Top-Down Mass Analysis of Protein Tyrosine Nitration: Comparison of Electron Capture Dissociation with “Slow-Heating” Tandem Mass Spectrometry Methods

Tyrosine nitration in proteins is an important post-translational modification (PTM) linked to various pathological conditions. When multiple potential sites of nitration exist, tandem mass spectrometry (MS/MS) methods provide unique tools to locate the nitro-tyrosine(s) precisely. Electron capture dissociation (ECD) is a powerful MS/MS method, different in its mechanisms to the “slow-heating” threshold fragmentation methods, such as collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD). Generally, ECD provides more homogeneous cleavage of the protein backbone and preserves labile PTMs. However recent studies in our laboratory demonstrated that ECD of doubly charged nitrated peptides is inhibited by the large electron affinity of the nitro group, while CID efficiency remains unaffected by nitration. Here, we have investigated the efficiency of ECD versus CID and IRMPD for top-down MS/MS analysis of multiply charged intact nitrated protein ions of myoglobin, lysozyme, and cytochrome c in a commercial Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. CID and IRMPD produced more cleavages in the vicinity of the sites of nitration than ECD. However the total number of ECD fragments was greater than those from CID or IRMPD, and many ECD fragments contained the site(s) of nitration. We conclude that ECD can be used in the top-down analysis of nitrated proteins, but precise localization of the sites of nitration may require either of the “slow-heating” methods

Electron Capture Dissociation Mass Spectrometry of Tyrosine Nitrated Peptides

In vivo protein nitration is associated with many disease conditions that involve oxidative stress and inflammatory response. The modification involves addition of a nitro group at the position ortho to the phenol group of tyrosine to give 3-nitrotyrosine. To understand the mechanisms and consequences of protein nitration, it is necessary to develop methods for identification of nitrotyrosine-containing proteins and localization of the sites of modification.Here, we have investigated the electron capture dissociation (ECD) and collision-induced association (CID) behavior of 3-nitrotyrosine-containing peptides. The presence of nitration did not affect the CID behavior of the peptides. For the doubly-charged peptides, addition of nitration severely inhibited the production of ECD sequence fragments. However, ECD of the triply-charged nitrated peptides resulted in some singly-charged sequence fragments. ECD of the nitrated peptides is characterized by multiple losses of small neutral species including hydroxyl radicals, water and ammonia. The origin of the neutral losses has been investigated by use of activated ion (AI) ECD. Loss of ammonia appears to be the result of non-covalent interactions between the nitro group and protonated lysine side-chains

Bidimensional Tandem Mass Spectrometry for Selective Identification of Nitration Sites in Proteins.

Nitration of protein tyrosine residues is very often regarded as a molecular signal of peroxynitrite formation during development, oxidative stress, and aging. However, protein nitration might also have biological functions comparable to protein phosphorylation, mainly in redox signaling and in signal transduction. The major challenge in the proteomic analysis of nitroproteins is the need to discriminate modified proteins, usually occurring at substoichiometric levels from the large amount of nonmodified proteins. Moreover, precise localization of the nitration site is often required to fully describe the biological process. Existing methodologies essentially rely on immunochemical techniques either using 2D-PAGE fractionation in combination with western blot analyses or exploiting immunoaffinity procedures to selectively capture nitrated proteins. Here we report a totally new approach involving dansyl chloride labeling of the nitration sites that rely on the enormous potential of MSn analysis. The tryptic digest from the entire protein mixture is directly analyzed by MS on a linear ion trap mass spectrometer. Discrimination between nitro- and unmodified peptide is based on two selectivity criteria obtained by combining a precursor ion scan and an MS3 analysis. This new procedure was successfully applied to the identification of 3-nitrotyrosine residues in complex protein mixtures

Mononitration of durene

Reaction conditions under which the nitration of durene give predominantly, and in high yield, mononitrodurene (I), rather than dinitrodurene were found. The nitrating agent was No2+RF6-. Nitration with nitrosulfuric acid also gave mononitrodurene; however, byproducts such as 2.3.5.6-Me4C6HCH2C6H2Me3-2,4,5, were also formed. The NO2 PF6- gave an intermediate additional compound with durene. The reaction carried out with 3.6-dideuterodurene in D2O shows no isotope effect

