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.     

DeepNitro: Prediction of Protein Nitration and Nitrosylation Sites by Deep Learning

Protein nitration and nitrosylation are essential post-translational modifications(PTMs)involved in many fundamental cellular processes. Recent studies have revealed that excessive levels of nitration and nitrosylation in some critical proteins are linked to numerous chronic diseases.Therefore, the identification of substrates that undergo such modifications in a site-specific manner is an important research topic in the community and will provide candidates for targeted therapy. In this study, we aimed to develop a computational tool for predicting nitration and nitrosylation sites in proteins. We first constructed four types of encoding features, including positional amino acid distributions, sequence contextual dependencies, physicochemical properties, and position-specificscoring features, to represent the modified residues. Based on these encoding features, we established a predictor called DeepNitro using deep learning methods for predicting protein nitration and nitrosylation. Using n-fold cross-validation, our evaluation shows great AUC values for DeepNitro, 0.65 for tyrosine nitration, 0.80 for tryptophan nitration, and 0.70 for cysteine nitrosylation, respectively,demonstrating the robustness and reliability of our tool. Also, when tested in the independent dataset, DeepNitro is substantially superior to other similar tools with a 7%à42% improvement in the prediction performance. Taken together, the application of deep learning method and novel encoding schemes, especially the position-specific scoring feature, greatly improves the accuracy of nitration and nitrosylation site prediction and may facilitate the prediction of other PTM sites. DeepNitro is implemented in JAVA and PHP and is freely available for academic research at http://deepnitro.renlab.org.     

Effect of nitric oxide donor and gamma irradiation on modifications of ERK and JNK in murine peritoneal macrophages

Mitogen activated protein kinases (MAPKs) play an important role in activation, differentiation and proliferation of macrophages. Macrophages, upon activation, produce large amounts of nitric oxide that inhibit the growth of variety of microorganisms and tumor cells. This nitric oxide which is known to interfere with tyrosine phosphorylation may result in changes in the pattern of activation of MAPKs. In a previous study we have found that tyrosine phosphorylation of MAPKs was completely abolished in the presence of nitric oxide donor and radiation but this did not affect the function of macrophages. In this study the other post translational modifications namely nitration and ubiquitination of JNK and ERK have been looked at. Both ERK and JNK were found to be nitrated. However, there was no increase in ubiquitination of ERK and JNK, indicating that ubiquitination, in this case was not a natural consequence of nitration and may serve in signaling. Additionally, when the nitration was extensive, phosphorylation was also inhibited. The activation of substrates of ERK and JNK were looked at to determine the consequences of such modifications. Inhibition of phosphorylation and extensive nitration of JNK did not prevent activation of its substrate, c-jun. This study indicates that ERK and JNK may be under regulation by different type of modifications in macrophages.     

Effect of nitric oxide donor and gamma irradiation on modifications of ERK and JNK in murine peritoneal macrophages

Mitogen activated protein kinases (MAPKs) play an important role in activation, differentiation and proliferation of macrophages. Macrophages, upon activation, produce large amounts of nitric oxide that inhibit the growth of variety of microorganisms and tumor cells. This nitric oxide which is known to interfere with tyrosine phosphorylation may result in changes in the pattern of activation of MAPKs. In a previous study we have found that tyrosine phosphorylation of MAPKs was completely abolished in the presence of nitric oxide donor and radiation but this did not affect the function of macrophages. In this study the other post translational modifications namely nitration and ubiquitination of JNK and ERK have been looked at. Both ERK and JNK were found to be nitrated. However, there was no increase in ubiquitination of ERK and JNK, indicating that ubiquitination, in this case was not a natural consequence of nitration and may serve in signaling. Additionally, when the nitration was extensive, phosphorylation was also inhibited. The activation of substrates of ERK and JNK were looked at to determine the consequences of such modifications. Inhibition of phosphorylation and extensive nitration of JNK did not prevent activation of its substrate, c-jun. This study indicates that ERK and JNK may be under regulation by different type of modifications in macrophages.     

