Protein tyrosine nitration is triggered by nerve growth factor during neuronal differentiation of PC12 cells

Nitric oxide (NO) is a signaling molecule implicated in a spectrum of cellular processes including neuronal differentiation. The signaling pathway triggered by NO in physiological processes involves the activation of soluble guanylate cyclase and S-nitrosylation of proteins, and, as recently proposed, nitration of tyrosine residues in proteins. However, little is known about the mechanisms involved and the target proteins for endogenous NO during the progression of neuronal differentiation. To address this question, we investigated the presence, localization, and subcellular distribution of nitrated proteins during neurotrophin-induced differentiation of PC12 cells. We find that some proteins show basal levels of tyrosine nitration in PC12 cells grown in the absence of nerve growth factor (NGF) and that nitration levels increase significantly after 2 days of incubation with this neurotrophin. Nitrated proteins accumulate over a period of several days in the presence of NGF. We demonstrate that this nitration is coupled to activation of nitric oxide synthase. The subcellular distribution of nitrated proteins changes during PC12 cell differentiation, displaying a shift from the cytosolic to the cytoskeletal fraction and we identified alpha-tubulin as the major target of nitration in PC12 cells by N-terminal sequence and MALDI-TOF analyses. We conclude that tyrosine nitration of proteins could be a novel molecular mechanism involved in the signaling pathway by which NO modulates NGF-induced differentiation in PC12 cells.     

3-nitrotyrosine attenuates respiratory syncytial virus infection in human bronchial epithelial cell line

3-nitrotyrosine (NO2Tyr), an L-tyrosine derivative during nitrative stress, can substitute the COOH-terminal tyrosine of alpha-tubulin, posttranslationally altering microtubular functions. Because infection of the cells by respiratory syncytial virus (RSV) may require intact microtubules, we tested the hypothesis that NO2Tyr would inhibit RSV infection and intracellular signaling via nitrotyrosination of alpha-tubulin. A human bronchial epithelial cell line (BEAS-2B) was incubated with RSV with or without NO2Tyr. The release of chemokines and viral particles and activation of interferon regulatory factor-3 (IRF-3) were measured. Incubation with NO2Tyr increased nitrotyrosinated alpha-tubulin, and NO2Tyr colocalized with microtubules. RSV-infected cells released viral particles, RANTES, and IL-8 in a time- and dose-dependent manner, and intracellular RSV proteins coprecipitated with alpha-tubulin. NO2Tyr attenuated the RSV-induced release of RANTES, IL-8, and viral particles by 50-90% and decreased alpha-tubulin-associated RSV proteins. 3-chlorotyrosine, another L-tyrosine derivative, had no effects. NO2Tyr also inhibited the RSV-induced shift of the unphosphorylated form I of IRF-3 to the phosphorylated form II. Pre-exposure of the cells to NO(2) (0.15 ppm, 4 h), which produced diffuse protein tyrosine nitration, did not affect RSV-induced release of RANTES, IL-8, or viral particles. NO2Tyr did not affect the potential of viral spreading to the neighboring cells since the RSV titers were not decreased when the uninfected cells were cocultured with the preinfected cells in NO2Tyr-containing medium. These results indicate that NO2Tyr, by replacing the COOH-terminal tyrosine of alpha-tubulin, attenuated RSV infection, and the inhibition appeared to occur at the early stages of RSV infection.     

Nitration of the tyrosyl radical in ribonucleotide reductase by nitrogen dioxide: a gamma radiolysis study

Nitrogen dioxide is a product of peroxynitrite homolysis and peroxidase-catalyzed oxidation of nitrite. It is of great importance in protein tyrosine nitration because most nitration pathways end with the addition of *NO2 to a one-electron-oxidized tyrosine. The rate constant of this radical addition reaction is high with free tyrosine-derived radicals. However, little is known of tyrosine radicals in proteins. In this paper, we have used *NO2 generated by gamma radiolysis to study the nitration of the R2 subunit of ribonucleotide reductase, which contains a long-lived tyrosyl radical on Tyr122. Most of the nitration occurred on Tyr122, but nonradical tyrosines were also modified. In addition, peptidic bonds close to nitrated Tyr122 could be broken. Nitration at Tyr122 was not observed with a radical-free metR2 protein. The estimated rate constant of the Tyr122 radical reaction with *NO2 was of 3 x 10(4) M(-1) s(-1), thus several orders of magnitude lower than that of a radical on free tyrosine. Nitration rate of other tyrosine residues in R2 was even lower, with an estimated value of 900 M(-1) s(-1). This study shows that protein environment can significantly reduce the reactivity of a tyrosyl radical. In ribonucleotide reductase, the catalytically active radical residue is very efficiently protected against nitrogen oxide attack and subsequent nitration.     

