<p> Peroxynitrite is formed in blood vessels upon reaction of superoxide anion with nitric oxide (NO). It can oxidize proteins and thiols and nitrosylate free or protein bound thiols and tyrosine residues, thereby producing vascular dysfunction. Peroxynitrite therefore, may contribute to hypertension and cardiovascular diseases. We investigated the in vitro effects of commercially available peroxynitrite. De-endothelialized rings from the left descending coronary artery of pig were treated with peroxynitrite for 30 min, washed and then contracted with cyclopiazonic acid (CPA) or by membrane depolarization with KCl. Tissues pre-treated with peroxynitrite showed inhibition of the CPA-induced contraction with an IC50 of ≈100 uM but there was no effect on KCl-induced contraction. Peroxynitrite is stable only at alkaline pH and it may decompose to form superoxide and NO. However, including superoxide dismutase + catalase along with peroxynitrite did not change its effect.</p> <p> Next, we used the same protocol to compare the effects of peroxynitrite and NO generating agents: 3-morpholino sydnonimine (SIN-1), s-nitroso-N-acetylpenicilliamine (SNAP), sodium nitroprusside (SNP) and spermine nonoate. The effectiveness of these agents to inhibit the CPA-induced contraction was SNAP > spermine nonoate ≥ SIN-1 > SNP. SNAP was the most effective in inhibiting the KCl-induced contraction with spermine nononoate being less effective and SIN-1 and SNP not producing any significant inhibition. We further investigated the effect of SNAP. Catalase, superoxide dismutase or CPTIO (a NO scavenger) did not prevent the effects of SNAP on the KCl or the CPA-induced contractions. The guanylate cyclase inhibitor ODQ, partially reversed the effects of only low concentrations of SNAP. Thus, pretreatment with NO generating agents such as SNAP and spermine NONOate appear to be more effective in inhibiting the contraction of the pig coronary artery than with peroxynitrite or the peroxynitrite generating agent SIN-1. Since SIN-1, SNAP, SNP and NONOates produce different amounts of peroxynitrite, nitric oxide and S-nitrosylation products, their effects may be used to delineate the molecular basis of the actions of peroxynitrite and NO on the arterial function.</p> / Thesis / Master of Science (MSc)

Many common cancers, including breast, prostate and lung cancers, have a propensity to metastasize to bone. Although these cancers go undetected in their native tissues, bone metastases often produce excruciating pain, the etiology of which is poorly understood. Cancer-induced bone pain (CIBP) is not well-controlled with existing medications, severely compromising patient quality of life. While CIBP is multifaceted, increased level of the excitatory neurotransmitter glutamate in the bone-tumor microenvironment may contribute to the pain state. Here, we demonstrate for the first time a relationship between reactive oxygen/nitrogen species, glutamate in the bone-tumor microenvironment and pain behaviors. The murine mammary adenocarcinoma cell line 66.1 is found to release glutamate via the cystine/glutamate antiporter system xc⁻. In a syngeneic model of breast CIBP in which 66.1 cells are inoculated into the femur intramedullary space, administration of sulfasalazine, an established system xc⁻ inhibitor and anti-inflammatory agent, reduces femur glutamate level and attenuates CIBP-related behaviors. Peroxynitrite, a reactive nitrogen species known to be generated in breast tumors, is shown to drive 66.1 system xc⁻ functional expression and tumor cell glutamate release. The elimination of peroxynitrite with the redox modulators FeTMPyP or SRI10 not only modulates tumor cell system xc⁻ functional expression in vitro and in vivo, significantly altering glutamate levels, but also assuages CIBP. In sum, we demonstrate that pharmacological inhibition of system xc⁻ transport attenuates CIBP-related behaviors. These data support a role for tumor-derived glutamate in CIBP and validate system xc⁻ an analgesic target in this pain state.

A família Tiol Peroxidases (TPxs - Peroxirredoxinas e Glutationa peroxidases) purificadas definitivamente reduzem peróxidos rapidamente (peroxinitrito, ONOOH/ONOO; peróxido de hidrogênio, H2O2), mas nenhuma evidência direta desta atividade foi demonstrada em células vivas. Isto é particularmente importante pois o ciclo catalítico da atividade peróxido redutase de TPxs depende de sucessivas reações de trocas de tióis que podem limitar a velocidade de redução do peróxido. Neste trabalho, esta questão foi investigada em Saccharomyces cerevisiae (Sc) por meio de cinética de competição com um indicador fluorescente que é específico para ONOO (ácido borônico de cumarina; CBA), com a expectativa de que quanto maior a atividade peroxinitrito redutase, menor a oxidação do indicador. Também foi investigado o papel de duas peroxirredoxinas (Prxs) específicas na remoção deste peróxido. O estudo mostrou que a oxidação do indicador CBA dependente de ONOO foi sempre significativamente maior em células de Saccharomyces cerevisiae deficientes em TPxs (cepa 8) relativo a cepa nativa (WT). Além disso, a transfecção do gene que codifica a Prx mais abundante em Saccharomyces cerevisiae (Tsa1) na cepa 8 diminui parcialmente a oxidação de CBA. Além disso, a oxidação de CBA foi maior na cepa deficiente apenas da peroxirredoxina Tsa1 (a mais abundante da família) relativo à cepa WT, mostrando a relevância desta isoforma especificamente. De forma adversa, a oxidação de CBA na cepa deficiente da peroxirredoxina Tsa2 foi semelhante à cepa WT. Também, foi constatado que o processo de remoção de ONOO é catalítico (e não estequiométrico) para crescentes fluxos de peroxinitrito em todas as cepas e condições utilizadas no estudo. Finalmente, o estudo sugere que células possuem sistemas catalíticos peroxinitrito redutase redundantes, já que a própria cepa 8 apresenta e pode modular esta atividade. Estes resultados confirmam a expectativa da relevância de TPxs na remoção de ONOO e por extensão de outros peróxidos biologicamente relevantes e são a primeira evidência direta e em tempo real da atividade peroxinitrito redutase de TPxs em células. / The purified Thiol Peroxidases family (TPxs - Peroxiredoxins and Glutathione peroxidases) rapidly reduces peroxides (peroxynitrite, ONOOH/ONOO-, hydrogen peroxide, H2O2), but no direct evidence of this activity has been demonstrated in living cells. This is particularly important since the catalytic cycle of the TPxs peroxide reductase activity depends on successive thiol exchange reactions, which may limit the rate of peroxide reduction. In this work, this question was investigated in Saccharomyces cerevisiae (Sc) by competition kinetics using a fluorescent indicator that is specific for ONOO- (coumarin boronic acid; CBA). It is expected that the higher the peroxynitrite reductase activity, the lower the oxidation of the indicator. The role of two specific peroxiredoxins (Prxs) in the removal of this peroxide has also been investigated. The study showed that the oxidation of ONOO- dependent CBA indicator was always significantly higher in TPxs-deficient Saccharomyces cerevisiae cells (strain 8) compared to the native strain (WT). In addition, the transfection of the gene encoding the most abundant Prx into Saccharomyces cerevisiae (Tsa1) in the 8 strain partially diminishes CBA oxidation. Besides that, CBA oxidation was greater in the deficient strain only of the peroxiredoxin Tsa1 (the most abundant in the family) compared to the WT strain, showing the relevance of this isoform specifically. On the other hand, CBA oxidation in the deficient strain of the Tsa2 peroxiredoxin was similar to the WT strain. Also, it was found that the ONOO- removal process is catalytic (and not stoichiometric) for increasing peroxynitrite fluxes in all strains and conditions used in the study. Finally, the study suggests that cells have redundant peroxynitrite reductase catalytic systems, since the 8 strain itself presents and can modulate this activity. These results confirm the expectation of the relevance of TPxs in the removal of ONOO- and by extension of other biologically relevant peroxides and are the first direct and real-time evidence of peroxynitrite reductase activity of TPxs in cells.

Os oxidantes derivados do peroxinitrito e das peroxidases, como mieloperoxidase (MPO), e os danos que ocasionam em proteínas vêm sendo muito estudados pela sua relevância em processos inflamatórios. Neste trabalho, as proteínas RNase e lisozima foram empregadas como alvos de oxidação e nitração mediadas por peroxinitrito e MPO/H<SUB.2O2/NO2-. Experimentos de EPR indicaram que as oxidações envolvem a formação de radicais protéicos sendo que os principais foram caracterizados como RNase-tirosila e lisozima-tirosila exposto e não exposto ao solvente, respectivamente. Estimativas do rendimento de radicais protéicos e produtos nitrados nos pHs 5,4, 6,4 e 7,4 mostrou que o peroxinitrito e o sistema MPO/H<SUB.2O2/NO2- são oxidantes mais efetivos nos pHs 7,4 e 5,4, respectivamente. Na condição ótima para cada oxidante foram identificados produtos de oxidação/nitração de resíduos de Tyr e Trp por HPLC-UV/MS-ESI. Para localização dos resíduos modificados nas estruturas das proteínas tratadas, elas foram digeridas com tripsina e os peptídeos resultantes submetidos a análise por HPLC/MS-MALDI-ToF. Desses resultados pode-se concluir que a RNase foi nitrada preferencialmente nos fragmentos contendo o(s)resíduo(s) Tyr115 > Tyr92/97 > Tyr73/76 por peroxinitrito e em praticamente todos os resíduos de tirosina por MPOH<SUB.2O2/NO2-. No caso da lisozima, o peroxinitrito oxidou principalmente o fragmento contendo os resíduos Trp62/63 que se mostrou nitrado e oxidado a dímero e quinurenina. Já o sistema MPO/H<SUB.2O2/NO2- nitrou o fragmento contendo os resíduos Tyr23/28 e nitrou e oxidou a dímeros e quinurenina o fragmento contendo os resíduos Trp62/63. As relações entre a acessibilidade dos resíduos específicos nas estruturas terciárias e a formação de produtos de oxidação/nitração são discutidas. Também, a possível importância da oxidação de resíduos de triptofano em agregação de proteínas é enfatizada. Paralelamente, examinou-se os efeitos do nitróxido tempol sobre a nitração da RNase mediada por MPO ou HRP/H<SUB.2O2/NO2- em condições de máxima nitração. De fato, as interações de tempol com peroxidases eram pouco conhecidas apesar da eficiência do nitróxido em reduzir a injúria e os níveis de 3-nitrotirosina em proteínas de tecidos de animais submetidos a condições inflamatórias. Foram determinadas as constantes de velocidade da reação do tempol com os intermediários oxidantes da MPO e HRP e também, o consumo de reagentes e a formação de produtos. A simulação dos resultados experimentais indicou que o tempol inibe a nitração da RNase mediada por peroxidases principalmente pela sua capacidade de reagir rapidamente com o &#8226;NO2 com formação de nitrito e cátion oxamônio que, por sua vez, recicla para tempol reagindo com H2O2 para produzir O2. / The oxidants derived from peroxynitrite and peroxidase enzymes, such as myeloperoxidase (MPO), and the lesions they promote in proteins are being extensively investigated because of their relevance in inflammatory processes. Here, the proteins RNase and lysozyme were employed as targets of oxidations/nitrations mediated by peroxynitrite and MPO/H<SUB.2O2/NO2-. EPR experiments showed that the oxidations produced protein radicals of which the prominent ones were characterized as RNase-tyrosyl and lysozyme-tyrosil solvent-exposed and non-exposed, respectively. Estimates of protein radical and nitrated product yields at pH 5.4, 6.4 and 7.4 indicated that peroxynitrite and MPO/H<SUB.2O2/NO2- were more effective oxidants at pH 7.4 and 5.4, respectively. At the best condition for each oxidant, the oxidation/nitration products of Tyr and Trp residues were identified by HPLC-UV/ESI-MS analysis. The site of oxidation in the protein structures were identified by HPLC/MALDI-ToF-MS analysis of tryptic digests after oxidative treatment. From these results, it was concluded that RNase was nitrated mainly in Tyr115 > Tyr92/97 > Tyr62/63 by peroxynitrite and in all Tyr by MPO/H<SUB.2O2/NO2-. In the case of lysozyme, peroxynitrite modified mainly Trp62/63 that resulted nitrated and oxidized to a dimer and kynurenine. The MPO/H<SUB.2O2/NO2- system promoted the nitration of Tyr23/Trp28 and nitration and oxidation to dimer and kynurenine of Trp62/63. The relationships between residue accessibility in the structure of the proteins and their oxidation/nitration are discussed. The possible importance of Trp oxidation in protein aggregation is emphasized. In parallel, the effects of the nitroxide tempol upon RNase nitration mediated by MPO or HRP/H<SUB.2O2/NO2- was examined. Indeed, the interactions of tempol with peroxidases have been little investigated although the nitroxide is very efficient in reducing injury and 3-nitrotyrosine protein levels in tissues of animals submitted to inflammatory conditions. The second order rate constants of tempol reactions with the ferryl oxidants of MPO and HRP were determined. The consumption of reactants and formation of products were also determined. Computer simulation of the results indicated that tempol inhibits RNAse nitration mediated by peroxidases mainly because of its capability to rapidly react with &#8226;NO2 with formation of nitrite and the oxammonium cation, which, in turn, recycles back to tempol, by reacting with H2O2 to produce O2.

Methylglyoxal (MG) is a reactive á-oxoaldehyde and a glucose metabolite. Previous studies in our laboratory have shown that MG induces the production of reactive oxygen species (ROS), such as superoxide (O2.-), nitric oxide (NO) and peroxynitrite (ONOO-), in vascular smooth muscle cells (VSMCs, A-10 cells). However, the effect of endogenous MG and mechanisms of MG-induced oxidative stress have not been thoroughly explored. The present study investigated fructose (a precursor of MG)- induced ONOO- formation in A-10 cells and whether this process was mediated via endogenous MG formation; roles of MG in regulating mitochondrial ROS (mtROS) production and mitochondrial functions in A-10 cells; and effect of MG on neutrophils in patients with type 2 diabetes mellitus (T2DM). Fructose induced intracellular production of MG in a concentration- and time- dependent manner. A significant increase in the production of NO, O2.−, and ONOO− was observed in the cells exposed to fructose or MG. Fructose- or MG-induced ONOO− generation was significantly inhibited by MG scavengers and by O2.− or NO inhibitors. The data showed that fructose treatment increased the formation of ONOO− via increased NO and O2.− production in A-10 cells, and this effect was directly mediated by an elevated intracellular concentration of MG. By inhibiting complex III and manganese superoxide dismutase activities, MG induced mitochondrial overproduction of O2.-, and mitochondrial ONOO- further. MG also reduced mitochondrial ATP synthesis, indicating the dysfunction of mitochondria. In addition, MG increased plasma NO levels in patients with T2DM, which reflected the oxidative status in those patients. MG-induced oxidative stress in patients with T2DM significantly enhanced levels of cytokines released from neutrophils. Moreover, the neutrophils from T2DM patients showed a greater proclivity for apoptosis, which was further increased by in vitro MG treatment. Our data demonstrate that MG-induced oxidative damage, particularly ONOO- production, contributes to the pathogenesis of T2DM and its vascular complications.

Methylglyoxal (MG) is a reactive á-oxoaldehyde and a glucose metabolite. Previous studies in our laboratory have shown that MG induces the production of reactive oxygen species (ROS), such as superoxide (O2.-), nitric oxide (NO) and peroxynitrite (ONOO-), in vascular smooth muscle cells (VSMCs, A-10 cells). However, the effect of endogenous MG and mechanisms of MG-induced oxidative stress have not been thoroughly explored. The present study investigated fructose (a precursor of MG)- induced ONOO- formation in A-10 cells and whether this process was mediated via endogenous MG formation; roles of MG in regulating mitochondrial ROS (mtROS) production and mitochondrial functions in A-10 cells; and effect of MG on neutrophils in patients with type 2 diabetes mellitus (T2DM). Fructose induced intracellular production of MG in a concentration- and time- dependent manner. A significant increase in the production of NO, O2.−, and ONOO− was observed in the cells exposed to fructose or MG. Fructose- or MG-induced ONOO− generation was significantly inhibited by MG scavengers and by O2.− or NO inhibitors. The data showed that fructose treatment increased the formation of ONOO− via increased NO and O2.− production in A-10 cells, and this effect was directly mediated by an elevated intracellular concentration of MG. By inhibiting complex III and manganese superoxide dismutase activities, MG induced mitochondrial overproduction of O2.-, and mitochondrial ONOO- further. MG also reduced mitochondrial ATP synthesis, indicating the dysfunction of mitochondria. In addition, MG increased plasma NO levels in patients with T2DM, which reflected the oxidative status in those patients. MG-induced oxidative stress in patients with T2DM significantly enhanced levels of cytokines released from neutrophils. Moreover, the neutrophils from T2DM patients showed a greater proclivity for apoptosis, which was further increased by in vitro MG treatment. Our data demonstrate that MG-induced oxidative damage, particularly ONOO- production, contributes to the pathogenesis of T2DM and its vascular complications.

