Peroxynitrite oxidation of tubulin sulfhydryls inhibits microtubule polymerization

Considerable evidence both in vitro and in vivo implicates protein damage by peroxynitrite as a probable mechanism of cell death. Herein, we report that treatment of bovine brain microtubule protein, composed of tubulin and microtubule-associated proteins, with peroxynitrite led to a dose-dependent inhibition of microtubule polymerization. The extent of cysteine oxidation induced by peroxynitrite correlated well with inhibition of microtubule polymerization. Disulfide bonds between the subunits of the tubulin heterodimer were detected by Western blot as a result of peroxynitrite-induced cysteine oxidation. Addition of disulfide reducing agents including dithiothreitol and beta-mercaptoethanol restored a significant portion of the polymerization activity that was lost following peroxynitrite addition. Thus, peroxynitrite-induced disulfide bonds are at least partially responsible for the observed inhibition of polymerization. Sodium bicarbonate protected microtubule protein from the peroxynitrite-induced inhibition of polymerization. Tyrosine nitration of microtubule protein by 1 mM peroxynitrite increased approximately twofold when sodium bicarbonate was present whereas the extent of cysteine oxidation decreased from 7.5 to 6.3 mol cysteine/mol tubulin. These results indicate that cysteine oxidation of tubulin by peroxynitrite, rather than tyrosine nitration, is the primary mechanism of inhibition of microtubule polymerization.     

Reversible inhibition of mammalian glutamine synthetase by tyrosine nitration

The effect of tyrosine nitration on mammalian GS activity and stability was studied in vitro. Peroxynitrite at a concentration of 5 micro mol/l produced tyrosine nitration and inactivation of GS, whereas 50 micro mol/l peroxynitrite additionally increased S-nitrosylation and carbonylation and degradation of GS by the 20S proteasome. (-)Epicatechin completely prevented both, tyrosine nitration and inactivation of GS by peroxynitrite (5 micro mol/l). Further, a putative "denitrase" activity restored the activity of peroxynitrite (5 micro mol/l)-treated GS. The data point to a potential regulation of GS activity by a reversible tyrosine nitration. High levels of oxidative stress may irreversibly damage and predispose the enzyme to proteasomal degradation.     

Tyrosine nitration impairs mammalian aldolase A activity

Protein tyrosine nitration increases in vivo as a result of oxidative stress and is elevated in numerous inflammatory-associated diseases. Mammalian fructose-1,6-bisphosphate aldolases are tyrosine nitrated in lung epithelial cells and liver, as well as in retina under different inflammatory conditions. Using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we now show that aldolase A is nitrated in human skin fibroblasts. To reveal the consequences of tyrosine nitration, we studied the impact of peroxynitrite on the glycolytic functions of aldolase A. A peroxynitrite concentration-dependent decrease in fructose-1,6-bisphosphate cleavage activity was observed with a concomitant increase in nitrotyrosine immunoreactivity. Both V(max) and the K(m) for fructose-1,6-bisphosphate decreased after incubation with peroxynitrite. Aldolase nitrotyrosine immunoreactivity diminished following carboxypeptidase Y digestion, demonstrating that tyrosine residues in the carboxyl-terminal region of aldolase are major targets of nitration. Aldolase A contains a carboxyl-terminal tyrosine residue, Tyr(363), that is critical for its catalytic activity. Indeed, tandem mass spectrometric analysis of trypsin-digested aldolase showed that Tyr(363) is the most susceptible to nitration, with a modification of Tyr(342) occurring only after nitration of Tyr(363). These tyrosine nitrations likely result in altered interactions between the carboxyl-terminal region and enzyme substrate or reaction intermediates causing the decline in activity. The results suggest that tyrosine nitration of aldolase A can contribute to an impaired cellular glycolytic activity.     

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.     

