RESUMO

The photochemical nitrating agent 5-methyl-1,4-dinitro-1H-imidazole (DNI) has been recently described as an effective tool for nitrating tyrosine residues in proteins under 390 nm irradiation (Long T. et al., 2021). Herein, we describe the one-step synthesis of DNI from the precursor 4-methyl-5-nitro-1H-imidazole with good yield (66%) and high purity (>99%). Spectral analysis of DNI reveals two maximum peaks (228 and 290 nm) with maximum nitration yields and kinetics occurring at 290 nm. Electron paramagnetic resonance (EPR)- and mass spectrometry (MS)- spin trapping analysis evidenced the formation of nitrogen dioxide (•NO2) upon irradiation of DNI, implying the homolysis of the N-N bond in the DNI molecule. Irradiation of DNI at 290, 390 nm, or UVA light (315-400 nm), produced tyrosine nitration, with yields approaching ca. 30% with respect to DNI at 290 nm exposure. Indeed, using alpha-synuclein as a model protein, the main protein post-translational modification triggered by DNI was the generation of 3-nitrotyrosine as shown by MS analysis. Additionally, the formation of di-tyrosine was also observed. Finally, intracellular •NO2 production upon DNI photolysis in bovine aortic endothelial cells was evidenced by the nitration of the tyrosine analog probe p-hydroxyphenylacetic acid (PHPA) and cellular protein tyrosine nitration.

Assuntos

Células Endoteliais , Dióxido de Nitrogênio , Animais , Bovinos , Células Endoteliais/metabolismo , Tirosina/metabolismo , Nitratos/metabolismo , Imidazóis

RESUMO

The interaction between cytochrome c and cardiolipin is a relevant process in the mitochondrial redox homeostasis, playing roles in the mechanism of electron transfer to cytochrome c oxidase and also modulating cytochrome c conformation, reactivity and function. Peroxynitrite is a widespread nitrating agent formed in mitochondria under oxidative stress conditions, and can result in the formation of tyrosine nitrated cytochrome c. Some of the nitro-cytochrome c species undergo conformational changes at physiological pH and increase its peroxidase activity. In this work we evaluated the influence of cardiolipin on peroxynitrite-mediated cytochrome c nitration yields and site-specificity. Our results show that cardiolipin enhances cytochrome c nitration by peroxynitrite and targets it to heme-adjacent Tyr67. Cytochrome c nitration also modifies the affinity of protein with cardiolipin. Using a combination of experimental techniques and computer modeling, it is concluded that structural modifications in the Tyr67 region are responsible for the observed changes in protein-derived radical and tyrosine nitration levels, distribution of nitrated proteoforms and affinity to cardiolipin. Increased nitration of cytochrome c in presence of cardiolipin within mitochondria and the gain of peroxidatic activity could then impact events such as the onset of apoptosis and other processes related to the disruption of mitochondrial redox homeostasis.

Assuntos

Cardiolipinas/metabolismo , Cardiolipinas/farmacologia , Citocromos c/química , Citocromos c/metabolismo , Nitratos/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Tirosina/metabolismo , Animais , Sítios de Ligação , Cavalos , Cinética , Modelos Moleculares , Ácido Peroxinitroso/metabolismo , Conformação Proteica/efeitos dos fármacos , Especificidade por Substrato

