Protein tyrosine nitration of mitochondrial carbamoyl phosphate synthetase 1 and its functional consequences.
ABSTRACT: Mitochondria are the primary locus for the generation of reactive nitrogen species including peroxynitrite and subsequent protein tyrosine nitration. Protein tyrosine nitration may have important functional and biological consequences such as alteration of enzyme catalytic activity. In the present study, mouse liver mitochondria were incubated with peroxynitrite, and the mitochondrial proteins were separated by 1D and 2D gel electrophoresis. Nitrotyrosinylated proteins were detected with an anti-nitrotyrosine antibody. One of the major proteins nitrated by peroxynitrite was carbamoyl phosphate synthetase 1 (CPS1) as identified by LC-MS protein analysis and Western blotting. The band intensity of nitration normalized to CPS1 was increased in a peroxynitrite concentration-dependent manner. In addition, CPS1 activity was decreased by treatment with peroxynitrite in a peroxynitrite concentration- and time-dependent manner. The decreased CPS1 activity was not recovered by treatment with reduced glutathione, suggesting that the decrease of the CPS1 activity is due to tyrosine nitration rather than cysteine oxidation. LC-MS analysis of in-gel digested samples, and a Popitam-based modification search located 5 out of 36 tyrosine residues in CPS1 that were nitrated. Taken together with previous findings regarding CPS1 structure and function, homology modeling of mouse CPS1 suggested that nitration at Y1450 in an ?-helix of allosteric domain prevents activation of CPS1 by its activator, N-acetyl-l-glutamate. In conclusion, this study demonstrated the tyrosine nitration of CPS1 by peroxynitrite and its functional consequence. Since CPS1 is responsible for ammonia removal in the urea cycle, nitration of CPS1 with attenuated function might be involved in some diseases and drug-induced toxicities associated with mitochondrial dysfunction.
Project description:The peroxynitrite anion is a potent oxidizing agent, formed by the diffusion-limited combination of nitric oxide and superoxide, and its production under physiological conditions is associated with the pathologies of a number of inflammatory and neurodegenerative diseases. Nitration of Escherichia coli iron superoxide dismutase (Fe-SOD) by peroxynitrite was investigated, and demonstrated by spectral changes and electrospray mass spectroscopic analysis. HPLC and mass studies of the tryptic digests of the mono-nitrated Fe-SOD indicated that tyrosine-34 was the residue most susceptible to nitration by peroxynitrite. Exclusive nitration of this residue occurred when Fe-SOD was exposed to a cumulative dose of 0.4 mM peroxynitrite. Unlike with human Mn-SOD, this single modification did not inactivate E. coli Fe-SOD at pH 7.4. When Fe-SOD was exposed to higher concentrations of peroxynitrite (7 mM), eight tyrosine residues per subunit of the protein, of the nine available, were nitrated without loss of catalytic activity of the enzyme. The pK(a) of nitrated tyrosine-34 was determined to be 7.95+/-0.15, indicating that the peroxynitrite-modified enzyme appreciably maintains its protonation state under physiological conditions.
Project description:The nitric oxide-mediated actions are mostly due to cyclic GMP (cGMP) formation, but cGMP-independent mechanisms, such as tyrosine nitration, have been suggested as potential signaling pathways modulating the NO-induced responses. However, the mechanisms that lead to tyrosine nitration in platelets are poorly studied, and the protein targets of nitration have not been identified in these cells. Therefore, we have used the model of platelet adhesion to fibrinogen-coated plates to investigate the cGMP-independent mechanisms of the NO-donor sodium nitroprusside (SNP) that leads to inhibition of platelet adhesion. SNP concentration-dependently inhibited platelet adhesion, as observed at 15-min and 60-min adhesion. Additionally, SNP markedly increased the cGMP levels, and the soluble guanylate inhibitor ODQ nearly abolished the SNP-mediated cGMP elevations in all experimental conditions used. Nevertheless, ODQ failed to affect the adhesion inhibition obtained with 1.0 mM SNP at 15 min. On the other hand, superoxide dismutase or peroxynitrite (ONOO(-)) scavenger epigallocatechin gallate significantly reversed the inhibition of platelet adhesion by SNP (1 mM, 15 min). Western blot analysis in SNP (1 mM, 15 min)-treated platelets showed a single tyrosine-nitrated protein with an apparent mass of approximately 105 kDa. Nanospray LC-MS/MS identified the human alpha-actinin 1 cytoskeletal isoform (P12814) as the protein contained in the nitrated SDS gel band. Thus, tyrosine nitration of alpha-actinin, through ONOO(-) formation, may be a key modulatory mechanism to control platelet adhesion.
