Reactive Oxygen Species in Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) includes chronic bronchitis and emphysema. Environmental exposure, primarily cigarette smoking, can cause high oxidative stress and is the main factor of COPD development. Cigarette smoke also contributes to the imbalance of oxidant/antioxidant due to exogenous reactive oxygen species (ROS). Moreover, endogenously released ROS during the inflammatory process and mitochondrial dysfunction may contribute to this disease progression. ROS and reactive nitrogen species (RNS) can oxidize different biomolecules such as DNA, proteins, and lipids leading to epithelial cell injury and death. Various detoxifying enzymes and antioxidant defense systems can be involved in ROS removal. In this review, we summarize the main findings regarding the biological role of ROS, which may contribute to COPD development, and cytoprotective mechanisms against this disease progression

Reactive Oxygen Species in Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) includes chronic bronchitis and emphysema. Environmental exposure, primarily cigarette smoking, can cause high oxidative stress and is the main factor of COPD development. Cigarette smoke also contributes to the imbalance of oxidant/antioxidant due to exogenous reactive oxygen species (ROS). Moreover, endogenously released ROS during the inflammatory process and mitochondrial dysfunction may contribute to this disease progression. ROS and reactive nitrogen species (RNS) can oxidize different biomolecules such as DNA, proteins, and lipids leading to epithelial cell injury and death. Various detoxifying enzymes and antioxidant defense systems can be involved in ROS removal. In this review, we summarize the main findings regarding the biological role of ROS, which may contribute to COPD development, and cytoprotective mechanisms against this disease progression

Enzymology of the key steps regulating Peroxiredoxin / Sulfiredoxin system in the context of redox cell signaling

Les peroxyrédoxines (Prx) sont des peroxydases à thiol, ubiquitaires, qui jouent un rôle central dans la physiologie du peroxyde d’hydrogène. Une famille de Prx dite "2-Cys-Prx typique" possède une propriété unique de suroxydation de la Cys catalytique sous forme acide sulfinique, qui constitue un mécanisme de régulation des fonctions des 2-Cys-Prx typiques en tant que peroxydase, capteur de peroxyde ou protéine chaperon. La réduction des 2-Cys-Prx typiques suroxydées est catalysée par la Sulfirédoxine (Srx), une sulfinyl réductase ATP-dépendante dont la constante catalytique est de l’ordre de 1-2 min-1, une valeur faible qui doit être corrélée au rôle de Srx dans la régulation redox. L’objectif de ce travail était d’analyser l’enzymologie de la régulation du système Prx/Srx au niveau, du processus de suroxydation des 2-Cys-Prx typiques, de l’étape limitante de la Srx, et de son recyclage par les systèmes redox cellulaires. Dans un premier temps, nous avons caractérisé les deux étapes du cycle catalytique de la 2-Cys-Prx typique majeure de S. cerevisiae Tsa1, dont la compétition contrôle la sensibilité à la suroxydation, par une stratégie combinant cinétiques rapides, système enzymatique couplé et modélisation cinétique. Ces travaux suggèrent que cette compétition est contrôlée par une réorganisation conformationnelle au cours du cycle catalytique de la Tsa1. Dans un second temps, l’étude de la première étape du mécanisme catalytique de Srx, qui consiste en l’activation ATP-dépendante du groupement acide sulfinique de la 2 Cys-Prx a permis, i) de montrer que l’étape limitante de la réaction catalysée par Srx était associée au processus chimique de transfert de phosphate, et ii) de proposer un modèle d’assemblage du complexe Michaelien Prx/Srx/ATP formé lors de ce processus. Enfin, par une approche combinant cinétiques enzymatiques in vitro et génétique de la levure in vivo, nous avons établi que le mécanisme de recyclage des Srx à 1 Cys existant chez les plantes ou les mammifères implique le rôle du glutathion comme réducteur cellulaire, contrairement à la Srx de S. cerevisiae qui est recyclée par le système thiorédoxine. De façon inattendue, la spécificité du glutathion dans ce mécanisme est assurée par un événement de reconnaissance au sein du complexe Prx/SrxThe peroxiredoxins (Prx) are ubiquitous thiol peroxidases, which play a central role in the physiology of hydrogen peroxide. A subclass of Prx called "typical 2-Cys-Prx" has a unique property to hyperoxidize the catalytic Cys into the sulfinic acid form, which acts as a regulation mechanism of their functions, as peroxidase, peroxide sensor or protein chaperone. The reduction of the overoxidized form is catalyzed by sulfiredoxin (Srx), an ATP-dependent sulfinyl reductase whose catalytic constant is about 1-2 min-1, a low value that must be correlated to the role of Srx in redox regulation. The aim of this study was to analyze the enzymology of the regulation of the Prx/Srx system at three diffrents points of control: the hyper-oxidation process of typical 2-Cys-Prx, the rate-limiting step of the Srx mechanism and the recycling step of Srx by the cellular thiol redox systems. We have first characterized the competition mechanism between the two steps of the catalytic mechanism of the major typical 2-Cys-Prx of S. cerevisiae, Tsa1, through a strategy combining rapid kinetics, coupled enzyme system and kinetic modelling analysis. This work suggests that the sensitivity to hyper-oxidation is controlled by a conformational reorganization during the catalytic cycle of Tsa1. Next, the study of the first step of Srx catalytic mechanism, which involves the ATP-dependent activation of the sulfinic acid form of typical 2-Cys Prx i) has shown that the rate-limiting step is associated with the chemical phosphate transfer process, and ii) provided an assembly model of the Michaelien complex Prx/Srx/ATP, formed during this process. Finally, through the combination of in vitro enzyme kinetics and in vivo yeast genetic tools, we established that the recycling mechanism of one Cys Srx, existing in plants or mammals, involves the glutathione (GSH) as reducer in cells, contrary to the Srx from S. cerevisiae, which is recycled by the Thioredoxin system. Unexpectedly, our study suggests that GSH binds the thiolsulfinate complex, confirming the role of GSH as the primary reducing system of 1-Cys-Sr

