NO-derived Reactive Nitrogen Species Research Articles

S-Nitrosylation of the virulence regulator AphB promotes Vibrio cholerae pathogenesis.

Vibrio cholerae is the etiologic agent of the severe human diarrheal disease cholera. To colonize mammalian hosts, this pathogen must defend against host-derived toxic compounds, such as nitric oxide (NO) and NO-derived reactive nitrogen species (RNS). RNS can covalently add an NO group to a reactive cysteine thiol on target proteins, a process called protein S-nitrosylation, which may affect bacterial stress responses. To better understand how V. cholerae regulates nitrosative stress responses, we profiled V. cholerae protein S-nitrosylation during RNS exposure. We identified an S-nitrosylation of cysteine 235 of AphB, a LysR-family transcription regulator that activates the expression of tcpP, which activates downstream virulence genes. Previous studies show that AphB C235 is sensitive to O2 and reactive oxygen species (ROS). Under microaerobic conditions, AphB formed dimer and directly repressed transcription of hmpA, encoding a flavohemoglobin that is important for NO resistance of V. cholerae. We found that tight regulation of hmpA by AphB under low nitrosative stress was important for V. cholerae optimal growth. In the presence of NO, S-nitrosylation of AphB abolished AphB activity, therefore relieved hmpA expression. Indeed, non-modifiable aphBC235S mutants were sensitive to RNS in vitro and drastically reduced colonization of the RNS-rich mouse small intestine. Finally, AphB S-nitrosylation also decreased virulence gene expression via debilitation of tcpP activation, and this regulation was also important for V. cholerae RNS resistance in vitro and in the gut. These results suggest that the modulation of the activity of virulence gene activator AphB via NO-dependent protein S-nitrosylation is critical for V. cholerae RNS resistance and colonization.

Nitric Oxide in Macrophage Immunometabolism: Hiding in Plain Sight.

Nitric Oxide (NO) is a soluble endogenous gas with various biological functions like signaling, and working as an effector molecule or metabolic regulator. In response to inflammatory signals, immune myeloid cells, like macrophages, increase production of cytokines and NO, which is important for pathogen killing. Under these proinflammatory circumstances, called “M1”, macrophages undergo a series of metabolic changes including rewiring of their tricarboxylic acid (TCA) cycle. Here, we review findings indicating that NO, through its interaction with heme and non-heme metal containing proteins, together with components of the electron transport chain, functions not only as a regulator of cell respiration, but also a modulator of intracellular cell metabolism. Moreover, diverse effects of NO and NO-derived reactive nitrogen species (RNS) involve precise interactions with different targets depending on concentration, temporal, and spatial restrictions. Although the role of NO in macrophage reprogramming has been in evidence for some time, current models have largely minimized its importance. It has, therefore, been hiding in plain sight. A review of the chemical properties of NO, past biochemical studies, and recent publications, necessitates that mechanisms of macrophage TCA reprogramming during stimulation must be re-imagined and re-interpreted as mechanistic results of NO exposure. The revised model of metabolic rewiring we describe here incorporates many early findings regarding NO biochemistry and brings NO out of hiding and to the forefront of macrophages immunometabolism.

CD4+ T cells indirectly kill tumor cells via induction of cytotoxic macrophages in mouse models.

