Molecular Targets Of Nitric Oxide Research Articles

Tyrosine Nitration of Flagellins: a Response of Sinorhizobium meliloti to Nitrosative Stress.

Rhizobia are bacteria which can either live as free organisms in the soil or interact with plants of the legume family with, as a result, the formation of root organs called nodules in which differentiated endosymbiotic bacteria fix atmospheric nitrogen to the plant's benefit. In both lifestyles, rhizobia are exposed to nitric oxide (NO) which can be perceived as a signaling or toxic molecule. NO can act at the transcriptional level but can also modify proteins by S-nitrosylation of cysteine or nitration of tyrosine residues. However, only a few molecular targets of NO have been described in bacteria and none of them have been characterized in rhizobia. Here, we examined tyrosine nitration of Sinorhizobium meliloti proteins induced by NO. We found three tyrosine-nitrated proteins in S. meliloti grown under free-living conditions, in response to an NO donor. Two nitroproteins were identified by mass spectrometry and correspond to flagellins A and B. We showed that one of the nitratable tyrosines is essential to flagellin function in motility.IMPORTANCE Rhizobia are found as free-living bacteria in the soil or in interaction with plants and are exposed to nitric oxide (NO) in both environments. NO is known to have many effects on animals, plants, and bacteria where only a few molecular targets of NO have been described so far. We identified flagellin A and B by mass spectrometry as tyrosine-nitrated proteins in Sinorhizobium melilotiin vivo We also showed that one of the nitratable tyrosines is essential to flagellin function in motility. The results enhanced our understanding of NO effects on rhizobia. Identification of bacterial flagellin nitration opens a new possible role of NO in plant-microbe interactions.

Nitrosothiol-Trapping-Based Proteomic Analysis of S-Nitrosylation in Human Lung Carcinoma Cells.

Nitrosylation of cysteines residues (S-nitrosylation) mediates many of the cellular effects of nitric oxide in normal and diseased cells. Recent research indicates that S-nitrosylation of certain proteins could play a role in tumor progression and responsiveness to therapy. However, the protein targets of S-nitrosylation in cancer cells remain largely unidentified. In this study, we used our recently developed nitrosothiol trapping approach to explore the nitrosoproteome of human A549 lung carcinoma cells treated with S-nitrosocysteine or pro-inflammatory cytokines. Using this approach, we identified about 300 putative nitrosylation targets in S-nitrosocysteine-treated A549 cells and approximately 400 targets in cytokine-stimulated cells. Among the more than 500 proteins identified in the two screens, the majority represent novel targets of S-nitrosylation, as revealed by comparison with publicly available nitrosoproteomic data. By coupling the trapping procedure with differential thiol labeling, we identified nearly 300 potential nitrosylation sites in about 150 proteins. The proteomic results were validated for several proteins by an independent approach. Bioinformatic analysis highlighted important cellular pathways that are targeted by S-nitrosylation, notably, cell cycle and inflammatory signaling. Taken together, our results identify new molecular targets of nitric oxide in lung cancer cells and suggest that S-nitrosylation may regulate signaling pathways that are critically involved in lung cancer progression.

Possible role of glutamine synthetase in the NO signaling response in root nodules by contributing to the antioxidant defenses