The nitration of tau protein in neurone-like PC12 cells

Tyrosine nitration of proteins is emerging as a post-translational modification playing a role in physiological conditions. Looking for the molecular events triggered by nitric oxide in nerve growth factor-induced neuronal differentiation, we now find that nitration occurs on the microtubule-associated protein tau. In differentiated PC12 cells, we have identified as tau a nitrated protein that co-immunoprecipitates with alpha-tubulin and indicated that the modified protein is associated with the cytoskeleton but it is confined to a restricted cell region. This paper supplies the first evidence that nitration of tau occurs in a physiological process and suggests that it could play a role in neuronal differentiation.     

Tyrosine Nitration is a Novel Post-translational Modification Occurring on the Neural Intermediate Filament Protein Peripherin

The biological implication of protein tyrosine nitration in signaling pathways triggered by nitric oxide is recently emerging. Here we report for the first time that nitrotyrosination occurs in the neural intermediate filament protein peripherin. In neuron-like PC12 cells, nitrated peripherin is associated with the cytoskeleton fraction, its level increases during the progression of NGF-induced differentiation and the nitrated protein remains closely associated with stable microtubules. Tyr 17 and Tyr 376 were identified by MALDI-TOF analyses as two specific residues endogenously nitrated. Finally, peripherin nitration is not restricted to PC12 cells but it is also present in vivo in rat brain. Gabriella Tedeschi and Graziella Cappelletti contributed equally to this work.     

Tyrosine nitration in prostaglandin H(2) synthase

In this study, we investigated the effects of various nitrogen oxide (NO(x)) species on the extent of prostaglandin H(2) synthase-1 (PGHS-1) nitration in purified protein and in vascular smooth muscle cells. We also examined PGHS-1 activity under these conditions and found the degree of nitration to correlate inversely with enzyme activity. In addition, since NO(x) species are thought to invoke damage during the pathogenesis of atherosclerosis, we examined human atheromatous tissue for PGHS-1 nitration. Both peroxynitrite and tetranitromethane induced Tyr nitration of purified PGHS-1, whereas 1-hydroxy-2-oxo-3-(N-methyl-aminopropyl)-3-methyl-1-triazene (NOC-7; a nitric oxide-releasing compound) did not. Smooth muscle cells treated with peroxynitrite showed PGHS-1 nitration. The extent of nitration by specific NO(x) species was determined by electrospray ionization mass spectrometry. Tetranitromethane was more effective than peroxynitrite, NOC-7, and nitrogen dioxide at nitrating a tyrosine-containing peptide (12%, 5%, 1%, and <1% nitration, respectively). Nitrogen dioxide and, to a lesser extent, peroxynitrite, induced dityrosine formation. Using UV/Vis spectroscopy, it was estimated that the reaction of PGHS-1 with excess peroxynitrite yielded two nitrated tyrosines/PGHS-1 subunit. Finally, atherosclerotic tissue obtained from endarterectomy patients was shown to contain nitrated PGHS-1. Thus, prolonged exposure to elevated levels of peroxynitrite may cause oxidative damage through tyrosine nitration.     

