Maurizio Minetti

Peroxynitrite modulates tyrosine-dependent signal transduction pathway of human erythrocyte band 3.

Peroxynitrite, the product of the reaction between nitric oxide and superoxide anion, is able to nitrate protein tyrosines. If this modification occurs on phosphotyrosine kinase substrates, it can down‐regulate cell signaling. We investigated the effects of peroxynitrite on band 3‐mediated signal transduction of human erythrocytes. Peroxynitrite treatment induced two different responses. At low concentrations (10–100 μM) it stimulated a metabolic response, leading to 1) a reversible inhibition of phosphotyrosine phosphatase activity, 2) a rise of tyrosine phosphorylation in the 22K cytoplasmic domain of band 3, 3) the release of glyceraldehyde 3‐phosphate dehydrogenase from the membrane, and 4) the enhancement of lactate production. At high concentrations (200–1000 μM), peroxynitrite induced 1) cross‐linking of membrane proteins, 2) inhibition of band 3 tyrosine phosphorylation, 3) nitration of tyrosines in the 22K cytoplasmic domain of band 3, 4) binding of hemoglobin to the membrane, 5) irreversible inhibition of phosphotyrosine kinase activity, 6) massive methemoglobin production, and 7) irreversible inhibition of lactate production. Our results demonstrate that at concentrations that could conceivably be achieved in vivo (10–100 μM), peroxynitrite behaves like other oxidants, i.e., it stimulates band 3 tyrosine phosphorylation and increases glucose metabolism. Thus, one plausible physiologic effect of peroxynitrite is the up‐regulation of signaling through the reversible inhibition of phosphotyrosine phosphatase activity. At high concentrations of peroxynitrite, the tyrosine phosphorylation ceases in parallel with the nitration of band 3 tyrosines, but at these concentrations phosphotyrosine kinase activity and glycolysis are also irreversibly inhibited. Thus, at least in red blood cells, the postulated down‐regulation of signaling by peroxynitrite cannot merely be ascribed to the nitration of tyrosine kinase targets.—Mallozzi, C., Di Stasi, A. M. M., Minetti, M. Peroxynitrite modulates tyrosine‐dependent signal transduction pathway of human erythrocyte band 3. FASEB J. 11, 1281–1290 (1997)

Role of Ascorbate and Protein Thiols in the Release of Nitric oxide from S-Nitroso-Albumin and S-Nitroso-Glutathione in Human Plasma

In this work we investigated the stability in aerobic plasma of two naturally occurring S-nitrosothiols, the S-nitroso adduct of serum albumin (S-NO-albumin) and the S-nitroso adduct of glutathione (S-NO-glutathione). In contrast to their behavior in physiological buffers, in which they are stable, in plasma these S-nitrosothiols showed a slow but continuous release of .NO. In the presence of red blood cells, the .NO was quantitatively oxidized to NO3- with stoichiometric formation of methemoglobin. In the absence of red blood cells, the principal oxidation product was NO2- with small amounts of NO3- (about 1/5 of the amount of NO2-). The release of .NO was also proven by spin trapping experiments with 2-(4-Carboxyphenyl)4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide which, when added to plasma in the presence of S-NO-glutathione, was transformed into 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl. Both dialysable and nondialysable compounds are involved in the release of .NO from S-nitrosothiols. Ascorbate and the thiol group of serum albumin are the plasma components mainly involved in the release of .NO, while endogenous L-cysteine and glutathione play a minor role due to their relative low concentrations. However, in contrast to the thiol-dependent release that is known to induce the formation of disulfides, the ascorbate-dependent release of .NO from S-NO-glutathione resulted in the formation of free sulfhydryls. Our results suggest that in plasma the .NO release from S-NO-albumin and S-NO-glutathione may be regulated by heterolytic NO+ transfer and reductive activation to .NO, rather than by homolytic decomposition of labile S-nitrosothiols.

gp120 HIV envelope glycoprotein increases the production of nitric oxide in human monocyte-derived macrophages.

The effect of recombinant gp120 HIV envelope glycoprotein on the generation of free radicals by monocyte‐derived macrophages (MDM) was measured by EPR spin trapping with 5,5 ‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO). After 1 day in culture, MDM produced a spin trap adduct of DMPO with hyperfine splitting constants superimposable on those of DMPO‐OH. The addition of gp120 to MDM increased the production of DMPO‐OH and after 1 h, the amount of DMPO‐OH produced by 40 μg/ml gp120 was about 300% that of untreated MDM. The use of selective inhibitors suggested the participation of the nitric oxide/l‐arginine oxidative pathway, but did not provide evidence for trapping of hydroxyl radical or other oxygen free radicals. The specificity of gp120 was proven by two different anti‐gp120 antibodies that either in‐hibited (polyclonal) or increased (monoclonal) the production of free radicals. Dexamethasone inhibited the effect of gp120, suggesting the possible involvement of an inducible nitric oxide (NO·) synthase. Moreover, treatment of MDM with gp120 for 15 h increased in a dose‐dependent manner the production of NO2 ‐, a stable end product of NO·. Soluble CD4 did not modify the intensity of the DMPO‐OH adduct, whereas yeast mannan and Ca2+‐chelators abolished the increase in the DMPO‐OH signal induced by gp120. These data suggest the possible involvement of mannose‐specific endocytotic lectin of MDM. The reaction of DMPO with sodium nitroprusside, an organic nitrate that releases NO·, also produced DMPO‐OH. Our findings indicate that gpl20 increases free radical production from MDM as detected by spin‐trapping methods, and that the spin trap adduct results from a reaction involving NO· or closely related oxidized derivatives. J. Leukoc. Biol. 55: 175–182; 1994.