Nitric oxide (NO) is emerging as an important regulatory player in the Rhizobium-legume symbiosis. The occurrence of NO during several steps of the symbiotic interaction suggests an important, but yet unknown, signaling role of this molecule for root nodule formation and functioning. The identification of the molecular targets of NO is key for the assembly of the signal transduction cascade that will ultimately help to unravel NO function. We have recently shown that the key nitrogen assimilatory enzyme glutamine synthetase (GS) is a molecular target of NO in root nodules of Medicago truncatula, being post-translationally regulated by tyrosine nitration in relation to nitrogen fixation. In functional nodules of M. truncatula NO formation has been located in the bacteroid containing cells of the fixation zone, where the ammonium generated by bacterial nitrogenase is released to the plant cytosol and assimilated into the organic pools by plant GS. We propose that the NO-mediated GS post-translational inactivation is connected to nitrogenase inhibition induced by NO and is related to metabolite channeling to boost the nodule antioxidant defenses. Glutamate, a substrate for GS activity is also the precursor for the synthesis of glutathione (GSH), which is highly abundant in root nodules of several plant species and known to play a major role in the antioxidant defense participating in the ascorbate/GSH cycle. Existing evidence suggests that upon NO-mediated GS inhibition, glutamate could be channeled for the synthesis of GSH. According to this hypothesis, GS would be involved in the NO-signaling responses in root nodules and the NO-signaling events would meet the nodule metabolic pathways to provide an adaptive response to the inhibition of symbiotic nitrogen fixation by reactive nitrogen species.

Nitric oxide-sphingolipid interplays in plant signalling: a new enigma from the Sphinx?

Nitric oxide (NO) emerged as one of the major signaling molecules operating during plant development and plant responses to its environment. Beyond the identification of the direct molecular targets of NO, a series of studies considered its interplay with other actors of signal transduction and the integration of NO into complex signaling networks. Beside the close relationships between NO and calcium or phosphatidic acid signaling pathways that are now well-established, recent reports paved the way for interplays between NO and sphingolipids (SLs). This mini-review summarizes our current knowledge of the influence NO and SLs might exert on each other in plant physiology. Based on comparisons with examples from the animal field, it further indicates that, although SL–NO interplays are common features in signaling networks of eukaryotic cells, the underlying mechanisms and molecular targets significantly differ.

INTRACELLULAR TARGETS OF PROAPOPTOTIC INFLUENCE OF GASEOUS TRANSMITTERS

Main molecular targets of nitric oxide, hydrogen sulfide and carbon monoxide proapoptotic action in Jurkat cells were determined in this study. Decrease of mitochondrial transmembrane potential was shown during all three gases action. Reason of this event is the Bcl-2 family members disbalance. Proapoptotic proteins release after mitochondrion membranes permeabilisation could be abolished by protein xIAP inhibition of caspase -9 and -3 activity during NO and CO application.

Glutamine Synthetase Is a Molecular Target of Nitric Oxide in Root Nodules of Medicago truncatula and Is Regulated by Tyrosine Nitration

Nitric oxide (NO) is emerging as an important regulatory player in the Rhizobium-legume symbiosis, but its biological role in nodule functioning is still far from being understood. To unravel the signal transduction cascade and ultimately NO function, it is necessary to identify its molecular targets. This study provides evidence that glutamine synthetase (GS), a key enzyme for root nodule metabolism, is a molecular target of NO in root nodules of Medicago truncatula, being regulated by tyrosine (Tyr) nitration in relation to active nitrogen fixation. In vitro studies, using purified recombinant enzymes produced in Escherichia coli, demonstrated that the M. truncatula nodule GS isoenzyme (MtGS1a) is subjected to NO-mediated inactivation through Tyr nitration and identified Tyr-167 as the regulatory nitration site crucial for enzyme inactivation. Using a sandwich enzyme-linked immunosorbent assay, it is shown that GS is nitrated in planta and that its nitration status changes in relation to active nitrogen fixation. In ineffective nodules and in nodules fed with nitrate, two conditions in which nitrogen fixation is impaired and GS activity is reduced, a significant increase in nodule GS nitration levels was observed. Furthermore, treatment of root nodules with the NO donor sodium nitroprusside resulted in increased in vivo GS nitration accompanied by a reduction in GS activity. Our results support a role of NO in the regulation of nitrogen metabolism in root nodules and places GS as an important player in the process. We propose that the NO-mediated GS posttranslational inactivation is related to metabolite channeling to boost the nodule antioxidant defenses in response to NO.