Nitration of cytoskeletal proteins in the chicken embryo chorioallantoic membrane

Protein tyrosine nitration is one of the post-translational modifications that alter the biological function of proteins. Two important mechanisms are involved: peroxynitrite formation and myeloperoxidase or eosinophil peroxidase (EPO) activity. In the present work we studied the nitration of proteins in the in vivo system of chicken embryo chorioallantoic membrane (CAM). 3-Nitrotyrosine was detected only in the insoluble fraction of the CAM homogenate. By immunoprecipitation, Western blot analysis, and double immunofluorescence, we identified two major polypeptides that were nitrated: actin and alpha-tubulin. Quantification of actin and alpha-tubulin nitration revealed that they are differentially nitrated during normal development of the chicken embryo CAM. After irradiation, although they were both increased, they required different time periods to return to the physiological levels of nitration. It seems that both peroxynitrite formation and EPO activity are involved in the in vivo tyrosine nitration of cytoskeletal proteins. These data suggest that tyrosine nitration of cytoskeletal proteins has a physiological role in vivo, which depends on the protein involved and is differentially regulated.     

Mass spectrometric identification of tryptophan nitration sites on proteins in peroxynitrite-treated lysates from PC12 cells

One of the important sites of peroxynitrite action that affects cellular function is known to be nitration of tyrosine residues. However, tryptophan residues could be another target of peroxynitrite-dependent modification of protein function, as we have shown previously using a model protein (F. Yamakura et al., J. Biochem. 138:57-69; 2005). Here, we report the identification of several proteins that allowed us to determine the position of nitrotryptophan in their amino acid sequences in a more complex system. We modified lysates from PC12 cells with and without nerve growth factor (NGF) by treatment with peroxynitrite (0.98 or 4.9 mM). Western blot analyses using anti-6-nitrotryptophan antibody showed several immunoreactive bands and spots, which were subsequently subjected to trypsin digestion and LC-ESI-MS-MS analysis. We identified several tryptic peptides including nitrotryptophan residues, which were derived from L-lactate dehydrogenase A, malate dehydrogenase 1, M2 pyruvate kinase, and heat-shock protein 90 α, in peroxynitrite-treated lysates from PC12 cells, and l-lactate dehydrogenase A, malate dehydrogenase 1, transaldorase, and lactoylglutathione lyase, in peroxynitrite-treated lysates from NGF/PC12 cells. The molar ratio of 3-nitrotyrosine to 6-nitrotryptophan in protease-digested PC12 cell lysates treated with peroxynitrite was determined to be 5.8 to 1 by using an HPLC-CoulArray system. This is the first report to identify several specific sites of nitrated tryptophan on proteins in a complex system treated with peroxynitrite and to compare the susceptibility of nitration between tryptophan and tyrosine residues of the proteins.     

Up-regulation of protein tyrosine nitration in methamphetamine-induced neurotoxicity through DDAH/ADMA/NOS pathway

Protein tyrosine nitration is an important post-translational modification mediated by nitric oxide (NO) associated oxidative stress, occurring in a variety of neurodegenerative diseases. In our previous study, an elevated level of dimethylarginine dimethylaminohydrolase 1 (DDAH1) protein was observed in different brain regions of acute methamphetamine (METH) treated rats, indicating the possibility of an enhanced expression of protein nitration that is mediated by excess NO through the DDAH1/ADMA (Asymmetric Dimethylated l-arginine)/NOS (Nitric Oxide Synthase) pathway. In the present study, proteomic methods, including stable isotope labeling with amino acids in cell culture (SILAC) and two dimensional electrophoresis, were used to determine the relationship between protein nitration and METH induced neurotoxicity in acute METH treated rats and PC12 cells. We found that acute METH administration evokes a positive activation of DDAH1/ADMA/NOS pathway and results in an over-production of NO in different brain regions of rat and PC12 cells, whereas the whole signaling could be repressed by DDAH1 inhibitor N^ω-(2-methoxyethyl)-arginine (l-257). In addition, enhanced expressions of 3 nitroproteins were identified in rat striatum and increased levels of 27 nitroproteins were observed in PC12 cells. These nitrated proteins are key factors for Cdk5 activation, cytoskeletal structure, ribosomes function, etc. l-257 also displayed significant protective effects against METH-induced protein nitration, apoptosis and cell death. The overall results illustrate that protein nitration plays a significant role in the acute METH induced neurotoxicity via the activation of DDAH1/ADMA/NOS pathway.     