Inactivation of glutathione S-transferases by nitric oxide-derived oxidants: exploring a role for tyrosine nitration

Reactive intermediates derived from nitric oxide ((*)NO) are thought to play a contributing role in disease states associated with inflammation and infection. We show here that glutathione S-transferases (GSTs), principal enzymes responsible for detoxification of endogenous and exogenous electrophiles, are susceptible to inactivation by reactive nitrogen species (RNS). Treatment of isolated GSTs or rat liver homogenates with either peroxynitrite, the myeloperoxidase/hydrogen peroxide/nitrite system, or tetranitromethane, resulted in loss of GST activity with a concomitant increase in the formation of protein-associated 3-nitrotyrosine (NO(2)Tyr). This inactivation was only partially (<25%) reversible by dithiothreitol, and exposure of GSTs to hydrogen peroxide or S-nitrosoglutathione was only partially inhibitory (<25%) and did not result in protein nitration. Thus, irreversible modifications such as tyrosine nitration may have contributed to GST inactivation by RNS. Since all GSTs contain a critical, highly conserved, active-site tyrosine residue, we postulated that this Tyr residue might present a primary target for nitration by RNS, thus leading to enzyme inactivation. To directly investigate this possibility, we analyzed purified mouse liver GST-mu, following nitration by several RNS, by trypsin digestion, HPLC separation, and matrix-assisted laser desorption/ionization-time of flight analysis, to determine the degree of tyrosine nitration of individual Tyr residues. Indeed, nitration was found to occur preferentially on several tyrosine residues located in and around the GST active site. However, RNS concentrations that resulted in near complete GST inactivation only caused up to 25% nitration of even preferentially targeted tyrosine residues. Hence, nitration of active-site tyrosine residues may contribute to GST inactivation by RNS, but is unlikely to fully account for enzyme inactivation. Overall, our studies illustrate a potential mechanism by which RNS may promote (oxidative) injury by environmental pollutants in association with inflammation.     

Protein nitration as footprint of oxidative stress-related nitric oxide signaling pathways in developing Ciona intestinalis

Developmental processes in the ascidian Ciona intestinalis depend on a complex interplay of events including, during metamorphosis, a caspase-dependent apoptosis which is regulated by the nitric oxide (NO)-cGMP signaling pathway. Herein we disclose an alternate NO-mediated signaling pathway during Ciona development which appears to be critically dependent on local redox control. Evidence in support of this conclusion includes: (a) inhibitors of NO synthase (NOS) and scavengers of NO-derived nitrating agents markedly decrease the rate of Ciona metamorphosis; (b) an NO donor or peroxynitrite caused an opposite effect; (c) increased protein nitration is observed at larva stage. Integrated proteomic and immunochemical methodologies identified nitrated tyrosine residues in ERK and snail. Overall, these results point to protein nitration as a hitherto overlooked NO-dependent regulatory mechanism in Ciona which is specifically triggered by elevated ROS production during developmental processes.     

Reprint of "oxidative alterations of cyclooxygenase during atherogenesis" [Prostag. Oth. Lipid. M. 80 (2006) 1-14

Nitric oxide (*NO) and eicosanoids are critical mediators of physiological and pathophysiological processes. They include inflammation and atherosclerosis. *NO production and eicosanoid synthesis become disrupted during atherosclerosis and thus, it is important to understand the mechanisms that may contribute to this outcome. We, and others, have shown that nitrogen oxide (NOx) species modulate cyclooxygenase (COX; also known as prostaglandin H2 synthase) activity and alter eicosanoid production. We have determined that peroxynitrite (ONOO-) has multiple effects on COX activity. ONOO- can provide the peroxide tone necessary for COX activation, such that simultaneous exposure of COX to its arachidonic acid substrate and ONOO- results in increased eicosanoid production. Alternatively, in the absence of arachidonic acid, ONOO- can modify COX through nitration of an essential tyrosine residue (Tyr385) such that it is incapable of catalysis. In this regard, we have shown that COX nitration occurs in human atherosclerotic tissue and in aortic lesions from ApoE-/- mice kept on a high fat diet. Additionally, we have demonstrated that Tyr nitration in ApoE-/- mice is dependent on the inducible form of NO synthase (iNOS). Under conditions where ONOO- persists and arachidonic acid is not immediately available, the cell may try to correct the situation by responding to ONOO- and releasing arachidonic acid via a signaling pathway to favor COX activation. Other post-translational modifications of COX by NOx species include S-nitrosation of cysteine (Cys) residues (which may have an activating effect) and Cys oxidation. The central focus of this review will include a discussion of how NOx species alter COX activity at the molecular level and how these modifications may contribute to altered eicosanoid output during atherosclerosis and lesion development.     