RESUMO

Oxidative post-translational modification of proteins by molecular oxygen (O2)- and nitric oxide (•NO)-derived reactive species is a usual process that occurs in mammalian tissues under both physiological and pathological conditions and can exert either regulatory or cytotoxic effects. Although the side chain of several amino acids is prone to experience oxidative modifications, tyrosine residues are one of the preferred targets of one-electron oxidants, given the ability of their phenolic side chain to undergo reversible one-electron oxidation to the relatively stable tyrosyl radical. Naturally occurring as reversible catalytic intermediates at the active site of a variety of enzymes, tyrosyl radicals can also lead to the formation of several stable oxidative products through radical-radical reactions, as is the case of 3-nitrotyrosine (NO2Tyr). The formation of NO2Tyr mainly occurs through the fast reaction between the tyrosyl radical and nitrogen dioxide (•NO2). One of the key endogenous nitrating agents is peroxynitrite (ONOO-), the product of the reaction of superoxide radical (O2•-) with •NO, but ONOO--independent mechanisms of nitration have been also disclosed. This chemical modification notably affects the physicochemical properties of tyrosine residues and because of this, it can have a remarkable impact on protein structure and function, both in vitro and in vivo. Although low amounts of NO2Tyr are detected under basal conditions, significantly increased levels are found at pathological states related with an overproduction of reactive species, such as cardiovascular and neurodegenerative diseases, inflammation and aging. While NO2Tyr is a well-established stable oxidative stress biomarker and a good predictor of disease progression, its role as a pathogenic mediator has been laboriously defined for just a small number of nitrated proteins and awaits further studies.

Assuntos

Radicais Livres/metabolismo , Proteínas/metabolismo , Tirosina/análogos & derivados , Radicais Livres/química , Humanos , Oxirredução , Processamento de Proteína Pós-Traducional , Proteínas/química , Tirosina/química

RESUMO

Peroxiredoxins (Prx) are enzymes that efficiently reduce hydroperoxides through active participation of cysteine residues (CP, CR). The first step in catalysis, the reduction of peroxide substrate, is fast, 107 - 108 M-1s-1 for human Prx2. In addition, the high intracellular concentration of Prx positions them not only as good antioxidants but also as central players in redox signaling pathways. These biological functions can be affected by post-translational modifications that could alter the peroxidase activity and/or interaction with other proteins. In particular, inactivation by hyperoxidation of CP, which occurs when a second molecule of peroxide reacts with the CP in the sulfenic acid form, modulates their participation in redox signaling pathways. The higher sensitivity to hyperoxidation of some Prx has been related to the presence of structural motifs that disfavor disulfide formation at the active site, making the CP sulfenic acid more available for hyperoxidation or interaction with a redox protein target. We previously reported that treatment of human Prx2 with peroxynitrite results in tyrosine nitration, a post-translational modification on non-catalytic residues, yielding a more active peroxidase with higher resistance to hyperoxidation. In this work, studies on various mutants of hPrx2 confirm that the presence of the tyrosyl side-chain of Y193, belonging to the C-terminal YF motif of eukaryotic Prx, is necessary to observe the increase in Prx2 resistance to hyperoxidation. Moreover, our results underline the critical role of this structural motif on the rate of disulfide formation that determines the differential participation of Prx in redox signaling pathways.

Assuntos

Oxirredução , Peroxirredoxinas/genética , Processamento de Proteína Pós-Traducional/genética , Tirosina/genética , Domínio Catalítico/genética , Cisteína/genética , Dissulfetos/química , Humanos , Mutação/genética , Nitratos/metabolismo , Peroxidase/genética , Peróxidos/metabolismo , Peroxirredoxinas/efeitos dos fármacos , Peroxirredoxinas/metabolismo , Ácido Peroxinitroso/farmacologia , Transdução de Sinais/efeitos dos fármacos

