The nitration of proteins in platelets

Nitric oxide has many important physiological functions, but it may also form an important oxidant, peroxynitrite, as a consequence of its reaction with superoxide anions. Peroxynitrite is capable of nitrating the aromatic amino acids in proteins, particularly tyrosine. Nitrated proteins are found in tissues of a variety of diseases where inflammation occurs. However, our recent work suggests that more selective nitration of specific proteins may occur during normal physiological processes, such as platelet activation by collagen. It is not yet clear what role this may play in the normal cell biology, but there is potential to be a role in signal transduction mechanisms, possibly by influencing tyrosine phosphorylation or dephosphorylation.     

Signal transduction by protein tyrosine nitration: competition or cooperation with tyrosine phosphorylation-dependent signaling events?

This review article is an attempt to stimulate a discussion on the significance of protein tyrosine nitration to cellular signaling and its relationships with protein tyrosine phosphorylation. Initially, it provides basic information on growth factor and oxidants as modulators/mediators of tyrosine phosphorylation-dependent signal transduction pathways. The effects of exogenous and endogenous tyrosine nitration on such pathways were examined by reviewing published and unpublished observations. From an initial perspective that tyrosine nitration was a toxic manifestation of nitric oxide, the concept evolved to a protein modification that could also function in cellular signaling events, possibly cooperating with tyrosine phosphorylation.     

Peroxynitrite inhibits T lymphocyte activation and proliferation by promoting impairment of tyrosine phosphorylation and peroxynitrite-driven apoptotic death

Peroxynitrite (ONOO-) is a potent oxidizing and nitrating agent produced by the reaction of nitric oxide with superoxide. It readily nitrates phenolic compounds such as tyrosine residues in proteins, and it has been demonstrated that nitration of tyrosine residues in proteins inhibits their phosphorylation. During immune responses, tyrosine phosphorylation of key substrates by protein tyrosine kinases is the earliest of the intracellular signaling pathways following activation through the TCR complex. This work was aimed to evaluate the effects of ONOO- on lymphocyte tyrosine phosphorylation, proliferation, and survival. Additionally, we studied the generation of nitrating species in vivo and in vitro during immune activation. Our results demonstrate that ONOO-, through nitration of tyrosine residues, is able to inhibit activation-induced protein tyrosine phosphorylation in purified lymphocytes and prime them to undergo apoptotic cell death after PHA- or CD3-mediated activation but not upon phorbol ester-mediated stimulation. We also provide evidence indicating that peroxynitrite is produced during in vitro immune activation, mainly by cells of the monocyte/macrophage lineage. Furthermore, immunohistochemical studies demonstrate the in vivo generation of nitrating species in human lymph nodes undergoing mild to strong immune activation. Our results point to a physiological role for ONOO- as a down-modulator of immune responses and also as key mediator in cellular and tissue injury associated with chronic activation of the immune system.     

Effects of peroxynitrite-induced protein tyrosine nitration on insulin-stimulated tyrosine phosphorylation in HepG2 cells

Tyrosine nitration by peroxynitrite can affect signal transduction pathways involving tyrosine phosphorylation. The present study was undertaken to investigate the effects of peroxynitrite-induced protein tyrosine nitration on insulin-stimulated tyrosine phosphorylation in HepG2 cells. We show here that exposure of HepG2 cells to peroxynitrite led to a dose-dependent increase in tyrosine nitration of cellular proteins, mainly membrane and nuclear proteins. Furthermore, peroxynitrite induced differential responses in tyrosine phosphorylation of membrane proteins as well as cytosolic proteins according to peroxynitrite concentrations used. Our findings indicate at low concentrations peroxynitrite upregulates the insulin signaling and may operate as a signaling molecule, but at higher concentrations peroxynitrite downregulates the insulin signaling and may be involved in insulin resistance, suggesting peroxynitrite plays a dual role in regulation of the insulin signaling.     

