Protein tyrosine nitration--functional alteration or just a biomarker?

Protein 3-nitrotyrosine is a posttranslational modification found in many pathological conditions from acute to chronic diseases. Could 3-nitrotyrosine formation participate on the basis of these diseases or is it just a marker connected with the associated nitroxidative stress? In vitro and in vivo data, including proteomic research, show that protein tyrosine nitration is a selective process where only a small amount of proteins is found nitrated and one or a few tyrosine residues are modified in each. Accumulating data suggest a strong link between protein 3-nitrotyrosine and the mechanism involved in disease development. In this review, we analyze the factors determining protein 3-nitrotyrosine formation, the functional and biological outcome associated with protein tyrosine nitration, and the fate of the nitrated proteins.     

Bioinformatics analysis reveals biophysical and evolutionary insights into the 3-nitrotyrosine post-translational modification in the human proteome

Protein 3-nitrotyrosine is a post-translational modification that commonly arises from the nitration of tyrosine residues. This modification has been detected under a wide range of pathological conditions and has been shown to alter protein function. Whether 3-nitrotyrosine is important in normal cellular processes or is likely to affect specific biological pathways remains unclear. Using GPS-YNO2, a recently described 3-nitrotyrosine prediction algorithm, a set of predictions for nitrated residues in the human proteome was generated. In total, 9.27 per cent of the proteome was predicted to be nitratable (27 922/301 091). By matching the predictions against a set of curated and experimentally validated 3-nitrotyrosine sites in human proteins, it was found that GPS-YNO2 is able to predict 73.1 per cent (404/553) of these sites. Furthermore, of these sites, 42 have been shown to be nitrated endogenously, with 85.7 per cent (36/42) of these predicted to be nitrated. This demonstrates the feasibility of using the predicted dataset for a whole proteome analysis. A comprehensive bioinformatics analysis was subsequently performed on predicted and all experimentally validated nitrated tyrosine. This found mild but specific biophysical constraints that affect the susceptibility of tyrosine to nitration, and these may play a role in increasing the likelihood of 3-nitrotyrosine to affect processes, including phosphorylation and DNA binding. Furthermore, examining the evolutionary conservation of predicted 3-nitrotyrosine showed that, relative to non-nitrated tyrosine residues, 3-nitrotyrosine residues are generally less conserved. This suggests that, at least in the majority of cases, 3-nitrotyrosine is likely to have a deleterious effect on protein function and less likely to be important in normal cellular function.     

Nitrative stress through formation of 8-nitroguanosine: insights into microbial pathogenesis.

Reactive oxygen and nitrogen species, respectively, mediate oxidative and nitrative stresses by means of oxidation and nitration of various biomolecules including proteins, lipids, and nucleic acids. We have observed nitric oxide (NO)-dependent formation of 8-nitroguanosine and 3-nitrotyrosine during microbial infection, and we determined that both 8-nitroguanosine and 3-nitrotyrosine are useful biomarkers of nitrative stress. Of importance, however, is the great difference in biological characteristics of these two nitrated compounds. 8-Nitroguanosine has unique biochemical and pharmacological properties such as redox activity and mutagenic potential, which 3-nitrotyrosine does not. In this review, we discuss the mechanism of nitrative stress occurring during microbial infections, with special emphasis on biological functions of 8-nitroguanosine formed via NO during the host response to pathogens. These findings provide insights into NO-mediated pathogenesis not only of viral infections but also of many other diseases.     