Nitration of PECAM-1 ITIM tyrosines abrogates phosphorylation and SHP-2 binding

Platelet-endothelial cell adhesion molecule-1 (PECAM-1) is a cell adhesion molecule with a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) that, when phosphorylated, binds Src homology 2 domain-containing protein-tyrosine phosphatase (SHP-2). PECAM-1 is expressed at endothelial cell junctions where exposure to inflammatory intermediates may result in post-translational amino acid modifications that affect protein structure and function. Reactive nitrogen species (RNS), which are produced at sites of inflammation, nitrate tyrosine residues, and several proteins modified by tyrosine nitration have been found in diseased tissue. We show here that the RNS, peroxynitrite, induced nitration of both full-length cellular PECAM-1 and a purified recombinant PECAM-1 cytoplasmic domain. Mass spectrometric analysis of tryptic fragments revealed quantitative nitration of ITIM tyrosine 686. A synthetic peptide containing 3-nitrotyrosine at position 686 could not be phosphorylated nor bind SHP-2. These data suggest that ITIM tyrosine nitration may represent a mechanism for modulating phosphotyrosine-dependent signal transduction pathways.     

Effects of nitric oxide synthase-3 overexpression on post-translational modifications and cell survival in HepG2 cells

Hepatocarcinoma is the fifth most common neoplasm and the third cause of cancer-related death. The development of genetic- and/or molecular-based therapies is urgently required. The administration of high doses of nitric oxide (NO) promotes cell death in hepatocytes. NO contributes to cell signaling by inducing oxidative/nitrosative-dependent post-translational modifications. The aim of the present study was to investigate protein modifications and its relation with alteration of cell proliferation and death in hepatoma cells. Increased intracellular NO production was achieved by stable nitric oxide synthase-3 (NOS-3) overexpression in HepG2 cells. We assessed the pattern of nitration, nitrosylation and carbonylation of proteins by proteomic analysis. The results showed that NOS-3 cell overexpression increased oxidative stress, which affected proteins mainly involved in cell protein folding. Carbonylation also altered metabolism, as well as immune and antioxidant responses. The interaction of nitrosative and oxidative stress generated tyrosine nitration, which affected the tumor marker Serpin B3, ATP synthesis and cytoskeleton. All these effects were associated with a decrease in chaperone activity, a reduction in cell proliferation and an increased cell death. Our study showed that alteration of nitration, nitrosylation and carbonylation pattern of proteins by NO-dependent oxidative/nitrosative stress was related to a reduction of cell survival in a hepatoma cell line.     

Intervention by nitric oxide, NO, and its oxide derivatives particularly in mammals

Nitric oxide (NO) is a natural and stable free radical produced in soil and water by the bacteriological reduction of nitrites and nitrates and in animals by the enzyme oxidation of L-arginine. NO is biosynthesised by finely regulated enzymatic systems called NO-synthases and readily diffuses through tissues. It reacts rapidly with hemoproteins and iron-sulphur centers to form nitrosylated compounds. It oxidises more slowly to form nitrogen oxides that nitrosate thiols into thionitrite. NO is transported in these various forms and released spontaneously or through yet unclear mechanisms into most cells; it also regulates oxygen consumption at the mitochondrial respiratory chain level through interaction with cytochrome oxidase. In the cardiovascular system, NO lowers blood pressure by activating a hemoprotein, the guanylate cyclase present in muscle cells; through such interaction it acts also as a neuromediator and neuromodulator in the nervous system. However, many of NO's roles result from rapid coupling to other radicals; for example, it reacts with the superoxide anion (O2-) to form oxoperoxinitrate (ONOO-, also known as peroxynitrite). This strong oxidant of metallic centers, thiols, and antioxidants is also able to convert tyrosine to 3-nitrotyrosine and to act upon tyrosine residues contained in proteins. The biological aspects of the roles of NO are presented with particular respect to the rapid interactions of NO with hemoproteins' iron and other radicals. Concurrently, NO oxidation enables nitrosation reactions primarily of thiols but ultimately of nucleic bases. The thionitrite function (R-S-NO) thus formed and the dimerisation and nitration of tyrosine residues are protein post-translational modifications that are being investigated in animals.     

Peroxynitrite oxidizes erythrocyte membrane band 3 protein and diminishes its anion transport capacity

We describe an altered membrane band 3 protein-mediated anion transport in erythrocytes exposed to peroxynitrite, and relate the loss of anion transport to cell damage and to band 3 oxidative modifications. We found that peroxynitrite down-regulate anion transport in a dose dependent relation (100-300 μmoles/l). Hemoglobin oxidation was found at all peroxynitrite concentrations studied. A dose-dependent band 3 protein crosslinking and tyrosine nitration were also observed. Band 3 protein modifications were concomitant with a decrease in transport activity. ( - )-Epicatechin avoids band 3 protein nitration but barely affects its transport capacity, suggesting that both processes are unrelated. N-acetyl cysteine partially reverted the loss of band 3 transport capacity. It is concluded that peroxynitrite promotes a decrease in anion transport that is partially due to the reversible oxidation of band 3 cysteine residues. Additionally, band 3 tyrosine nitration seems not to be relevant for the loss of its anion transport capacity.     