Peroxynitrite-mediated lipid oxidation and nitration: Mechanisms and consequences

Lipid oxidation and nitration represents a novel area of research of relevance in the understanding of inflammatory processes. Peroxynitrite, the product of the diffusion-limited reaction between nitric oxide and superoxide anion, mediates oxidative modifications in lipid systems including cell membranes and lipoproteins. In this review, we discuss the mechanisms of lipid oxidation and nitration by peroxynitrite as well as the influence of physiological molecules and cell targets to redirect peroxynitrite reactivity. We also provide evidence to support that oxidation/nitration of lipids results in the formation of novel signaling modulators of key lipid-metabolizing enzymes.     

Tyrosine nitration as a key event of signal transduction that regulates functional state of the cell

This review is dedicated to the role of nitration of proteins by tyrosine residues in physiological and pathological conditions. First of all, we analyze the biochemical evidence of peroxynitrite formation and reactions that lead to its formation, types of posttranslational modifications (PTMs) induced by reactive nitrogen species, as well as three biological pathways of tyrosine nitration. Then, we describe two possible mechanisms of protein nitration that are involved in intracellular signal transduction, as well as its interconnection with phosphorylation/dephosphorylation of tyrosine. Next part of the review is dedicated to the role of proteins nitration in different pathological conditions. In this section, special attention is devoted to the role of nitration in changes of functional properties of actin—protein that undergoes PTMs both in normal and pathological conditions. Overall, this review is devoted to the main features of protein nitration by tyrosine residue and the role of this process in intracellular signal transduction in basal and pathological conditions.     

Peroxynitrite signaling in human erythrocytes: synergistic role of hemoglobin oxidation and band 3 tyrosine phosphorylation

Peroxynitrite crosses the red blood cell (RBC) membrane and reacts with hemoglobin (Hb) producing mainly metHb, which is reduced back to ferrousHb by NADH- and NADPH-dependent reductases. Peroxynitrite also induces band 3 (B3) tyrosine phosphorylation, a signaling pathway believed to activate glucose metabolism. This study was aimed to decipher the relationship between these two peroxynitrite-dependent processes. Peroxynitrite induced a burst of the hexose monophosphate shunt (HMS), revealed by NMR studies, and a burst of the glycolytic pathway, measured by lactate production. The HMS plays a prominent role in membrane signaling, as demonstrated by B3 phosphotyrosine inhibition by the glycolytic pathway inhibitor 2-deoxy-glucose (2DG) and activation by dehydroepiandrosterone (DHEA), an inhibitor of HMS. Peroxynitrite-induced B3 tyrosine phosphorylation was paralleled by the inhibition of membrane-associated phosphotyrosine phosphatase (PTP) activity, which was protected by 2DG but not DHEA. Interestingly, heme poisoning with CO inhibited peroxynitrite-dependent Hb oxidation and lactate production but did not affect PTP down regulation. These results suggest two distinct and concurrent effects of peroxynitrite: one mediated by Hb which, likely in its oxidized state, binds more strongly to B3, and another mediated by PTP-dependent B3 phosphorylation. Both effects are directed towards a surge in glucose utilization.     

Tyrosine phosphorylation/dephosphorylation regulates peroxynitrite-mediated peptide nitration

Proteins are targets of reactive nitrogen species such as peroxynitrite and nitrogen dioxide. Among the various amino acids in proteins, tyrosine and tryptophan residues are especially susceptible to attack by reactive nitrogen species. On the other hand, protein tyrosine phosphorylation has gained much attention in respect to cellular regulatory events and signal transduction. Peroxynitrite-mediated nitration of peptide YPPPPPW and phosphopeptide pYPPPPPW were studied at pH 7.4. The predominant nitrated products were separated and identified by reverse phase high performance liquid chromatography coupled with electrospray ionization mass spectrometry (LC-MS). The nitration sites were established by tandem electrospray ionization-mass spectrometry (LC-MS/MS). A regulatory effect of tyrosine phosphorylation/dephosphorylation on peptide nitration was observed. YPPPPPW was predominantly nitrated at tyrosine residue while pYPPPPPW was nitrated at tryptophan one. Our results can help in understanding the biochemical significance of the relationship of tyrosine phosphorylation and nitration in proteins.     