It is well recognized that CD4+ T cells may play an important role in immunosurveillance and immunotherapy against cancer. However, the details of how these cells recognize and eliminate the tumor cells remain incompletely understood. For the past 25years, we have focused on how CD4+ T cells reject multiple myeloma cells in a murine model (MOPC315). In our experimental system, the secreted tumor-specific antigen is taken up by tumor-infiltrating macrophages that process it and present a neoepitope [a V region-derived idiotypic (Id) peptide] on MHC class II molecules to Th1 cells. Stimulated Th1 cells produce IFNγ, which activates macrophages in a manner that elicits an M1-like, tumoricidal phenotype. Through an inducible nitric oxide synthetase (iNOS)-dependent mechanism, the M1 macrophages secrete nitric oxide (NO) that diffuses into neighboring tumor cells. Inside the tumor cells, NO-derived reactive nitrogen species, including peroxynitrite, causes nitrosylation of proteins and triggers apoptosis by the intrinsic apoptotic pathway. This mode of indirect tumor recognition by CD4+ T cells operates independently of MHC class II expression on cancer cells. However, secretion of the tumor-specific antigen, and uptake and MHCII presentation on macrophages, is required for rejection. Similar mechanisms can also be observed in a B-lymphoma model and in the unrelated B16 melanoma model. Our findings reveal a novel mechanism by which CD4+ T cells kill tumor cells indirectly via induction of intratumoral cytotoxic macrophages. The data suggest that induction of M1 polarization of tumor-infiltrating macrophages, by CD4+ T cells or through other means, could serve as an immunotherapeutic strategy.

Interaction between antioxidants and reactive nitrogen species during pepper fruit ripening

Pepper fruit is of great interest for human nutrition due to its high content in vitamins A and, mainly, C. At ripening, decreases of the nitric oxide (NO) levels occur in peppers, accompanied by increases of nitrated and nitrosated proteins, two post-translational modifications favored by the NO-derived reactive nitrogen species (RNS). Catalase is the main enzyme which undergoes nitration at ripening and, in in vitro assays with RNS, it was found that this enzyme was inactivated. Besides, catalase from pepper fruits displays atypical molecular features comparing to other plant catalases. Recently, it was proved that NO delays ripening of peppers and that it interacts with a number of antioxidants thus involving changes in the nutritional properties of fruits. Treatment of pepper fruits with NO increased their content of ascorbate about 40%, and this was due to an induction of the galactono-lactone dehydrogenase (GalLDH) gene together a higher activity of GalLDH, the last enzyme of the ascorbate biosynthetic pathway in plants. At present, high throughput approaches based on RNA-Seq analysis have provided a set of genes affected by treatment of pepper fruits with NO, what could be used to gain added values through biotechnological approaches.

Crosstalk between Nitric Oxide and MPK1/2 Mediates Cold Acclimation-induced Chilling Tolerance in Tomato.

The participation of nitric oxide (NO) in the responses of plants towards biotic and abiotic stresses is well established. However, the mechanism involved particularly in cold acclimation-induced chilling tolerance remains elusive. Here we show the cold acclimation induced-chilling tolerance was associated with inductions of nitrate reductase (NR)-dependent NO production, S-nitrosylated glutathione reductase (GSNOR) activity and mitogen-activated protein kinases MPK1/2 activation in tomato plants. Silencing of NR resulted in decreased GSNOR activity and MPK1/2 activation, which subsequently compromised cold acclimation-induced chilling tolerance. By contrast, silencing of GSNOR caused decreased NR activity, increased NO accumulation and MPK1/2 activation, and enhanced cold acclimation-induced chilling tolerance. Furthermore, co-silencing of MPK1 and MPK2 attenuated the NR-dependent NO production and cold acclimation-induced tolerance to chilling. Results from present study suggest the importance of MPK1/2 for the induction of NR-dependent NO generation, while the accumulation of nitrosylated glutathione from NO-derived reactive nitrogen species could potentially S-nitrosylate NR. These findings provide new insight into the crosstalk of NO and MPK1/2 in cold acclimation-induced chilling tolerance in tomato plants.

Nitrated Fatty Acids Reverse Cigarette Smoke-Induced Alveolar Macrophage Activation and Inhibit Protease Activity via Electrophilic S-Alkylation.

