Tyrosine Modifications Research Articles

Effects of cigarette smoke on salivary protein tyrosine nitration

IntroductionNitration of tyrosine and tyrosine-containing proteins and their roles in pathophysiology have just recently been reviewed. Despite low yields of tyrosine modifications, nitration of tyrosine residues may inactivate important proteins. Nitrotyrosine can be formed by various nitrating agents, including peroxynitrite. Thus, the occurrence of nitrotyrosine-containing proteins in vivo should be regarded as a general indication of tissue damage induced by reactive nitrogen species such as peroxynitrite. This strongly suggests that peroxynitrite could be formed in vivo under certain pathophysiological conditions.ObjectiveOur aim in this study was to elucidate the effect of cigarette smoke (CS) on nitrotyrosine formation in saliva proteins.MethodsWe exposed saliva to CS, in vitro, and used Western Blotting (WB) and monoclonal anti-nitrotyrosine antibody to assess the level of saliva protein nitration.ResultsAs saliva contains extensive amounts of nitrites, it was no surprise that at basal levels, saliva proteins, albumin, and α-amylase all were already nitrated. The WB also revealed that with continuous exposure to CS the tyrosine nitration of both albumin and α-amylase is declining significantly after 3 h. A quite similar effect was seen after exposure to aldehydes, but to a less extent as compared to CS. Exposure of nitrotyrosine-modified bovine serum albumin (BSA-N) to aldehydes, produced a similar effect, meaning a decrease in tyrosine nitration.ConclusionsThese findings might be explained by the possible ability of CS aldehydes to reduce protein-bound nitro group to an amine. Another proposed mechanism is that CS unsaturated aldehydes react with proteins mainly through Michael addition reaction; leading to the generation of stable aldehyde-protein adducts (APA). Thus, it may react with nitro groups of saliva proteins, like albumin or α-amylase, to generate APA, which ultimately, may not be recognized by our antibody. Another possible mechanism, is interaction between the aldehyde group with the hydroxyl group of the 3-nitrotyrosine, forming a hemiacetal, which is not recognized by the antibody. This mechanism might explain the difference in the 'denitration' effects caused by the saturated aldehyde acetaldehyde, which exists in large amounts in CS, and unsaturated aldehydes. Therefore, it is possible that the main player in the CS smoke "denitration" effect on salivary proteins is the aldehyde group and not the double bond of unsaturated aldehydes.

CSF Neutrophils Are Implicated in the Development of Vasospasm in Subarachnoid Hemorrhage

Cerebral vasospasm is a significant cause of morbidity in patients after aneurysmal subarachnoid hemorrhage (aSAH). There are few effective treatments. The search for new treatments has focused predominantly on dilating cerebral blood vessels. Growing evidence supports a role for inflammation in its pathogenesis but no potential target for intervention has emerged. CSF and clinical information from patients with aSAH were collected. Additionally, tyrosine modifications by stable isotope dilution HPLC with online tandem mass spectrometry were quantified in CSF samples. We report an association between neutrophil accumulation in the cerebrospinal fluid of patients with aSAH and the development of vasospasm. In particular, CSF neutrophil content of >62% on the third day after aSAH is an independent predictor of the later development of vasospasm (OR 6.8, 95% CI 2.0-23.3, P = 0.002). Further, activity of myeloperoxidase and NADPH oxidase is elevated in aSAH suggesting a role for modification of CSF proteins by reactive oxidant species. Neutrophil percentage is an independent predictor of vasospasm in aSAH patients, days prior to its onset suggesting a role of neutrophils in vasospasm. The activity of neutrophil enzymes is also increased suggesting a mechanism for blood vessel damage. Inflammation mediated by neutrophils is a potential target for therapies in vasospasm. More study is necessary to determine the mechanism by which neutrophils damage cerebral blood vessels.

Nitrosative protein tyrosine modifications: biochemistry and functional significance

Nitrosative modifications regulate cellular signal transduction and pathogenesis of inflammatory responses and neurodegenerative diseases. Protein tyrosine nitration is a biomarker of oxidative stress and also influences protein structure and function. Recent advances in mass spectrometry have made it possible to identify modified proteins and specific modified amino acid residues. For analysis of nitrated peptides with low yields or only a subset of peptides, affinity 'tags' can be bait for 'fishing out' target analytes from complex mixtures. These tagged peptides are then extracted to a solid phase, followed by mass analysis. In this review, we focus on protein tyrosine modifications caused by nitrosative stresses and proteomic methods for selective enrichment and identification of nitrosative protein modifications.

Using Phage Display to Select Antibodies Recognizing Post-translational Modifications Independently of Sequence Context

Many cellular activities are controlled by post-translational modifications, the study of which is hampered by the lack of specific reagents due in large part to their ubiquitous and non-immunogenic nature. Although antibodies against specifically modified sequences are relatively easy to obtain, it is extremely difficult to derive reagents recognizing post-translational modifications independently of the sequence context surrounding the modification. In this study, we examined the possibility of selecting such antibodies from large phage antibody libraries using sulfotyrosine as a test case. Sulfotyrosine is a post-translational modification important in many extracellular protein-protein interactions, including human immunodeficiency virus infection. After screening almost 8000 selected clones, we were able to isolate a single specific single chain Fv using two different selection strategies, one of which included elution with tyrosine sulfate. This antibody was able to recognize sulfotyrosine independently of its sequence context in test peptides and a number of different natural proteins. Antibody reactivity was lost by antigen treatment with sulfatase or preincubation with soluble tyrosine sulfate, indicating its specificity. The isolation of this antibody signals the potential of phage antibody libraries in the derivation of reagents specific for post-translational modifications, although the extensive screening required indicates that such antibodies are extremely rare.

Tyrosine Modifications and Inactivation of Active Site Manganese Superoxide Dismutase Mutant (Y34F) by Peroxynitrite

Recent studies from this laboratory have demonstrated that human manganese superoxide dismutase (MnSOD) is a target for tyrosine nitration in several chronic inflammatory diseases including chronic organ rejection, arthritis, and tumorigenesis. Furthermore, we demonstrated that peroxynitrite (ONOO−) is the only known biological oxidant competent to inactivate enzymatic activity, nitrate critical tyrosine residues, and induce dityrosine formation in MnSOD. To elucidate the differential contributions of tyrosine nitration and oxidation during enzymatic inactivation, we now compare ONOO−treatment of native recombinant human MnSOD (WT-MnSOD) and a mutant, Y34F-MnSOD, in which tyrosine 34 (the residue most susceptible to ONOO−-mediated nitration) was mutated to phenylalanine. Both WT-MnSOD (IC50= 65 μM, 15 μM MnSOD) and Y34F-MnSOD (IC50= 55 μM, 15 μM Y34F) displayed similar dose-dependent sensitivity to ONOO−-mediated inactivation. Compared to WT-MnSOD, the Y34F-MnSOD mutant demonstrated significantly less efficient tyrosine nitration and enhanced formation of dityrosine following treatment with ONOO−. Collectively, these results suggest that complete inactivation of MnSOD by ONOO−can occur independent of the active site tyrosine residue and includes not only nitration of critical tyrosine residues but also tyrosine oxidation and subsequent formation of dityrosine.

Peroxynitrite Induces Covalent Dimerization of Epidermal Growth Factor Receptors in A431 Epidermoid Carcinoma Cells