Peroxynitrite Induces Tyrosine Nitration and Modulates Tyrosine Phosphorylation of Synaptic Proteins

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 (> or =50 microM) 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 microM 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 nitrated 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 (> or =50 microM) 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.     

Src redox regulation: there is more than meets the eye

Src-family kinases are critically involved in the control of cytoskeleton organization and in the generation of integrin-dependent signaling responses, inducing tyrosine phosphorylation of many signaling and cytoskeletal proteins. Activity of the Src family of tyrosine kinases is tightly controlled by inhibitory phosphorylation of a carboxy-terminal tyrosine residue, inducing an inactive conformation through binding with its SH2 domain. Dephosphorylation of C-ter tyrosine, as well as its deletion of substitution with phenylalanine in oncogenic Src kinases, leads to autophosphorylation at a tyrosine in the activation loop, thereby leading to enhanced Src activity. Beside this phophorylation/dephosphorylation circuitry, cysteine oxidation has been recently reported as a further mechanism of enzyme activation. Mounting evidence describes Src activation via its redox regulation as a key outcome in several circumstances, including growth factor and cytokines signaling, integrin-mediated cell adhesion and motility, membrane receptor cross-talk as well in cell transformation and tumor progression. Among the plethora of data involving Src kinase in physiological and pathophysiological processes, this review will give emphasis to the redox component of the regulation of this master kinase.     

Queuine mediated inhibition in phosphorylation of tyrosine phosphoproteins in cancer

Protein phosphorylation or dephosphorylation is the most important regulatory switch of signal transduction contributing to control of cell proliferation. The reversibility of phosphorylation and dephosphorylation is due to the activities of kinases and phosphatase, which determine protein phosphorylation level of cell under different physiological and pathological conditions. Receptor tyrosine kinase (RTK) mediated cellular signaling is precisely coordinated and tightly controlled in normal cells which ensures regulated mitosis. Deregulation of RTK signaling resulting in aberrant activation in RTKs leads to malignant transformation. Queuine is one of the modified base of tRNA which participates in down regulation of tyrosine kinase activity. The guanine analogue queuine is a nutrient factor to eukaryotes and occurs as free base or modified nucleoside queuosine into the first anticodon position of specific tRNAs. The tRNAs are often queuine deficient in cancer and fast proliferating tissues. The present study is aimed to investigate queuine mediated inhibition in phosphorylation of tyrosine phosphorylated proteins in lymphoma bearing mouse. The result shows high level of cytosolic and membrane associated tyrosine phosphoprotein in DLAT cancerous mouse liver compared to normal. Queuine treatments down regulate the level of tyrosine phosphoproteins, which suggests that queuine is involved in regulation of mitotic signaling pathways.     

Activation pattern of mitogen-activated protein kinases elicited by peroxynitrite: attenuation by selenite supplementation

Peroxynitrite is a mediator of toxicity in pathological processes in vivo and causes damage by oxidation and nitration reactions. Here, we report a differential induction of mitogen-activated protein kinases (MAPKs) in WB-F344 rat liver epithelial cells by peroxynitrite. For the exposure of cultured cells with peroxynitrite, we employed a newly developed infusion method. At 6.5 microM steady-state concentration, the activation of p38 MAPK was immediate, while JNK1/2 and ERK1/2 were activated 60 min and 15 min subsequent to 3 min of exposure to peroxynitrite, respectively. Protein-bound 3-nitrotyrosine was detected. When cells were grown in a medium supplemented with sodium selenite (1 microM) for 48 h, complete protection was afforded against the activation of p38 and against nitration of tyrosine residues. These data suggest a new role for peroxynitrite in activating signal transduction pathways capable of modulating gene expression. Further, the abolition of the effects of peroxynitrite by selenite supplementation suggests a protective role of selenium-containing proteins.     

