Presence Of Nitrated Proteins Research Articles

Early events following phosphorus restriction involve changes in proteome and affects nitric oxide metabolism in soybean leaves

Phosphorus (P) is a macronutrient with structural and regulatory functions, essential for energy transfer. Under limited P availability, plant cells respond to internal signals, adjusting their metabolic pathways in order to reorganize physiological priorities. This work was conducted with the aim to explore the initial changes following P deprivation, before neither growth nor photosynthesis was strongly affected. The first unifoliate leaves of nine-day-old soybean plants (Glycine max, cv. Williams 82) were analyzed. Plants grown hydroponically under control conditions (+P, nutrient solution with 500 μM H2PO4−) were compared with those restricted in phosphate (-P, without H2PO4− in the nutrient solution). No visible symptoms of P-deficiency, such as changes in pigment intensity and leaf number or size, were observed up to 48 h of P deprivation. Neither fresh weight nor shoot/root FW ratio was affected in plants as a consequence of early P-restriction. Shotgun proteomic analyses of leaves from plants exposed to 24 h of P-deprivation, revealed that a total of 202 proteins were exclusively detected and 232 increased their relative abundance under –P conditions. The proteins affected belong mainly to the catalytic activity group according to Gene Ontology Consortium including proteins like 6-phosphogluconate dehydrogenase (from pentose phosphate pathway), pyrophosphate-fructose 6-phosphate 1-phosphotransferase subunit alpha, alpha-1,4 glucan phosphorylases (from carbohydrate metabolic processes), and other enzymes involved in metabolic processes such as glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and fructose-bisphosphate aldolase (from gycolisis), and nitrate reductase (from nitrogen assimilation). Early events in leaves also involve higher levels of the bioactive molecule nitric oxide (NO), detected employing confocal laser microscopy, accompanied with a parallel increase in nitrate reductase activity. Furthermore, the presence of nitrated proteins was detected using shotgun liquid chromatography–mass spectrometry (LCMS/MS). This post-translational protein modification, affected proteins mainly related to photosynthesis (chlorophyll a–b binding protein, ribulose bisphosphate carboxylase), and some other metabolic processes (glutamine synthetase, methionine synthase, lipoxygenase). This methodology allowed the confirmation of nitration sites in proteins previously described as putatively nitrated. Taken together the data presented suggest important metabolic changes, including nitric oxide metabolism, during the first hours of P deprivation.

P-153 - Nuclear translocation and alternative conformation of cytochrome c after peroxynitrite-mediated challenge to cells

Cytochrome c (cyt c) is a mitochondrial heme-containing protein that functions as an electron carrier in the mitochondrial respiratory chain and as a pro-apoptotic mediator in the cytosol. A monoclonal antibody (mAb 1D3) was generated that recognizes a region in cyt c surrounding residue Pro44 in an alternative conformation of the protein (such as that present after cyt c nitration Tyr74). Previous studies shown that in a peroxynitrite-treated Hela cell model, cyt c translocate to the cellular nuclei as detected by the 1D3 suggesting its oxidative modification. The molecular interactions of cyt c in this unusual location are not known. The aim of this study was to gain insights into the identification of the possible partners of oxidatively-modified and/or nitrated cyt c with nuclear proteins. Herein, we employed four cell lines (Hela, Vero, BAECs and B16F1), exposed the cultures to peroxynitrite, and evaluated general redox status by flow cytometry, and the presence of nitrated proteins by SILAC technology 24 hours after treatment. In all cases, cyt c was shown in the cellular nuclei. The identification of the cyt c interacting proteins following oxidative modification is under way using cellular fractionation, 2D-gel electrophoresis and mass spectrometry.