Aromatic nitrations by mixed acid. Slow liquid-liquid reaction regime

Aromatic nitrations by mixed acid have been selected as a specific case of a heterogeneous liquid-liquid reaction. An extensive experimental programme has been followed using adiabatic and heat-flow calorimetry and pilot reactor experiments, supported by chemical analysis. A series of nitration experiments has been carried out to study the influences of different initial and operating conditions such as temperature, stirring speed and sulphuric acid concentration. In parallel, a mathematical model to predict the overall conversion rate has been developed. In this paper the mathematical modelling and the implementation and experimental validation for benzene, toluene and chlorobenzene mononitration in the kinetic control regime (slow liquid-liquid reaction) are presented and discussed

Nitro-fatty acid formation and metabolism

Nitro-fatty acids (NO 2 -FA) are pleiotropic modulators of redox signaling pathways. Their effects on inflammatory signaling have been studied in great detail in cell, animal and clinical models primarily using exogenously administered nitro-oleic acid. While we know a considerable amount regarding NO 2 -FA signaling, endogenous formation and metabolism is relatively unexplored. This review will cover what is currently known regarding the proposed mechanisms of NO 2 -FA formation, dietary modulation of endogenous NO 2 -FA levels, pathways of NO 2 -FA metabolism and the detection of NO 2 -FA and corresponding metabolites.Fil: Buchan, Gregory J.. University of Pittsburgh; Estados UnidosFil: Bonacci, Gustavo Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Fazzari, Marco. University of Pittsburgh; Estados Unidos. Fondazione Ri.Med; ItaliaFil: Salvatore, Sonia Rosana. University of Pittsburgh; Estados UnidosFil: Gelhaus Wendell, Stacy. University of Pittsburgh; Estados Unido

Striatal neuroinflammation promotes parkinsonism in rats

The specific role of neuroinflammation in the pathogenesis of Parkinson's disease remains to be fully elucidated. By infusing lipopolysaccharide (LPS) into the striatum, we investigated the effect of neuroinflammation on the dopamine nigrostriatal pathway. Here, we report that LPS-induced neuroinflammation in the striatum causes progressive degeneration of the dopamine nigrostriatal system, which is accompanied by motor impairments resembling parkinsonism. Our results indicate that neurodegeneration is associated with defects in the mitochondrial respiratory chain related to extensive S-nitrosylation/nitration of mitochondrial proteins. Mitochondrial injury was prevented by treatment of L-N^6^-(l-iminoethyl)-lysine, an inducible nitric oxide synthase (iNOS) inhibitor, suggesting that iNOS-derived NO is responsible for mitochondrial dysfunction. Furthermore, the nigral dopamine neurons exhibited intracytoplasmic [alpha]-synuclein and ubiquitin accumulation. These results demonstrate that degeneration of nigral dopamine neurons by neuroinflammation is associated with mitochondrial malfunction induced by NO-mediated S-nitrosylation/nitration of mitochondrial proteins

Nitric oxide from inflammatory origin impairs neural stem cell proliferation by inhibiting epidermal growth factor receptor signaling

Neuroinflammation is characterized by activation of microglial cells, followed by production of nitric oxide (NO), which may have different outcomes on neurogenesis, favoring or inhibiting this process. In the present study, we investigated how the inflammatory mediator NO can affect proliferation of neural stem cells (NSCs), and explored possible mechanisms underlying this effect. We investigated which mechanisms are involved in the regulation of NSC proliferation following treatment with an inflammatory stimulus (lipopolysaccharide plus IFN-gamma), using a culture system of subventricular zone (SVZ)-derived NSCs mixed with microglia cells obtained from wild-type mice (iNOS(+/+)) or from iNOS knockout mice (iNOS(-/-)). We found an impairment of NSC cell proliferation in iNOS(+/+) mixed cultures, which was not observed in iNOS(-/-) mixed cultures. Furthermore, the increased release of NO by activated iNOS(+/+) microglial cells decreased the activation of the ERK/MAPK signaling pathway, which was concomitant with an enhanced nitration of the EGF receptor. Preventing nitrogen reactive species formation with MnTBAP, a scavenger of peroxynitrite (ONOO-), or using the ONOO- degradation catalyst FeTMPyP cell proliferation and ERK signaling were restored to basal levels in iNOS(+/+) mixed cultures. Moreover, exposure to the NO donor NOC-18 (100 mu M), for 48 h, inhibited SVZ-derived NSC proliferation. Regarding the antiproliferative effect of NO, we found that NOC-18 caused the impairment of signaling through the ERK/MAPK pathway, which may be related to increased nitration of the EGF receptor in NSC. Using MnTBAP nitration was prevented, maintaining ERK signaling, rescuing NSC proliferation. We show that NO from inflammatory origin leads to a decreased function of the EGF receptor, which compromised proliferation of NSC. We also demonstrated that NO-mediated nitration of the EGF receptor caused a decrease in its phosphorylation, thus preventing regular proliferation signaling through the ERK/MAPK pathway.Foundation for Science and Technology, (FCT, Portugal); COMPETE; FEDER [PEst-C/SAU/LA0001/2013-2014, PEst-OE/EQB/LA0023/2013-2014, PTDC/SAU-NEU/102612/2008, PTDC/NEU-OSD/0473/2012]; FCT, Portugal [SERH/BPD/78901/2011, SERH/BD/38127/2007, SFRH/BD/77903/2011, SFRH/BD/79308/2011]info:eu-repo/semantics/publishedVersio