Nitrated fatty acids (NFAs), endogenous products of nonenzymatic reactions of NO-derived reactive nitrogen species with unsaturated fatty acids, exhibit substantial anti-inflammatory activities. They are both reversible electrophiles and peroxisome proliferator-activated receptor γ (PPARγ) agonists, but the physiological implications of their electrophilic activity are poorly understood. We tested their effects on inflammatory and emphysema-related biomarkers in alveolar macrophages (AMs) of smoke-exposed mice. NFA (10-nitro-oleic acid or 12-nitrolinoleic acid) treatment downregulated expression and activity of the inflammatory transcription factor NF-κB while upregulating those of PPARγ. It also downregulated production of inflammatory cytokines and chemokines and of the protease cathepsin S (Cat S), a key mediator of emphysematous septal destruction. Cat S downregulation was accompanied by decreased AM elastolytic activity, a major mechanism of septal destruction. NFAs downregulated both Cat S expression and activity in AMs of wild-type mice, but only inhibited its activity in AMs of PPARγ knockout mice, pointing to a PPARγ-independent mechanism of enzyme inhibition. We hypothesized that this mechanism was electrophilic S-alkylation of target Cat S cysteines, and found that NFAs bind directly to Cat S following treatment of intact AMs and, as suggested by in silico modeling and calculation of relevant parameters, elicit S-alkylation of Cys25 when incubated with purified Cat S. These results demonstrate that NFAs’ electrophilic activity, in addition to their role as PPARγ agonists, underlies their protective effects in chronic obstructive pulmonary disease (COPD) and support their therapeutic potential in this disease.

Protein S-nitrosylation in Plasmodium falciparum

Due to its life in different hosts and environments, the human malaria parasite Plasmodium falciparum is exposed to oxidative and nitrosative challenges. Nitric oxide (NO) and NO-derived reactive nitrogen species can constitute nitrosative stress and play a major role in NO-related signaling. However, the mode of action of NO and its targets in P. falciparum have hardly been characterized. Protein S-nitrosylation (SNO), a posttranslational modification of protein cysteine thiols, has emerged as a principal mechanism by which NO exerts diverse biological effects. Despite its potential importance, SNO has hardly been studied in human malaria parasites. Using a biotin-switch approach coupled to mass spectrometry, we systemically studied SNO in P. falciparum cell extracts. We identified 319 potential targets of SNO that are widely distributed throughout various cellular pathways. Glycolysis in the parasite was found to be a major target, with glyceraldehyde-3-phosphate dehydrogenase being strongly inhibited by S-nitrosylation of its active site cysteine. Furthermore, we show that P. falciparum thioredoxin 1 (PfTrx1) can be S-nitrosylated at its nonactive site cysteine (Cys43). Mechanistic studies indicate that PfTrx1 possesses both denitrosylating and transnitrosylating activities mediated by its active site cysteines and Cys43, respectively. This work provides first insights into the S-nitrosoproteome of P. falciparum and suggests that the malaria parasite employs the thioredoxin system to deal with nitrosative challenges. Our results indicate that SNO may influence a variety of metabolic processes in P. falciparum and contribute to our understanding of NO-related signaling processes and cytotoxicity in the parasites.

Plasmodium falciparum: Nitric oxide modulates heme speciation in isolated food vacuoles

Nitric oxide (NO) and NO-derived reactive nitrogen species (RNS) are present in the food vacuole (FV) of Plasmodium falciparum trophozoites. The product of PFL1555w, a putative cytochrome b 5, localizes in the FV membrane, similar to what was previously observed for the product of PF13_0353, a putative cytochrome b 5 reductase. These two gene products may contribute to NO generation by denitrification chemistry from nitrate and/or nitrite present in the erythrocyte cytosol. The possible coordination of NO to heme species present in the food vacuole was probed by resonance Raman spectroscopy. The spectroscopic data revealed that in situ generated NO interacts with heme inside the intact FVs to form ferrous heme nitrosyl complexes that influence intra-vacuolar heme solubility. The formation of heme nitrosyl complexes within the FV is a previously unrecognized factor that could affect the equilibrium between soluble and crystallized heme within the FV in vivo.

Plasmodium falciparum: Food vacuole localization of nitric oxide-derived species in intraerythrocytic stages of the malaria parasite

Nitric oxide (NO) has diverse biological functions. Numerous studies have documented NO’s biosynthetic pathway in a wide variety of organisms. Little is known, however, about NO production in intraerythrocytic Plasmodium falciparum. Using diaminorhodamine-4-methyl acetoxymethylester (DAR-4M AM), a fluorescent indicator, we obtained direct evidence of NO and NO-derived reactive nitrogen species (RNS) production in intraerythrocytic P. falciparum parasites, as well as in isolated food vacuoles from trophozoite stage parasites. We preliminarily identified two gene sequences that might be implicated in NO synthesis in intraerythrocytic P. falciparum. We showed localization of the protein product of one of these two genes, a molecule that is structurally similar to a plant nitrate reductase, in trophozoite food vacuole membranes. We confirmed previous reports on the antiproliferative effect of NOS (nitric oxide synthase) inhibitors in P. falciparum cultures; however, we did not obtain evidence that NOS inhibitors had the ability to inhibit RNS production or that there is an active NOS in mature forms of the parasite. We concluded that a nitrate reductase activity produce NO and NO-derived RNS in or around the food vacuole in P. falciparum parasites. The food vacuole is a critical parasitic compartment involved in hemoglobin degradation, heme detoxification and a target for antimalarial drug action. Characterization of this relatively unexplored synthetic activity could provide important clues into poorly understood metabolic processes of the malaria parasite.

Beneficial Actions of Polyunsaturated Fatty Acids in Cardiovascular Diseases: But, How and Why?

Omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and to a limited extent ω- 6 fatty acids: arachidonic acid (AA), γ-linolenic acid (GLA) and dihomo-GLA (DGLA), prevent cardiovascular disease, thrombosis and atherosclerosis, reduce cardiac arrhythmias, lower plasma cholesterol and triglycerides, lower high blood pressure and improve endothelial function. These beneficial actions of the fatty acids could be attributed to their ability to augment endothelial nitric oxide generation, inhibit HMG-CoA reductase and angiotensin converting enzyme (ACE) activities, block the production of pro-inflammatory cytokines, and modulate telomerase activity. AA, EPA, and DHA form precursors to anti-inflammatory molecules: lipoxins and resolvins that help in wound healing, inhibit the actions of pro-inflammatory eicosanoids, and suppress the production of free radicals and enhance nitric oxide (NO) generation. In addition, these fatty acids react with NO and NO-derived reactive nitrogen species to form nitroalkene derivative of fatty acids called as nitrolipids. Nitrolipids serve as potent ligands for peroxisome proliferator activated receptors (PPARs), inhibit platelet activation through elevation of cyclic AMP levels; suppress superoxide generation, degranulation, and integrin expression by human neutrophils, and induce vasorelaxation in a concentration dependent manner by releasing NO. Nitrolipids are formed at the sites of inflammation in significant amounts and increased by oxidative inflammatory reactions. Furthermore, ω-3 and ω -6 fatty acids interact with each other to enhance the formation of prostaglandin E1 (PGE1), prostacyclin (PGI2), and PGI3, which are potent platelet anti-aggregators and vasodilators. Thus, GLA, DGLA, AA, EPA, and DHA when present in optimum amounts in the tissues, especially in endothelial cells, myocardium, platelets, and neutrophils, may aid in the prevention of cardiovascular diseases. Keywords: Polyunsaturated fatty acids, eicosapentaenoic acid, docosahexaenoic acid, arachidonic acid, coronary heart disease, lipoxins, prostaglandins, leukotrienes, resolvins, linoleic acid

Does peroxynitrite sustain nuclear factor-κB?