RESUMO

Mitochondria are main sites of peroxynitrite formation. While at low concentrations mitochondrial peroxynitrite has been associated with redox signaling actions, increased levels can disrupt mitochondrial homeostasis and lead to pathology. Peroxiredoxin 3 is exclusively located in mitochondria, where it has been previously shown to play a major role in hydrogen peroxide reduction. In turn, reduction of peroxynitrite by peroxiredoxin 3 has been inferred from its protective actions against tyrosine nitration and neurotoxicity in animal models, but was not experimentally addressed so far. Herein, we demonstrate the human peroxiredoxin 3 reduces peroxynitrite with a rate constant of 1 × 107 M-1 s-1 at pH 7.8 and 25 °C. Reaction with hydroperoxides caused biphasic changes in the intrinsic fluorescence of peroxiredoxin 3: the first phase corresponded to the peroxidatic cysteine oxidation to sulfenic acid. Peroxynitrite in excess led to peroxiredoxin 3 hyperoxidation and tyrosine nitration, oxidative post-translational modifications that had been previously identified in vivo. A significant fraction of the oxidant is expected to react with CO2 and generate secondary radicals, which participate in further oxidation and nitration reactions, particularly under metabolic conditions of active oxidative decarboxylations or increased hydroperoxide formation. Our results indicate that both peroxiredoxin 3 and 5 should be regarded as main targets for peroxynitrite in mitochondria.

Assuntos

Mitocôndrias/metabolismo , Oxidantes/metabolismo , Peroxirredoxina III/genética , Peroxirredoxinas/genética , Dióxido de Carbono/metabolismo , Cisteína/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Cinética , Oxirredução , Peroxirredoxina III/metabolismo , Ácido Peroxinitroso/metabolismo , Processamento de Proteína Pós-Traducional/genética , Transdução de Sinais/genética

RESUMO

Reactive oxygen and nitrogen species are important microbicidal agents and are also involved in lymphocyte unresponsiveness during experimental infections. Many of the biological effects attributed to nitric oxide are mediated by peroxynitrites, which induce the nitration of immune cells, among others. Our group has demonstrated that nitric oxide is involved in the suppressive activity of myeloid-derived suppressor cells in Trypanosoma cruzi-infected mice, with a higher number of CD8+ T cells suffering surface-nitration compared to uninfected controls. Studying the functional and phenotypic features of peripheral CD8+ T cells from chagasic patients and human cells experimentally infected with T. cruzi, we found that different regulatory mechanisms impaired the effector functions of T cytotoxic population from seropositive patients. Peripheral leukocytes from chagasic patients showed increased nitric oxide production concomitant with increased tyrosine nitration of CD8+ T cells. Additionally, this cytotoxic population exhibited increased apoptotic rate, loss of the TCRζ-chain, and lower levels of CD107a, a marker of degranulation. Strikingly, IL-6 stimulation of in vitro-infected peripheral blood mononuclear cells obtained from healthy donors, blunted T. cruzi-induced nitration of CD3+CD8+ cells, and increased their survival. Furthermore, the treatment of these cultures with an IL-6 neutralizing antibody increased the percentage of T. cruzi-induced CD8+ T cell nitration and raised the release of nitric oxide. The results suggest that the under-responsiveness of cytotoxic T cell population observed in the setting of long-term constant activation of the immune system could be reverted by the pleiotropic actions of IL-6, since this cytokine improves its survival and effector functions.

RESUMO

Peroxiredoxins are cys-based peroxidases that function in peroxide detoxification and H2O2-induced signaling. Human Prx2 is a typical 2-Cys Prx arranged as pentamers of head-to-tail homodimers. During the catalytic mechanism, the active-site cysteine (CP) cycles between reduced, sulfenic and disulfide state involving conformational as well as oligomeric changes. Several post-translational modifications were shown to affect Prx activity, in particular CP overoxidation which leads to inactivation. We have recently reported that nitration of Prx2, a post-translational modification on non-catalytic tyrosines, unexpectedly increases its peroxidase activity and resistance to overoxidation. To elucidate the cross-talk between this post-translational modification and the enzyme catalysis, we investigated the structural changes of Prx2 after nitration. Analytical ultracentrifugation, UV absorption, circular dichroism, steady-state and time-resolved fluorescence were used to connect catalytically relevant redox changes with tyrosine nitration. Our results show that the reduced nitrated Prx2 structurally resembles the disulfide-oxidized native form of the enzyme favoring a locally unfolded conformation that facilitates disulfide formation. These results provide structural basis for the kinetic analysis previously reported, the observed increase in activity and the resistance to overoxidation of the peroxynitrite-treated enzyme.