Post-translational modifications of superoxide dismutase

Post-translational modifications of proteins control many biological processes through the activation, inactivation, or gain-of-function of the proteins. Recent developments in mass spectrometry have enabled detailed structural analyses of covalent modifications of proteins and also have shed light on the post-translational modification of superoxide dismutase. In this review, we introduce some covalent modifications of superoxide dismutase, nitration, phosphorylation, glutathionylaion, and glycation. Nitration has been the most extensively analyzed modification both in vitro and in vivo. Reaction of human Cu,Zn superoxide dismutase (SOD) with reactive nitrogen species resulted in nitration of a single tryptophan residue to 6-nitrotryptophan, which could be a new biomarker of a formation of reactive nitrogen species. On the other hand, tyrosine 34 of human MnSOD was exclusively nitrated to 3-nitrotyrosine and almost completely inactivated by the reaction with peroxynitrite. The nitrated MnSOD has been found in many diseases caused by ischemia/reperfusion, inflammation, and others and may have a pivotal role in the pathology of the diseases. Most of the post-translational modifications have given rise to a reduced activity of SOD. Since phosphorylation and nitration of SOD have been shown to have a possible reversible process, these modifications may be related to a redox signaling process in cells. Finally we briefly introduce a metal insertion system of SOD, focusing particularly on the iron misincorporation of nSOD, as a part of post-translational modifications.     

Use of a novel double-sandwich enzyme-linked immunosorbent assay method for assaying chondroitin sulfate proteoglycans that bear 3-nitrotyrosine core protein modifications, a previously unrecognized proteoglycan modification in hydrocephalus

3-Nitrotyrosine is a useful marker for nitric oxide-mediated tissue injury. However, which proteins are preferred peroxynitrite modification targets is unclear. Chondroitin sulfate proteoglycans (CSPGs) abnormally accumulate in cerebrospinal fluid of human neonates with hydrocephalus and may be a target for peroxynitrite modification. We examined (1). whether CSPG core protein can be modified by peroxynitrite in vitro; (2). to what degree in comparison to bovine serum albumin (BSA), the most commonly used nitrated protein standard; (3). whether nitrated CSPGs can be measured directly in biological samples; and (4). whether nitrated proteoglycan concentrations in cerebrospinal fluid correlate with disease. In vitro nitration of bovine aggrecan was performed by exposure to different peroxynitrite concentrations, and 3-nitrotyrosine products were measured. Bovine serum albumin (BSA) nitration was also performed in comparison. A larger percentage of tyrosine residues were nitrated in aggrecan than in BSA under all conditions tested. An enzyme-linked immunosorbent assay (ELISA) for 3-nitrotyrosine consistently overestimated aggrecan nitration when nitrated BSA was used as the standard. This is important as most current assays of nitration in biological samples use nitrated BSA as the standard. Therefore, if nitrated CPSGs were a substantial portion of the nitrated proteins in a sample, total nitrated protein content would be overestimated. Aggrecan retained its function of binding hyaluronic acid despite substantial nitration. A double-sandwich ELISA was developed for nitrated CSPGs in biological samples, using nitrated aggrecan as standard. [Nitrated CSPG] was found to be significantly elevated in preterm hydrocephalus cerebrospinal fluid (P<0.02), but correlated poorly with cerebrospinal fluid [nitric oxide] (P>0.069), suggesting that nitrated CSPG and NO levels may be independant markers of tissue injury. Peroxynitrite-mediated protein tyrosine nitration is a previously unrecognized modification of CSPGs, and may reflect level of brain injury in hydrocephalus.     

Analysis of nitrated proteins by nitrotyrosine-specific affinity probes and mass spectrometry

Tyrosine nitration is a well-established protein modification that occurs in disease states associated with oxidative stress and increased nitric oxide synthase activity. Nitration of specific tyrosine residues has been reported to affect protein structure and function, suggesting that 3-nitrotyrosine formation may not only be a disease marker but may also be involved in the pathogenesis of some diseases and in normal regulatory processes. It has been, however, difficult to identify sites of nitration. We describe a method that combines specific isolation of nitrated proteins with mass spectrometric determination of the amino acid sequence and the site of nitration of individual proteins. A complex protein mixture, e.g., serum or cell lysate, was enriched for nitrotyrosine-containing proteins by immunoprecipitation with antinitrotyrosine antibodies. The nitrotyrosines were then reduced to aminotyrosines with a strong reducing agent in parallel in-gel and in-solution procedures. Using nitrated human serum albumin as a model, we reduced the disulfide bonds with dithiothreitol and alkylated the free sulfhydryl groups with iodoacetamide. The nitrotyrosines were next reduced to aminotyrosines with sodium dithionite, and-at pH 5.0-cleavable biotin tags were selectively attached to the aminotyrosines and the albumin was then digested with trypsin. The biotinylated tryptic peptides were purified on a streptavidin affinity column and identified by mass spectrometry. We have also purified nitrated human serum albumin from an enriched sample of SJL mouse plasma and confirmed its identity by peptide mass fingerprinting and MASCOT.     