Oxidative modification of low-density lipoprotein: lipid peroxidation by myeloperoxidase in the presence of nitrite

Oxidative modification of low-density lipoprotein (LDL) is a pivotal process in early atherogenesis and can be brought about by myeloperoxidase (MPO), which is capable of reacting with nitrite, a NO metabolite. We studied MPO-mediated formation of conjugated dienes in isolated human LDL in dependence on the concentrations of nitrite and chloride. This reaction was strongly stimulated by low concentrations (5-50 microM) of nitrite which corresponds to the reported concentration in the arterial vessel wall. Under these conditions no protein tyrosine nitration occurred; this reaction required much higher nitrite concentrations (100 microM-1 mM). Chloride neither supported lipid peroxidation alone nor was its presence mandatory for the effect of nitrite. We propose a prominent role of lipid peroxidation for the proatherogenic action of the MPO/nitrite system, whereas peroxynitrite may be competent for protein tyrosine nitration of LDL. Monomeric and oligomeric flavan-3-ols present in cocoa products effectively counteracted, at micromolar concentrations, the MPO/nitrite-mediated lipid peroxidation of LDL. Flavan-3-ols also suppressed protein tyrosine nitration induced by MPO/nitrite or peroxynitrite as well as Cu2+-mediated lipid peroxidation of LDL. This multi-site protection by (-)-epicatechin or other flavan-3-ols against proatherogenic modification of LDL may contribute to the purported beneficial effects of dietary flavan-3-ols for the cardiovascular system.     

The effects of nitronium ion on nitration, carbonylation and coagulation of human fibrinogen

The effect of nitronium ion on nitration, carbonylation and coagulation of human fibrinogen (Fg) in vitro was investigated. We observed that nitration of tyrosine, induced by NO2BF4 (0.01 mmol/l), was increased. No changes in carbonylation by NO2BF4 (0.01 mmol/l) were noticed. Mentioned alterations were associated with amplified coagulation of Fg. Higher concentrations of NO2BF4 (1 and 0.1 mmol/l) triggered growth of nitration and carbonylation of Fg, but led to inhibition of polymerization. Slight nitration may be responsible for increase, whereas sizable nitration and oxidation may lead to inhibition of Fg coagulation.     

Redox Proteomics Uncovers Peroxynitrite-sensitive Proteins That Help Escherichia coli to Overcome Nitrosative Stress

Peroxynitrite is a highly reactive chemical species with antibacterial properties that are synthesized in immune cells. In a proteomic approach, we identified specific target proteins of peroxynitrite-induced modifications in Escherichia coli. Although peroxynitrite caused a fairly indiscriminate nitration of tyrosine residues, reversible modifications of protein thiols were highly specific. We used a quantitative redox proteomic method based on isotope-coded affinity tag chemistry and identified four proteins consistently thiol-modified in cells treated with peroxynitrite as follows: AsnB, FrmA, MaeB, and RidA. All four were required for peroxynitrite stress tolerance in vivo. Three of the identified proteins were modified at highly conserved cysteines, and MaeB and FrmA are known to be directly involved in the oxidative and nitrosative stress response in E. coli. In in vitro studies, we could show that the activity of RidA, a recently discovered enamine/imine deaminase, is regulated in a specific manner by the modification of its single conserved cysteine. Mutation of this cysteine 107 to serine generated a constitutively active protein that was not susceptible to peroxynitrite.     

Tyrosine nitration of c-SRC tyrosine kinase in human pancreatic ductal adenocarcinoma

During pancreatic tumorigenesis, the equilibrium between cell survival and cell death is altered, allowing aggressive neoplasia and resistance to radiation and chemotherapy. Local oxidative stress is one mechanism regulating programmed cell death and growth and may contribute to both tumor progression and suppression. Our recent in situ immunohistochemical studies demonstrated that levels of total nitrotyrosine, a footprint of the reactive nitrogen species peroxynitrite, are elevated in human pancreatic ductal adenocarcinomas. In this study, quantitative HPLC-EC techniques demonstrated a 21- to 97-fold increase in the overall levels of nitrotyrosine of human pancreatic tumor extracts compared to normal pancreatic extracts. Western blot analysis of human pancreatic tumor extracts showed that tyrosine nitration was restricted to a few specific proteins. Immunoprecipitation coupled with Western analysis identified c-Src tyrosine kinase as a target of both tyrosine nitration and tyrosine phosphorylation. Peroxynitrite treatment of human pancreatic carcinoma cells in vitro resulted in increased tyrosine nitration and tyrosine phosphorylation of c-Src kinase, increased (>2-fold) c-Src kinase activity, and increased association between c-Src kinase and its downstream substrate cortactin. Collectively, these observations suggest that peroxynitrite-mediated tyrosine nitration and tyrosine phosphorylation of c-Src kinase may lead to enhanced tyrosine kinase signaling observed during pancreatic ductal adenocarcinoma growth and metastasis.     