植物细胞一氧化氮信号转导研究进展

一氧化氮(nitric oxide, NO)作为重要的信号分子, 调控植物的种子萌发、根形态建成和花器官发生等许多生长发育过程, 并参与气孔运动的调节以及植物对多种非生物胁迫和病原体侵染的应答过程。已经知道, 精氨酸依赖的NOS途径和亚硝酸盐依赖的NR途径是植物细胞NO产生的主要酶促合成途径。NO及其衍生物能够直接修饰底物蛋白的金属基团、半胱氨酸和酪氨酸残基, 通过金属亚硝基化、巯基亚硝基化和Tyr-硝基化等化学修饰方式, 调节靶蛋白的活性, 并影响cGMP和Ca²⁺信使系统等下游信号途径, 调控相应的生理过程。最新的一些研究结果也显示, MAPK级联系统与NO信号转导途径之间存在复杂的交叉调控。此外, 作为活跃的小分子信号, NO和活性氧相互依赖并相互影响, 共同介导了植物的胁迫应答和激素响应过程。文章综述了植物NO信号转导研究领域中一些新的研究进展, 对NO与活性氧信号途径间的交叉作用等也作了简要介绍。     

Peroxynitrite signaling: receptor tyrosine kinases and activation of stress-responsive pathways

Peroxynitrite, generated for example in inflammatory processes, is capable of nitrating and oxidizing biomolecules, implying a considerable impact on the integrity of cellular structures. Cells respond to stressful conditions by the activation of signaling pathways, including receptor tyrosine kinase-dependent pathways such as mitogen-activated protein kinases and the phosphoinositide-3-kinase/Akt pathway. Peroxynitrite affects signaling pathways by nitration as well as by oxidation: while nitration of tyrosine residues by peroxynitrite modulates signaling processes relying on tyrosine phosphorylation and dephosphorylation, oxidation of phosphotyrosine phosphatases may lead to an alteration in the tyrosine phosphorylation/dephosphorylation balance. The flavanol (-)-epicatechin is a potent inhibitor of tyrosine nitration and may be employed as a tool to distinguish signaling effects due to tyrosine nitration from those that are due to oxidation reactions.     

Peroxynitrite oxidizes erythrocyte membrane band 3 protein and diminishes its anion transport capacity

We describe an altered membrane band 3 protein-mediated anion transport in erythrocytes exposed to peroxynitrite, and relate the loss of anion transport to cell damage and to band 3 oxidative modifications. We found that peroxynitrite down-regulate anion transport in a dose dependent relation (100–300 μmoles/l). Hemoglobin oxidation was found at all peroxynitrite concentrations studied. A dose-dependent band 3 protein crosslinking and tyrosine nitration were also observed. Band 3 protein modifications were concomitant with a decrease in transport activity. ( ? )-Epicatechin avoids band 3 protein nitration but barely affects its transport capacity, suggesting that both processes are unrelated. N-acetyl cysteine partially reverted the loss of band 3 transport capacity. It is concluded that peroxynitrite promotes a decrease in anion transport that is partially due to the reversible oxidation of band 3 cysteine residues. Additionally, band 3 tyrosine nitration seems not to be relevant for the loss of its anion transport capacity.     

Free radical-mediated oxidation of free amino acids and amino acid residues in proteins

Summary. We summarize here results of studies designed to elucidate basic mechanisms of reactive oxygen (ROS)-mediated oxidation of proteins and free amino acids. These studies have shown that oxidation of proteins can lead to hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, nitrosylation of sulfhydryl groups, sulfoxidation of methionine residues, chlorination of aromatic groups and primary amino groups, and to conversion of some amino acid residues to carbonyl derivatives. Oxidation can lead also to cleavage of the polypeptide chain and to formation of cross-linked protein aggregates. Furthermore, functional groups of proteins can react with oxidation products of polyunsaturated fatty acids and with carbohydrate derivatives (glycation/glycoxidation) to produce inactive derivatives. Highly specific methods have been developed for the detection and assay of the various kinds of protein modifications. Because the generation of carbonyl derivatives occurs by many different mechanisms, the level of carbonyl groups in proteins is widely used as a marker of oxidative protein damage. The level of oxidized proteins increases with aging and in a number of age-related diseases. However, the accumulation of oxidized protein is a complex function of the rates of ROS formation, antioxidant levels, and the ability to proteolytically eliminate oxidized forms of proteins. Thus, the accumulation of oxidized proteins is also dependent upon genetic factors and individual life styles. It is noteworthy that surface-exposed methionine and cysteine residues of proteins are particularly sensitive to oxidation by almost all forms of ROS; however, unlike other kinds of oxidation the oxidation of these sulfur-containing amino acid residues is reversible. It is thus evident that the cyclic oxidation and reduction of the sulfur-containing amino acids may serve as an important antioxidant mechanism, and also that these reversible oxidations may provide an important mechanism for the regulation of some enzyme functions.     