Specific electrochemical nitration of horse heart myoglobin

Earlier findings on electronitration of hen egg-white lysozyme demonstrated a product which was mononitrated at Tyr23, by ion-exchange chromatography, absorbance at 430 nm, dithionite reduction, and Edman sequencing of a nitrated proteolytic peptide. However, the whole protein was not sequenced; therefore, although the enzyme remained active upon nitration, reaction at other residues could not be completely eliminated. This study has now been extended to the redox protein myoglobin. We demonstrate the novel electronitration (electrooxidation in the presence of nitrite) of a specific tyrosine residue in horse heart myoglobin and also in apomyoglobin. Production of the yellow chromophore, 3-nitrotyrosine (3-NT), was apparent in apomyoglobin from A430 but was masked in holomyoglobin by the Soret band. In both cases, the presence of 3-NT in the electronitrated samples was further indicated by the binding of antibody to 3-NT in Western blots. High-resolution electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry revealed a reaction product at [M + 45] (consistent with substitution of NO2 for H), indicating that the nitration reaction is the only reaction occurring which gives rise to a change in mass in the electrooxidation. Fragmentation mass spectrometry identified the nitration site as Tyr103, with no nitration at Tyr146. The procedure may be useful in preparing model nitrated proteins for the study of disease mechanisms.     

NMDA receptor stimulation induces temporary alpha-tubulin degradation signaled by nitric oxide-mediated tyrosine nitration in the nervous system of Sepia officinalis

Biochemical and immunohistochemical evidence is reported, showing basal protein nitration in specific regions of the optic lobes of Sepia officinalis, mainly in the fiber layers of the plexiform zone. SDS-PAGE analysis of optic lobe extracts revealed an intense 3-nitrotyrosine immunoreactive band identified as alpha-tubulin by immunoprecipitation and partial purification. Stimulation of NMDA receptors resulted in a selective decrease in alpha-tubulin levels within 30 min with partial recovery after 4 h. The effect was suppressed by the NO synthase (NOS) inhibitor L-nitroarginine. Incubation of optic lobes with free 3-nitrotyrosine resulted likewise in a selective loss of alpha-tubulin, due apparently to incorporation of the amino acid into the C-terminus of detyrosinated alpha-tubulin to give the nitrated protein purportedly more susceptible to degradation. Overall, these results point to a novel potential physiologic role of NO and free 3-nitrotyrosine in the control of the alpha-tubulin tyrosination/detyrosination cycle and turnover in Sepia nervous tissue.     

Electrochemical nitration of myoglobin at tyrosine 103: Structure and stability

Nitration in proteins is a physiologically relevant process and the formation of 3-nitrotyrosine was first proposed as an in vivo marker of the production of reactive nitrogen species in oxidative stress. No studies have been published on structural changes associated with nitration of myoglobin. To address this deficiency the electrochemical nitration of equine skeletal muscle (Mb) at amino acid tyrosine 103 has been investigated for the evaluation and characterization of structural and thermal stability changes. Y103 in Mb is one of the most exposed tyrosine residues and it is also close to the heme group. Effects of Y103 nitration on the secondary and tertiary structure of Y103 have been studied by UV–Vis, circular dichroism, fluorescence and NMR spectroscopy and by electrochemical studies. At physiological pH, subtle changes were observed involving slight loosening of the tertiary structure and conformational exchange processes. Thermal stability of the nitrated protein was found to be reduced by 5 °C for the nitrated Mb compared with the native Mb at physiological pH. Altogether, NMR data indicates that nitrated Mb has a very similar tertiary structure to that of native Mb, although with a slightly open conformation.     