Nitrergic modulation of an oviposition digging rhythm in locusts

In locusts, a central pattern generator underlies the rhythmic movements of the ovipositor valves that serve to drive the abdomen into damp soil in order to lay eggs. We have investigated the role of nitric oxide (NO) in the control of this oviposition digging rhythm. NO increases the frequency of the rhythm by acting via sGC to elevate cGMP, which in turn acts via PKG. Increasing exogenous NO levels using the NO donors SNAP and PAPANONOate increased the cycle frequency of the fictive digging rhythm, as did increasing endogenous NO by bath application of the substrate for NOS, l-arginine. On the other hand, application of the NO scavenger PTIO decreased the cycle frequency, indicating that NO must normally exert a continuous and dynamic role on the central pattern generator underlying the oviposition rhythm. Inhibiting the main molecular target of NO, soluble guanylate cyclase, with ODQ reduced the cycle frequency of the rhythm, suggesting that NO mediated its effects via sGC and cyclic GMP. Further evidence for this was produced by bath application of 8-Br-cGMP, which increased the frequency of the rhythm. Bath application of the generic protein kinase inhibitor and a selective PKG inhibitor, H-7 and KT-5823, respectively, reduced the frequency of the rhythm, suggesting that PKG acted as a target for cGMP. Thus, we conclude that NO plays a key role in regulating the frequency of the central pattern generator controlling rhythmic egg-laying movements in locusts by acting via sGC/cGMP-PKG.

Coordinated induction of iNOS–VEGF–KDR–eNOS after resveratrol consumption: A potential mechanism for resveratrol preconditioning of the heart

Existing evidence indicates that resveratrol, a red wine and grape-derived polyphenolic antioxidant, can pharmacologically precondition the heart in a nitric oxide (NO)-dependent manner. To further explore the role of NO in resveratrol-mediated cardioprotection, the induction for the expression of the potential molecular targets of NO including VEGF and KDR as well as iNOS and eNOS were examined by Western blot analysis and immunohistochemistry. Two groups of rats were studied, one group of animals was fed resveratrol for 7 days while the other group was given water only. After 1, 3, 5 and 7 days, the rats were sacrificed and the expression of the proteins was examined by Western blot analysis. Western blot detected an overexpression of iNOS and VEGF within 24 h of resveratrol treatment while the induction of KDR was not increased until after 3 days and eNOS expression after 5 days of resveratrol treatment. These expressions were further increased after 7 days of resveratrol treatment, when the rats were sacrificed for the isolated working heart preparation. Resveratrol provided cardioprotection as evidenced by superior post-ischemic ventricular recovery, reduced myocardial infarct size and decreased number of apoptotic cardiomyocytes. Immunohistochemistry was performed in the hearts at baseline, and at the end of 30-min ischemia/2-h reperfusion. The hearts obtained from resveratrol-treated rats revealed enhanced expression for iNOS, eNOS and VEGF and KDR compared to control hearts at the end of reperfusion. The results of this study demonstrate that resveratrol leads to a coordinated upregulation of iNOS–VEGF–KDR–eNOS, which is likely to play a role in resveratrol-mediated cardioprotection.

HIF-1α-prolyl hydroxylase: molecular target of nitric oxide in the hypoxic signal transduction pathway

We have investigated inhibitory mechanisms of hypoxic activation of HIF-1α by nitric oxide (NO). Using a Hep3B cell-derived cell line, HRE7 cells, we found that the inhibition of HIF-1α activity by NO requires a substantial amount of oxygen, albeit at a lower level. We further investigated the effect of NO on the binding activity of the von Hippel–Lindau tumor suppressor protein (pVHL) to the N-terminal activation domain (NAD) overlapping the oxygen-dependent degradation domain (ODD) of HIF-1α, because this reaction involves prolyl hydroxylation in NAD that requires oxygen. Although we could not detect any binding activity when NAD was incubated with whole cell extracts from cells treated with CoCl 2 or desferrioxamine, the binding capacity was manifested when Hep3B cells were treated together with NO. This activation was also observed when whole cell extracts from CoCl 2-treated cells were incubated with NO. The prolyl hydroxylase from Hep3B cells treated with CoCl 2 was partially purified about 80-fold, and several enzymatic properties were examined. The enzyme required ferrous ion and 2-oxoglutaric acid. Strong activation of the prolyl hydroxylase by NO was observed without further addition of ferrous ion.