Enzymologie des étapes clés de régulation du système Peroxyrédoxine / Sulfirédoxine dans le contexte de la signalisation cellulaire redox

The peroxiredoxins (Prx) are ubiquitous thiol peroxidases, which play a central role in the physiology of hydrogen peroxide. A subclass of Prx called "typical 2-Cys-Prx" has a unique property to hyperoxidize the catalytic Cys into the sulfinic acid form, which acts as a regulation mechanism of their functions, as peroxidase, peroxide sensor or protein chaperone. The reduction of the overoxidized form is catalyzed by sulfiredoxin (Srx), an ATP-dependent sulfinyl reductase whose catalytic constant is about 1-2 min-1, a low value that must be correlated to the role of Srx in redox regulation. The aim of this study was to analyze the enzymology of the regulation of the Prx/Srx system at three diffrents points of control: the hyper-oxidation process of typical 2-Cys-Prx, the rate-limiting step of the Srx mechanism and the recycling step of Srx by the cellular thiol redox systems. We have first characterized the competition mechanism between the two steps of the catalytic mechanism of the major typical 2-Cys-Prx of S. cerevisiae, Tsa1, through a strategy combining rapid kinetics, coupled enzyme system and kinetic modelling analysis. This work suggests that the sensitivity to hyper-oxidation is controlled by a conformational reorganization during the catalytic cycle of Tsa1. Next, the study of the first step of Srx catalytic mechanism, which involves the ATP-dependent activation of the sulfinic acid form of typical 2-Cys Prx i) has shown that the rate-limiting step is associated with the chemical phosphate transfer process, and ii) provided an assembly model of the Michaelien complex Prx/Srx/ATP, formed during this process. Finally, through the combination of in vitro enzyme kinetics and in vivo yeast genetic tools, we established that the recycling mechanism of one Cys Srx, existing in plants or mammals, involves the glutathione (GSH) as reducer in cells, contrary to the Srx from S. cerevisiae, which is recycled by the Thioredoxin system. Unexpectedly, our study suggests that GSH binds the thiolsulfinate complex, confirming the role of GSH as the primary reducing system of 1-Cys-SrxLes peroxyrédoxines (Prx) sont des peroxydases à thiol, ubiquitaires, qui jouent un rôle central dans la physiologie du peroxyde d’hydrogène. Une famille de Prx dite "2-Cys-Prx typique" possède une propriété unique de suroxydation de la Cys catalytique sous forme acide sulfinique, qui constitue un mécanisme de régulation des fonctions des 2-Cys-Prx typiques en tant que peroxydase, capteur de peroxyde ou protéine chaperon. La réduction des 2-Cys-Prx typiques suroxydées est catalysée par la Sulfirédoxine (Srx), une sulfinyl réductase ATP-dépendante dont la constante catalytique est de l’ordre de 1-2 min-1, une valeur faible qui doit être corrélée au rôle de Srx dans la régulation redox. L’objectif de ce travail était d’analyser l’enzymologie de la régulation du système Prx/Srx au niveau, du processus de suroxydation des 2-Cys-Prx typiques, de l’étape limitante de la Srx, et de son recyclage par les systèmes redox cellulaires. Dans un premier temps, nous avons caractérisé les deux étapes du cycle catalytique de la 2-Cys-Prx typique majeure de S. cerevisiae Tsa1, dont la compétition contrôle la sensibilité à la suroxydation, par une stratégie combinant cinétiques rapides, système enzymatique couplé et modélisation cinétique. Ces travaux suggèrent que cette compétition est contrôlée par une réorganisation conformationnelle au cours du cycle catalytique de la Tsa1. Dans un second temps, l’étude de la première étape du mécanisme catalytique de Srx, qui consiste en l’activation ATP-dépendante du groupement acide sulfinique de la 2 Cys-Prx a permis, i) de montrer que l’étape limitante de la réaction catalysée par Srx était associée au processus chimique de transfert de phosphate, et ii) de proposer un modèle d’assemblage du complexe Michaelien Prx/Srx/ATP formé lors de ce processus. Enfin, par une approche combinant cinétiques enzymatiques in vitro et génétique de la levure in vivo, nous avons établi que le mécanisme de recyclage des Srx à 1 Cys existant chez les plantes ou les mammifères implique le rôle du glutathion comme réducteur cellulaire, contrairement à la Srx de S. cerevisiae qui est recyclée par le système thiorédoxine. De façon inattendue, la spécificité du glutathion dans ce mécanisme est assurée par un événement de reconnaissance au sein du complexe Prx/Sr