Superoxide Dismutase Catalyzes Nitration of Tyrosines by Peroxynitrite in the Rod and Head Domains of Neurofilament-L

Abstract: Superoxide dismutase (SOD) catalyzes the nitration of specific tyrosine residues in proteins by peroxynitrite (ONOO⁻), which may be the damaging gain-of-function resulting from mutations to SOD associated with familial amyotrophic lateral sclerosis (ALS). We found that disassembled neurofilament-L (light subunit) was more susceptible to tyrosine nitration catalyzed by SOD in vitro. Neurofilament-L was selectively nitrated compared with the majority of other proteins present in brain homogenates. Assembled neurofilament-L was more resistant to nitration, suggesting that the susceptible tyrosine residues were protected by intersubunit contacts in assembled neurofilaments. Electrospray mass spectrometry of trypsin-digested neurofilament-L showed that tyrosine 17 in the head region and tyrosines 138, 177, and 265 in α-helical coil regions of the rod domain of neurofilament-L were particularly susceptible to SOD-catalyzed nitration. Nitrated neurofilament-L inhibited the assembly of unmodified neurofilament subunits, suggesting that the affected tyrosines are located in regions important for intersubunit contacts. Neurofilaments are major structural proteins expressed in motor neurons and known to be important for their survival in vivo. We suggest that SOD-catalyzed nitration of neurofilament-L may have a significant role in the pathogenesis of ALS.     

Effect of methionine sulfoxide reductase B1 (SelR) gene silencing on peroxynitrite-induced F-actin disruption in human lens epithelial cells

F-actin plays a crucial role in fundamental cellular processes, and is extremely susceptible to peroxynitrite attack due to the high abundance of tyrosine in the peptide. Methionine sulfoxide reductase (Msr) B1 is a selenium-dependent enzyme (selenoprotein R) that may act as a reactive oxygen species (ROS) scavenger. However, its function in coping with reactive nitrogen species (RNS)-mediated stress and the physiological significance remain unclear. Thus, the present study was conducted to elucidate the role and mechanism of MsrB1 in protecting human lens epithelial (hLE) cells against peroxynitrite-induced F-actin disruption. While exposure to high concentrations of peroxynitrite and gene silencing of MsrB1 by siRNA alone caused disassembly of F-actin via inactivation of extracellular signal-regulated kinase (ERK) in hLE cells, the latter substantially aggravated the disassembly of F-actin triggered by the former. This aggravation concurred with elevated nitration of F-actin and inactivation of ERK compared with that induced by the peroxynitrite treatment alone. In conclusion, MsrB1 protected hLE cells against the peroxynitrite-induced F-actin disruption, and the protection was mediated by inhibiting the resultant nitration of F-actin and inactivation of ERKs.     

Kinetics of superoxide dismutase- and iron-catalyzed nitration of phenolics by peroxynitrite.

Superoxide dismutase and Fe3+EDTA catalyzed the nitration by peroxynitrite (ONOO-) of a wide range of phenolics including tyrosine in proteins. Nitration was not mediated by a free radical mechanism because hydroxyl radical scavengers did not reduce either superoxide dismutase or Fe3+EDTA-catalyzed nitration and nitrogen dioxide was not a significant product from either catalyst. Rather, metal ions appear to catalyze the heterolytic cleavage of peroxynitrite to form a nitronium-like species (NO2+). The calculated energy for separating peroxynitrous acid into hydroxide ion and nitronium ion is 13 kcal.mol-1 at pH 7.0. Fe3+EDTA catalyzed nitration with an activation energy of 12 kcal.mol-1 at a rate of 5700 M-1.s-1 at 37 degrees C and pH 7.5. The reaction rate of peroxynitrite with bovine Cu,Zn superoxide dismutase was 10(5) M-1.s-1 at low superoxide dismutase concentrations, but the rate of nitration became independent of superoxide dismutase concentration above 10 microM with only 9% of added peroxynitrite yielding nitrophenol. We propose that peroxynitrite anion is more stable in the cis conformation, whereas only a higher energy species in the trans conformation can fit in the active site of Cu,Zn superoxide dismutase. At high superoxide dismutase concentrations, phenolic nitration may be limited by the rate of isomerization from the cis to trans conformations of peroxynitrite as well as by competing pathways for peroxynitrite decomposition. In contrast, Fe3+EDTA appears to react directly with the cis anion, resulting in greater nitration yields.     