Assuntos

Dissulfetos/química , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/ultraestrutura , Nitrocompostos/química , Ácido Peroxinitroso/química , Sítios de Ligação , Oxirredução , Ligação Proteica , Conformação Proteica

RESUMO

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.

Assuntos

Eritrócitos/enzimologia , Proteínas de Homeodomínio/metabolismo , Nitrogênio/metabolismo , Estresse Oxidativo/fisiologia , Ácido Peroxinitroso/metabolismo , Animais , Domínio Catalítico , Echinococcus granulosus/enzimologia , Ativação Enzimática/fisiologia , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Peróxido de Hidrogênio/metabolismo , Oxirredução , Processamento de Proteína Pós-Traducional/fisiologia , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologia , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Tirosina/metabolismo

RESUMO

Renal complications in diabetes are severe and may lead to renal insufficiency. Early alterations in tight junction (TJ) proteins in diabetic nephropathy (DN) have not been explored and the role of oxidative stress in their disassembly has been poorly characterized. We investigated the expression and distribution of TJ proteins: claudin-5 in glomeruli (GL), occludin and claudin-2 in proximal tubules (PTs), and ZO-1 and claudin-1, -4, and -8 in distal tubules (DTs) of rats 21 days after streptozotocin injection. Redox status along the nephron segments was evaluated. Diabetes increased kidney injury molecule-1 expression. Expression of sodium glucose cotransporters (SGLT1 and SGLT2) and facilitative glucose transporter (GLUT2) was induced. Increased oxidative stress was present in GL and PTs and to a lesser extent in DTs (measured by superoxide production and PKCß2 expression), owing to NADPH oxidase activation and uncoupling of the endothelial nitric oxide synthase-dependent pathway. Claudin-5, occludin, and claudin-2 expression was decreased, whereas claudin-4 and -8 expression increased. ZO-1 was redistributed from membrane to cytosol. Increased nitration of tyrosine residues in claudin-2 was found, which might contribute to decrement of this protein in proximal tubule. In contrast, occludin was not nitrated. We suggest that loss of claudin-2 is associated with increased natriuresis and that loss of glomerular claudin-5 might explain early presence of proteinuria. These findings suggest that oxidative stress is related to alterations in TJ proteins in the kidney that are relevant to the pathogenesis and progression of DN and for altered sodium regulation in diabetes.

Assuntos

Claudinas/metabolismo , Nefropatias Diabéticas/metabolismo , Estresse Oxidativo/fisiologia , Animais , Western Blotting , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/metabolismo , Feminino , Imunofluorescência , Imunoprecipitação , Oxirredução , Ratos , Ratos Wistar , Proteínas de Junções Íntimas/metabolismo

RESUMO

Peroxynitrite is the product of the diffusion-controlled reaction of nitric oxide and superoxide radicals. Peroxynitrite, a reactive short-lived peroxide with a pKa of 6.8, is a good oxidant and nucleophile. It also yields secondary free radical intermediates such as nitrogen dioxide and carbonate radicals. Much of nitric oxide- and superoxide-dependent cytotoxicity resides on peroxynitrite, which affects mitochondrial function and triggers cell death via oxidation and nitration reactions. Peroxynitrite is an endogenous toxicant but is also a cytotoxic effector against invading pathogens. The biological chemistry of peroxynitrite is modulated by endogenous antioxidant mechanisms and neutralized by synthetic compounds with peroxynitrite-scavenging capacity.

Assuntos

Oxidantes/química , Ácido Peroxinitroso/química , Animais , Humanos , Concentração de Íons de Hidrogênio , Mitocôndrias/metabolismo , Óxido Nítrico/química , Nitrogênio/química , Oxirredução , Superóxido Dismutase/química , Superóxidos/química , Tirosina/química