Submolecular adventures of brain tyrosine: what are we searching for now?

This overview summarizes recent findings on the role of tyrosyl radical (TyrO(*)) in the multitudinous neurochemical systems of brain, and theorizes on the putative role of TyrO(*) in neurological disorders [Parkinson's disease (PD), Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS)]. TyrO(*) and tyrosine per se can interact with reactive oxygen species (ROS) and reactive nitrogen species (RNS) via radical mechanisms and chain propagating reactions. The concentration of TyrO(*), ROS and RNS can increase dramatically under conditions of generalized stress: oxidative, nitrative or reductive as well, and this can induce damage directly (by lipid peroxidation) or indirectly (by proteins oxidation and/or nitration), potentially causing apoptotic neuronal cell death or autoschizis.Evidence of lesion-induced neuronal oxidative stress includes the presence of protein peroxides (TyrOOH), DT (o,o'-dityrosine) and 3-NT (3-nitrotyrosine). Mechanistic details of protein- and enzymatic oxidation/nitration in vivo remain unresolved, although recent in vitro data strongly implicate free radical pathways via TyrO(*). Nitration/denitration processes can be pathological, but they also may play: 1). a signal transduction role, because nitration of tyrosine residues through TyrO(*) formation can modulate, as well the phosphorylation (tyrosine kinases activity) and/or tyrosine hydroxylation (tyrosine hydroxylase inactivation), leading to consequent dopamine synthesis failure and increased degradation of target proteins, respectively; 2). a role of "blocker" for radical-radical reactions (scavenging of NO(*), NO(*)(2) and CO(3)(*-) by TyrO(*)); 3). a role of limiting factors for peroxynitrite formation, by lowering O(2)(*-) formation, which is strongly linked to the pathogenesis of neural diseases.It is still not known if tyrosine oxidation/nitration via TyrO(*) formation is 1). a footprint of generalized stress and neuronal disorders, or 2). an important part of O(2)(*-) and NO(*) metabolism, or 3). merely a part of integral processes for maintaining of neuronal homeostasis. The full answer to these questions should be of top research priority, as the problem of increased free radical formation in brain and/or imbalance of the ratios ROS/RNS/TyrO(*) may be all important in defining whether oxidative stress is the critical determinant of tissue and neural cell injury that leads to pathological end-points.     

蛋白组氨酸磷酸酶研究进展

主要概括磷酸酶的种类,原核细胞磷酸组氨酸生物功能及调控,哺乳动物组氨酸残基磷酸化、去磷酸化,以及组氨酸磷酸酶及其底物的最新研究进展. 信号转导在生长发育及细胞功能中起极其重要的作用. 无论在原核还是真核细胞,蛋白质磷酸化是细胞内信号转导的关键机制. 研究最多的可逆的真核蛋白磷酸化,主要发生在含有羟基的丝氨酸、苏氨酸和酪氨酸残基上. 不同的激酶和磷酸酶受不同机制的调节,而调节过程中出现的差异是人类很多疾病的潜在基础. 与大量有关羟基磷酸化氨基酸的报道相比,有关氨基磷酸化氨基酸的报道甚少. 据估计,自然界中存在的磷酸组氨酸比磷酸酪氨酸多10 ~ 100倍,但不如磷酸丝氨酸丰富. 虽然对脊椎动物蛋白质中存在磷酸组氨酸的认识可以追溯到20世纪60年代初, 但由于研究手段的限制,至今对脊椎动物蛋白组氨酸激酶及组氨酸磷酸酶的结构及功能知之甚少. 但是,近几年的研究有突破性的发现,克隆和重组表达哺乳动物组氨酸磷酸酶为研究氨基磷酸化氨基酸的生物功能翻开新的一章.     

Specific dephosphorylation of Janus Kinase 2 by protein tyrosine phosphatases

Many protein kinases are activated through phosphorylation of an activation loop thereby turning on downstream signaling pathways. Activation of JAK2, a nonreceptor tyrosine kinase with an important role in growth factor and cytokine signaling, requires phosphorylation of the 1007 and 1008 tyrosyl residues. Dephosphorylation of these two sites by phosphatases presumably inactivates the enzyme, but the underlying mechanism is not known. In this study, we employed MALDI‐TOF/TOF and triple quadrupole mass spectrometers to analyze qualitatively and quantitatively the dephosphorylation process by using synthetic peptides derived from the tandem autophosphorylation sites (Y1007 and Y1008) of human JAK2. We found that tyrosine phosphatases catalyzed the dephosphorylation reaction sequentially, but different enzymes exhibited different selectivity. Protein tyrosine phosphatase 1B caused rapid dephosphorylation of Y1008 followed by Y1007, while SHP1 and SHP2 selectively dephosphorylated Y1008 only, and yet HePTP randomly removed a single phosphate from either Y1007 or Y1008, leaving behind mono‐phosphorylated peptides. The specificity of dephosphorylation was further confirmed by molecular modeling. The data reveal multiple modes of JAK2 regulation by tyrosine phosphatases, reflecting a complex, and intricate interplay between protein phosphorylation and dephosphorylation.     

Inhibition of tyrosine phosphatases antagonizes CD95-mediated apoptosis.

Ligation of the CD95 receptor resulted in a transient increase of cellular tyrosine phosphorylation. The inhibition of protein tyrosine phosphatases by pervanadate, a potent activator of B cells and T cells through the induction of tyrosine phosphorylation and downstream signaling events in the activation cascade, antagonized CD95-triggered apoptosis. Pervanadate exerted its inhibitory effect only during the early phase of apoptosis prior to the CD95-induced decrease of the mitochondrial transmembrane potential. Inhibition of tyrosine phosphatases delayed the cleavage and activation of caspase-8 and caspase-3 and antagonized the tyrosine dephosphorylation of the CD95 receptor-associated phosphoproteins p61 and p89/92. In contrast, ligation of the tumor necrosis factor (TNF) receptor resulted in a continuous tyrosine dephosphorylation of cellular proteins. Pervanadate-induced tyrosine phosphorylation increased the TNF-alpha-induced cytotoxicity and NF-kappaB activation, suggesting that it stimulates early signaling events prior to the separation of the two signaling pathways.     

Peroxynitrite formation and function in plants

Peroxynitrite (ONOO⁻) is a reactive nitrogen species formed when nitric oxide (NO) reacts with the superoxide anion (O₂⁻). It was first identified as a mediator of cell death in animals but was later shown to act as a positive regulator of cell signaling, mainly through the posttranslational modification of proteins by tyrosine nitration. In plants, peroxynitrite is not involved in NO-mediated cell death and its physiological function is poorly understood. However, it is emerging as a potential signaling molecule during the induction of defense responses against pathogens and this could be mediated by the selective nitration of tyrosine residues in a small number of proteins. In this review we discuss the general role of tyrosine nitration in plants and evaluate recent evidence suggesting that peroxynitrite is an effector of NO-mediated signaling following pathogen infection.     