Reactive Oxygen Species in Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) includes chronic bronchitis and emphysema. Environmental exposure, primarily cigarette smoking, can cause high oxidative stress and is the main factor of COPD development. Cigarette smoke also contributes to the imbalance of oxidant/antioxidant due to exogenous reactive oxygen species (ROS). Moreover, endogenously released ROS during the inflammatory process and mitochondrial dysfunction may contribute to this disease progression. ROS and reactive nitrogen species (RNS) can oxidize different biomolecules such as DNA, proteins, and lipids leading to epithelial cell injury and death. Various detoxifying enzymes and antioxidant defense systems can be involved in ROS removal. In this review, we summarize the main findings regarding the biological role of ROS, which may contribute to COPD development, and cytoprotective mechanisms against this disease progression

Evidence That Glutathione and the Glutathione System Efficiently Recycle 1-Cys Sulfiredoxin In Vivo

International audienceAims: Typical 2-Cys peroxiredoxins (2-Cys Prxs) are Cys peroxidases that undergo inactivation by hyperoxidation of the catalytic Cys, a modification reversed by ATP-dependent reduction by sulfiredoxin (Srx). Such an attribute is thought to provide regulation of 2-Cys Prxs functions. The initial steps of the Srx catalytic mechanism lead to a Prx/Srx thiolsulfinate intermediate that must be reduced to regenerate Srx. In Saccharomyces cerevisiae Srx, the thiolsulfinate is resolved by an extra Cys (Cys48) that is absent in mammalian, plant, and cyanobacteria Srxs (1-Cys Srxs). We have addressed the mechanism of reduction of 1-Cys Srxs using S. cerevisiae Srx mutants lacking Cys48 as a model. Results: We have tested the recycling of Srx by glutathione (GSH) by a combination of in vitro steady-state and single-turnover kinetic analyses, using enzymatic coupled assays, Prx fluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and reverse-phase chromatography coupled to mass spectrometry. We demonstrate that GSH reacts directly with the thiolsulfinate intermediate, by following saturation kinetics with an apparent dissociation constant of 34 mu M, while producing S-glutathionylated Srx as a catalytic intermediate which is efficiently reduced by the glutaredoxin/glutathione reductase system. Total cellular depletion of GSH impacted the recycling of Srx, confirming in vivo that GSH is the physiologic reducer of 1-Cys Srx. Innovation: Our study suggests that GSH binds to the thiolsulfinate complex, thus allowing non-rate limiting reduction. Such a structural recognition of GSH enables an efficient catalytic reduction, even at very low GSH cellular levels. Conclusion: This study provides both in vitro and in vivo evidence of the role of GSH as the primary reducer of 1-Cys Srxs. Antioxid. Redox Signal. 22, 731-743

Dynamics of a key conformational transition in the mechanism of peroxiredoxin sulfinylation

International audiencePeroxiredoxins from the Prx1 subfamily (Prx) are moonlighting peroxidases that operate in peroxide signaling and are regulated by sulfinylation. Prxs offer a major model of protein−thiol oxidative modification. They react with H 2 O 2 to form a sulfenic acid intermediate that either engages into a disulfide bond, committing the enzyme into its peroxidase cycle, or again reacts with peroxide to produce a sulfinic acid that inactivates the enzyme. Sensitivity to sulfinylation depends on the kinetics of these two competing reactions and is critically influenced by a structural transition from a fully folded (FF) to locally unfolded (LU) conformation. Analysis of the reaction of the Tsa1 Saccharomyces cerevisiae Prx with H 2 O 2 by Trp fluorescencebased rapid kinetics revealed a process linked to the FF/LU transition that is kinetically distinct from disulfide formation and suggested that sulfenate formation facilitates local unfolding. Use of mutants of distinctive sensitivities and of different peroxide substrates showed that sulfinylation sensitivity is not coupled to the resolving step kinetics but depends only on the sulfenic acid oxidation and FF-to-LU transition rate constants. In addition, stabilization of the active site FF conformation, the determinant of sulfinylation kinetics, is only moderately influenced by the Prx C-terminal tail dynamics that determine the FF → LU kinetics. From these two parameters, the relative sensitivities of Prxs toward hyperoxidation with different substrates can be predicted, as confirmed by in vitro and in vivo patterns of sulfinylation