Inducible nitric oxide synthase (NOS2) expressed in septic patients is nitrated on selected tyrosine residues: implications for enzymic activity.
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ABSTRACT:
SUBMITTER: Lanone S
PROVIDER: S-EPMC1222810 | biostudies-other | 2002 Sep
REPOSITORIES: biostudies-other
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Project description:Nitric oxide synthase 2, inducible (Nos2) expression is necessary for the microbicidal activity of macrophages. However, NOS2 over-activation causes multiple inflammatory disorders, suggesting a tight gene regulation is necessary. Using cytosolic flagellin as a model for inflammasome-dependent NOS2 activation, we discovered a surprising new role for NLRC4/caspase-1 axis in regulating chromatin accessibility of the Nos2 promoter. We found that activation of two independent mechanisms is necessary for NOS2 expression by cytosolic flagellin: caspase-1 and NF-κB activation. NF-κB activation was necessary, but not sufficient, for NOS2 expression. Conversely, caspase-1 was necessary for NOS2 expression, but dispensable for NF-κB activation, indicating that this protease acts downstream NF-κB activation. We demonstrated that epigenetic regulation of Nos2 by caspase-1 involves cleavage of the chromatin regulator PARP1 (also known as ARTD1) and chromatin accessibility of the NF-κB binding sites located at the Nos2 promoter. Remarkably, caspase-1-mediated Nos2 transcription and NO production contribute to the resistance of macrophages to Salmonella typhimurium infection. Our results uncover the molecular mechanism behind the constricted regulation of Nos2 expression and open new therapeutic opportunities based on epigenetic activities of caspase-1 against infectious and inflammatory diseases.
| S-EPMC5288713 | biostudies-literature
Project description:Significance"Nitroproteomic" is under active development, as 3-nitrotyrosine in proteins constitutes a footprint left by the reactions of nitric oxide-derived oxidants that are usually associated to oxidative stress conditions. Moreover, protein tyrosine nitration can cause structural and functional changes, which may be of pathophysiological relevance for human disease conditions. Biological protein tyrosine nitration is a free radical process involving the intermediacy of tyrosyl radicals; in spite of being a nonenzymatic process, nitration is selectively directed toward a limited subset of tyrosine residues. Precise identification and quantitation of 3-nitrotyrosine in proteins has represented a "tour de force" for researchers. Recent Advances: A small number of proteins are preferential targets of nitration (usually less than 100 proteins per proteome), contrasting with the large number of proteins modified by other post-translational modifications such as phosphorylation, acetylation, and, notably, S-nitrosation. Proteomic approaches have revealed key features of tyrosine nitration both in vivo and in vitro, including selectivity, site specificity, and effects in protein structure and function.Critical issuesIdentification of 3-nitrotyrosine-containing proteins and mapping nitrated residues is challenging, due to low abundance of this oxidative modification in biological samples and its unfriendly behavior in mass spectrometry (MS)-based technologies, that is, MALDI, electrospray ionization, and collision-induced dissociation.Future directionsThe use of (i) classical two-dimensional electrophoresis with immunochemical detection of nitrated proteins followed by protein ID by regular MS/MS in combination with (ii) immuno-enrichment of tyrosine-nitrated peptides and (iii) identification of nitrated peptides by a MIDAS™ experiment is arising as a potent methodology to unambiguously map and quantitate tyrosine-nitrated proteins in vivo. Antioxid. Redox Signal. 26, 313-328.
| S-EPMC5326983 | biostudies-literature
Project description:Several lines of evidence support a pathophysiological role of immunity in atherosclerosis. Tyrosine-nitrated proteins, a footprint of oxygen- and nitrogen-derived oxidants generated by cells of the immune system, are enriched in atheromatous lesions and in circulation of patients with coronary artery disease (CAD). However, the consequences of possible immune reactions triggered by the presence of nitrated proteins in subjects with clinically documented atherosclerosis have not been explored.Specific immunoglobulins that recognize 3-nitrotyrosine epitopes were identified in human lesions, as well as in circulation of patients with CAD. The levels of circulating immunoglobulins against 3-nitrotyrosine epitopes were quantified in patients with CAD (n=374) and subjects without CAD (non-CAD controls, n=313). A 10-fold increase in the mean level of circulating immunoglobulins against protein-bound 3-nitrotyrosine was documented in patients with CAD (3.75±1.8 ?g antibody Eq/mL plasma versus 0.36±0.8 ?g antibody Eq/mL plasma), and was strongly associated with angiographic evidence of significant CAD.The results of this cross-sectional study suggest that posttranslational modification of proteins via nitration within atherosclerotic plaque-laden arteries and in circulation serve as neo-epitopes for the elaboration of immunoglobulins, thereby providing an association between oxidant production and the activation of the immune system in CAD.