Dopamine prevents nitration of tyrosine hydroxylase by peroxynitrite and nitrogen dioxide: is nitrotyrosine formation an early step in dopamine neuronal damage?

Peroxynitrite and nitrogen dioxide (NO2) are reactive nitrogen species that have been implicated as causal factors in neurodegenerative conditions. Peroxynitrite-induced nitration of tyrosine residues in tyrosine hydroxylase (TH) may even be one of the earliest biochemical events associated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced damage to dopamine neurons. Exposure of TH to peroxynitrite or NO2 results in nitration of tyrosine residues and modification of cysteines in the enzyme as well as inactivation of catalytic activity. Dopamine (DA), its precursor 3,4-dihydroxyphenylalanine, and metabolite 3,4-dihydroxyphenylacetic acid completely block the nitrating effects of peroxynitrite and NO2 on TH but do not relieve the enzyme from inhibition. o-Quinones formed in the reaction of catechols with either peroxynitrite or NO2 react with cysteine residues in TH and inhibit catalytic function. Using direct, real-time evaluation of tyrosine nitration with a green fluorescent protein-TH fusion protein stably expressed in intact cells (also stably expressing the human DA transporter), DA was also found to prevent NO2-induced nitration while leaving TH activity inhibited. These results show that peroxynitrite and NO2 react with DA to form quinones at the expense of tyrosine nitration. Endogenous DA may therefore play an important role in determining how DA neurons are affected by reactive nitrogen species by shifting the balance of their effects away from tyrosine nitration and toward o-quinone formation.     

Effects of peroxynitrite-induced protein tyrosine nitration on insulin-stimulated tyrosine phosphorylation in HepG2 cells

Tyrosine nitration by peroxynitrite can affect signal transduction pathways involving tyrosine phosphorylation. The present study was undertaken to investigate the effects of peroxynitrite-induced protein tyrosine nitration on insulin-stimulated tyrosine phosphorylation in HepG2 cells. We show here that exposure of HepG2 cells to peroxynitrite led to a dose-dependent increase in tyrosine nitration of cellular proteins, mainly membrane and nuclear proteins. Furthermore, peroxynitrite induced differential responses in tyrosine phosphorylation of membrane proteins as well as cytosolic proteins according to peroxynitrite concentrations used. Our findings indicate at low concentrations peroxynitrite upregulates the insulin signaling and may operate as a signaling molecule, but at higher concentrations peroxynitrite downregulates the insulin signaling and may be involved in insulin resistance, suggesting peroxynitrite plays a dual role in regulation of the insulin signaling.     

Evidence for peroxynitrite-mediated modifications to p53 in human gliomas: possible functional consequences

Based on previous findings of increased nitric oxide synthase (NOS) expression in human gliomas (4), we hypothesized that peroxynitrite, a highly reactive metabolite of nitric oxide (NO) and superoxide (O(*-)(2)), might be increased in these tumors in vivo. Here we demonstrate that nitrotyrosine (a footprint of peroxynitrite protein modification) is present in human malignant gliomas. Furthermore, we show that p53, a key tumor suppressor protein, has evidence of peroxynitrite-mediated modifications in gliomas in vivo. Experiments in vitro demonstrate that peroxynitrite treatment of recombinant wild-type p53 at physiological concentrations results in formation of higher molecular weight aggregates, tyrosine nitration, and loss of specific DNA binding. Peroxynitrite treatment of human glioma cell lysates similarly resulted in selective tyrosine nitration of p53 and was also associated with loss of p53 DNA binding ability. These data indicate that tyrosine nitration of proteins occurs in human gliomas in vivo, that p53 may be a target of peroxynitrite in these tumors, and that physiological concentrations of peroxynitrite can result in a loss of p53 DNA binding ability in vitro. These findings raise the possibility that peroxynitrite may contribute to loss of wild-type p53 functional activity in gliomas by posttranslational protein modifications.     