Tyrosine Nitration of Voltage-dependent Anion Channels in Cardiac Ischemia-reperfusion: Reduction by Peroxynitrite Scavenging

Excess superoxide (O2−) and nitric oxide (NO) forms peroxynitrite (ONOO−) during cardiac ischemia reperfusion (IR) injury, which in turn induces protein tyrosine nitration (tyr-N). Mitochondria are both a source of and target for ONOO−. Our aim was to identify specific mitochondrial proteins that display enhanced tyr-N after cardiac IR injury, and to explore whether inhibiting O2−/ONOO− during IR decreases mitochondrial protein tyr-N and consequently improves cardiac function. We show here that IR increased tyr-N of 35 and 15 kDa mitochondrial proteins using Western blot analysis with 3-nitrotyrosine antibody. Immunoprecipitation (IP) followed by LC–MS/MS identified 13 protein candidates for tyr-N. IP and Western blot identified and confirmed that the 35 kDa tyr-N protein is the voltage-dependent anion channel (VDAC). Tyr-N of native cardiac VDAC with IR was verified on recombinant (r) VDAC with exogenous ONOO−. We also found that ONOO− directly enhanced rVDAC channel activity, and rVDAC tyr-N induced by ONOO− formed oligomers. Resveratrol (RES), a scavenger of O2−/ONOO−, reduced the tyr-N levels of both native and recombinant VDAC, while L-NAME, which inhibits NO generation, only reduced tyr-N levels of native VDAC. O2− and ONOO− levels were reduced in perfused hearts during IR by RES and L-NAME and this was accompanied by improved cardiac function. These results identify tyr-N of VDAC and show that reducing ONOO− during cardiac IR injury can attenuate tyr-N of VDAC and improve cardiac function

The alkaline transition of cytochrome c revisited: Effects of electrostatic interactions and tyrosine nitration on the reaction dynamics

Here we investigated the effect of electrostatic interactions and of protein tyrosine nitration of mammalian cytochrome c on the dynamics of the so-called alkaline transition, a pH- and redox-triggered conformational change that implies replacement of the axial ligand Met80 by a Lys residue. Using a combination of electrochemical, time-resolved SERR spectroelectrochemical experiments and molecular dynamics simulations we showed that in all cases the reaction can be described in terms of a two steps minimal reaction mechanism consisting of deprotonation of a triggering group followed by ligand exchange. The pK a alk values of the transition are strongly modulated by these perturbations, with a drastic downshift upon nitration and an important upshift upon establishing electrostatic interactions with a negatively charged model surface. The value of pK a alk is determined by the interplay between the acidity of a triggering group and the kinetic constants for the forward and backward ligand exchange processes. Nitration of Tyr74 results in a change of the triggering group from Lys73 in WT Cyt to Tyr74 in the nitrated protein, which dominates the pK a alk downshift towards physiological values. Electrostatic interactions, on the other hand, result in strong acceleration of the backward ligand exchange reaction, which dominates the pK a alk upshift. The different physicochemical conditions found here to influence pK a alk are expected to vary depending on cellular conditions and subcellular localization of the protein, thus determining the existence of alternative conformations of Cyt in vivo.Fil: Oviedo Rouco, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Castro, Maria Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Álvarez Paggi, Damián Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Spedalieri, Ana Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Tortora, Verónica. Universidad de la República; UruguayFil: Tomasina, Florencia. Universidad de la República; UruguayFil: Radi, Rafael. Universidad de la República; UruguayFil: Murgida, Daniel Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentin