Tyr Nitration Research Articles

H2S-Generating Cytosolic L-Cysteine Desulfhydrase and Mitochondrial D-Cysteine Desulfhydrase from Sweet Pepper (Capsicum annuum L.) Are Regulated During Fruit Ripening and by Nitric Oxide.

Aims: Pepper fruit is a horticultural product worldwide consumed that has great nutritional and economic relevance. Besides the phenotypical changes that undergo pepper fruit during ripening, there are many associated modifications at transcriptomic, proteomic, biochemical, and metabolic levels. Nitric oxide (NO) and hydrogen sulfide (H2S) are recognized signal molecules that can exert regulatory functions in diverse plant processes. This study aims at analyzing the interrelationship between NO and H2S during fruit ripening. Results: Our data indicate that the H2S-generating cytosolic L-cysteine desulfhydrase (LCD) and the mitochondrial D-cysteine desulfhydrase (DCD) activities are downregulated during ripening but this effect was reverted after NO treatment of fruits. Innovation and Conclusion: Using as a model the non-climacteric pepper fruits at different ripening stages and under an NO-enriched atmosphere, the activity of the H2S-generating LCD and DCD was analyzed. LCD and DCD activities were downregulated during ripening, but this effect was reverted after NO treatment of fruits. The analysis of LCD activity by non-denaturing polyacrylamide gel electrophoresis (PAGE) allowed identifying three isozymes designated CaLCD I to CaLCD III, which were differentially modulated by NO and strictly dependent on pyridoxal 5'-phosphate (PLP). In vitro analyses of green fruit samples in the presence of different compounds including NO donors, peroxynitrite (ONOO-), and reducing agents such as reduced glutathione (GSH) and L-cysteine (L-Cys) triggered an almost 100% inhibition of CaLCD II and CaLCD III. This redox adaptation process of both enzymes could be cataloged as a hormesis phenomenon. The protein tyrosine (Tyr) nitration (an NO-promoted post-translational modification) of the recombinant LCD was corroborated by immunoblot and by mass spectrometry (MS) analyses. Among the 11 Tyr residues present in this enzyme, MS of the recombinant LCD enabled us to identify that Tyr82 and Tyr254 were nitrated by ONOO-, this occurring near the active center on the enzyme, where His237 and Lys260 together with the cofactor PLP are involved. These data support the relationship between NO and H2S during pepper fruit ripening, since LCD and DCD are regulated by NO during this physiological event, and this could also be extrapolated to other plant species.

Photo-tuneable protein nitration by sensitiser tris(bipyridine)-Ruthenium(II) chloride complex

Post-translational modifications (PTMs) of proteins are a diverse source of variability that impacts on their functions, localisation, regulation, and lifetime. However, one of the main pitfalls in their study is that they appear in rather low frequencies and/or are only transiently observed. To overcome this issue and ease the study in vitro of stress-related protein PTMs, several methods have been proposed to model stress conditions and chemically introduce them. These techniques employ the combination of peroxides with transition metal ions or haem-containing proteins, as well as other possibilities such as peroxy radicals or UV radiation. However, their control, reproducibility and undesired secondary reactions that reduce the process yield are often a matter of concern. Here we introduce a photo-tuneable method that selectively targets nitration of aromatic residues. We initially present the adaptation of an oxidation method based on the photosensitiser tris(2,2′-bipyridine)-Ruthenium(II) chloride complex and ammonium persulfate, in which we employ an alternative radical neutralisation/trapping pathway that uses nitrite ions for the nitration of free l-Tyrosine and L-Tryptophan amino acids. After analysing the effect of several factors, we report the application of the photo-tuneable protein nitration (PTPN) method to four different model proteins in which we evaluate the nitration and oxidation of residues in each case. A mass spectrometry label-free quantitation of Tyr and Trp nitration is also described in order to compare the degree of modification and the accessibility of these residues. The method described could be employed to scale up the production of proteins with a selected range of oxidative PTMs for their characterisation, the assessment of their pathophysiological roles, and the development of detection and quantification methods to validate these PTMs as novel biomarkers associated with oxidative stress-related pathologies, such as in cardiovascular or neurodegenerative diseases.

Tyr-nitration in maize CDKA;1 results in lower affinity for ATP binding

Cyclin-dependent kinase A (CDKA) is a key component for cell cycle progression. The catalytic kinase activity depends on the protein's ability to form an active complex with cyclins and on phosphoregulatory mechanisms. Cell cycle arrest and plant growth impairment under abiotic stress have been linked to different molecular processes triggered by increased levels of reactive oxygen and nitrogen species (ROS and RNS). Among these, posttranslational modifications (PTMs) of key proteins such as CDKA;1 may be of significance. Herein, isolated maize embryo axes were subjected to sodium nitroprusside (SNP) as an inductor of nitrosative conditions to evaluate if CDKA;1 protein was a target for RNS. A high degree of protein nitration was detected; this included the specific Tyr-nitration of CDKA;1. Tyr15 and Tyr19, located at the ATP-binding site, were the selective targets for nitration according to both in silico analysis using the predictive software GPS-YNO2, and in vitro mass spectrometry studies of recombinant nitrated ZmCDKA;1. Spectrofluorometric measurements demonstrated a reduction of ZmCDKA;1-NO2 affinity for ATP. From these results, we conclude that Tyr nitration in CDKA;1 could act as an active modulator of cell cycle progression during redox stress.

Identification and quantification of sites of nitration and oxidation in the key matrix protein laminin and the structural consequences of these modifications

Laminin is a major protein of the basement membrane (BM), a specialized extracellular matrix (ECM) of the artery wall. The potent oxidizing and nitrating agent peroxynitrous acid (ONOOH) is formed at sites of inflammation, and data implicate ONOOH in ECM damage and cardiovascular disease. Co-localization of 3-nitrotyrosine, a product of ONOOH-mediated tyrosine (Tyr) modification, and laminin has been reported in human atherosclerotic lesions. The sites and consequences of 3-nitrotyrosine (and related nitrated tryptophan) formation on laminin, it's self-assembly and cell interactions are poorly understood. In this study murine laminin-111 was exposed to ONOOH (1–500-fold molar excess). Nitration sites were mapped and quantified using LC-MS/MS. Mono-nitration was detected at 148 sites (126 Tyr, 22 Trp), and di-nitration at 14 sites. Label-free quantification showed enhanced nitration with increasing oxidant doses. Tyr nitration was ∼10-fold greater than at Trp. CO2 modulated damage in a site-specific manner, with most sites less extensively nitrated. 119 mono-nitration sites were identified with CO2 present, and no unique sites were detected. 23 di-nitration sites were detected, with 15 unique to the presence of CO2. Extensive modification was detected at sites involved in cell adhesion, protein-protein interactions and self-polymerization. Tyr-145 on the γ1 chain was extensively nitrated, and endothelial cells exhibited decreased adhesion to a nitrated peptide modelling this site. Modification of residues involved in self-polymerization interfered with the formation of ordered polymers as detected by scanning electron microscopy. These laminin modifications may contribute to endothelial cell dysfunction and modulate ECM structure and assembly, and thereby contribute to atherogenesis.

Blocking Tyr265 nitration of protein phosphatase 2A attenuates nitrosative stress-induced endothelial dysfunction in renal microvessels.

Protein tyrosine (Tyr) nitration, the covalent addition of a nitro group (•NO2) to Tyr residues, is emerging as a candidate mechanism of endothelial dysfunction. Previous studies have shown that Tyr nitration is primarily induced by nitrosative stress, a process characterized by the production of reactive nitrogen species, especially peroxynitrite anion (ONOO-), which is considered a secondary product of NO in the presence of superoxide radicals (O2•-). However, the impact of nitrosative stress-induced Tyr nitration on endothelial dysfunction has not been thoroughly elucidated to date. We developed an endothelial dysfunction model, a process called "endothelial-to-mesenchymal transition (EndMT)," and evaluated the production of NO, O2•-, and protein nitration during EndMT. The results showed that TGF-β1 stimulation induced EndMT and elevated endothelial NO and O2•- production as well as nitration of the catalytic subunit of protein phosphatase (PP)2A. Mass spectrometry analysis showed that Tyr265 was the nitration site in the catalytic subunit of protein phosphatase (PP)2A, and this Tyr nitration increased PP2A activity and disrupted endothelial integrity. To devise an endothelial-targeted anti-PP2Ac nitration strategy, a mimic peptide, tyrosine 265 wild type (Y265WT), conjugated with the cell-penetrating peptide HIV-1 TAT protein (TAT) was synthesized. PP2Ac nitration and PP2A activity were significantly inhibited by pretreatment with TAT-265WT, and the integrity of endothelial cells was maintained. Furthermore, injection of TAT-265WT attenuated renal nitration formation and caused anticapillary rarefaction in a unilateral urethral obstructive nephropathy model. Taken together, these results offer preclinical proof of concept for TAT-265WT as a tractable agent to protect against nitrosative stress-induced endothelial dysfunction in renal microvessels.-Deng,Y., Cai, Y., Liu, L., Lin, X., Lu, P., Guo, Y., Han, M., Xu, G. Blocking Tyr265 nitration of protein phosphatase 2A attenuates nitrosative stress-induced endothelial dysfunction in renal microvessels.

218 - Tyrosine nitration regulates signaling pathways involved in Neurofibromatosis type 2-associated Schwannoma growth

Neurofibromatosis type 2 (NF2) is a genetic disorder of the nervous system caused by inactivation of the merlin tumor suppressor gene. NF2 patients develop bilateral vestibular schwannomas (VS) and other nervous system tumors throughout their life for which there is no effective drug treatment. Production of the oxidant peroxynitrite occurs in pathological conditions and leads to tyrosine (Tyr) nitration of proteins. Although Tyr nitration is found in multiple tumor types, its role in tumorigenesis is unknown. We discovered that VS and human merlin-deficient (MD) Schwann cells show significantly increased levels of peroxynitrite and Tyr nitration compared to normal Schwann cells. Notably, prevention of tyrosine nitration selectively decreases MD-Schwann cell survival. Our goal is to identify nitrated proteins that support VS growth as new potential tumor-specific therapeutic targets, enabling the development of safe pharmacological strategies for NF2 schwannomas. This is the first focused effort to determine the signaling pathways regulated by tyrosine nitration in pathological conditions. By performing immunoprecipitations using a polyclonal anti-nitrotyrosine antibody followed by mass spectrometry analysis and western blot, we identified a limited number of proteins endogenously nitrated in VS from NF2 patients, and in mouse and human MD-Schwann cells. A group of the identified proteins play a role in pathways that are dysregulated in NF2. Further, using complementary phospho-kinase arrays, we found that prevention of tyrosine nitration using urate and edaravone has an impact on several of these key signaling pathways. The identification of the specific nitrated proteins that promote schwannoma growth could provide exceptional novel targets for the treatment of NF2 and possibly other tumors of the nervous system as well. Supported by DoD, NRP, NIA W81XWH-17-1-0409 (to MCF).

Peroxynitrite Exposure of CXCL12 Impairs Monocyte, Lymphocyte and Endothelial Cell Chemotaxis, Lymphocyte Extravasation in vivo and Anti-HIV-1 Activity.

CXCL12 is a chemotactic cytokine that attracts many different cell types for homeostasis and during inflammation. Under stress conditions, macrophages and granulocytes produce factors such as peroxynitrite as a consequence of their oxidative response. After short incubations of CXCL12 with peroxynitrite, the gradual nitration of Tyr7, Tyr61, or both Tyr7 and Tyr61 was demonstrated with the use of mass spectrometry, whereas longer incubations caused CXCL12 degradation. Native CXCL12 and the nitrated forms, [3-NT61]CXCL12 and [3-NT7/61]CXCL12, were chemically synthesized to evaluate the effects of Tyr nitration on the biological activity of CXCL12. All CXCL12 forms had a similar binding affinity for heparin, the G protein-coupled chemokine receptor CXCR4 and the atypical chemokine receptor ACKR3. However, nitration significantly enhanced the affinity of CXCL12 for chondroitin sulfate. Internalization of CXCR4 and β-arrestin 2 recruitment to CXCR4 was significantly reduced for [3-NT7/61]CXCL12 compared to CXCL12, whereas β-arrestin 2 recruitment to ACKR3 was similar for all CXCL12 variants. [3-NT7/61]CXCL12 was weaker in calcium signaling assays and in in vitro chemotaxis assays with monocytes, lymphocytes and endothelial cells. Surprisingly, nitration of Tyr61, but not Tyr7, partially protected CXCL12 against cleavage by the specific serine protease CD26. In vivo, the effects were more pronounced compared to native CXCL12. Nitration of any Tyr residue drastically lowered lymphocyte extravasation to joints compared to native CXCL12. Finally, the anti-HIV-1 activity of [3-NT7]CXCL12 and [3-NT7/61]CXCL12 was reduced, whereas CXCL12 and [3-NT61]CXCL12 were equally potent. In conclusion, nitration of CXCL12 occurs readily upon contact with peroxynitrite and specifically nitration of Tyr7 fully reduces its in vitro and in vivo biological activities.

Identification and characterization of a bacterial cytochrome P450 monooxygenase catalyzing the 3-nitration of tyrosine in rufomycin biosynthesis

Rufomycin is a circular heptapeptide with anti-mycobacterial activity and is produced by Streptomyces atratus ATCC 14046. Its structure contains three non-proteinogenic amino acids, N-dimethylallyltryptophan, trans-2-crotylglycine, and 3-nitrotyrosine (3NTyr). Although the rufomycin structure was already reported in the 1960s, its biosynthesis, including 3NTyr generation, remains unclear. To elucidate the rufomycin biosynthetic pathway, we assembled a draft genome sequence of S. atratus and identified the rufomycin biosynthetic gene cluster (ruf cluster), consisting of 20 ORFs (rufA-rufT). We found a putative heptamodular nonribosomal peptide synthetase encoded by rufT, a putative tryptophan N-dimethylallyltransferase encoded by rufP, and a putative trimodular type I polyketide synthase encoded by rufEF Moreover, the ruf cluster contains an apparent operon harboring putative cytochrome P450 (rufO) and nitric oxide synthase (rufN) genes. A similar operon, txtDE, is responsible for the formation of 4-nitrotryptophan in thaxtomin biosynthesis; the cytochrome P450 TxtE catalyzes the 4-nitration of Trp. Therefore, we hypothesized that RufO should catalyze the Tyr 3-nitration. Disruption of rufO abolished rufomycin production by S. atratus, which was restored when 3NTyr was added to the culture medium of the disruptant. Recombinant RufO protein exhibited Tyr 3-nitration activity both in vitro and in vivo Spectroscopic analysis further revealed that RufO recognizes Tyr as the substrate with a dissociation constant of ∼0.1 μm These results indicate that RufO is an unprecedented cytochrome P450 that catalyzes Tyr nitration. Taken together with the results of an in silico analysis of the ruf cluster, we propose a rufomycin biosynthetic pathway in S. atratus.

Post-translational Modifications in Regulation of Chloroplast Function: Recent Advances.

Post-translational modifications (PTMs) of proteins enable fast modulation of protein function in response to metabolic and environmental changes. Phosphorylation is known to play a major role in regulating distribution of light energy between the Photosystems (PS) I and II (state transitions) and in PSII repair cycle. In addition, thioredoxin-mediated redox regulation of Calvin cycle enzymes has been shown to determine the efficiency of carbon assimilation. Besides these well characterized modifications, recent methodological progress has enabled identification of numerous other types of PTMs in various plant compartments, including chloroplasts. To date, at least N-terminal and Lys acetylation, Lys methylation, Tyr nitration and S-nitrosylation, glutathionylation, sumoylation and glycosylation of chloroplast proteins have been described. These modifications impact DNA replication, control transcriptional efficiency, regulate translational machinery and affect metabolic activities within the chloroplast. Moreover, light reactions of photosynthesis as well as carbon assimilation are regulated at multiple levels by a number of PTMs. It is likely that future studies will reveal new metabolic pathways to be regulated by PTMs as well as detailed molecular mechanisms of PTM-mediated regulation.

