RESUMO
The nitrone spin trap 5,5dimethyl1pyrroline Noxide (DMPO) dampens endotoxin-induced and TLR4-driven priming of macrophages, but the mechanism remains unknown. The available information suggests a direct binding of DMPO to the TIR domain, which is shared between TLRs. However, TLR2-TIR domain is the only TLR that have been crystallized. Our in silico data show that DMPO binds to four specific residues in the BB-loop within the TLR2-TIR domain. Our functional analysis using hTLR2.6-expressing HEKs cells showed that DMPO can block zymosan-triggered-TLR2-mediated NF-κB activation. However, DMPO did not affect the overall TLR2-MyD88 protein-protein interaction. DMPO binds to the BB-loop in the TIR-domain and dampens downstream signaling without affecting the overall TIR-MyD88 interaction. These data encourage the use of DMPO-derivatives as potential mechanism-based inhibitors of TLR-triggered inflammation.
Assuntos
Óxidos N-Cíclicos/metabolismo , Inflamação/metabolismo , Óxidos de Nitrogênio/metabolismo , Transdução de Sinais , Marcadores de Spin , Receptor 2 Toll-Like/metabolismo , Animais , Óxidos N-Cíclicos/química , Células HEK293 , Humanos , Inflamação/imunologia , Camundongos , Simulação de Dinâmica Molecular , Fator 88 de Diferenciação Mieloide/química , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/química , NF-kappa B/metabolismo , Óxidos de Nitrogênio/química , Ligação Proteica , Domínios Proteicos , Células RAW 264.7 , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 2 Toll-Like/químicaRESUMO
BACKGROUND AND AIM: Oxidative stress arising from inflammatory processes is a serious cause of cell and tissue damage. Tempol is an efficient antioxidant with superoxide dismutase-like activity. The purpose of this paper is to address the inhibition of protein disulfide isomerase (PDI), an essential redox chaperone whose active sites contain the Cys-Gly-His-Cys (CXXC) motif, by the nitroxide Tempol. RESULTS: In the presence of Tempol (5-120 µM), the reductase activity of PDI was reversibly affected both in vitro and in activated mice neutrophils, with an IC50 of 22.9 ± 10.8 µM. Inhibitory activity was confirmed by using both the insulin method and fluorescent formation of eosin-glutathione (E-GSH). The capacity of Tempol to bind the enzyme was determined by EPR and mass spectrometry. EPR Tempol signal decreased in the presence of PDI while remained unaffected when PDI thiols were previously blocked with NEM. When total protein was analyzed, 1 and 4 molecules of Tempol were bound to the protein. However, only one was found to be covalently bound to PDI at the a'active site. More specifically, Cys400 was modified by Tempol. CONCLUSION: We have shown that the nitroxide Tempol acts as an inhibitor of PDI through covalent binding to the Cys400 of the protein structure. Since PDI is coupled with the assembly of the NADPH oxidase complex of phagocytes, these findings reveal a novel action of Tempol that presents potential clinical applications for therapeutic intervention to target PDI knockdown in pathological processes in which this protein is engaged.
Assuntos
Óxidos N-Cíclicos/metabolismo , Cisteína/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Domínio Catalítico , Óxidos N-Cíclicos/química , Cisteína/química , Espectroscopia de Ressonância de Spin Eletrônica , Dissulfeto de Glutationa/química , Dissulfeto de Glutationa/metabolismo , Masculino , Camundongos , Neutrófilos/enzimologia , Ligação Proteica , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/química , Marcadores de Spin , Espectrometria de Massas em TandemRESUMO
Leishmania amazonensis is a protozoan parasite that occurs in many areas of Brazil and causes skin lesions. Using this parasite, our group showed the activation of Na+/K+ ATPase through a signaling cascade that involves the presence of heme and protein kinase C (PKC) activity. Heme is an important biomolecule that has pro-oxidant activity and signaling capacity. Reactive oxygen species (ROS) can act as second messengers, which are required in various signaling cascades. Our goal in this work is to investigate the role of hydrogen peroxide (H2O2) generated in the presence of heme in the Na+/K+ ATPase activity of L. amazonensis. Our results show that increasing concentrations of heme stimulates the production of H2O2 in a dose-dependent manner until a concentration of 2.5 µM heme. To confirm that the effect of heme on the Na+/K+ ATPase is through the generation of H2O2, we measured enzyme activity using increasing concentrations of H2O2 and, as expected, the activity increased in a dose-dependent manner until a concentration of 0.1 µM H2O2. To investigate the role of PKC in this signaling pathway, we observed the production of H2O2 in the presence of its activator phorbol 12-myristate 13-acetate (PMA) and its inhibitor calphostin C. Both showed no effect on the generation of H2O2. Furthermore, we found that PKC activity is increased in the presence of H2O2, and that in the presence of calphostin C, H2O2 is unable to activate the Na+/K+ ATPase. 100 µM of Mito-TEMPO was capable of abolishing the stimulatory effect of heme on Na+/K+ ATPase activity, indicating that mitochondria might be the source of the hydrogen peroxide production induced by heme. The modulation of L. amazonensis Na+/K+ ATPase by H2O2 opens new possibilities for understanding the signaling pathways of this parasite.
Assuntos
Heme/metabolismo , Peróxido de Hidrogênio/farmacologia , Leishmania/efeitos dos fármacos , Leishmania/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Catalase/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Óxidos N-Cíclicos/metabolismo , Óxidos N-Cíclicos/farmacologia , Ativação Enzimática/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Proteína Quinase C/metabolismo , Proteólise , Transdução de Sinais/efeitos dos fármacosRESUMO
Arsenopyrite (FeAsS) is one of the earth's primary mineral sources of As, yet its effects on cell damage remain largely unknown. This paper addresses the question whether FeAsS induces lipid peroxidation (LP), a major indicator of oxidative stress. Screening and monitoring of LP was conducted using Thiobarbituric Acid Reactive Substances (TBARSs) assay. The lipid source was supernatant of rat brain homogenates. The formation of TBARS by FeAsS was rapid and took place just after 10 min. Maximum TBARS levels (ca. 14 nmol TBARS per mg of protein) were observed after 1h and remained constant thereafter. Suspension fraction separations showed that dissolved and structural components contributed to LP. The formation of TBARS by soluble As, As(III) or As(V), compared to basal levels. The initiation of LP by FeAsS was consistent with a mechanism initiated by the Fe(3+)/O(2)(-) redox system, and differed initiated by Fe(2+)/O(2). The effectiveness of FeAsS and FeSO(4) as inducer compared, and surpassed that of AAPH. On the other hand, the initiation of LP by FeAsS is consistent with a mechanism initiated by perferryl ion and Fe(3+)/O(2)(-), and differs from the mechanism characteristic of FeSO(4) initiated by the Fe(2+)/O(2) redox system. Proposedly, FeAsS surfaces contain a mixture of Fe(3+) and Fe(2+) that, along with O(2) and O(2)(-), participate in multiple mechanisms of electron transfer. EPR determinations show decreases in DMPO-OH adduct signal in FeAsS suspensions after adding desferrioxamine-B (DFO-B), consistent with the idea that DFO-B serves as a radical scavenger.
Assuntos
Antioxidantes/farmacologia , Desferroxamina/farmacologia , Substâncias Perigosas/toxicidade , Compostos de Ferro/toxicidade , Minerais/toxicidade , Estresse Oxidativo , Sulfetos/toxicidade , Animais , Arsenicais , Óxidos N-Cíclicos/metabolismo , Peroxidação de Lipídeos , Masculino , Oxirredução , Ratos , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to monitor membrane dynamic changes in erythrocytes subjected to oxidative stress with hydrogen peroxide (H2O2). The lipid spin label, 5-doxyl stearic acid, responded to dramatic reductions in membrane fluidity, which was correlated with increases in the protein content of the membrane. Membrane rigidity, associated with the binding of hemoglobin (Hb) to the erythrocyte membrane, was also indicated by a spin-labeled maleimide, 5-MSL, covalently bound to the sulfhydryl groups of membrane proteins. At 2% hematocrit, these alterations in membrane occurred at very low concentrations of H2O2 (50 µM) after only 5 min of incubation at 37°C in azide phosphate buffer, pH 7.4. Lipid peroxidation, suggested by oxidative hemolysis and malondialdehyde formation, started at 300 µM H2O2 (for incubation of 3 h), which is a concentration about six times higher than those detected with the probes. Ascorbic acid and α-tocopherol protected the membrane against lipoperoxidation, but did not prevent the binding of proteins to the erythrocyte membrane. Moreover, the antioxidant (+)-catechin, which also failed to prevent the cross-linking of cytoskeletal proteins with Hb, was very effective in protecting erythrocyte ghosts from lipid peroxidation induced by the Fenton reaction. This study also showed that EPR spectroscopy can be useful to assess the molecular dynamics of red blood cell membranes in both the lipid and protein domains and examine oxidation processes in a system that is so vulnerable to oxidation.
Assuntos
Humanos , Antioxidantes/farmacologia , Membrana Eritrocítica/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Ácido Ascórbico/farmacologia , Catequina/farmacologia , Óxidos N-Cíclicos/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Membrana Eritrocítica/química , Membrana Eritrocítica/fisiologia , Hemólise , Concentração de Íons de Hidrogênio , Hemoglobinas/metabolismo , Peróxido de Hidrogênio/metabolismo , Fluidez de Membrana/efeitos dos fármacos , Estresse Oxidativo/fisiologia , alfa-Tocoferol/farmacologiaRESUMO
Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to monitor membrane dynamic changes in erythrocytes subjected to oxidative stress with hydrogen peroxide (H(2)O(2)). The lipid spin label, 5-doxyl stearic acid, responded to dramatic reductions in membrane fluidity, which was correlated with increases in the protein content of the membrane. Membrane rigidity, associated with the binding of hemoglobin (Hb) to the erythrocyte membrane, was also indicated by a spin-labeled maleimide, 5-MSL, covalently bound to the sulfhydryl groups of membrane proteins. At 2% hematocrit, these alterations in membrane occurred at very low concentrations of H(2)O(2) (50 µM) after only 5 min of incubation at 37°C in azide phosphate buffer, pH 7.4. Lipid peroxidation, suggested by oxidative hemolysis and malondialdehyde formation, started at 300 µM H(2)O(2) (for incubation of 3 h), which is a concentration about six times higher than those detected with the probes. Ascorbic acid and α-tocopherol protected the membrane against lipoperoxidation, but did not prevent the binding of proteins to the erythrocyte membrane. Moreover, the antioxidant (+)-catechin, which also failed to prevent the cross-linking of cytoskeletal proteins with Hb, was very effective in protecting erythrocyte ghosts from lipid peroxidation induced by the Fenton reaction. This study also showed that EPR spectroscopy can be useful to assess the molecular dynamics of red blood cell membranes in both the lipid and protein domains and examine oxidation processes in a system that is so vulnerable to oxidation.
Assuntos
Antioxidantes/farmacologia , Membrana Eritrocítica/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Ácido Ascórbico/farmacologia , Catequina/farmacologia , Óxidos N-Cíclicos/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Membrana Eritrocítica/química , Membrana Eritrocítica/fisiologia , Hemoglobinas/metabolismo , Hemólise , Humanos , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Fluidez de Membrana/efeitos dos fármacos , Estresse Oxidativo/fisiologia , alfa-Tocoferol/farmacologiaRESUMO
The angiotensin I-converting enzyme (ACE) converts the decapeptide angiotensin I (Ang I) into angiotensin II by releasing the C-terminal dipeptide. A novel approach combining enzymatic and electron paramagnetic resonance (EPR) studies was developed to determine the enzyme effect on Ang I containing the paramagnetic 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) at positions 1, 3, 8, and 9. Biological assays indicated that TOAC(1)-Ang I maintained partly the Ang I activity, and that only this derivative and the TOAC(3)-Ang I were cleaved by ACE. Quenching of Tyr(4) fluorescence by TOAC decreased with increasing distance between both residues, suggesting an overall partially extended structure. However, the local bend known to be imposed by the substituted diglycine TOAC is probably responsible for steric hindrance, not allowing the analogues containing TOAC at positions 8 and 9 to act as substrates. In some cases, although substrates and products differ by only two residues, the difference between their EPR spectral lineshapes allows monitoring the enzymatic reaction as a function of time.
Assuntos
Óxidos N-Cíclicos/metabolismo , Peptidil Dipeptidase A/metabolismo , Animais , Óxidos N-Cíclicos/química , Espectroscopia de Ressonância de Spin Eletrônica , Feminino , Cobaias , Íleo/enzimologia , Cinética , Espectrometria de Massas , Peptídeos/química , Peptídeos/metabolismo , Ratos , Espectrometria de Fluorescência , Especificidade por Substrato , Útero/enzimologiaRESUMO
N-Terminally and internally labeled analogues of the hormones angiotensin (AII, DRVYIHPF) and bradykinin (BK, RPPGFSPFR) were synthesized containing the paramagnetic amino acid 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC). TOAC replaced Asp1 (TOAC1-AII) and Val3 (TOAC3-AII) in AII and was inserted prior to Arg1 (TOAC0-BK) and replacing Pro3 (TOAC3-BK) in BK. The peptide conformational properties were examined as a function of trifluoroethanol (TFE) content and pH. Electron paramagnetic resonance spectra were sensitive to both variables and showed that internally labeled analogues yielded rotational correlation times (tauC) considerably larger than N-terminally labeled ones, evincing the greater freedom of motion of the N-terminus. In TFE, tauC increased due to viscosity effects. Calculation of tau(Cpeptide)/tau(CTOAC) ratios indicated that the peptides acquired more folded conformations. Circular dichroism spectra showed that, except for TOAC1-AII in TFE, the N-terminally labeled analogues displayed a conformational behavior similar to that of the parent peptides. In contrast, under all conditions, the TOAC3 derivatives acquired more restricted conformations. Fluorescence spectra of AII and its derivatives were especially sensitive to the ionization of Tyr4. Fluorescence quenching by the nitroxide moiety was much more pronounced for TOAC3-AII. The conformational behavior of the TOAC derivatives bears excellent correlation with their biological activity, since, while the N-terminally labeled peptides were partially active, their internally labeled counterparts were inactive [Nakaie, C. R., et al., Peptides 2002, 23, 65-70]. The data demonstrate that insertion of TOAC in the middle of the peptide chain induces conformational restrictions that lead to loss of backbone flexibility, not allowing the peptides to acquire their receptor-bound conformation.
Assuntos
Angiotensina II/química , Bradicinina/química , Óxidos N-Cíclicos/química , Marcadores de Spin , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Animais , Bradicinina/metabolismo , Bradicinina/farmacologia , Dicroísmo Circular , Óxidos N-Cíclicos/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Concentração de Íons de Hidrogênio , Conformação Proteica , Espectrometria de Fluorescência , Relação Estrutura-AtividadeRESUMO
Uric acid has been considered to be an efficient scavenger of peroxynitrite but the reaction between urate and peroxynitrite has been only partially characterized. Also, previous studies have indicated that urate may increase peroxynitrite-mediated oxidation of low density lipoprotein (LDL). Here, we examined the reaction between urate and peroxynitrite by combining kinetic, oxygen consumption, spin trapping, and product identification studies; in parallel, we tested the effect of urate upon peroxynitrite-mediated lipid oxidation. Our results demonstrated that urate reacts with peroxynitrite with an apparent second order rate constant of 4.8 x 10(2) M(-1). s(-1) in a complex process, which is accompanied by oxygen consumption and formation of allantoin, alloxan, and urate-derived radicals. The main radical was identified as the aminocarbonyl radical by the electrospray mass spectra of its 5, 5-dimethyl-l-pyrroline N-oxide adduct. Mechanistic studies suggested that urate reacts with peroxynitrous acid and with the radicals generated from its decomposition to form products that can further react with peroxynitrite anion. These many reactions may explain the reported efficiency of urate in inhibiting some peroxynitrite-mediated processes. Production of the aminocarbonyl radical, however, may propagate oxidative reactions. We demonstrated that this radical is likely to be the species responsible for the effects of urate in amplifying peroxynitrite-mediated oxidation of liposomes and LDL, which was monitored by the formation of lipid peroxides and thiobarbituric acid-reactive substances. The aminocarbonyl radical was not detectable during urate attack by other oxidants and consequently it is unlikely to be responsible for all previously described prooxidant effects of uric acid.
Assuntos
Radicais Livres/metabolismo , Metabolismo dos Lipídeos , Nitratos/metabolismo , Oxidantes/metabolismo , Ácido Úrico/metabolismo , Alantoína/metabolismo , Aloxano/metabolismo , Óxidos N-Cíclicos/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Sequestradores de Radicais Livres/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Peróxidos Lipídicos/metabolismo , Lipoproteínas LDL/metabolismo , Lipossomos/metabolismo , Ácido Nitroso/metabolismo , Oxirredução , Oxigênio/metabolismo , Ácido Peroxinitroso , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
The structural properties of rat liver microsomes were studied by physical and kinetic methods. The microsomes and the lipids extracted from the microsomes were labeled with 16-doxyl-stearic acid- and N-phenyl-1-naphthylamine. The electron spin resonance spectra and the fluorescence intensities were respectively determined at different temperatures from approximately 10 to 40 C. Both methods suggested the absence of a transition temperature indicative of a phase change in the bulk of the lipids of the microsomes in the temperature range studied. The fluidity of the lipid bilayer increased smoothly with the temperature. The Arrhenius plots of the NADH-ferricyanide reductase, NADH-cyt.c reductase, delta 9 desaturase, delta 6 desaturase and palmitic elongation to stearic acid also indicated the absence of a detectable change of phase from crystalline to liquid crystalline in the boundary lipids of these enzymes from 10 C to 40 C. The transference of electrons from the NADH-cyt.b5 reductase to the cyt.b5 is the rate limiting step in the first parts of the electron transport chain. However, the delta 9 desaturase is the rate limiting step of all the series of reactions involved in the delta 9 fatty acid desaturation. Similar conclusions may be extended to the delta 6 desaturation of fatty acids. The physical state of the lipids surrounding the desaturating system would be different from boundary lipids of the cyt.P450 system.