Inhibition of Peroxynitrite-Mediated Oxidation of Dopamine by Flavonoid and Phenolic Antioxidants and Their Structural Relationships

The interaction between peroxynitrite and dopamine and the inhibition of this reaction by plant-derived antioxidants have been investigated. Peroxynitrite promoted the oxidation of dopamine to 6-hydroxyindole-5-one as characterised by HPLC and photodiode array spectra, akin to the products of the tyrosinase-dopamine reaction, but no evidence of dopamine nitration was obtained. Although peroxynitrite did not cause nitration of dopamine in vitro, the catecholamine is capable of inhibiting the formation of 3-nitrotyrosine from peroxynitrite-mediated nitration of tyrosine. The plant-derived phenolic compounds, caffeic acid and catechin, inhibited peroxynitrite-mediated oxidation of dopamine. This effect is attributed to the ability of catechol-containing antioxidants to reduce peroxynitrite through electron donation, resulting in their oxidation to the corresponding o-quinones. The antioxidant effect of caffeic acid and catechin was comparable to that of the endogenous antioxidant, glutathione. In contrast, the structurally related monohydroxylated hydroxycinnamates, p-coumaric acid and ferulic acid, which inhibit tyrosine nitration through a mechanism of competitive nitration, did not inhibit peroxynitrite-induced dopamine oxidation. The findings of the present study suggest that certain plant-derived phenolics can inhibit dopamine oxidation.     

Reversible inhibition of mammalian glutamine synthetase by tyrosine nitration

The effect of tyrosine nitration on mammalian GS activity and stability was studied in vitro. Peroxynitrite at a concentration of 5 micro mol/l produced tyrosine nitration and inactivation of GS, whereas 50 micro mol/l peroxynitrite additionally increased S-nitrosylation and carbonylation and degradation of GS by the 20S proteasome. (-)Epicatechin completely prevented both, tyrosine nitration and inactivation of GS by peroxynitrite (5 micro mol/l). Further, a putative "denitrase" activity restored the activity of peroxynitrite (5 micro mol/l)-treated GS. The data point to a potential regulation of GS activity by a reversible tyrosine nitration. High levels of oxidative stress may irreversibly damage and predispose the enzyme to proteasomal degradation.     

Scavenging of peroxynitrite by oxyhemoglobin and identification of modified globin residues

Peroxynitrite is a strong oxidant involved in cell injury. In tissues, most of peroxynitrite reacts preferentially with CO(2) or hemoproteins, and these reactions affect its fate and toxicity. CO(2) promotes tyrosine nitration but reduces the lifetime of peroxynitrite, preventing, at least in part, membrane crossing. The role of hemoproteins is not easily predictable, because the heme intercepts peroxynitrite, but its oxidation to ferryl species and tyrosyl radical(s) may catalyze tyrosine nitration. The modifications induced by peroxynitrite/CO(2) on oxyhemoglobin were determined by mass spectrometry, and we found that alphaTyr42, betaTyr130, and, to a lesser extent, alphaTyr24 were nitrated. The suggested nitration mechanism is tyrosyl radical formation by long-range electron transfer to ferrylhemoglobin followed by a reaction with (*)NO(2). Dityrosine (alpha24-alpha42) and disulfides (beta93-beta93 and alpha104-alpha104) were also detected, but these cross-linkings were largely due to modifications occurring under the denaturing conditions employed for mass spectrometry. Moreover, immunoelectrophoretic techniques showed that the 3-nitrotyrosine content of oxyhemoglobin sharply increased only in molar excess of peroxynitrite, thus suggesting that this hemoprotein is not a catalyst of nitration. The noncatalytic role may be due to the formation of the nitrating species (*)NO(2) mainly in molar excess of peroxynitrite. In agreement with this hypothesis, oxyhemoglobin strongly inhibited tyrosine nitration of a target dipeptide (Ala-Tyr) and of membrane proteins from ghosts resealed with oxyhemoglobin. Erythrocytes were poor inhibitors of Ala-Tyr nitration on account of the membrane barrier. However, at the physiologic hematocrit, Ala-Tyr nitration was reduced by 65%. This "sink" function was facilitated by the huge amount of band 3 anion exchanger on the cell membrane. We conclude that in blood oxyhemoglobin is a peroxynitrite scavenger of physiologic relevance.