We read with interest the article by Hattori et al. [1] published in Cardiovascular Research in July, 2004. The authors showed almost complete suppression of lipopolysaccharide (LPS)-induced nuclear factor-κB (NF-κB) activation by nitric oxide (NO) in vascular smooth muscle cells. They showed that this effect of NO was not mediated through the second messenger cGMP or through peroxynitrite, an NO-derived reactive nitrogen species (RNS). Instead, the authors demonstrated sustained activation of LPS-induced NF-κB by peroxynitrite. Although the molecular mechanisms were not very clear, we considered this finding as a further step to explain the existing controversies regarding NO-mediated regulation of NF-κB activity. Actually, we are trying to explain some of our in vivo findings in the light of this in vitro evidence. However, a detailed report by Park et al. [2] published in March, 2005, compelled us to reanalyze the article by Hattori et al. [1] and to mention some essential points. Park et al. …

Nitric Oxide in Salmonella and Escherichia coli Infections.

This review discusses the role that nitric oxide (NO) and its congeners play on various stages in the pathophysiology of Escherichia coli and Salmonella infections, with special emphasis on the regulatory pathways that lead to high NO synthesis, the role of reactive nitrogen species (RNS) in host resistance, and the bacterial molecular targets and defense mechanisms that protect enteric bacteria against the nitrosative stress encountered in diverse host anatomical sites. In general, NO can react directly with prosthetic groups containing transition metal centers, with other radicals, or with sulfhydryl groups in the presence of an electron acceptor. Binding to iron complexes is probably the best characterized direct reaction of NO in biological systems. The targets of RNS are numerous. RNS can facilitate oxidative modifications including lipid peroxidation, hydroxylation, and DNA base and protein oxidation. In addition, RNS can inflict nitrosative stress through the nitrosation of amines and sulfhydryls. Numerous vital bacterial molecules can be targeted by NO. It is therefore not surprising that enteropathogenic bacteria are armed with a number of sensors to coordinate the protective response to nitrosative stress, along with an assortment of antinitrosative defenses that detoxify, repair, or avoid the deleterious effects of RNS encountered within the host. NO and NO-derived RNS play important roles in innate immunity to Salmonella and E. coli. Enzymatic NO production by NO synthases can be enhanced by microbial and other inflammatory stimuli and it exerts direct antimicrobial actions as well as immunomodulatory and vasoregulatory effects.

Early cell-specific changes in nitric oxide synthases, reactive nitrogen species formation, and ubiquitinylation during diabetes-related bladder remodeling.

Urinary bladder dysfunction in diabetes is a well-recognized phenomenon but the mechanisms involved and initiating events are not clear. The physiological production of nitric oxide (NO) plays an important role in bladder tone and local immune defense, and recent studies have shown that NO-derived reactive nitrogen species occur in many settings of chronic disease, including other diabetic complications. Here, we investigated the early time-dependent and cell-specific changes in the nitric oxide synthase isoforms (NOS1, 2, and 3), peroxynitrite, and ubiquitinylation in the well-documented streptozotocin-induced rat model of diabetes. Immunohistochemical methods and automated digital imaging were used for the measurement of morphometric and histochemical analysis of the bladder tissue regions. Region-specific 3-nitrotyrosine (a biomarker of NO dysregulation and reactive nitrogen species formation) and ubiquitinylated protein prevalence (marker of proteasomal activity) were also investigated. Immunohistochemistry revealed early, time-dependent, and cell-specific alterations in the three isoforms of NOS. We also observed region-specific increases in protein nitration, demonstrating first-time evidence of reactive nitrogen species formation in this setting. The changes in nitration did not pattern changes in NOS2 induction or tissue ubiquitinylation, and these alterations preceded any detectable changes in bladder structure (3 days vs 2 weeks) in this same animal preparation. These data demonstrate that selective and regionally distinct changes in nitric oxide production and impaired nitric oxide control are early events during diabetic cystopathy and that mechanisms leading to increased oxidative stress and proteasomal activation may be key participants leading to organ dysfunction in this setting.

Functional complementation in yeast reveals a protective role of chloroplast 2-Cys peroxiredoxin against reactive nitrogen species.

The importance of nitric oxide (NO) as a signaling molecule to various plant physiological and pathophysiological processes is becoming increasingly evident. However, little is known about how plants protect themselves from nitrosative and oxidative damage mediated by NO and NO-derived reactive nitrogen species (RNS). Peroxynitrite, the product of the reaction between NO and superoxide anion, is considered to play a central role in RNS-induced cytotoxicity, as a result of its potent ability to oxidize diverse biomolecules. Employing heterologous expression in bacteria and yeast, we investigated peroxynitrite-scavenging activity in plants of 2-Cys peroxiredoxin (2CPRX), originally identified as a hydroperoxide-reducing peroxidase that is ubiquitously distributed among organisms. The putative mature form of a chloroplast-localized 2CPRX from Arabidopsis thaliana was overproduced in Escherichia coli as an amino-terminally hexahistidine-tagged fusion protein. The purified recombinant 2CPRX, which was catalytically active as peroxidase, efficiently prevented the peroxynitrite-induced oxidation of a sensitive compound. We also examined in vivo the ability of the Arabidopsis 2CPRX to complement the 2CPRX deficiency of a Saccharomyces cerevisiae mutant. Functional expression in the mutant strain of the Arabidopsis 2CPRX not only increased cellular tolerance to hydrogen peroxide, but also complemented the hypersensitive growth defect induced by nitrite-mediated cytotoxicity. The complemented cells significantly enhanced the capacity to reduce RNS-mediated oxidative damages. The results presented here demonstrate a new role of plant 2CPRX as a critical determinant of the resistance to RNS, and support the existence of a plant enzymatic basis for RNS metabolism.

Pathophysiology of nitric oxide and related species: free radical reactions and modification of biomolecules

Since its initial discovery as an endogenously produced bioactive mediator, nitric oxide ( •NO) has been found to play a critical role in the cellular function of nearly all organ systems. Furthermore, aberrant production of •NO or reactive nitrogen species (RNS) derived from •NO, has been implicated in a number of pathological conditions, such as acute lung disease, atherosclerosis and septic shock. While •NO itself is fairly non-toxic, secondary RNS are oxidants and nitrating agents that can modify both the structure and function of numerous biomolecules both in vitro, and in vivo. The mechanisms by which RNS mediate toxicity are largely dictated by its unique reactivity. The study of how reactive nitrogen species (RNS) derived from •NO interact with biomolecules such as proteins, carbohydrates and lipids, to modify both their structure and function is an area of active research, which is lending major new insights into the mechanisms underlying their pathophysiological role in human disease. In the context of •NO-dependent pathophysiology, these biochemical reactions will play a major role since they: (i) lead to removal of •NO and decreased efficiency of •NO as an endothelial-derived relaxation factor (e.g. in hypertension, atherosclerosis) and (ii) lead to production of other intermediate species and covalently modified biomolecules that cause injury and cellular dysfunction during inflammation. Although the physical and chemical properties of •NO and •NO-derived RNS are well characterised, extrapolating this fundamental knowledge to a complicated biological environment is a current challenge for researchers in the field of •NO and free radical research. In this review, we describe the impact of •NO and •NO-derived RNS on biological processes primarily from a biochemical standpoint. In this way, it is our intention to outline the most pertinent and relevant reactions of RNS, as they apply to a diverse array of pathophysiological states. Since reactions of RNS in vivo are likely to be vast and complex, our aim in this review is threefold: (i) address the major sources and reactions of •NO-derived RNS in biological systems, (ii) describe current knowledge regarding the functional consequences underlying •NO-dependent covalent modification of specific biomolecules, and (iii) to summarise and critically evaluate the available evidence implicating these reactions in human pathology. To this end, three areas of special interest have been chosen for detailed description, namely, formation and role of S-nitrosothiols, modulation of lipid oxidation/nitration by RNS, and tyrosine nitration mechanisms and consequences.