Tau is Endogenously Nitrated in Mouse Brain: Identification of a Tyrosine Residue Modified In vivo by NO

Nitration of tau protein is normally linked to neurodegeneration but, until now, no comprehensive information is available regarding tau nitration in healthy subjects. It has been previously reported that in differentiated PC12 cells, tau co-immunoprecipitated with alpha-tubulin is nitrated at tyrosine residues and that this post-translation modification doesn’t impair the association of tau with the cytoskeleton. The present paper is focused on the identification of tyrosine residues endogenously modified in tau from PC12 cells and reports for the first time that tau is also nitrated in vivo in normal mouse brain and that one tyrosine is endogenously modified.     

Epitope motif of an anti‐nitrotyrosine antibody specific for tyrosine‐nitrated peptides revealed by a combination of affinity approaches and mass spectrometry

Nitration of tyrosine residues has been shown to be an important oxidative modification in proteins and has been suggested to play a role in several diseases such as atherosclerosis, asthma, lung and neurodegenerative diseases. Detection of nitrated proteins has been mainly based on the use of nitrotyrosine‐specific antibodies. In contrast, only a small number of nitration sites in proteins have been unequivocally identified by MS. We have used a monoclonal 3‐NT‐specific antibody, and have synthesized a series of tyrosine‐nitrated peptides of prostacyclin synthase (PCS) in which a single specific nitration site at Tyr‐430 had been previously identified upon reaction with peroxynitrite 17 . The determination of antibody‐binding affinity and specificity of PCS peptides nitrated at different tyrosine residues (Tyr‐430, Tyr‐421, Tyr‐83) and sequence mutations around the nitration sites provided the identification of an epitope motif containing positively charged amino acids (Lys and/or Arg) N‐terminal to the nitration site. The highest affinity to the anti‐3NT‐antibody was found for the PCS peptide comprising the Tyr‐430 nitration site with a K_D of 60 nM determined for the peptide, PCS(424‐436‐Tyr‐430NO₂); in contrast, PCS peptides nitrated at Tyr‐421 and Tyr‐83 had substantially lower affinity. ELISA, SAW bioaffinity, proteolytic digestion of antibody‐bound peptides and affinity‐MS analysis revealed highest affinity to the antibody for tyrosine‐nitrated peptides that contained positively charged amino acids in the N‐terminal sequence to the nitration site. Remarkably, similar N‐terminal sequences of tyrosine‐nitration sites have been recently identified in nitrated physiological proteins, such as eosinophil peroxidase and eosinophil‐cationic protein. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

The impact of metal catalysis on protein tyrosine nitration by peroxynitrite

In a series of heme and non-heme proteins the nitration of tyrosine residues was assessed by complete pronase digestion and subsequent HPLC-based separation of 3-nitrotyrosine. Bolus addition of peroxynitrite caused comparable nitration levels in all tested proteins. Nitration mainly depended on the total amount of tyrosine residues as well as on surface exposition. In contrast, when superoxide and nitrogen monoxide (NO) were generated at equal rates to yield low steady-state concentrations of peroxynitrite, metal catalysis seemed to play a dominant role in determining the sensitivity and selectivity of peroxynitrite-mediated tyrosine nitration in proteins. Especially, the heme-thiolate containing proteins cytochrome P450(BM-3) (wild type and F87Y variant) and prostacyclin synthase were nitrated with high efficacy. Nitration by co-generated NO/O(2)(-) was inhibited in the presence of superoxide dismutase. The NO source alone only yielded background nitration levels. Upon changing the NO/O(2)(-) ratio to an excess of NO, a decrease in nitration in agreement with trapping of peroxynitrite and derived radicals by NO was observed. These results clearly identify peroxynitrite as the nitrating species even at low steady-state concentrations and demonstrate that metal catalysis plays an important role in nitration of protein-bound tyrosine.     

Time course and site(s) of cytochrome c tyrosine nitration by peroxynitrite

Cytochrome c-dependent electron transfer and apoptosome activation require protein-protein binding, which are mainly directed by conformational and specific electrostatic interactions. Cytochrome c contains four highly conserved tyrosine residues, one internal (Tyr67), one intermediate (Tyr48), and two more accessible to the solvent (Tyr74 and Tyr97). Tyrosine nitration by biologically-relevant intermediates could influence cytochrome c structure and function. Herein, we analyzed the time course and site(s) of tyrosine nitration in horse cytochrome c by fluxes of peroxynitrite. Also, a method of purifying each (nitrated) cytochrome c product by cation-exchange HPLC was developed. A flux of peroxynitrite caused the time-dependent formation of different nitrated species, all less positively charged than the native form. At low accumulated doses of peroxynitrite, the main products were two mononitrated cytochrome c species at Tyr97 and Tyr74, as shown by peptide mapping and mass spectrometry analysis. At higher doses, all tyrosine residues in cytochrome c were nitrated, including dinitrated (i.e., Tyr97 and Tyr67 or Tyr74 and Tyr67) and trinitrated (i.e., Tyr97, Tyr74, and Tyr67) forms of the protein, with Tyr67 well represented in dinitrated species and Tyr48 being the least prone to nitration. All mono-, di-, and trinitrated cytochrome c species displayed an increased peroxidase activity. Nitrated cytochrome c in Tyr74 and Tyr67, and to a lesser extent in Tyr97, was unable to restore the respiratory function of cytochrome c-depleted mitochondria. The nitration pattern of cytochrome c in the presence of tetranitromethane (TNM) was comparable to that obtained with peroxynitrite, but with an increased relative nitration yield at Tyr67. The use of purified and well-characterized mono- and dinitrated cytochrome c species allows us to study the influence of nitration of specific tyrosines in cytochrome c functions. Moreover, identification of cytochrome c nitration sites in vivo may assist in unraveling the chemical nature of proximal reactive nitrogen species.     

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.     

Post-translational tyrosine nitration of eosinophil granule toxins mediated by eosinophil peroxidase

Nitration of tyrosine residues has been observed during various acute and chronic inflammatory diseases. However, the mechanism of tyrosine nitration and the nature of the proteins that become tyrosine nitrated during inflammation remain unclear. Here we show that eosinophils but not other cell types including neutrophils contain nitrotyrosine-positive proteins in specific granules. Furthermore, we demonstrate that the human eosinophil toxins, eosinophil peroxidase (EPO), major basic protein, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), and the respective murine toxins, are post-translationally modified by nitration at tyrosine residues during cell maturation. High resolution affinity-mass spectrometry identified specific single nitration sites at Tyr349 in EPO and Tyr33 in both ECP and EDN. ECP and EDN crystal structures revealed and EPO structure modeling suggested that the nitrated tyrosine residues in the toxins are surface exposed. Studies in EPO(-/-), gp91phox(-/-), and NOS(-/-) mice revealed that tyrosine nitration of these toxins is mediated by EPO in the presence of hydrogen peroxide and minute amounts of NOx. Tyrosine nitration of eosinophil granule toxins occurs during maturation of eosinophils, independent of inflammation. These results provide evidence that post-translational tyrosine nitration is unique to eosinophils.     

Posttranslational nitrotyrosination of alpha-tubulin induces cell cycle arrest and inhibits proliferation of vascular smooth muscle cells

Hyperproliferation of vascular smooth muscle cells is a hallmark of atherosclerosis and related vascular complications. Microtubules are important for many aspects of mammalian cell responses including growth, migration and signaling. alpha-Tubulin, a component of the microtubule cytoskeleton, is unique amongst cellular proteins in that it undergoes a reversible posttranslational modification whereby the C-terminal tyrosine residue is removed (Glu-tubulin) and re-added (Tyr-tubulin). Whereas the reversible detyrosination/tyrosination cycle of alpha-tubulin has been implicated in regulating various aspects of cell biology, the precise function of this posttranslational modification has remained poorly characterized. Herein, we provide evidence suggesting that alpha-tubulin detyrosination is a required event in the proliferation of vascular smooth muscle cells. Proliferation of rat aortic smooth muscle cells in response to serum was temporally associated with the detyrosination of alpha-tubulin, but not acetylation of alpha-tubulin; Glu-tubulin reached maximal levels between 12 and 18h following cell cycle initiation. Inclusion of 3-nitro-l-tyrosine (NO(2)Tyr) in the culture medium resulted in the selective nitrotyrosination of alpha-tubulin, that was paralleled by decreased elaboration of Glu-tubulin, decreased expression of cyclins A and E, decreased association of the microtubule plus-end binding protein EB1, and inhibited cell proliferation. Nitrotyrosination of alpha-tubulin did not induce necrotic or apoptotic death of rat aortic smooth muscle cells, but instead led to cell cycle arrest at the G(1)/S boundary coincident with decreased DNA synthesis. Collectively, these results suggest that the C-terminus of alpha-tubulin and its detyrosination are functionally important as a molecular switch that regulates cell cycle progression in vascular smooth muscle cells.     