Dynamics of protein nitration in cells and mitochondria

Nitric oxide is a precursor of reactive nitrating species such as peroxynitrite and nitrogen dioxide that modify proteins to generate 3-nitrotyrosine. Many diseases are associated with increased levels of protein-bound nitrotyrosine, and this is used as a marker for oxidative damage. However, the regulation of protein nitration and its role in cell function are unclear. We demonstrate that biological protein nitration can be a specific and dynamic process. Proteins were nitrated in distinct temporal patterns in cells undergoing inflammatory activation, and protein denitration and renitration occurred rapidly in respiring mitochondria. The targets of protein nitration varied over time, which may reflect their sensitivity to nitration, expression pattern, or turnover. The dynamic nature of the nitration process was revealed by denitration and renitration of proteins occurring within minutes in mitochondria that were subject to hypoxiaanoxia and reoxygenation. Our results have implications that are particularly important for ischemia-reperfusion injury.     

Protein nitration as footprint of oxidative stress-related nitric oxide signaling pathways in developing Ciona intestinalis

Developmental processes in the ascidian Ciona intestinalis depend on a complex interplay of events including, during metamorphosis, a caspase-dependent apoptosis which is regulated by the nitric oxide (NO)-cGMP signaling pathway. Herein we disclose an alternate NO-mediated signaling pathway during Ciona development which appears to be critically dependent on local redox control. Evidence in support of this conclusion includes: (a) inhibitors of NO synthase (NOS) and scavengers of NO-derived nitrating agents markedly decrease the rate of Ciona metamorphosis; (b) an NO donor or peroxynitrite caused an opposite effect; (c) increased protein nitration is observed at larva stage. Integrated proteomic and immunochemical methodologies identified nitrated tyrosine residues in ERK and snail. Overall, these results point to protein nitration as a hitherto overlooked NO-dependent regulatory mechanism in Ciona which is specifically triggered by elevated ROS production during developmental processes.     

Oxygen-insensitive nitroreductases of Escherichia coli do not reduce 3-nitrotyrosine

The oxygen-insensitive nitroreductases nfsA and nfsB are known to reduce para-nitrated aromatic compounds. We tested the hypothesis that these nitroreductases are capable of reducing 3-nitrotyrosine in proteins and peptides, as well as in free amino acids using wild-type and nfsA nfsB mutant strains of Escherichia coli. E. coli homogenates were incubated with nitrated proteins and the level of 3-nitrotyrosine immunoreactivity was assayed by Western blotting. Assay conditions that allow the nitroreductases to rapidly reduce nitrofurantoin did not result in the modification of 3-nitrotyrosine in protein, peptide, or free amino acid. Stimulation of nfsA nfsB activity with paraquat had no effect on 3-nitrotyrosine reduction. Nonlethal exposure of E. coli to peroxynitrite/CO(2) resulted in the reproducible nitration of tyrosine residues in endogenous proteins. The degree of 3-nitrotyrosine immunoreactivity over the 2-h postexposure period did not differ between mutant and wild-type strains. These results indicate that the nfsA and nfsB enzymes do not reduce 3-nitrotyrosine.     

Crystal structure of nitrated human manganese superoxide dismutase: mechanism of inactivation

A cellular consequence of the reaction of superoxide and nitric oxide is enhanced peroxynitrite levels. Reaction of peroxynitrite with manganese superoxide dismutase (MnSOD) causes nitration of the active-site residue Tyr34 and nearly complete inhibition of catalysis. We report the crystal structures at 2.4 A resolution of human MnSOD nitrated by peroxynitrite and the unmodified MnSOD. A comparison of these structures showed no significant conformational changes of active-site residues or solvent displacement. The side chain of 3-nitrotyrosine 34 had a single conformation that extended toward the manganese with O1 of the nitro group within hydrogen-bonding distance (3.1 A) of Nepsilon2 of the second-shell ligand Gln143. Also, nitration of Tyr34 caused a weakening, as evidenced by the lengthening, of a hydrogen bond between its phenolic OH and Gln143, part of an extensive hydrogen-bond network in the active site. Inhibition of catalysis can be attributed to a steric effect of 3-nitrotyrosine 34 that impedes substrate access and binding, and alteration of the hydrogen-bond network that supports proton transfer in catalysis. It is also possible that an electrostatic effect of the nitro group has altered the finely tuned redox potential necessary for efficient catalysis, although the redox potential of nitrated MnSOD has not been measured.     

Rapid and selective oxygen-regulated protein tyrosine denitration and nitration in mitochondria

Growing evidence connects a cumulative formation of 3-nitrotyrosyl adducts in proteins as a marker for oxidative damage with the pathogenesis of various diseases and pathological conditions associated with oxidative stress. A physiological signaling role for protein nitration has also been suggested. Controlled "denitration" would be essential for such a contribution of protein nitration to cellular regulatory processes. Thus, we further characterized such a potentially controlled, reversible tyrosine nitration that occurs in respiring mitochondria during oxygen deprivation followed by reoxygenation, which we recently discovered. Mitochondria constitute cellular centers of protein nitration and are leading candidates for a "nitrative" regulation. Mitochondria are capable of completely eliminating 3-nitrotyrosyl adducts during 20 min of hypoxia-anoxia and undergoing a selective partial reduction after only 5 min. This denitration is independent of protein degradation but depends on the oxygen tension. Reoxygenation re-establishes protein tyrosine nitration patterns that are almost identical to the pattern that occurs before hypoxia-anoxia, with nitration levels that depend on the duration of hypoxia-anoxia. The identified mitochondrial targets of this process are critical for energy and antioxidant homeostasis and, therefore, cell and tissue viability. This cycle of protein nitration and denitration shows analogies to protein phosphorylation, and we demonstrate that the cycle meets most of the criteria for a cellular signaling mechanism. Taken together, our data reveal that protein tyrosine nitration in mitochondria can be controlled, is target-selective and rapid, and is dynamic enough to serve as a nitrative regulatory signaling process that likely affects cellular energy, redox homeostasis, and pathological conditions when these features become disturbed.     

Nitration Transforms a Sensitive Peroxiredoxin 2 into a More Active and Robust Peroxidase

Peroxiredoxins (Prx) are efficient thiol-dependent peroxidases and key players in the mechanism of H₂O₂-induced redox signaling. Any structural change that could affect their redox state, oligomeric structure, and/or interaction with other proteins could have a significant impact on the cascade of signaling events. Several post-translational modifications have been reported to modulate Prx activity. One of these, overoxidation of the peroxidatic cysteine to the sulfinic derivative, inactivates the enzyme and has been proposed as a mechanism of H₂O₂ accumulation in redox signaling (the floodgate hypothesis). Nitration of Prx has been reported in vitro as well as in vivo; in particular, nitrated Prx2 was identified in brains of Alzheimer disease patients. In this work we characterize Prx2 tyrosine nitration, a post-translational modification on a noncatalytic residue that increases its peroxidase activity and its resistance to overoxidation. Mass spectrometry analysis revealed that treatment of disulfide-oxidized Prx2 with excess peroxynitrite renders mainly mononitrated and dinitrated species. Tyrosine 193 of the YF motif at the C terminus, associated with the susceptibility toward overoxidation of eukaryotic Prx, was identified as nitrated and is most likely responsible for the protection of the peroxidatic cysteine against oxidative inactivation. Kinetic analyses suggest that tyrosine nitration facilitates the intermolecular disulfide formation, transforming a sensitive Prx into a robust one. Thus, tyrosine nitration appears as another mechanism to modulate these enzymes in the complex network of redox signaling.     

Proteomic identification of tyrosine nitration targets in kidney of spontaneously hypertensive rats

Nitrosative and oxidative stress are implicated in the development of hypertension. Events in the renal medulla may play a key role in the development and progression of hypertension. This may arise through disruption of nitric oxide signalling in the medulla and be accompanied by enhanced nitrosative and oxidative stress as indicated by the presence of proteins containing 3-nitrotyrosine. Here we demonstrate enhanced protein nitration in the medulla of spontaneously hypertensive rats. We have identified several nitrated proteins with both varied subcellular location and functional roles. These proteins are involved in nitric oxide signalling, antioxidant defense and energy metabolism. Moreover, increased nitration was observed in conjunction with enhanced oxidative damage as evidenced by the presence of protein carbonyl oxidative stress biomarkers. Our results suggest that kidney medulla is subject to enhanced nitrosative and oxidative stress, and that resulting protein modifications may contribute to the progression of hypertension.     

Myoglobin-catalyzed tyrosine nitration: no need for peroxynitrite.

The nitration of tyrosine residues in protein to yield 3-nitrotyrosine derivatives has been suggested to represent a specific footprint for peroxynitrite formation in vivo. However, recent studies suggest that certain hemoproteins such as peroxidases catalyze the H(2)O(2)-dependent nitration of tyrosine to yield 3-nitrotyrosine in a peroxynitrite-independent reaction. Because 3-nitrotyrosine has been shown to be present in the postischemic myocardium, we wished to assess the ability of myoglobin to catalyze the nitration of tyrosine in vitro. We found that myoglobin catalyzed the oxidation of nitrite and promoted the nitration of tyrosine. Both nitrite oxidation and tyrosine nitration were H(2)O(2)-dependent and required the formation of ferryl (Fe(+4)) myoglobin. In addition, nitrite oxidation and tyrosine nitration were pH-dependent with a pH optimum of approximately 6.0. Taken together, these data suggest that the acidic pH and low oxygen tension produced during myocardial ischemia will facilitate myoglobin-catalyzed, peroxyntrite-independent formation of 3-nitrotyrosine.     

Immunological detection of nitrosative stress-mediated modified Tamm-Horsfall glycoprotein (THP) in calcium oxalate stone formers.

The generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in hyperoxaluric condition has been proved experimentally. This may result in the formation of the cytotoxic metabolite peroxynitrite, which is capable of causing lipid peroxidation and protein modification. The presence of nitrotyrosine in proteins has been associated with several pathological conditions. The present study investigated the presence of nitrotyrosine in the stone formers Tamm-Horsfall glycoprotein (THP). In vitro nitration of control THP was carried out using peroxynitrite. New Zealand white rabbits were immunized with peroxynitrated THP at 15-day intervals. Antisera collected following the third immunization were assayed for antibody titres using solid-phase ELISA. Antibodies were purified by affinity chromatography. The carbonyl content of control, stone formers and nitrated THP were determined. Western blotting was carried with control, stone formers and nitrated THPs. Immunodiffusion studies demonstrated cross-reaction with nitrated bovine serum albumin. Significant amounts (p < 0.001) of carbonyl content were present in both stone formers and nitrated THPs. Western blot analysis confirmed the presence of nitrated amino acid 3-nitrotyrosine in stone formers, which could bring about structural and functional modifications of THP in hyperoxaluric patients. A cross-reaction with nitrated bovine serum albumin confirms that the raised antibody has certain paratopes similar to the epitope of nitrated protein molecules. Detection of 3-nitrotyrosine in stone formers THP indicates that it is one of the key factors influencing the conversion of THP to a structurally and immunologically altered form during calcium oxalate stone formation.     

Tyrosine nitration: Localisation, quantification, consequences for protein function and signal transduction

The nitration of free tyrosine or protein tyrosine residues generates 3-nitrotyrosine the detection of which has been utilised as a footprint for the in vivo formation of peroxynitrite and other reactive nitrogen species. The detection of 3-nitrotyrosine by analytical and immunological techniques has established that tyrosine nitration occurs under physiological conditions and levels increase in most disease states. This review provides an updated, comprehensive and detailed summary of the tissue, cellular and specific protein localisation of 3-nitrotyrosine and its quantification. The potential consequences of nitration to protein function and the pathogenesis of disease are also examined together with the possible effects of protein nitration on signal transduction pathways and on the metabolism of proteins.     

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.