Project description:Protein tyrosine nitration is a post-translational modification mediated by reactive nitrogen species (RNS) that is associated with nitro-oxidative damage. No information about this process is available in relation to higher plants during development and senescence. Using pea plants at different developmental stages (ranging from 8 to 71 days), tyrosine nitration in the main organs (roots, stems, leaves, flowers, and fruits) was analysed using immunological and proteomic approaches. In the roots of 71-day-old senescent plants, nitroproteome analysis enabled the identification a total of 16 nitrotyrosine-immunopositive proteins. Among the proteins identified, NADP-isocitrate dehydrogenase (ICDH), an enzyme involved in the carbon and nitrogen metabolism, redox regulation, and responses to oxidative stress, was selected to evaluate the effect of nitration. NADP-ICDH activity fell by 75% during senescence. Analysis showed that peroxynitrite inhibits recombinant cytosolic NADP-ICDH activity through a process of nitration. Of the 12 tyrosines present in this enzyme, mass spectrometric analysis of nitrated recombinant cytosolic NADP-ICDH enabled this study to identify the Tyr392 as exclusively nitrated by peroxynitrite. The data as a whole reveal that protein tyrosine nitration is a nitric oxide-derived PTM prevalent throughout root development and intensifies during senescence.
Project description:Nitration of tyrosine and other aromatic amino acid residues in proteins occurs in the setting of inflammatory, neurodegenerative, and cardiovascular diseases-importantly, this modification has been implicated in the pathogenesis of diverse diseases and the physiological process of aging. To understand the biological consequences of aromatic nitration in both health and disease, it is critical to molecularly identify the proteins that undergo nitration, specify their cognate modification sites and quantify their extent of nitration. To date, unbiased identification of nitrated proteins has often involved painstaking 2D-gel electrophoresis followed by Western Blotting with an anti-nitrotyrosine antibody for detection. Apart from being relatively slow and laborious, this method suffers from limited coverage, the potential for false-positive identifications, and failure to reveal specific amino acid modification sites. To overcome these shortcomings, we have developed a solid-phase, chemical-capture approach for unbiased and high-throughput discovery of nitrotyrosine and nitrotryptophan sites in proteins. Utilizing this method, we have successfully identified several endogenously nitrated proteins in rat brain and a total of 244 nitrated peptides from 145 proteins following in vitro exposure of rat brain homogenates to the nitrating agent peroxynitrite (1 mM). As expected, Tyr residues constituted the great majority of peroxynitrite-mediated protein nitration sites; however, we were surprised to discover several brain proteins that contain nitrated Trp residues. By incorporating a stable-isotope labeling step, this new Aromatic Nitration Site IDentification (ANSID) method was also adapted for relative quantification of nitration site abundances in proteins. Application of the ANSID method offers great potential to advance our understanding of the role of protein nitration in disease pathogenesis and normal physiology.
