syndrome (DS), we tested the hypothesis that deamidated proteins may build up in trisomic patients. Blood samples were obtained from children with karyotypically confirmed full trisomy 21 and from age-matched healthy controls. Using recombinant PIMT as a probe, we demonstrated a dramatic rise of L-isoaspartates in erythrocyte membrane proteins from DS patients. The content of D-aspartate was also significantly increased. The integrity of the repair system was checked by evaluating methionine transport, PIMT specific activity, and intracellular concentrations of adenosylmethionine and adenosylhomocysteine. The overall methylation pathway was directly monitored by incubating fresh red blood cells with methyl-labeled methionine; a three-fold increase of protein methyl esters was detected in trisomic children. Deamidated species include ankyrin, band 4.1, band 4.2 and the integral membrane protein band 3; ankyrin and band 4.1 were significantly hypermethylated in DS. When DS red blood cells were subjected to oxidative treatment in vitro, the increase of protein deamidation paralleled lipid peroxidation and free radical generation. We observed a similar pattern in Epstein-Barr virus B-lymphocytes from trisomic patients. In conclusion, our findings support the hypothesis that protein instability at asparagine sites is a biochemical feature of DS, presumably depending upon the oxidative microenvironment. The possible pathophysiological implications are discussed.

The ‘Mediterranean’ variant of glucose-6-phosphate dehydrogenase (G6PD) deficiency is due to the C563CT point mutation, leading to replacement of Ser with Phe at position 188, resulting in acute haemolysis triggered by oxidants. Previous work has shown increased formation of altered aspartate residues in membrane proteins during cell ageing and in response to oxidative stress in normal erythrocytes. These abnormal residues are specifically recognized by the repair enzyme l-isoaspartate (d-aspartate) protein O-methyltransferase (PCMT; EC 2.1.1.77). The aim of this work was to study the possible involvement of protein aspartate damage in the mechanism linking the G6PD defect and erythrocyte injury, through oxidative stress. Patients affected by G6PD deficiency (Mediterranean variant) were selected. In situ methylation assays were performed by incubating intact erythrocytes in the presence of methyl-labelled methionine. Altered aspartate residues were detected in membrane proteins by methyl ester quantification. We present here evidence that, in G6PD-deficient erythrocytes, damaged residues are significantly increased in membrane proteins, in parallel with the decay of pyruvate kinase activity, used as a cell age marker. Erythrocytes from patients were subjected to oxidative stress in vitro, by treatment with t-butylhydroperoxide, monitored by a rise in concentration of both methaemoglobin and thiobarbituric acid-reactive substances. l-Isoaspartate residues increased dramatically in G6PD-deficient erythrocytes in response to such treatment, compared with baseline conditions. The increased susceptibility of G6PD-deficient erythrocytes to membrane protein aspartate damage in response to oxidative stress suggests the involvement of protein deamidation/isomerization in the mechanisms of cell injury and haemolysis.

Hydroxytyrosol, the major representative phenolic compound of virgin olive oil, is a dietary component. Its possible protective effect on hydrogen peroxide (H(2)O(2))-induced oxidative alterations was investigated in human erythrocytes. Cells were pretreated with micromolar hydroxytyrosol concentrations and then exposed to H(2)O(2) over different time intervals. Subsequently, erythrocytes were analyzed for oxidative hemolysis and lipid peroxidation. Our data demonstrate that hydroxytyrosol prevents both oxidative alterations, therefore, providing protection against peroxide-induced cytotoxicity in erythrocytes. The effect of oxidative stress on erythrocyte membrane transport systems, as well as the protective role of hydroxytyrosol, also were investigated in conditions of nonhemolytic mild H(2)O(2) treatment. Under these experimental conditions, a marked decrease in the energy-dependent methionine and leucine transport is observable; this alteration is quantitatively prevented by hydroxytyrosol pretreatment. On the other hand, the energy-independent glucose transport is not affected by the oxidative treatment. The reported data give new experimental support to the hypothesis of a protective role played by nonvitamin antioxidant components of virgin olive oil on oxidative stress in human systems.

Resveratrol (3,5,4′-trihydroxystilbene) is a naturally occurring phytoalexin, found in grapes and wine, which has been reported to exert a variety of important pharmacological effects. We have investigated the activity of resveratrol on proliferation and differentiation of the promyelocitic cell line HL-60. A concentration as low as 30 μM causes a complete arrest of proliferation and a rapid induction of differentiation towards a myelo-monocytic phenotype. Analyses by flow cytometry showed the absence of the G2/M peak and the accumulation of cells in G1 and S phases. Moreover, at the concentrations employed, a very low amount of apoptotic cells was evidenced. A detailed biochemical analysis demonstrated that the G1 phase of the cell division cycle engine was completely unmodified by resveratrol addition, thus indicating that the G1 → S transition occurs normally. Conversely, after only 24 h treatment, a significant increase of cyclins A and E could be observed along with the accumulation of cdc2 in the inactive phosphorylated form. These data demonstrate that resveratrol causes a complete and reversible cell cycle arrest at the S phase checkpoint.

Plants produce an extraordinary array of low molecular mass natural products endowed with biological activity. Among these molecules, resveratrol (3,5,4’-trihydroxystilbene) has been identified as an inhibitor of carcinogenesis with a pleiotropic mode of action. Extensive literature on its anticancer activity, performed in cellular models, suggests a potential antiproliferative and apoptogenic use of the stilbene. Similarly, studies on implanted cancers and chemical-induced tumors confirm a potential chemotherapeutical interest of the compound. Moreover, recent intriguing studies have demonstrated, in mice, that the negative effects (insulin resistance and hyperglycemia) of a high-fat diet might be prevented by resveratrol treatment. Despite these promising observations, only few clinical trials have been performed on the compound due to the scarce interest of pharmaceutical industry. We suggest that resveratrol might be considered an interesting anticancer compound in association with more specific target-oriented drugs.

The potential protective effects of oleuropein, a dietary antioxidant of olive oil, has been investigated in the isolated rat heart. The organs were subjected to 30 minutes of no-flow global ischemia and then reperfused. At different time intervals, the coronary effluent was collected and assayed for creatine kinase activity as well as for reduced and oxidized glutathione. In addition, the extent of lipid peroxidation was evaluated by measuring thiobarbituric acid reactive substance concentration in cardiac muscle. Pretreatment with 20 microg/g oleuropein before ischemia resulted in a significant decrease in creatine kinase and reduced glutathione release in the perfusate. The protective effect of oleuropein against the post-ischemic oxidative burst was investigated by measuring the release, in the coronary effluent, of oxidized glutathione, a sensitive marker of heart’s exposure to oxidative stress. Reflow in ischemic hearts was accompanied by a prompt release of oxidized glutathione; in ischemic hearts pretreated with oleuropein, this release was significantly reduced. Membrane lipid peroxidation was also prevented by oleuropein. The reported data provide the first experimental evidence of a direct cardioprotective effect of oleuropein in the acute events that follow coronary occlusion, likely because of its antioxidant properties. This finding strengthens the hypothesis that the nutritional benefit of olive oil in the prevention of coronary heart disease can be also related to the high content of oleuropein and its derivatives. Moreover, our data, together with the well documented antithrombotic and antiatherogenic activity of olive oil polyphenols, indicate these antioxidants as possible therapeutic tools for the pharmacological treatment of coronary heart disease as well as in the case of cardiac surgery, including transplantation.