| S-EPMC3501999 | biostudies-literature
Project description:XT-I (xylosyltransferase I) is the initial enzyme in the post-translational biosynthesis of glycosaminoglycan chains in proteoglycans. To gain insight into the structure-function relationship of the enzyme, a soluble active form of human XT-I was expressed in High Five insect cells with an apparent molecular mass of 90 kDa. Analysis of the electrophoretic mobility of the protein under non-reducing and reducing conditions indicated that soluble XT-I does not form homodimers through disulphide bridges. In addition, the role of the cysteine residues was investigated by site-directed mutagenesis combined with chemical modifications of XT-I by N-phenylmaleimide. Replacement of Cys471 or Cys574 with alanine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of soluble recombinant XT-I by forming disulphide bonds. On the other hand, N-phenylmaleimide treatment showed no effect on wild-type XT-I but strongly inactivated the cysteine mutants in a dose-dependant manner, indicating that seven intramolecular disulphide bridges are formed in wild-type XT-I. The inhibitory effect of UDP on the XT-I activity of C561A (Cys561-->Ala) mutant enzyme was significantly reduced compared with all other tested cysteine mutants. In addition, we tested for binding to UDP-agarose beads. The inactive mutants revealed no significantly different nucleotide-binding properties. Our study demonstrates that recombinant XT-I is organized as a monomer with no free thiol groups and strongly suggests that the catalytic activity does not depend on the presence of free thiol groups, furthermore, we identified five cysteine residues which are critical for enzyme activity.
| S-EPMC1134786 | biostudies-literature
Project description:Nitric oxide (NO) mediates host resistance to severe malaria and other infectious diseases. NO production and mononuclear cell expression of the NO producing enzyme-inducible nitric oxide synthase (NOS2) have been associated with protection from severe falciparum malaria. The purpose of this study was to identify single nucleotide polymorphisms (SNPs) and haplotypes in the NOS2 promoter, to identify associations of these haplotypes with malaria severity and to test the effects of these polymorphisms on promoter activity. We identified 34 SNPs in the proximal 7.3 kb region of the NOS2 promoter and inferred NOS2 promoter haplotypes based on genotyping 24 of these SNPs in a population of Tanzanian children with and without cerebral malaria. We identified 71 haplotypes; 24 of these haplotypes comprised 82% of the alleles. We determined whether NOS2 promoter haplotypes were associated with malaria severity in two groups of subjects from Dar es Salaam (N = 185 and N = 250) and in an inception cohort of children from Muheza-Tanga, Tanzania (N = 883). We did not find consistent associations of NOS2 promoter haplotypes with malaria severity or malarial anemia, although interpretation of these results was potentially limited by the sample size of each group. Furthermore, cytokine-induced NOS2 promoter activity determined using luciferase reporter constructs containing the proximal 7.3 kb region of the NOS2 promoter and the G-954C or C-1173T SNPs did not differ from NOS2 promoter constructs that lacked these polymorphisms. Taken together, these studies suggest that the relationship between NOS2 promoter polymorphisms and malaria severity is more complex than previously described.
| S-EPMC2939908 | biostudies-literature
Project description:The activation of cultured Raw 264.7 murine macrophages with interferon gamma and lipopolysaccharide results in the expression of inducible nitric oxide synthase (i_NOS) and the subsequent production of nitric oxide. In the present study, the i-NOS expressed in these activated cells was characterized for possible post-translational protein modification by endogenous tyrosine protein kinases. Western-blot analysis using phosphotyrosine antibodies revealed that i-NOS was phosphorylated on tyrosine residues and that this was an early event coinciding with the appearance of newly synthesized i-NOS. A brief exposure of activated cells to vanadate, a tyrosine phosphatase inhibitor, significantly increased the level of i-NOS tyrosine phosphorylation, suggesting that tyrosine phosphatases are dynamically involved in the regulation of this process. Vanadate treatment of activated cells also resulted in a rapid increase in enzyme activity, occurring within 5 min of exposure. Taken together, these results demonstrate that tyrosine kinases and phosphatases are involved in the post-translational modification of i-NOS and may potentially play a role in modulating the functional activity of the enzyme in macrophages.