Project description:Increased O(2)(*-) and NO production is a key mechanism of mitochondrial dysfunction in myocardial ischemia/reperfusion injury. In complex II, oxidative impairment and enhanced tyrosine nitration of the 70 kDa FAD-binding protein occur in the post-ischemic myocardium and are thought to be mediated by peroxynitrite (OONO(-)) in vivo [Chen, Y.-R., et al. (2008) J. Biol. Chem. 283, 27991-28003]. To gain deeper insights into the redox protein thiols involved in OONO(-)-mediated oxidative post-translational modifications relevant in myocardial infarction, we subjected isolated myocardial complex II to in vitro protein nitration with OONO(-). This resulted in site-specific nitration at the 70 kDa polypeptide and impairment of complex II-derived electron transfer activity. Under reducing conditions, the gel band of the 70 kDa polypeptide was subjected to in-gel trypsin/chymotrypsin digestion and then LC-MS/MS analysis. Nitration of Y(56) and Y(142) was previously reported. Further analysis revealed that C(267), C(476), and C(537) are involved in OONO(-)-mediated S-sulfonation. To identify the disulfide formation mediated by OONO(-), nitrated complex II was alkylated with iodoacetamide. In-gel proteolytic digestion and LC-MS/MS analysis were conducted under nonreducing conditions. The MS/MS data were examined with MassMatrix, indicating that three cysteine pairs, C(306)-C(312), C(439)-C(444), and C(288)-C(575), were involved in OONO(-)-mediated disulfide formation. Immuno-spin trapping with an anti-DMPO antibody and subsequent MS was used to define oxidative modification with protein radical formation. An OONO(-)-dependent DMPO adduct was detected, and further LC-MS/MS analysis indicated C(288) and C(655) were involved in DMPO binding. These results offered a complete profile of OONO(-)-mediated oxidative modifications that may be relevant in the disease model of myocardial infarction.
Project description:Low temperature (LT) negatively affects plant growth and development via the alteration of the metabolism of reactive oxygen and nitrogen species (ROS and RNS). Among RNS, tyrosine nitration, the addition of an NO2 group to a tyrosine residue, can modulate reduced nicotinamide-dinucleotide phosphate (NADPH)-generating systems and, therefore, can alter the levels of NADPH, a key cofactor in cellular redox homeostasis. NADPH also acts as an indispensable electron donor within a wide range of enzymatic reactions, biosynthetic pathways, and detoxification processes, which could affect plant viability. To extend our knowledge about the regulation of this key cofactor by this nitric oxide (NO)-related post-translational modification, we analyzed the effect of tyrosine nitration on another NADPH-generating enzyme, the NADP-malic enzyme (NADP-ME), under LT stress. In Arabidopsis thaliana seedlings exposed to short-term LT (4 °C for 48 h), a 50% growth reduction accompanied by an increase in the content of superoxide, nitric oxide, and peroxynitrite, in addition to diminished cytosolic NADP-ME activity, were found. In vitro assays confirmed that peroxynitrite inhibits cytosolic NADP-ME2 activity due to tyrosine nitration. The mass spectrometric analysis of nitrated NADP-ME2 enabled us to determine that Tyr-73 was exclusively nitrated to 3-nitrotyrosine by peroxynitrite. The in silico analysis of the Arabidopsis NADP-ME2 protein sequence suggests that Tyr73 nitration could disrupt the interactions between the specific amino acids responsible for protein structure stability. In conclusion, the present data show that short-term LT stress affects the metabolism of ROS and RNS, which appears to negatively modulate the activity of cytosolic NADP-ME through the tyrosine nitration process.
Project description:We have recently demonstrated that asymmetric dimethylarginine (ADMA) induces the translocation of endothelial nitric-oxide synthase (eNOS) to the mitochondrion via a mechanism that requires protein nitration. Thus, the goal of this study was elucidate how eNOS redistributes to mitochondria and to identify the nitrated protein responsible for this event. Our data indicate that exposure of pulmonary arterial endothelial cells to ADMA enhanced eNOS phosphorylation at the Akt1-dependent phosphorylation sites Ser(617) and Ser(1179). Mutation of these serine residues to alanine (S617A and S1179A) inhibited nitration-mediated eNOS translocation to the mitochondria, whereas the phosphormimic mutations (S617D and S1179D) exhibited increased mitochondrial redistribution in the absence of ADMA. The overexpression of a dominant-negative Akt1 also attenuated ADMA-mediated eNOS mitochondrial translocation. Furthermore, ADMA enhanced Akt1 nitration and increased its activity. Mass spectrometry identified a single nitration site in Akt1 located at the tyrosine residue (Tyr(350)) located within the client-binding domain. Replacement of Tyr(350) with phenylalanine abolished peroxynitrite-mediated eNOS translocation to mitochondria. We also found that in the absence of ADMA, eNOS translocation decreased mitochondrial oxygen consumption and superoxide production without altering cellular ATP level. This suggests that under physiologic conditions, eNOS translocation enhances mitochondria coupling. In conclusion, we have identified a new mechanism by which eNOS translocation to mitochondria is regulated by the phosphorylation of eNOS at Ser(617) and Ser(1179) by Akt1 and that this is enhanced when Akt1 becomes nitrated at Tyr(350).