Role of peroxynitrite-modified biomolecules in the etiopathogenesis of systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease characterized by autoantibodies directed against various biomolecules. The initial immunogens that drive the development of SLE are unknown, but characteristics of the immune response in SLE suggest that it is an antigen-driven response, and a chromatin antigen could be one of the immunogens for the production of antinuclear antibodies (ANA) in SLE. Other factors implicated in the pathogenesis of SLE include nitrogen-free radicals such as nitric oxide and peroxynitrite. The free radical-mediated damage to proteins results in the modification of amino acid residues, cross-linking of side chains and fragmentation. The tyrosine residues in proteins are susceptible to attack by various reactive nitrogen intermediates, including peroxynitrite to form 3-nitrotyrosine (3-NT). The presence of nitrated proteins in vivo indicates that peptides derived from the proteolytic degradation of modified proteins could serve as neoantigens. Histones are highly conserved proteins that are rich in basic amino acids lysine and arginine. Autoantibodies against histones and anti-DNA antibodies are present in SLE. The anti-DNA autoantibodies coexist with anti-histone autoantibodies and may react with chromatin-associated histones and histone complexes. Elevated levels of reactive nitrogen species (RNS) in SLE patients suggest a possible role in the pathogenesis of the disease. The alteration of proteins resulting from photomodification or peroxynitrite could lead to the development of antibodies. Therefore, the modified proteins or photoadducts could have important implications in autoimmunity, and understanding the pathophysiology of peroxynitrite-modified biomolecules could lead to a better understanding of autoimmune phenomenon in SLE.

Immunoglobulins Against Tyrosine-Nitrated Epitopes in Coronary Artery Disease

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 μg antibody Eq/mL plasma versus 0.36±0.8 μg 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.

Identification of tyrosine nitration in UCH‐L1 and GAPDH

Protein tyrosine nitration is a post-translational modification commonly used as a marker of cellular oxidative stress associated with numerous pathophysiological conditions. We focused on ubiquitin carboxyl terminal hydrolase-L1 (UCH-L1) and glyceraldehyde-3-phosphate (GAPDH) which are high-abundant brain proteins that have been identified to be highly susceptible to oxidative modification. Both UCH-L1 and GAPDH have been linked to the pathogenesis of Alzheimer's and Parkinson's disease, however specific nitration sites have not been elucidated. Identification of specific nitration sites and quantitation of endogenous nitrated proteins are important in correlating this modification to disease pathology. In this study, purified UCH-L1 and GAPDH were nitrated in vitro with peroxynitrite and the presence of nitrated proteins was confirmed by anti-3-nitrotyrosine Western blots. Data-dependent LC-MS/MS analysis identified several distinct tyrosine nitration sites in UCH-L1 (Tyr-80) and GAPDH (Tyr-47, Tyr-92, and Tyr-312). Subsequent validation with synthetic peptides was conducted for selected nitropeptides. An LC-MS/MS method was developed for semi-quantitative determination of the synthetic nitropeptides: KGQEVSPKVY(*) (UCH-L1) and mFQY(*) DSTHGKF (GAPDH). The nitropeptides were detectable in the mid-attomole range and the peak area response was linear over three orders of magnitude. Targeted analysis of endogenous UCH-L1 and GAPDH nitration was then conducted in an in vivo second-hand smoke rat model to evaluate the utility of this approach.

Nitric oxide induces HIF-1α stabilization and expression of intestinal trefoil factor in the damaged rat jejunum and modulates ulcer healing

The induction of intestinal trefoil factor (ITF) has been reported to depend on hypoxia-inducible factor-1 (HIF-1). Nitric oxide modulates HIF-1 activity. The present study aims to analyze the role of nitric oxide in jejunum damage induced by indomethacin and its ability to modulate epithelial function through the expression of ITF. Rats received indomethacin (7.5 mg/kg, s.c., twice), and a time course analysis of damage was performed (24-96 h after the first administration). In these animals, the role of nitric oxide was analyzed by using 1400W, a selective iNOS activity inhibitor (5 mg/kg, i.p./day), on: (1) intestinal damage, (2) ulcer healing, (3) the presence of nitrated proteins in the jejunum and (4) the protein expression of inducible nitric oxide synthase (iNOS), HIF-1α and ITF. Indomethacin induced damage in the jejunum that was apparent at 24 h and peaked at 48-72 h. An increase in iNOS, HIF-1α, ITF and nitrated proteins was observed in the injured jejunum. Immunoprecipitation of HIF-1α allowed determination of the nitration/nitrosylation of this protein by using nitrotyrosine and nitrocysteine antibodies. Blockade of iNOS activity did not significantly modify damage or iNOS expression, but did significantly impede ITF induction, HIF-1α stabilization and HIF-1α detection with antibodies against nitrated proteins. In parallel to these results, pre-treatment with 1400W delayed the healing of the ulcer provoked by indomethacin. These results suggest that iNOS-derived NO is involved in HIF-1α stabilization, probably through S-nitrosylation, and ITF expression in goblet cells of the damaged jejunum of indomethacin-treated rats and mediates ulcer healing.