| S-EPMC1217140 | biostudies-other
Project description:Nitric oxide (NO) reacts with superoxide to produce peroxynitrite, a potent oxidant and reportedly exerts cytotoxic action. Herein we validated the hypothesis that interaction of NO with superoxide exerts protection against superoxide toxicity using macrophages from mice with a knockout (KO) of inducible NO synthase (NOS2) and superoxide dismutase 1 (SOD1), either individually or both. While no difference was observed in viability between wild-type (WT) and NOS2KO macrophages, SOD1KO and SOD1-and NOS2-double knockout (DKO) macrophages were clearly vulnerable and cell death was observed within four days. A lipopolysaccharide (LPS) treatment induced the formation of NOS2, which resulted in NO production in WT and these levels were even higher in SOD1KO macrophages. The viability of the DKO macrophages but not SOD1KO macrophages were decreased by the LPS treatment. Supplementation of NOC18, a NO donor, improved the viability of SOD1KO and DKO macrophages both with and without the LPS treatment. The NOS2 inhibitor nitro-l-arginine methyl ester consistently decreased the viability of LPS-treated SOD1KO macrophages but not WT macrophages. Thus, in spite of the consequent production of peroxynitrite in LPS-stimulated macrophages, the coordinated elevation of NO appears to exert anti-oxidative affects by coping with superoxide cytotoxicity upon conditions of inflammatory stimuli.
| S-EPMC7905073 | biostudies-literature
Project description:ObjectivesNitration of tyrosine residues in protein is a post-translational modification, which occurs under oxidative stress, and is associated with several neurodegenerative diseases. To understand the role of nitrated proteins in oxidative stress-induced cell death, we identified nitrated proteins and checked correlation of their nitration in glutamate-induced HT22 cell death.Materials and methodsNitrated proteins were detected by western blotting using an anti-nitrotyrosine antibody, extracted from matching reference 2-dimensional electrophoresis gels, and identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.ResultsGlutamate treatment induced apoptosis in HT22 cells, while reactive oxygen species (ROS) inhibitor or neuronal nitric oxide synthase (nNOS) inhibitor blocked glutamate-induced HT22 cell death. Nitration levels of 13 proteins were increased after glutamate stimulation; six of them were involved in regulation of energy production and two were related to apoptosis. The other nitrated proteins were associated with calcium signal modulation, ER dysfunction, or were of unknown function.ConclusionsThe 13 tyrosine-nitrated proteins were detected in these glutamate-treated HT22 cells. Results demonstrated that cell death, ROS accumulation and nNOS expression were related to nitration of protein tyrosine in the glutamate-stimulated cells.
| S-EPMC6496341 | biostudies-literature
Project description:We report direct electrochemistry of the iNOS heme domain in a DDAB film on the surface of a basal plane graphite electrode. Cyclic voltammetry reveals FeIII/II and FeII/I couples at -191 and -1049 mV (vs Ag/AgCl). Imidazole and carbon monoxide in solution shift the FeIII/II potential by +20 and +62 mV, while the addition of dioxygen results in large catalytic waves at the onset of FeIII reduction. Voltammetry at higher scan rates (with pH variations) reveals that the FeIII/II cathodic peak can be resolved into two components, which are attributable to FeIII/II couples of five- and six-coordinate hemes. Digital simulation of our experimental data implicates water dissociation from the heme as a gating mechanism for ET in iNOS.
| S-EPMC2552398 | biostudies-literature
Project description:Post-translational modifications (PTMs) alter the function and fate of proteins and cells in almost every conceivable way. Protein modifications can occur as a result of specific regulating actions of enzymes, such as tyrosine kinases phosphorylating tyrosine residues or by nonenzymatic reactions, such as oxidation related to oxidative stress and diseases. While many studies have addressed the multisite, dynamic, and network-like properties of PTMs, only little is known of the interplay of the same site modifications. In this work, we studied the enzymatic phosphorylation of oxidized tyrosine (l-DOPA) residues using synthetic insulin receptor peptides, in which tyrosine residues were replaced with l-DOPA. The phosphorylated peptides were identified by liquid chromatography-high-resolution mass spectrometry and the site of phosphorylation by tandem mass spectrometry. The results clearly show that the oxidized tyrosine residues are phosphorylated, displaying a specific immonium ion peak in the MS2 spectra. Furthermore, we detected this modification in our reanalysis (MassIVE ID: MSV000090106) of published bottom-up phosphoproteomics data. The modification, where both oxidation and phosphorylation take place at the same amino acid, has not yet been published in PTM databases. Our data indicate that there can be multiple PTMs that do not exclude each other at the same modification site.
| S-EPMC10243104 | biostudies-literature