A família Tiol Peroxidases (TPxs - Peroxirredoxinas e Glutationa peroxidases) purificadas definitivamente reduzem peróxidos rapidamente (peroxinitrito, ONOOH/ONOO; peróxido de hidrogênio, H2O2), mas nenhuma evidência direta desta atividade foi demonstrada em células vivas. Isto é particularmente importante pois o ciclo catalítico da atividade peróxido redutase de TPxs depende de sucessivas reações de trocas de tióis que podem limitar a velocidade de redução do peróxido. Neste trabalho, esta questão foi investigada em Saccharomyces cerevisiae (Sc) por meio de cinética de competição com um indicador fluorescente que é específico para ONOO (ácido borônico de cumarina; CBA), com a expectativa de que quanto maior a atividade peroxinitrito redutase, menor a oxidação do indicador. Também foi investigado o papel de duas peroxirredoxinas (Prxs) específicas na remoção deste peróxido. O estudo mostrou que a oxidação do indicador CBA dependente de ONOO foi sempre significativamente maior em células de Saccharomyces cerevisiae deficientes em TPxs (cepa 8) relativo a cepa nativa (WT). Além disso, a transfecção do gene que codifica a Prx mais abundante em Saccharomyces cerevisiae (Tsa1) na cepa 8 diminui parcialmente a oxidação de CBA. Além disso, a oxidação de CBA foi maior na cepa deficiente apenas da peroxirredoxina Tsa1 (a mais abundante da família) relativo à cepa WT, mostrando a relevância desta isoforma especificamente. De forma adversa, a oxidação de CBA na cepa deficiente da peroxirredoxina Tsa2 foi semelhante à cepa WT. Também, foi constatado que o processo de remoção de ONOO é catalítico (e não estequiométrico) para crescentes fluxos de peroxinitrito em todas as cepas e condições utilizadas no estudo. Finalmente, o estudo sugere que células possuem sistemas catalíticos peroxinitrito redutase redundantes, já que a própria cepa 8 apresenta e pode modular esta atividade. Estes resultados confirmam a expectativa da relevância de TPxs na remoção de ONOO e por extensão de outros peróxidos biologicamente relevantes e são a primeira evidência direta e em tempo real da atividade peroxinitrito redutase de TPxs em células. / The purified Thiol Peroxidases family (TPxs - Peroxiredoxins and Glutathione peroxidases) rapidly reduces peroxides (peroxynitrite, ONOOH/ONOO-, hydrogen peroxide, H2O2), but no direct evidence of this activity has been demonstrated in living cells. This is particularly important since the catalytic cycle of the TPxs peroxide reductase activity depends on successive thiol exchange reactions, which may limit the rate of peroxide reduction. In this work, this question was investigated in Saccharomyces cerevisiae (Sc) by competition kinetics using a fluorescent indicator that is specific for ONOO- (coumarin boronic acid; CBA). It is expected that the higher the peroxynitrite reductase activity, the lower the oxidation of the indicator. The role of two specific peroxiredoxins (Prxs) in the removal of this peroxide has also been investigated. The study showed that the oxidation of ONOO- dependent CBA indicator was always significantly higher in TPxs-deficient Saccharomyces cerevisiae cells (strain 8) compared to the native strain (WT). In addition, the transfection of the gene encoding the most abundant Prx into Saccharomyces cerevisiae (Tsa1) in the 8 strain partially diminishes CBA oxidation. Besides that, CBA oxidation was greater in the deficient strain only of the peroxiredoxin Tsa1 (the most abundant in the family) compared to the WT strain, showing the relevance of this isoform specifically. On the other hand, CBA oxidation in the deficient strain of the Tsa2 peroxiredoxin was similar to the WT strain. Also, it was found that the ONOO- removal process is catalytic (and not stoichiometric) for increasing peroxynitrite fluxes in all strains and conditions used in the study. Finally, the study suggests that cells have redundant peroxynitrite reductase catalytic systems, since the 8 strain itself presents and can modulate this activity. These results confirm the expectation of the relevance of TPxs in the removal of ONOO- and by extension of other biologically relevant peroxides and are the first direct and real-time evidence of peroxynitrite reductase activity of TPxs in cells.

Os oxidantes derivados do peroxinitrito e das peroxidases, como mieloperoxidase (MPO), e os danos que ocasionam em proteínas vêm sendo muito estudados pela sua relevância em processos inflamatórios. Neste trabalho, as proteínas RNase e lisozima foram empregadas como alvos de oxidação e nitração mediadas por peroxinitrito e MPO/H<SUB.2O2/NO2-. Experimentos de EPR indicaram que as oxidações envolvem a formação de radicais protéicos sendo que os principais foram caracterizados como RNase-tirosila e lisozima-tirosila exposto e não exposto ao solvente, respectivamente. Estimativas do rendimento de radicais protéicos e produtos nitrados nos pHs 5,4, 6,4 e 7,4 mostrou que o peroxinitrito e o sistema MPO/H<SUB.2O2/NO2- são oxidantes mais efetivos nos pHs 7,4 e 5,4, respectivamente. Na condição ótima para cada oxidante foram identificados produtos de oxidação/nitração de resíduos de Tyr e Trp por HPLC-UV/MS-ESI. Para localização dos resíduos modificados nas estruturas das proteínas tratadas, elas foram digeridas com tripsina e os peptídeos resultantes submetidos a análise por HPLC/MS-MALDI-ToF. Desses resultados pode-se concluir que a RNase foi nitrada preferencialmente nos fragmentos contendo o(s)resíduo(s) Tyr115 > Tyr92/97 > Tyr73/76 por peroxinitrito e em praticamente todos os resíduos de tirosina por MPOH<SUB.2O2/NO2-. No caso da lisozima, o peroxinitrito oxidou principalmente o fragmento contendo os resíduos Trp62/63 que se mostrou nitrado e oxidado a dímero e quinurenina. Já o sistema MPO/H<SUB.2O2/NO2- nitrou o fragmento contendo os resíduos Tyr23/28 e nitrou e oxidou a dímeros e quinurenina o fragmento contendo os resíduos Trp62/63. As relações entre a acessibilidade dos resíduos específicos nas estruturas terciárias e a formação de produtos de oxidação/nitração são discutidas. Também, a possível importância da oxidação de resíduos de triptofano em agregação de proteínas é enfatizada. Paralelamente, examinou-se os efeitos do nitróxido tempol sobre a nitração da RNase mediada por MPO ou HRP/H<SUB.2O2/NO2- em condições de máxima nitração. De fato, as interações de tempol com peroxidases eram pouco conhecidas apesar da eficiência do nitróxido em reduzir a injúria e os níveis de 3-nitrotirosina em proteínas de tecidos de animais submetidos a condições inflamatórias. Foram determinadas as constantes de velocidade da reação do tempol com os intermediários oxidantes da MPO e HRP e também, o consumo de reagentes e a formação de produtos. A simulação dos resultados experimentais indicou que o tempol inibe a nitração da RNase mediada por peroxidases principalmente pela sua capacidade de reagir rapidamente com o &#8226;NO2 com formação de nitrito e cátion oxamônio que, por sua vez, recicla para tempol reagindo com H2O2 para produzir O2. / The oxidants derived from peroxynitrite and peroxidase enzymes, such as myeloperoxidase (MPO), and the lesions they promote in proteins are being extensively investigated because of their relevance in inflammatory processes. Here, the proteins RNase and lysozyme were employed as targets of oxidations/nitrations mediated by peroxynitrite and MPO/H<SUB.2O2/NO2-. EPR experiments showed that the oxidations produced protein radicals of which the prominent ones were characterized as RNase-tyrosyl and lysozyme-tyrosil solvent-exposed and non-exposed, respectively. Estimates of protein radical and nitrated product yields at pH 5.4, 6.4 and 7.4 indicated that peroxynitrite and MPO/H<SUB.2O2/NO2- were more effective oxidants at pH 7.4 and 5.4, respectively. At the best condition for each