Biological selectivity and functional aspects of protein tyrosine nitration

The formation of nitric oxide in biological systems has led to the discovery of a number of post-translational protein modifications that could regulate protein function or potentially be utilized as transducers of nitric oxide signaling. Principal among the nitric oxide-mediated protein modifications are: the nitric oxide-iron heme binding, the S-nitrosylation of reduced cysteine residues, and the C-nitration of tyrosine and tryptophan residues. With the exception of the nitric oxide binding to heme iron proteins, the other two modifications appear to require secondary reactions of nitric oxide and the formation of nitrogen oxides. The rapid development of analytical and immunological methodologies has allowed for the quantification of S-nitrosylated and C-nitrated proteins in vivo revealing an apparent selectivity and specificity of the proteins modified. This review is primarily focused upon the nitration of tyrosine residues discussing parameters that may govern the in vivo selectivity of protein nitration, and the potential biological significance and clinical relevance of this nitric oxide-mediated protein modification.     

Determination of the structure of lipid vesicle-bound angiotensin II and angiotensin I

A mass spectrometry (MS)-based strategy was developed to determine the structure of lipid vesicle-bound angiotensin II (AII) and angiotensin I (AI). It involves hydrogen-deuterium exchange (HDX), chemical modifications (e.g., nitration of tyrosine, acetylation of free amino group), and ladder sequencing. HDX is also combined with tandem mass spectrometry (MS/MS) to provide structural details at individual amino acid residues. It was observed that a major portion of both of these peptide hormones interacts with the phospholipid head groups on the surface of the vesicles and that Tyr residue is embedded in the vesicles. Both peptides have a U-shaped structure in the lipid environment.     

Posttranslational modifications on protein kinase c isozymes. Effects of epinephrine and phorbol esters

The posttranslational modifications induced on PKC isozymes as result of their activation were investigated. Reciprocal immunoprecipitations followed by Western blot analysis demonstrated that all PKC isozymes expressed in rat hepatocytes are modified by tyrosine nitration and tyrosine phosphorylation in different ways upon exposure of cells to a direct PKC activator (TPA), or to an extracellular ligand known to activate PKC-dependent pathways (epinephrine). Our data demonstrate for the first time that all PKC isozymes are also dynamically modified by O-linked beta-N-acetylglucosamine (O-GlcNAc); the presence of this modification was confirmed in part by FT-ICR mass spectrometry analysis. Interestingly, the O-GlcNAc modified Ser or Thr were mapped at similar positions in several PKC isozymes. The biochemical meaning of these posttranslational modifications was investigated for PKC alpha and delta. It was found that the PKC phosphorylation status of both isozymes in tyrosine and serine residues seems to regulate directly the enzyme activity since catalytic inactivation correlate with dephosphorylation of Ser at the C-terminus autophosphorylation sites of each PKC isozyme, and with an increase in the level of tyrosine phosphorylation. Whereas none of the other posttranslational modifications showed per se a direct effect in PKC delta activity, increased tyrosine nitration and O-GlcNAc modifications correlate negatively with PKCalpha activity.     

植物细胞一氧化氮信号转导研究进展

一氧化氮（nitric oxide,NO）作为重要的信号分子,调控植物的种子萌发、根形态建成和花器官发生等许多生长发育过程,并参与气孔运动的调节以及植物对多种非生物胁迫和病原体侵染的应答过程。已经知道,精氨酸依赖的NOS途径和亚硝酸盐依赖的NR途径是植物细胞NO产生的主要酶促合成途径。NO及其衍生物能够直接修饰底物蛋白的金属基团、半胱氨酸和酪氨酸残基,通过金属亚硝基化、巯基亚硝基化和Tyr．硝基化等化学修饰方式,调节靶蛋白的活性,并影响cGMP和Ca2＋信使系统等下游信号途径,调控相应的生理过程。最新的一些研究结果也显示,MAPK级联系统与NO信号转导途径之间存在复杂的交叉调控。此外,作为活跃的小分子信号,NO和活性氧相互依赖并相互影响,共同介导了植物的胁迫应答和激素响应过程。文章综述了植物NO信号转导研究领域中一些新的研究进展,对NO与活性氧信号途径间的交叉作用等也作了简要介绍。