Nitric oxide-dependent generation of reactive species in sickle cell disease. Actin tyrosine induces defective cytoskeletal polymerization

The intermittent vascular occlusion occurring in sickle cell disease (SCD) leads to ischemia-reperfusion injury and activation of inflammatory processes including enhanced production of reactive oxygen species and increased expression of inducible nitric-oxide synthase (NOS2). Appreciating that impaired nitric oxide-dependent vascular function and the concomitant formation of oxidizing and nitrating species occur in concert with increased rates of tissue reactive oxygen species production, liver and kidney NOS2 expression, tissue 3-nitrotyrosine (NO(2)Tyr) formation and apoptosis were evaluated in human SCD tissues and a murine model of SCD. Liver and kidney NOS2 expression and NO(2)Tyr immunoreactivity were significantly increased in SCD mice and humans, but not in nondiseased tissues. TdT-mediated nick end-label (TUNEL) staining showed apoptotic cells in regions expressing elevated levels of NOS2 and NO(2)Tyr in all SCD tissues. Gas chromatography mass spectrometry analysis revealed increased plasma protein NO(2)Tyr content and increased levels of hepatic and renal protein NO(2)Tyr derivatives in SCD (21.4 +/- 2.6 and 37.5 +/- 7.8 ng/mg) versus wild type mice (8.2 +/- 2.2 and 10 +/- 1.2 ng/mg), respectively. Western blot analysis and immunoprecipitation of SCD mouse liver and kidney proteins revealed one principal NO(2)Tyr-containing protein of 42 kDa, compared with controls. Enzymatic in-gel digestion and MALDI-TOF mass spectrometry identified this nitrated protein as actin. Electrospray ionization and fragment analysis by tandem mass spectrometry revealed that 3 of 15 actin tyrosine residues are nitrated (Tyr(91), Tyr(198), and Tyr(240)) at positions that significantly modify actin assembly. Confocal microscopy of SCD human and mouse tissues revealed that nitration led to morphologically distinct disorganization of filamentous actin. In aggregate, we have observed that the hemoglobin point mutation of sickle cell disease that mediates hemoglobin polymerization defects is translated, via inflammatory oxidant reactions, into defective cytoskeletal polymerization.     

Benzene increases protein-bound 3-nitrotyrosine in bone marrow of B6C3F1 mice

Benzene, an environmental pollutant, is myelotoxic and leukemogenic in humans. The molecular mechanisms that can account for its biological effects have not been fully elucidated. We hypothesize that one of the underlying mechanism involves nitration of proteins by peroxynitrite and/or by bone marrow myeloperoxidase-dependent pathways in nitric oxide (NO) metabolism. Using 3-nitrotyrosine [Tyr(NO(2))] as a biomarker for NO-induced damage to proteins, we examined the effects of benzene on the levels of Tyr(NO(2)) in bone marrow in vivo. Groups of 8 weeks old B6C3F(1) male mice were given a single i.p. injection of benzene (50, 100, 200 or 400mg/kg bodyweight) in corn oil. The mice in control groups received either no treatment or a single injection of the vehicle. The mice were killed 1h after treatment and proteins were isolated from bone marrow, lung, liver and plasma. The proteins were enzymatically hydrolyzed; amino acids were separated and purified by high pressure liquid chromatography, derivatized, and quantified by electron capture-negative chemical ionization-gas chromatography/mass spectrometry (EC-NCI-GC/MS). In the GC/MS assay, 3-nitro-l-[(13)C(9)]tyrosine was used as an internal standard and l-[(2)H(4)]tyrosine served to monitor artifactual formation of 3-nitrotyrosine during sample preparation and analysis. We found that treatment of mice with benzene elevates nitration of tyrosine residues in bone marrow proteins. There was a dose (50-200mg benzene/kg b.w.)-dependent increase in protein-bound Tyr(NO(2)) formation (1.5- to 4.5-fold); however, the levels of Tyr(NO(2)) at 400mg benzene/kg b.w. were significantly higher than control but lower than that formed at 200mg benzene/kg b.w. The results of this study, for the first time, indicate that benzene increases protein-bound 3-Tyr(NO(2)) in bone marrow in vivo, and support our previous finding that benzene is metabolized to nitrated products in bone marrow of mice; collectively, these results may in part account for benzene-induced myelotoxicity.     

Protein tyrosine nitration of 15-hydroxy prostaglandin dehydrogenase in the human mast cell line LAD2

Mast cells (MC) play a pivotal role in allergic inflammation and nitric oxide (NO) is known to regulate MC function. One mechanism of NO mediated actions is the post-translational modification protein tyrosine nitration mediated by reactive nitrogen species. In this study we identified targets for nitration in the human mast cell line LAD2 after treatment with a nitric oxide donor and with peroxynitrite. Using two dimensional gel electrophoresis and western blot analyses with monoclonal and polyclonal antibodies we identified 15-hydroxy prostaglandin dehydrogenase (PGDH), a major prostaglandin catabolizing enzyme, as a target for nitration in LAD2. This is the first report on expression of this enzyme in MC and also the first report that PGDH is a target of protein tyrosine nitration. Since MC synthesize and metabolize many prostaglandins including prostaglandin E(2), the major substrate for PGDH, nitration of this prostaglandin catabolizing enzyme is likely functionally significant.     

Eosinophils are a major source of nitric oxide-derived oxidants in severe asthma: characterization of pathways available to eosinophils for generating reactive nitrogen species

Eosinophil recruitment and enhanced production of NO are characteristic features of asthma. However, neither the ability of eosinophils to generate NO-derived oxidants nor their role in nitration of targets during asthma is established. Using gas chromatography-mass spectrometry we demonstrate a 10-fold increase in 3-nitrotyrosine (NO(2)Y) content, a global marker of protein modification by reactive nitrogen species, in proteins recovered from bronchoalveolar lavage of severe asthmatic patients (480 +/- 198 micromol/mol tyrosine; n = 11) compared with nonasthmatic subjects (52.5 +/- 40.7 micromol/mol tyrosine; n = 12). Parallel gas chromatography-mass spectrometry analyses of bronchoalveolar lavage proteins for 3-bromotyrosine (BrY) and 3-chlorotyrosine (ClY), selective markers of eosinophil peroxidase (EPO)- and myeloperoxidase-catalyzed oxidation, respectively, demonstrated a dramatic preferential formation of BrY in asthmatic (1093 +/- 457 micromol BrY/mol tyrosine; 161 +/- 88 micromol ClY/mol tyrosine; n = 11 each) compared with nonasthmatic subjects (13 +/- 14.5 micromol BrY/mol tyrosine; 65 +/- 69 micromol ClY/mol tyrosine; n = 12 each). Bronchial tissue from individuals who died of asthma demonstrated the most intense anti-NO(2)Y immunostaining in epitopes that colocalized with eosinophils. Although eosinophils from normal subjects failed to generate detectable levels of NO, NO(2-), NO(3-), or NO(2)Y, tyrosine nitration was promoted by eosinophils activated either in the presence of physiological levels of NO(2-) or an exogenous NO source. At low, but not high (e.g., >2 microM/min), rates of NO flux, EPO inhibitors and catalase markedly attenuated aromatic nitration. These results identify eosinophils as a major source of oxidants during asthma. They also demonstrate that eosinophils use distinct mechanisms for generating NO-derived oxidants and identify EPO as an enzymatic source of nitrating intermediates in eosinophils.