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.     

Peroxynitrite oxidizes erythrocyte membrane band 3 protein and diminishes its anion transport capacity

We describe an altered membrane band 3 protein-mediated anion transport in erythrocytes exposed to peroxynitrite, and relate the loss of anion transport to cell damage and to band 3 oxidative modifications. We found that peroxynitrite down-regulate anion transport in a dose dependent relation (100-300 μmoles/l). Hemoglobin oxidation was found at all peroxynitrite concentrations studied. A dose-dependent band 3 protein crosslinking and tyrosine nitration were also observed. Band 3 protein modifications were concomitant with a decrease in transport activity. ( - )-Epicatechin avoids band 3 protein nitration but barely affects its transport capacity, suggesting that both processes are unrelated. N-acetyl cysteine partially reverted the loss of band 3 transport capacity. It is concluded that peroxynitrite promotes a decrease in anion transport that is partially due to the reversible oxidation of band 3 cysteine residues. Additionally, band 3 tyrosine nitration seems not to be relevant for the loss of its anion transport capacity.     

Peroxynitrite oxidizes erythrocyte membrane band 3 protein and diminishes its anion transport capacity

We describe an altered membrane band 3 protein-mediated anion transport in erythrocytes exposed to peroxynitrite, and relate the loss of anion transport to cell damage and to band 3 oxidative modifications. We found that peroxynitrite down-regulate anion transport in a dose dependent relation (100–300 μmoles/l). Hemoglobin oxidation was found at all peroxynitrite concentrations studied. A dose-dependent band 3 protein crosslinking and tyrosine nitration were also observed. Band 3 protein modifications were concomitant with a decrease in transport activity. ( ? )-Epicatechin avoids band 3 protein nitration but barely affects its transport capacity, suggesting that both processes are unrelated. N-acetyl cysteine partially reverted the loss of band 3 transport capacity. It is concluded that peroxynitrite promotes a decrease in anion transport that is partially due to the reversible oxidation of band 3 cysteine residues. Additionally, band 3 tyrosine nitration seems not to be relevant for the loss of its anion transport capacity.     

Dopamine-melanin protects against tyrosine nitration, tryptophan oxidation and Ca(2+)-ATPase inactivation induced by peroxynitrite

The effects of dopamine-melanin (DA-melanin), a synthetic model of neuromelanin, on peroxynitrite-mediated 3-nitrotyrosine formation, oxidation of tryptophan in bovine serum albumin and inactivation of erythrocyte membrane Ca(2+)-ATPase activity were investigated in the absence and in the presence of bicarbonate. DA-melanin inhibited nitration of free tyrosine, loss of tryptophan residues and Ca(2+)-ATPase inactivation by peroxynitrite in a dose dependent manner. In the presence of bicarbonate, this inhibitory effect was lower for nitration and insignificant for oxidative protein modifications. These results suggest that neuromelanin can protect against nitrating and oxidizing action of peroxynitrite but is a worse protector against the peroxynitrite-CO(2) adduct. As peroxynitrite may be a mediator of neurotoxic processes, the obtained results suggest that neuromelanin may be important as a physiological protector against peroxynitrite.     

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 microM, 15 microM MnSOD) and Y34F-MnSOD (IC50 = 55 microM, 15 microM 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 inactivates human-tissue inhibitor of metalloproteinase-4

Peroxynitrite, via post-translational modifications to target proteins, contributes to cardiovascular injury and cancer. Since tissue inhibitor of metalloproteinase-4 (TIMP-4), the activity of which is impaired in both pathological conditions, has several amino acid residues susceptible to peroxynitrite, we investigated its role as a potential target of peroxynitrite. Peroxynitrite-induced nitration and oligomerization of TIMP-4 attenuated its inhibitory activity against MMP-2 activity and endothelial or tumor cell invasiveness. Moreover, cell treatment with peroxynitrite promoted the nitration of endogenous TIMP-4. HPLC/ESI-MS/MS analysis of peroxynitrite-treated TIMP-4 showed modifications at Y114, Y195, Y188 and Y190. In conclusion, TIMP-4 nitration might be a potential mechanism contributing to cardiovascular disease and cancer.