Irreversible tyrosine modifications by inflammatory oxidants such as peroxynitrite (ONOO-) can affect signal transduction pathways involving tyrosine phosphorylation. The epidermal growth factor receptor (EGFR), a member of the c-ErbB receptor tyrosine kinase family, is involved in regulation of epithelial cell growth and differentiation, and possible modulation of EGFR-dependent signaling by ONOO- was studied. Exposure of epidermoid carcinoma A431 cells to 0.1-1.0 mM ONOO- resulted in tyrosine nitration on EGFR and other proteins but did not significantly affect EGFR tyrosine autophosphorylation. A high molecular mass tyrosine-phosphorylated protein (approximately 340 kDa) was detected in A431 cell lysates after exposure to ONOO-, most likely representing a covalently dimerized form of EGFR, based on immunoprecipitation and/or immunoblotting with alpha-EGFR antibodies and co-migration with ligand-induced EGFR dimers cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Covalent EGFR dimerization by ONOO- probably involved intermolecular dityrosine cross-linking and was enhanced after receptor activation with epidermal growth factor. Furthermore, irreversibly cross-linked EGFR was more extensively tyrosine-phosphorylated compared with the monomeric form, indicating that ONOO- preferentially cross-links activated EGFR. Exposure of A431 cells to ONOO- markedly reduced the kinetics of tyrosine phosphorylation of a downstream EGFR substrate, phospholipase C-gamma1, which may be related to covalent alterations in EGFR. Alteration of EGFR signaling by covalent EGFR dimerization by inflammatory oxidants such as ONOO- may affect conditions of increased EGFR activation such as epithelial repair or tumorigenesis.

Peroxynitrite-Mediated Inactivation of Manganese Superoxide Dismutase Involves Nitration and Oxidation of Critical Tyrosine Residues

Previous studies from our laboratory have demonstrated that the mitochondrial protein manganese superoxide dismutase is inactivated, tyrosine nitrated, and present as higher molecular mass species during human renal allograft rejection. To elucidate mechanisms whereby tyrosine modifications might result in loss of enzymatic activity and altered structure, the effects of specific biological oxidants on recombinant human manganese superoxide dismutase in vitro have been evaluated. Hydrogen peroxide or nitric oxide had no effect on enzymatic activity, tyrosine modification, or electrophoretic mobility. Exposure to either hypochlorous acid or tetranitromethane (pH 6) inhibited (approximately 50%) enzymatic activity and induced the formation of dityrosine and higher mass species. Treatment with tetranitromethane (pH 8) inhibited enzymatic activity 67% and induced the formation of nitrotyrosine. In contrast, peroxynitrite completely inhibited enzymatic activity and induced formation of both nitrotyrosine and dityrosine along with higher molecular mass species. Combination of real-time spectral analysis and electrospray mass spectroscopy revealed that only three (Y34, Y45, and Y193) of the nine total tyrosine residues in manganese superoxide dismutase were nitrated by peroxynitrite. Inspection of X-ray crystallographic data suggested that neighboring glutamate residues associated with two of these tyrosines may promote targeted nitration by peroxynitrite. Tyr34, which is present in the active site, appeared to be the most susceptible residue to peroxynitrite-mediated nitration. Collectively, these observations are consistent with previous results using chronically rejecting human renal allografts and provide a compelling argument supporting the involvement of peroxynitrite during this pathophysiologic condition.

Selenium-containing compounds inactivate peroxynitrite and protect DNA from single-strand breaks more effectively than their sulfur analogs

The aim of the present study was the investigation of peroxynitrite (ONOO−) effects on fibrinolysis in vitro and in silico. The exposure of human plasminogen to ONOO− (10–1000 μM) resulted in a decrease of t-PA-induced amidolytic activity of plasmin; the inhibitory effect was associated with the increasing level of 3-nitrotyrosine in plasminogen/plasmin molecule. Furthermore, ONOO− displayed both the ability to impair the t-PA-induced activation of plasminogen to plasmin, and to reduce the rate of fibrin lysis by plasmin. The susceptibility of plasminogen in blood plasma to nitrative action of ONOO− was revealed by the immunoprecipitation technique. To confirm the hypothesis that 3-nitrotyrosine generation is crucial for the impairment of plasmin activity, (−)-epicatechin, a polyphenolic antioxidant that selectively prevents tyrosine nitration, was used both for in vitro experiments as well as for in silico studies on ONOO−, ONOOH and (−)-epicatechin binding and plasminogen nitration. (−)-Epicatechin effectively protected plasminogen against ONOO−-induced inactivation and significantly reduced the level of 3-nitrotyrosine. The obtained results revealed tyrosine nitration as the most likely mechanism of the inhibitory effect of ONOO− on plasmin(ogen) functions. The possible role of tyrosine modifications was additionally confirmed by bioinformatics calculations with indication of nitration susceptible tyrosine residues.