2-(3,4-Dihydroxyphenyl)ethanol (DPE), a naturally occurring phenolic antioxidant molecule found in olive oil, has been reported to exert several biological and pharmacological activities. We studied the effect of this compound on the proliferation and survival of HL60 cell line. Concentrations from 50 to 100 microM DPE, comparable to its olive oil content, caused a complete arrest of HL60 cell proliferation and the induction of apoptosis. This was demonstrated by flow cytometric analyses, poly(ADP-ribose) polymerase cleavage, and caspase 3 activation. The apoptotic effect requires the presence of two ortho-hydroxyl groups on the phenyl ring, since tyrosol, 2-(4-hydroxyphenyl)ethanol, did not induce either cell growth arrest or apoptosis. DPE-dependent apoptosis is associated with an early release of cytochrome c from mitochondria which precedes caspase 8 activation, thus ruling out the engagement of cell death receptors in the apoptotic process. 2-(3,4-Dihydroxyphenyl)ethanol induced cell death in quiescent and differentiated HL60 cells, as well as in resting and activated peripheral blood lymphocytes, while did not cause cell death in two colorectal cell lines (HT-29 and CaCo2). These results suggest that DPE down-regulates the immunological response, thus explaining the well-known antinflammatory and chemopreventive effects of olive oil at the intestinal level.

Pyrrolidine dithiocarbamate (PDTC) is a synthetic antioxidant molecule, which has been recently proposed as an antitumoral agent on the basis of its capability of inducing apoptosis. We investigated the effect of PDTC on the proliferation and survival of the promyelocitic cell line HL-60. Concentration as low as 10 μM of PDTC induces a significant reduction of the growth rate and the contemporaneous activation of the apoptotic process. Programmed cell death was demonstrated by biochemical analyses, including the activation of procaspase 3 and the cleavage of poly(ADP-ribose) polymerase (PARP). PDTC-dependent apoptosis was associated with an early release of cytochrome c from mitochondria, while the involvement of pathways due to cell death receptors engagement was ruled out by detailed time-course analyses of caspases 3 and 8 activation. Moreover, no up-regulation of p21 CIP1 level, a pivotal cyclin-dependent kinase inhibitor, occurred at PDTC concentration able to induce apoptosis. Finally, in vitro incubation of purified mitochondria with PDTC demonstrated that the molecule is directly able to induce cytochrome c release from the intermembrane space, thus confirming that mitochondria are a primary cellular target of the molecule.

Protein L-isoaspartate (D-aspartate) O-methyltransferase (PCMT) is a ubiquitous enzyme with substrate specificity toward proteins and peptides containing altered aspartyl residues. These are generated from the spontaneous deamidation of normal L-asparaginyl residues and/or isomerization of L-aspartyl residues. Such degradation reactions occur both in vivo and in purified protein molecules. Because of its peculiar substrate specificity, PCMT has been proposed as a tool for identifying and characterizing the L-isoaspartyl-containing byproducts of natural and recombinant proteins. Data presented here show the usefulness of PCMT for the identification of these altered residues in food proteins and the occurrence of these at detectable levels in several proteins from various animal sources. In addition, we noted that ovalbumin, either as a purified protein or in the whole chicken egg, becomes a better substrate for this enzyme during prolonged storage, thus indicating a progressive accumulation of the altered amino acid residues over time. The protective effects of trehalose against the occurrence of such specific protein damage in ovalbumin stored for several days were also assessed. Protein L-isoaspartate (D-aspartate) O-methyltransferase (PCMT) is a ubiquitous enzyme with substrate specificity toward proteins and peptides containing altered aspartyl residues. These are generated from the spontaneous deamidation of normal L-asparaginyl residues and/or isomerization of L-aspartyl residues. Such degradation reactions occur both in vivo and in purified protein molecules. Because of its peculiar substrate specificity, PCMT has been proposed as a tool for identifying and characterizing the L-isoaspartyl-containing byproducts of natural and recombinant proteins. Data presented here show the usefulness of PCMT for the identification of these altered residues in food proteins and the occurence of these at detectable levels in several proteins from various various animal sources. In addition, we noted that ovalbumin, either as a purified protein or in the whole chicken egg, becomes a better substrate for this enzyme during prolonged storage, thus indicating a progressive accumulation of the altered amino acid residues over time. The protective effects of trehalose against the occurence of such specific protein damage in ovalbumin stored for several days were also assessed.