REVIEW ■ : Nitric Oxide: Actions and Pathological Roles

Nitric oxide is a unique biological messenger molecule. It mediates, in part, the immune functions of mac rophages ; it is produced by endothelial cells to mediate blood vessel relaxation; and it also serves as a neurotransmitter in the central and peripheral nervous system. Endothelial nitric oxide synthase and neuronal nitric oxide synthase are thought to be primarily constitutive, with activation induced by calcium entry into cells in the absence of protein synthesis, whereas the macrophage nitric oxide synthase is inducible with large increases in new nitric oxide synthase protein synthesis after immune stimulation. The molecular targets of nitric oxide are expanding, as are its physiological and pathophysiological roles in the nervous system. Nitric oxide may regulate neurotransmitter release, and it may play a key role in nervous system morpho genesis and synaptic plasticity and regulate gene expression. Under conditions of excessive formation, nitric oxide is emerging as an important neurotoxin in a variety of disorders of the nervous system. The Neuro scientist 1:7-18, 1995

Reciprocal modulation of aconitase activity and RNA-binding activity of iron regulatory factor by nitric oxide.

The recent discovery that nitric oxide (NO) is synthetized by many cell types has focused a lot of attention on this gaseous radical thus far considered as highly toxic. NO plays a role in major vital functions including the regulation of vascular tone, the non-specific immune defense and neurotransmission. This wide range of physiological functions has been extensively reviewed recently (Hibbs et al., 1990; Moncada et al., 1991; Nathan, 1992) and will not be developped. However, it is important to recall that many isoforms of NO synthases have been described and are coded by at least three genes (Nathan, 1992). Functionally, two main types exist: one is constitutively expressed and produces low amount of NO (picomolar range) in response to calcium elevation. The other one is induced at the transcriptional level after a lag of several hours and produces far larger amount of NO (nanomolar range). The main molecular target of NO produced in minute amounts by the constitutive NO synthase is a soluble, heme-dependent guanylate cyclase. Resulting increase of cGMP results in a cascade of events leading to physiological effects including vasodilation and synaptic plasticity (Moncada et al., 1991; Bredt and Snyder, 1992).KeywordsNitric OxideElectron Paramagnetic ResonanceTyrosyl RadicalAconitase ActivityClostridium PasteurianumThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Nitric oxide, a biological effector. Electron paramagnetic resonance detection of nitrosyl-iron-protein complexes in whole cells.

Nitric oxide has been used for more than 20 years as an electron paramagnetic resonance probe of oxygen binding sites in oxygen-carriers and oxygen-metabolizing metalloenzymes. The high reactivity of NO with oxygen and the superoxide anion and its high affinity for metalloproteins led biochemists to consider NO as a highly toxic compound for a living cell. This assertion has recently been reconsidered following a number of discoveries of great significance: the finding of the activation of guanylate cyclase by NO, the recognition that NO is the precursor of nitrite and nitrate ions released in the activation of macrophages by endotoxin and cytokines, evidence that NO is an Endothelium-Derived Relaxing Factor, and the discovery of NO-biosynthesis from L-arginine, a pathway common in various biological cell-to-cell signalling processes. It is now admitted that NO plays a key bioregulatory role within mammalian cells, between cells of different types and in the host defence response. In the present review we have attempted to give a general picture of what is known of the chemical, physical, biochemical and biophysical properties of NO among the various nitrogen oxides. We have focussed on the structural information that can be obtained by electron paramagnetic resonance spectroscopy of nitrosyl-metalloprotein complexes. Finally we have shown how molecular targets of nitric oxide can be characterized, within whole cells, by electron paramagnetic resonance spectroscopy.