Project description:Tyrosine nitration is a post-translational protein modification with potentially significant biological implications. In the present study we demonstrate, for the first time, that tyrosine residues of human inducible nitric oxide synthase (NOS2) can be nitrated by peroxynitrite in vitro, leading to a decreased activity. Moreover, we show that NOS2 expressed in a skeletal muscle from septic patients is nitrated on selective tyrosine residues belonging to a canonic sequence. This phenomenon could be an endogenous mechanism of in vivo modulation of NOS2 enzymic activity.
Project description:Protein tyrosine nitration has been observed in a variety of human diseases associated with oxidative stress, such as inflammatory, neurodegenerative, and cardiovascular conditions. However, the pathways leading to nitration of tyrosine residues are still unclear. Recent studies have shown that peroxynitrite (PN), produced by the reaction of superoxide and nitric oxide, can lead to protein nitration and inactivation. Tyrosine nitration may also be mediated by nitrogen dioxide produced by the oxidation of nitrite by peroxidases. Manganese superoxide dismutase (MnSOD), which plays a critical role in cellular defense against oxidative stress by decomposing superoxide within mitochondria, is nitrated and inactivated under pathological conditions. In this study, MnSOD is shown to catalyze PN-mediated self-nitration. Direct, spectroscopic observation of the kinetics of PN decay and nitrotyrosine formation (k(cat) = 9.3 × 10(2) M(-1) s(-1)) indicates that the mechanism involves redox cycling between Mn(2+) and Mn(3+), similar to that observed with superoxide. Distinctive patterns of tyrosine nitration within MnSOD by various reagents were revealed and quantified by MS/MS analysis of MnSOD trypsin digest peptides. These analyses showed that three of the seven tyrosine residues of MnSOD (Tyr34, Tyr9, and Tyr11) were the most susceptible to nitration and that the relative amounts of nitration of these residues varied widely depending upon the nature of the nitrating agent. Notably, nitration mediated by PN, in both the presence and absence of CO2, resulted in nitration of the active site tyrosine, Tyr34, while nitration by freely diffusing nitrogen dioxide led to surface nitration at Tyr9 and Tyr11. Flux analysis of the nitration of Tyr34 by PN-CO2 showed that the nitration rate coincided with the kinetics of the reaction of PN with CO2. These kinetics and the 20-fold increase in the efficiency of tyrosine nitration in the presence of CO2 suggest a specific role for the carbonate radical anion (•CO3(-)) in MnSOD nitration by PN. We also observed that the nitration of Tyr34 caused inactivation of the enzyme, while nitration of Tyr9 and Tyr11 did not interfere with the superoxide dismutase activity. The loss of MnSOD activity upon Tyr34 nitration implies that the responsible reagent in vivo is peroxynitrite, acting either directly or through the action of •CO3(-).
Project description:Endothelial nitric oxide synthase-derived NO and its derivative, peroxynitrite (ONOO(-)), suppresses oxygen consumption by nitration of mitochondrial proteins after reperfusion. However, very few nitrated proteins are identified to date. In this paper, ischemia/reperfusion (I/R) injury was induced in mouse heart by ligation and release of the left anterior descending coronary artery. Western blotting showed that tyrosine nitration was higher in I/R hearts. Nitrated proteins were identified by capillary-liquid chromatography-nanospray tandem mass spectrometry. A total of 23 proteins were identified as being nitrated after I/R and 10 of them were from mitochondria. The nitrated mitochondrial proteins included 4 subunits from the oxidative phosphorylation system (the 24 and the 30 kDa subunits of complex I, the Rieske ISP of complex III, and the alpha subunit of ATP synthase), five enzymes in the matrix, and voltage-dependent anion channel. In purified complex I treated with ONOO(-), 3-NT was identified locating at the residue of Y247 of the 30 kDa subunit and the residues of Y47, Y53 of the 49 kDa subunit. In conclusion, I/R induced protein nitration and mitochondrial proteins were the major targets. Selective nitration of proteins from the oxidative phosphorylation system at the beginning of reperfusion may contribute to the suppression of oxygen consumption.