Peroxynitrite induces senescence and apoptosis of red blood cells through the activation of aspartyl and cysteinyl proteases

Changes in the oxidative status of erythrocytes can reduce cell lifetime, oxygen transport, and delivery capacity to peripheral tissues and have been associated with a plethora of human diseases. Among reactive oxygen and nitrogen species of importance in red blood cell (RBC) homeostasis, superoxide and nitric oxide radicals play a key role. In the present work, we evaluated subcellular effects induced by peroxynitrite, the product of the fast reaction between superoxide and nitric oxide. Peroxynitrite induced 1) oxidation of oxyhemoglobin to methemoglobin, 2) cytoskeleton rearrangement, 3) ultrastructural alterations, and 4) altered expression of band‐3 and decreased expression of glycophorin A. With respect to control cells, this occurred in a significantly higher percentage of human RBC (∼40%). The presence of antioxidants inhibited these modifications. Furthermore, besides these senescence‐associated changes, other important modifications, absent in control RBC and usually associated with apoptotic cell death, were detected in a small but significant subset of peroxynitrite‐exposed RBC (∼7%). Active protease cathepsin E and μ‐calpain increased; activation of caspase 2 and caspase 3 was detected; and phosphatidylserine externalization, an early marker of apoptosis, was observed. Conversely, inhibition of cathepsin E, μ‐calpain, as well as caspase 2 and 3 by specific inhibitors resulted in a significant impairment of erythrocyte “apoptosis.” Altogether, these results indicate that peroxynitrite, a milestone of redox‐mediated damage in human pathology, can hijack human RBC toward senescence and apoptosis by a mechanism involving both cysteinyl and aspartyl proteases.

Peroxynitrite activates kinases of the SRC family and upregulates tyrosine phosphorylation signaling

The hypothesis that peroxynitrite may act as a signaling molecule able to upregulate protein tyrosine phosphorylation is discussed. This article focuses on the mechanisms for activating kinases of the src family, an important class of nonreceptor tyrosine kinases implicated in the regulation of cell communication, proliferation, migration, differentiation, and survival. Recent in vitro findings show that in erythrocytes, synaptosomes, and cerebellar primary culture cells peroxynitrite is able to inhibit phosphatases and to activate different members of the src kinase family through different mechanisms involving cysteine-dependent and -independent processes. The ability of nitrotyrosine-containing peptides with SH2 binding affinity to activate src kinases is also discussed.

Peroxynitrite Induces Tyrosine Nitration and Modulates Tyrosine Phosphorylation of Synaptic Proteins

Abstract : Peroxynitrite, the product of the radical‐radical reaction between nitric oxide and superoxide anion, is a potent oxidant involved in tissue damage in neurodegenerative disorders. We investigated the modifications induced by peroxynitrite in tyrosine residues of proteins from synaptosomes. Peroxynitrite treatment (≥50 μM) induced tyrosine nitration and increased tyrosine phosphorylation. Synaptophysin was identified as one of the major nitrated proteins and pp60src kinase as one of the major phosphorylated substrates. Further fractionation of synaptosomes revealed nitrated synaptophysin in the synaptic vesicles, whereas phosphorylated pp60src was enriched in the postsynaptic density fraction. Tyrosine phosphorylation was increased by treatment with 50‐500 μM peroxynitrite and decreased by higher concentrations, suggesting a possible activation/inactivation of kinases. Immunocomplex kinase assay proved that peroxynitrite treatment of synaptosomes modulated the pp60src autophosphorylation activity. The addition of bicarbonate (CO2 1.3 mM) produced a moderate enhancing effect on some nitrate proteins but significantly protected the activity of pp60src against peroxynitrite‐mediated inhibition so that at 1 mM peroxynitrite, the kinase was still more active than in untreated synaptosomes. The phosphotyrosine phosphatase activity of synaptosomes was inhibited by peroxynitrite (≥50 μM) but significantly protected by CO2. Thus, the increase of phosphorylation cannot be attributed to peroxynitrite‐mediated inhibition of phosphatases. We suggest that peroxynitrite may regulate the posttranslational modification of tyrosine residues in pre‐ and postsynaptic proteins. Identification of the major protein targets gives insight into the pathways possibly involved in neuronal degeneration associated with peroxynitrite overproduction.

Activation of src tyrosine kinases by peroxynitrite

In this study, we demonstrate that the phosphorylation activity of five tyrosine kinases of the src family from both human erythrocytes (lyn, hck and c‐fgr) and bovine synaptosomes (lyn and fyn) was stimulated by treatment with 30–250 μM peroxynitrite. This effect was not observed with syk, a non‐src family tyrosine kinase. Treatment of kinase immunoprecipitates with 0.01–10 μM peroxynitrite showed that the interaction of these enzymes with the oxidant also activated the src kinases. Higher concentrations of peroxynitrite inhibited the activity of all kinases, indicating enzyme inactivation. The addition of bicarbonate (1.3 mM CO2) did not modify the upregulation of src kinases but significantly protected the kinases against peroxynitrite‐mediated inhibition. Upregulation of src kinase activity by 1 μM peroxynitrite was 3.5–5‐fold in erythrocytes and 1.2–2‐fold in synaptosomes, but this could be the result, at least in part, of the higher basal level of src kinase activity in synaptosomes. Our results indicate that peroxynitrite can upregulate the tyrosine phosphorylation signal through the activation of src kinases.

Nitrotyrosine mimics phosphotyrosine binding to the SH2 domain of the src family tyrosine kinase lyn

The nitration of tyrosine residues in protein occurs through the action of reactive oxygen and nitrogen species and is considered a marker of oxidative stress under pathological conditions. The most active nitrating species so far identified is peroxynitrite, the product of the reaction between nitric oxide and superoxide anion. Previously, we have reported that in erythrocytes peroxynitrite irreversibly upregulates lyn, a tyrosine kinase of the src family. In this study we investigated the possible role of tyrosine nitration in the mechanism of lyn activation. We found that tyrosine containing peptides modelled either on the C‐terminal tail of src kinases or corresponding to the first 15 amino acids of human erythrocyte band 3 were able to activate lyn when the tyrosine was substituted with 3‐nitrotyrosine. The activity of nitrated peptides was shared with phosphorylated but not with unphosphorylated, chlorinated or scrambled peptides. Recombinant lyn src homology 2 (SH2) domain blocked the capacity of the band 3‐derived nitrotyrosine peptide to activate lyn and we demonstrated that this peptide specifically binds the SH2 domain of lyn. We propose that nitropeptides may activate src kinases through the displacement of the phosphotyrosine in the tail from its binding site in the SH2 domain. These observations suggest a new mechanism of peroxynitrite‐mediated signalling that may be correlated with the upregulation of tyrosine phosphorylation observed in several pathological conditions.

Peroxynitrite affects exocytosis and SNARE complex formation and induces tyrosine nitration of synaptic proteins

The reactive species peroxynitrite, formed via the near diffusion‐limited reaction of nitric oxide and superoxide anion, is a potent oxidant that contributes to tissue damage in neurodegenerative disorders. Peroxynitrite readily nitrates tyrosine residues in proteins, producing a permanent modification that can be immunologically detected. We have previously demonstrated that in the nerve terminal, nitrotyrosine immunoreactivity is primarily associated with synaptophysin. Here we identify two other presynaptic proteins nitrated by peroxynitrite, Munc‐18 and SNAP25, both of which are involved in sequential steps leading to vesicle exocytosis. To investigate whether peroxynitrite affects vesicle exocytosis, we used the fluorescent dye FM1‐43 to label a recycling population of secretory vesicles within the synaptosomes. Bolus addition of peroxynitrite stimulated exocytosis and glutamate release. Notably, these effects were strongly reduced in the presence of NaHCO3, indicating that peroxynitrite acts mainly intracellularly. Furthermore, peroxynitrite enhanced the formation of the sodium dodecyl sulfate‐resistant SNARE complex in a dose‐dependent manner (100–1000 µm) and induced the formation of 3‐nitrotyrosine in proteins of SNARE complex. These data suggest that modification(s) of synaptic vesicle proteins induced by peroxynitrite may affect protein–protein interactions in the docking/fusion steps, thus promoting exocytosis, and that, under excessive production of superoxide and nitric oxide, neurons may up‐regulate neuronal signaling.

Agglutinating activity of gliadin-derived peptides from bread wheat: Implications for coeliac disease pathogenesis

The PT-digest of bread wheat gliadin was very active in agglutinating undifferentiated human K562(S) cells. This activity was quantitatively, but not qualitatively, similar to that of Con A or WGA. Moreover, Con A-induced cell agglutination was inhibited by mannan and mannose, WGA-induced agglutination by NAG only, and cell agglutination induced by bread wheat gliadin peptides was inhibited by each of these three saccharides. Not only was mannan the most active saccharide in preventing cell agglutination induced by bread wheat gliadin peptides, but it was also able to dissociate agglutinated cells. As compared to the PT- digest of whole bread wheat gliadin, the digest obtained from purified A-gliadin was tenfold more active. The PT-digest of durum wheat gliadin did not show any agglutinating activity.