Protein carboxyl methyltransferase of type II selectively recognizes L‐isoaspartyl and D‐aspartyl residues spontaneously occurring in proteins and peptide substrates. Membrane protein methylation levels increase with erythrocyte aging in circulation, in parallel with the spontaneous formation of abnormal aspartyl sites, due to protein intrinsic instability. We found that enzymic methyl esterification of erythrocyte membrane proteins in hereditary spherocytosis, a model of cytoskeletal disarray, is significantly increased compared to normal red blood cells. This cannot be explained by an increase in mean age of spherocytes, which are on the contrary significantly younger than control cells. No differences in cytosolic methyltransferase specific activity, as well as in the intracellular concentrations of the methyl donor adenosylmethionine and/or of the methylation inhibitor adenosylhomocysteine were observed. We identified bands 2.1, 4.1 and 4.2 as the main targets for increased methylation, whose levels were correlated with the degree of spectrin deficiency associated with this anemia. Our findings indicate that membrane‐protein methyl esterification represents a marker of membrane structural alteration in vivo in spherocytosis. We hypothesize that either an increased accessibility of methylation sites normally not available to the methyltransferase, or accelerated formation of methyl‐accepting sites in membrane proteins are present in spherocytosis. Copyright © 1995, Wiley Blackwell. All rights reserved

Background Natural proteins undergo in vivo spontaneous post-biosynthetic deamidation of specific asparagine residues with isoaspartyl formation. Deamidated-isomerized molecules are both structurally and functionally altered. The enzyme isoaspartyl protein carboxyl-O-methyltransferase (PCMT; EC 2.1.1.77) has peculiar substrate specificity towards these deamidated proteins. It catalyzes methyl esterification of the free alpha-carboxyl group at the isoaspartyl site, thus initiating the repair of these abnormal proteins through the conversion of the isopeptide bond into a normal alpha-peptide bond. Deamidation occurs slowly during cellular and molecular aging, being accelerated by physical-chemical stresses brought to the living cells. Previous evidence supports a role of protein deamidation in the acquisition of susceptibility to apoptosis. Aim of this work was to shed a light on the role of PCMT in apoptosis clarifying the relevant mechanism(s). Methodology/principal findings Endothelial cells transiently transfected with various constructs of PCMT, i.e. overexpressing wild type PCMT or negative dominants, were used to investigate the role of protein methylation during apoptosis induced by oxidative stress (H(2)O(2); 0.1-0.5 mM range). Results show that A) Cells overexpressing "wild type" human PCMT were resistant to apoptosis, whereas overexpression of antisense PCMT induces high sensitivity to apoptosis even at low H(2)O(2) concentrations. B) PCMT protective effect is specifically due to its methyltransferase activity rather than to any other non-enzymatic interactions. In fact negative dominants, overexpressing PCMT mutants devoid of catalytic activity do not prevent apoptosis. C) Cells transfected with antisense PCMT, or overexpressing a PCMT mutant, accumulate isoaspartyl-containing damaged proteins upon H(2)O(2) treatment. Proteomics allowed the identification of proteins, which are both PCMT substrates and apoptosis effectors, whose deamidation occurs under oxidative stress conditions leading to programmed cell death. These proteins, including Hsp70, Hsp90, actin, and Bcl-xL, are recognized and methylated by PCMT, according to the general repair mechanism of this methyltransferase. Conclusion/significance Apoptosis can be modulated by "on/off" switch partitioning the amount of specific protein effectors, which are either in their active (native) or inactive (deamidated) molecular forms. Deamidated proteins can also be functionally restored through methylation. Bcl-xL provides a case for the role of PCMT in the maintenance of functional stability of this antiapoptotic protein.