oxidant, the oxidation/nitration products of Tyr and Trp residues were identified by HPLC-UV/ESI-MS analysis. The site of oxidation in the protein structures were identified by HPLC/MALDI-ToF-MS analysis of tryptic digests after oxidative treatment. From these results, it was concluded that RNase was nitrated mainly in Tyr115 > Tyr92/97 > Tyr62/63 by peroxynitrite and in all Tyr by MPO/H<SUB.2O2/NO2-. In the case of lysozyme, peroxynitrite modified mainly Trp62/63 that resulted nitrated and oxidized to a dimer and kynurenine. The MPO/H<SUB.2O2/NO2- system promoted the nitration of Tyr23/Trp28 and nitration and oxidation to dimer and kynurenine of Trp62/63. The relationships between residue accessibility in the structure of the proteins and their oxidation/nitration are discussed. The possible importance of Trp oxidation in protein aggregation is emphasized. In parallel, the effects of the nitroxide tempol upon RNase nitration mediated by MPO or HRP/H<SUB.2O2/NO2- was examined. Indeed, the interactions of tempol with peroxidases have been little investigated although the nitroxide is very efficient in reducing injury and 3-nitrotyrosine protein levels in tissues of animals submitted to inflammatory conditions. The second order rate constants of tempol reactions with the ferryl oxidants of MPO and HRP were determined. The consumption of reactants and formation of products were also determined. Computer simulation of the results indicated that tempol inhibits RNAse nitration mediated by peroxidases mainly because of its capability to rapidly react with &#8226;NO2 with formation of nitrite and the oxammonium cation, which, in turn, recycles back to tempol, by reacting with H2O2 to produce O2.

Upon responding to cellular stress, p53 protein becomes stabilised and acts as a transcription factor mainly resulting from phosphorylation and acetylation of the protein. Nitration of p53 protein is poorly characterised by comparison with phosphorylation and acetylation. The main aim of this work was to study the effects of nitration on p53 functional activities and on p53-MDM2 protein-protein interactions. Preliminary work was to characterise the nitration of p53 protein over-expressed in E. coli BL21(DE3) which was then purified by a series of column chromatography. GST-MDM2 protein along with control GST protein were also overexpressed in BL21 which were subsequently purified by a single step batch purification before subjected to nitration. Peroxynitrite, a nitrating agent used in this study, was generated in vitro. Preliminary nitration work was carried out using BSA as a model protein as it is easily nitrated owing to its high number of tyrosine residues (19 residues). The present results showed that p53 and GST-MDM2 proteins were hardly nitrated as no strong nitro-tyrosine signals were obtained. This might be due to these proteins, being overexpressed in E. coli, were not properly folded resulting in hidden/cryptic tyrosine residues of which making nitration difficult to achieve. Peroxynitrite was shown to have a degrading property, reducing protein levels of peroxynitrite-treated p53, GST-MDM2 and GST proteins. Immunoprecipitation studies of cancer cell lysates with different p53 status treated with peroxynitrite showed very weak signals of nitro-p53 protein in mutant p53 cells whereby no nitro-p53 protein signal in wild-type p53 MCF7 cells. In addition, NO donor GSNO-treated MCF7 cells showed weak nitro-p53 protein signals. / Ministry of Science, Technology and Innovation (MOSTI) of Malaysia

No description available.