Cells redox environment modulates BRCA1 expression and DNA homologous recombination repair

Cancer development and progression have been linked to oxidative stress, a condition characterized by unbalanced increase in ROS and RNS production. The main endogenous initiators of the redox imbalance in cancer cells are defective mitochondria, elevated NOX activity, and uncoupled NOS3. Traditionally, most attention has been paid to direct oxidative damage to DNA by certain ROS. However, increase in oxidative DNA lesions does not always lead to malignancy. Hence, additional ROS-dependent, pro-carcinogenic mechanisms must be important. Our recent study demonstrated that Tyr nitration of PP2A stimulates its activity and leads to downregulation of BRCA1 expression. This provides a mechanism for chromosomal instability essential for tumor progression. In the present work, we demonstrated that inhibition of ROS production by generating mitochondrial-electron-transport-deficient cell lines (ρ0 cells) or by inhibition of NOX activity with a selective peptide inhibitor significantly reduced PP2A Tyr nitration and its activity in different cancer cell lines. As a result of the decreased PP2A activity, BRCA1 expression was restored along with a significantly enhanced level of DNA HRR. We used TCGA database to analyze the correlation between expressions of the NOX regulatory subunits, NOS isoforms, and BRCA1 in the 3 cancer research studies: breast invasive carcinoma, ovarian cystadenocarcinoma, and lung adenocarcinoma. TCGA database analysis demonstrated that the high expression levels of most of the NOX regulatory subunits responsible for stimulation of NOX1-NOX4 were associated with significant downregulation of BRCA1 expression.

Redox Regulation Of Metabolic And Signaling Pathways By Thioredoxin And Glutaredoxin In Nitric Oxide Treated Hepatoblastoma Cells

NO has an antiproliferative action on HepG2 cells and Thioredoxin (Trx) and Glutaredoxin (Grx) have denitrosilase and deglutathionylase activities. To ascertain whether Trx and/or Grx systems intermediate the anti-proliferative effect of NO on hepatoblastoma cells by modulating the redox-state of key proteins. HepG2 cells overexpressing Nitric Oxide Synthase-3 (NOS-3) were transfected with specific siRNA to silence Trx1 and Grx1. The expression and thiolic redox state of proteins were determined by Western blot and redox mobility shift assay. Overexpression of NOS3 increased the levels and activities of proteins of the redoxin systems, Trx1, Grx1, TrxR1 and TxnIP, and the levels of signaling proteins (Akt1, pAkt1-Ser473, MapK, pMapK, Stat3, Fas). The thiolic redox state of Trx1, Grx1 and Akt1 shifted to more oxidized. Increases were also observed in Pro-apoptotic Caspase-3 fragment levels; caspase 3, 8 and 9 activities; antiapoptotic (Bcl-2); mitochondrial energetic (Aco2) and heme (Urod) metabolism; Glycolysis (Pkm2); and pentose phosphate pathway (Tkt). However, two cytosolic proteins related to iron (Aco1) and one carbon (Mat2) metabolism decreased markedly. Moreover, the redox state of Urod and Aco1 shifted to more oxidized cysteines. Trx1 or Grx1 silencing augmented Tyr nitration and diminished cell proliferation in WT cells, but attenuated the antiproliferative effect on NO, the increase of Fas, Akt1 and pAkt1-Ser473 and the oxidative modification of Akt1 in NOS3 cells. Trx1 and Grx1 exert contradictory influences on HepG2 cells. They are required for proliferation but they also contribute to antiproliferative effect of NO, associated to Akt1 redox changes.

Hyperammonemia in gene-targeted mice lacking functional hepatic glutamine synthetase

Urea cycle defects and acute or chronic liver failure are linked to systemic hyperammonemia and often result in cerebral dysfunction and encephalopathy. Although an important role of the liver in ammonia metabolism is widely accepted, the role of ammonia metabolizing pathways in the liver for maintenance of whole-body ammonia homeostasis in vivo remains ill-defined. Here, we show by generation of liver-specific Gln synthetase (GS)-deficient mice that GS in the liver is critically involved in systemic ammonia homeostasis in vivo. Hepatic deletion of GS triggered systemic hyperammonemia, which was associated with cerebral oxidative stress as indicated by increased levels of oxidized RNA and enhanced protein Tyr nitration. Liver-specific GS-deficient mice showed increased locomotion, impaired fear memory, and a slightly reduced life span. In conclusion, the present observations highlight the importance of hepatic GS for maintenance of ammonia homeostasis and establish the liver-specific GS KO mouse as a model with which to study effects of chronic hyperammonemia.

Leghemoglobin is nitrated in functional legume nodules in a tyrosine residue within the heme cavity by a nitrite/peroxide-dependent mechanism.

Protein tyrosine (Tyr) nitration is a post-translational modification yielding 3-nitrotyrosine (NO2 -Tyr). Formation of NO2 -Tyr is generally considered as a marker of nitro-oxidative stress and is involved in some human pathophysiological disorders, but has been poorly studied in plants. Leghemoglobin (Lb) is an abundant hemeprotein of legume nodules that plays an essential role as an O2 transporter. Liquid chromatography coupled to tandem mass spectrometry was used for a targeted search and quantification of NO2 -Tyr in Lb. For all Lbs examined, Tyr30, located in the distal heme pocket, is the major target of nitration. Lower amounts were found for NO2 -Tyr25 and NO2 -Tyr133. Nitrated Lb and other as yet unidentified nitrated proteins were also detected in nodules of plants not receiving NO3- and were found to decrease during senescence. This demonstrates formation of nitric oxide (˙NO) and NO2- by alternative means to nitrate reductase, probably via a ˙NO synthase-like enzyme, and strongly suggests that nitrated proteins perform biological functions and are not merely metabolic byproducts. In vitro assays with purified Lb revealed that Tyr nitration requires NO2-+H2 O2 and that peroxynitrite is not an efficient inducer of nitration, probably because Lb isomerizes it to NO3-. Nitrated Lb is formed via oxoferryl Lb, which generates nitrogen dioxide and tyrosyl radicals. This mechanism is distinctly different from that involved in heme nitration. Formation of NO2 -Tyr in Lb is a consequence of active metabolism in functional nodules, where Lb may act as a sink of toxic peroxynitrite and may play a protective role in the symbiosis.

Differential inhibition of Arabidopsis superoxide dismutases by peroxynitrite-mediated tyrosine nitration.

Despite the importance of superoxide dismutases (SODs) in the plant antioxidant defence system little is known about their regulation by post-translational modifications. Here, we investigated the in vitro effects of nitric oxide derivatives on the seven SOD isoforms of Arabidopsis thaliana. S-nitrosoglutathione, which causes S-nitrosylation of cysteine residues, did not influence SOD activities. By contrast, peroxynitrite inhibited the mitochondrial manganese SOD1 (MSD1), peroxisomal copper/zinc SOD3 (CSD3), and chloroplastic iron SOD3 (FSD3), but no other SODs. MSD1 was inhibited by up to 90% but CSD3 and FSD3 only by a maximum of 30%. Down-regulation of these SOD isoforms correlated with tyrosine (Tyr) nitration and both could be prevented by the peroxynitrite scavenger urate. Site-directed mutagenesis revealed that-amongst the 10 Tyr residues present in MSD1-Tyr63 was the main target responsible for nitration and inactivation of the enzyme. Tyr63 is located nearby the active centre at a distance of only 5.26 Å indicating that nitration could affect accessibility of the substrate binding pocket. The corresponding Tyr34 of human manganese SOD is also nitrated, suggesting that this might be an evolutionarily conserved mechanism for regulation of manganese SODs.

Site-specific Nitration of Apolipoprotein A-I at Tyrosine 166 Is Both Abundant within Human Atherosclerotic Plaque and Dysfunctional

We reported previously that apolipoprotein A-I (apoA-I) is oxidatively modified in the artery wall at tyrosine 166 (Tyr(166)), serving as a preferred site for post-translational modification through nitration. Recent studies, however, question the extent and functional importance of apoA-I Tyr(166) nitration based upon studies of HDL-like particles recovered from atherosclerotic lesions. We developed a monoclonal antibody (mAb 4G11.2) that recognizes, in both free and HDL-bound forms, apoA-I harboring a 3-nitrotyrosine at position 166 apoA-I (NO2-Tyr(166)-apoA-I) to investigate the presence, distribution, and function of this modified apoA-I form in atherosclerotic and normal artery wall. We also developed recombinant apoA-I with site-specific 3-nitrotyrosine incorporation only at position 166 using an evolved orthogonal nitro-Tyr-aminoacyl-tRNA synthetase/tRNACUA pair for functional studies. Studies with mAb 4G11.2 showed that NO2-Tyr(166)-apoA-I was easily detected in atherosclerotic human coronary arteries and accounted for ∼ 8% of total apoA-I within the artery wall but was nearly undetectable (>100-fold less) in normal coronary arteries. Buoyant density ultracentrifugation analyses showed that NO2-Tyr(166)-apoA-I existed as a lipid-poor lipoprotein with <3% recovered within the HDL-like fraction (d = 1.063-1.21). NO2-Tyr(166)-apoA-I in plasma showed a similar distribution. Recovery of NO2-Tyr(166)-apoA-I using immobilized mAb 4G11.2 showed an apoA-I form with 88.1 ± 8.5% reduction in lecithin-cholesterol acyltransferase activity, a finding corroborated using a recombinant apoA-I specifically designed to include the unnatural amino acid exclusively at position 166. Thus, site-specific nitration of apoA-I at Tyr(166) is an abundant modification within the artery wall that results in selective functional impairments. Plasma levels of this modified apoA-I form may provide insights into a pathophysiological process within the diseased artery wall.

Lipopolysaccharide-induced Lung Injury Involves the Nitration-mediated Activation of RhoA

Acute lung injury (ALI) is characterized by increased endothelial hyperpermeability. Protein nitration is involved in the endothelial barrier dysfunction in LPS-exposed mice. However, the nitrated proteins involved in this process have not been identified. The activation of the small GTPase RhoA is a critical event in the barrier disruption associated with LPS. Thus, in this study we evaluated the possible role of RhoA nitration in this process. Mass spectroscopy identified a single nitration site, located at Tyr(34) in RhoA. Tyr(34) is located within the switch I region adjacent to the nucleotide-binding site. Utilizing this structure, we developed a peptide designated NipR1 (nitration inhibitory peptide for RhoA 1) to shield Tyr(34) against nitration. TAT-fused NipR1 attenuated RhoA nitration and barrier disruption in LPS-challenged human lung microvascular endothelial cells. Further, treatment of mice with NipR1 attenuated vessel leakage and inflammatory cell infiltration and preserved lung function in a mouse model of ALI. Molecular dynamics simulations suggested that the mechanism by which Tyr(34) nitration stimulates RhoA activity was through a decrease in GDP binding to the protein caused by a conformational change within a region of Switch I, mimicking the conformational shift observed when RhoA is bound to a guanine nucleotide exchange factor. Stopped flow kinetic analysis was used to confirm this prediction. Thus, we have identified a new mechanism of nitration-mediated RhoA activation involved in LPS-mediated endothelial barrier dysfunction and show the potential utility of "shielding" peptides to prevent RhoA nitration in the management of ALI.

Sepiapterin Ameliorates Chemically Induced Murine Colitis and Azoxymethane-Induced Colon Cancer

The effects of modulating tetrahydrobiopterin (BH4) levels with a metabolic precursor, sepiapterin (SP), on dextran sodium sulfate (DSS)-induced colitis and azoxymethane (AOM)-induced colorectal cancer were studied. SP in the drinking water blocks DSS-induced colitis measured as decreased disease activity index (DAI), morphologic criteria, and recovery of Ca(2+)-induced contractility responses lost as a consequence of DSS treatment. SP reduces inflammatory responses measured as the decreased number of infiltrating inflammatory macrophages and neutrophils and decreased expression of proinflammatory cytokines interleukin 1β (IL-1β), IL-6, and IL-17A. High-performance liquid chromatography analyses of colonic BH4 and its oxidized derivative 7,8-dihydrobiopterin (BH2) are inconclusive although there was a trend for lower BH4:BH2 with DSS treatment that was reversed with SP. Reduction of colonic cGMP levels by DSS was reversed with SP by a mechanism sensitive to 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a specific inhibitor of the NO-sensitive soluble guanylate cyclase (sGC). ODQ abrogates the protective effects of SP on colitis. This plus the finding that SP reduces DSS-enhanced protein Tyr nitration are consistent with DSS-induced uncoupling of NOS. The results agree with previous studies that demonstrated inactivation of sGC in DSS-treated animals as being important in recruitment of inflammatory cells and in altered cholinergic signaling and colon motility. SP also reduces the number of colon tumors in AOM/DSS-treated mice from 7 to 1 per unit colon length. Thus, pharmacologic modulation of BH4 with currently available drugs may provide a mechanism for alleviating some forms of colitis and potentially minimizing the potential for colorectal cancer in patients with colitis.

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.

Inhibition of Arabidopsis O-Acetylserine(thiol)lyase A1 by Tyrosine Nitration

The last step of sulfur assimilation is catalyzed by O-acetylserine(thiol)lyase (OASTL) enzymes. OASTLs are encoded by a multigene family in the model plant Arabidopsis thaliana. Cytosolic OASA1 enzyme is the main source of OASTL activity and thus crucial for cysteine homeostasis. We found that nitrating conditions after exposure to peroxynitrite strongly inhibited OASTL activity. Among OASTLs, OASA1 was markedly sensitive to nitration as demonstrated by the comparative analysis of OASTL activity in nitrated crude protein extracts from wild type and different oastl mutants. Furthermore, nitration assays on purified recombinant OASA1 protein led to 90% reduction of the activity due to inhibition of the enzyme, as no degradation of the protein occurred under these conditions. The reduced activity was due to nitration of the protein because selective scavenging of peroxynitrite with epicatechin impaired OASA1 nitration and the concomitant inhibition of OASTL activity. Inhibition of OASA1 activity upon nitration correlated with the identification of a modified OASA1 protein containing 3-nitroTyr(302) residue. The essential role of the Tyr(302) residue for the catalytic activity was further demonstrated by the loss of OASTL activity of a Y302A-mutated version of OASA1. Inhibition caused by Tyr(302) nitration on OASA1 activity seems to be due to a drastically reduced O-acetylserine substrate binding to the nitrated protein, and also to reduced stabilization of the pyridoxal-5'-phosphate cofactor through hydrogen bonds. This is the first report identifying a Tyr nitration site of a plant protein with functional effect and the first post-translational modification identified in OASA1 enzyme.

Nitration of the Tumor Suppressor Protein p53 at Tyrosine 327 Promotes p53 Oligomerization and Activation

Previous studies demonstrate that nitric oxide (NO) promotes p53 transcriptional activity by a classical DNA damage responsive mechanism involving activation of ATM/ATR and phosphorylation of p53. These studies intentionally used high doses of NO donors to achieve the maximum DNA damage. However, lower concentrations of NO donors also stimulate rapid and unequivocal nuclear retention of p53 but apparently do not require ATM/ATR-dependent p53 phosphorylation or total p53 protein accumulation. To identify possible mechanisms for p53 activation at low NO levels, the role of Tyr nitration in p53 activation was evaluated. Low concentrations of the NO donor, DETA NONOate (<200 microM), exclusively nitrate Tyr327 within the tetramerization domain promoting p53 oligomerization, nuclear accumulation, and increased DNA-binding activity without p53 Ser15 phosphorylation. Molecular modeling indicates that nitration of one Tyr327 stabilizes the dimer by about 2.67 kcal mol(-1). Significant quantitative and qualitative differences in the patterns of p53-target gene modulation by low (50 microM), non-DNA-damaging and high (500 microM), DNA-damaging NO donor concentrations were shown. These results demonstrate a new posttranslational mechanism for modulating p53 transcriptional activity responsive to low NO concentrations and independent of DNA damage signaling.