Membrane proteins from human erythrocytes were methylated with purified protein methylase II (S-adenosylmethionine:protein-carboxyl O-methyltransferase, EC.2.1.1.24). The methylated proteins were analyzed by dodecyl sulfate/polyacrylamide gel electrophoresis. Monomeric and dimeric glycophorin A (NaIO4/Schiff-2 and NaIO4/Schiff-1 positive bands) and 'band 4.5' were identified as two major classes of methyl-acceptor polypeptides for protein methylase II. In rabbit erythrocyte membrane where glycophorin A is absent, 'band 4.5' was the only major methyl-acceptor protein component. Extracted and purified glycophorin A from human erythrocytes was also found to be an excellent substrate for protein methylase II with a Km of 35.7 microM. The role of erythrocyte membrane protein methylation is discussed with regard to membrane function.

1. 1. Evidence of the presence of a pyridoxal-5-phosphatc-dependcnt omithinc decarboxylase in human prostate gland is reported. 2. 2. Thiols are required for enzyme activity, which is inhibited by sulphydryl reagents. 3. 3. The p H curve exhibits an optimum p 7.0 and a reproducible shoulder at pH 8.o. 4. 4. The apparent Km for the substrate is o-13 mM. Putrcscine is a competitive inhibitor, with an apparent K t of 4-3 mM.

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°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.

1. Introduction The biochemical roles of S-adenosylmethionine as donor of methyl group, propylamine moiety and aminobutyryl side chain in a variety of reactions are well established [ 1,2]. Although the biological importance of SAM is widely recognized, the mecha- nisms of transport of this highly charged molecule in mammalian cells have not yet been investigated. An active transport system with high affinity towards SAM has been described in yeast cells, which accumulate very peculiarly the sulfonium compound into the vacuoles [3,4]

Adenosine deaminase (EC 3.5.4.4), purified from Aspergillus oryzae , is active in deaminating S -adenosylhomocysteine and its related thioethers, whereas the related sulfonium compound, S -adenosylmethionine, is not deaminated. By taking advantage of the different reactivity of the two compounds, a coupled optical enzyme assay for methyl transfer reactions has been developed. The amount of Ado-Hcy formed is calculated from the decrease in optical density at 265 nm, after addition of an excess of adenosine deaminase. The validity of the method has been tested with three purified enzymes, i.e., homocysteine methyltransferase, histamine methylase, and acetylserotonin methyltransferase. Some kinetic constants of these enzymes have been obtained. The procedure is highly accurate, reproducible, and relatively simple compared to the conventional radio-chemical methods currently in use.

In order to separate S -adenosyl-(5′)-3-methylthiopropylamine (decarboxylated AdoMet) from its biological precursor, S-adenosylmethionine (AdoMet), two procedures have been devised. The first method involves two sequential chromatographic steps on Dowex 50, H + form, and Dowex 1, OH − form, respectively. An alternate procedure takes advantage of the unusual heat stability of decarboxylated AdoMet, as compared to its precursor, and permits the purification of the sulfonium compound by single-step chromatography. The method utilizes an acid hydrolysis, at 100°C, of a mixture of the two compounds, resulting in a selective cleavage of AdoMet into homoserine and 5′-methylthioadenosine: A single-step chromatography on Dowex 50, Na + form, permits the separation of the unmodified S -adenosyl-(5′)-3-methylthiopropylamine from the hydrolytic products of AdoMet. The yield is quantitative, and the procedure is simple and highly reproducible.

1. Introduction The enzyme S-adenosylmethionine:protein car- boxyl-U-methyl transferase (EC 2.1 .1.24; protein methylase II) methyl esterifies the aspartyl and/or glutamyl residues of preformed proteins, using S-adenosylmethionine as the methyl donor [ 141. The protein carboxyl methyl esters formed are un- stable and undergo rapid hydrolysis at physiological pH and temperature yielding methanol [5,6]. Several membrane proteins were shown to be the endogenous substrates for the enzyme in both prokaryotic and eukaryotic cells [ 1,7--91 and a number of studies were done to determine the physiological roles of this selec- tive, post-translational, protein methylation in mem- brane functions. The high activity of PM II and the large amounts of its membrane-bound endogenous substrates in nervous and endocrine tissues suggest a role of this reaction in the synaptic function [lo] and neurosecretory processes [ 1 O-l 21. In