Cycle redox quinone-quinone réductase 2 et conséquences sur la production d'espèces oxygénées réactives dans le contexte cellulaire

Quinone reductase 2 or QR2 is an enzyme that, like its counterpart QR1, plays a role in detoxification of the highly reactives quinones by reducing them into hydroquinones. On one hand, it has been observed at the cellular and tissue level that the activity of this flavoprotein could have deleterious effects by triggering an overproduction of reactive oxygen species (ROS). On the other hand, overexpression or under expression of QR2 has been observed in some neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. In this context, this work focused on the study of reactive oxygen species produced during the quinone / QR2 redox cycle and their variations depending on the nature of the quinone, on both purified protein and cell models, in comparison to QR1. The redox properties of the substrates, co-substrates and inhibitors ok QR2 studied by electrochemistry allowed to classify them according to their capacity to be reduced. The enzymatic activity of the protein, either purified or intracellular, was followed by various methodologies (electron paramagnetic resonance, UV-visible and fluorescence spectroscopy, U(H)PLC-MS, confocal fluorescence microscopy). Production of superoxide radical is observed in the presence of cell lines overexpressing or not QR1 and QR2. Quinones are reduced enzymatically to form hydroquinones via the activity of quinone reductase (QR1 and QR2) and semiquinone via the activity of one electron reductases (e.g. CytP540 reductase). Reoxidation of these products is responsible for a greater or lesser production of the superoxide radical, according to the initial structure of the quinone and the affinity for different reductases. Menadione causes a higher production of cellular superoxide in the absence of QR1 and QR2. These analyzes have also shown that, like its counterpart QR1, QR2 is capable of reducing ortho-quinones including catecholquinones (aminochrome, dopachrome, adrenochrome) known for their neuronal toxicity.La quinone réductase 2 ou QR2 est une enzyme qui, comme son homologue QR1, joue un rôle de détoxification des quinones, molécules fortement réactives, en les réduisant en hydroquinones. Cependant, il a été observé au niveau cellulaire et tissulaire que l'activité de cette flavoprotéine pouvait avoir des effets délétères en déclenchant une surproduction d'espèces réactives de l'oxygène (ROS). D'autre part, on observe une surexpression ou une sous expression de QR2 dans certaines maladies neurodégénératives comme la maladie de Parkinson et la maladie d'Alzheimer. Dans ce contexte, ce travail a porté sur l'étude des espèces oxygénées réactives produites lors du cycle redox quinone / QR2 et leurs variations en fonction de la nature de la quinone, sur protéine purifiée et sur modèles cellulaires comparativement à QR1. Les propriétés d'oxydo-réduction des substrats, co-substrats et inhibiteurs de QR2 étudiées par électrochimie ont permis de les classer en fonction de leur capacité à être réduits. L'activité enzymatique de la protéine, qu'elle soit purifiée ou intracellulaire, a été suivie par différentes méthodologies (résonance paramagnétique électronique, spectroscopie UV-visible et de fluorescence, U(H)PLC-MS, microscopie confocale de fluorescence). La production du radical superoxyde est observée en présence de lignées cellulaires surexprimant ou non QR1 et QR2. Les quinones sont réduites enzymatiquement pour donner des hydroquinones via l'activité des quinones réductases (QR1 et QR2) et des semiquinones via l'activité de réductases à un électron (CytP540 réductase par exemple). La réoxydation de ces produits est responsable d'une production plus ou moins forte de radicaux superoxydes selon la structure initiale de la quinone et l'affinité pour les différentes réductases. La ménadione provoque une production cellulaire de superoxyde plus importante en l'absence de QR1 et QR2. Ces analyses ont également démontré que, comme son homologue QR1, QR2 est capable de réduire les ortho-quinones dont certaines catécholquinones (aminochrome, dopachrome, adrénochrome) reconnues pour leur toxicité neuronale

Quinone-quinone reductase 2 redox cycle and consequences on the production of reactive oxygen species in the cellular context

La quinone réductase 2 ou QR2 est une enzyme qui, comme son homologue QR1, joue un rôle de détoxification des quinones, molécules fortement réactives, en les réduisant en hydroquinones. Cependant, il a été observé au niveau cellulaire et tissulaire que l'activité de cette flavoprotéine pouvait avoir des effets délétères en déclenchant une surproduction d'espèces réactives de l'oxygène (ROS). D'autre part, on observe une surexpression ou une sous expression de QR2 dans certaines maladies neurodégénératives comme la maladie de Parkinson et la maladie d'Alzheimer. Dans ce contexte, ce travail a porté sur l'étude des espèces oxygénées réactives produites lors du cycle redox quinone / QR2 et leurs variations en fonction de la nature de la quinone, sur protéine purifiée et sur modèles cellulaires comparativement à QR1. Les propriétés d'oxydo-réduction des substrats, co-substrats et inhibiteurs de QR2 étudiées par électrochimie ont permis de les classer en fonction de leur capacité à être réduits. L'activité enzymatique de la protéine, qu'elle soit purifiée ou intracellulaire, a été suivie par différentes méthodologies (résonance paramagnétique électronique, spectroscopie UV-visible et de fluorescence, U(H)PLC-MS, microscopie confocale de fluorescence). La production du radical superoxyde est observée en présence de lignées cellulaires surexprimant ou non QR1 et QR2. Les quinones sont réduites enzymatiquement pour donner des hydroquinones via l'activité des quinones réductases (QR1 et QR2) et des semiquinones via l'activité de réductases à un électron (CytP540 réductase par exemple). La réoxydation de ces produits est responsable d'une production plus ou moins forte de radicaux superoxydes selon la structure initiale de la quinone et l'affinité pour les différentes réductases. La ménadione provoque une production cellulaire de superoxyde plus importante en l'absence de QR1 et QR2. Ces analyses ont également démontré que, comme son homologue QR1, QR2 est capable de réduire les ortho-quinones dont certaines catécholquinones (aminochrome, dopachrome, adrénochrome) reconnues pour leur toxicité neuronale.Quinone reductase 2 or QR2 is an enzyme that, like its counterpart QR1, plays a role in detoxification of the highly reactives quinones by reducing them into hydroquinones. On one hand, it has been observed at the cellular and tissue level that the activity of this flavoprotein could have deleterious effects by triggering an overproduction of reactive oxygen species (ROS). On the other hand, overexpression or under expression of QR2 has been observed in some neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. In this context, this work focused on the study of reactive oxygen species produced during the quinone / QR2 redox cycle and their variations depending on the nature of the quinone, on both purified protein and cell models, in comparison to QR1. The redox properties of the substrates, co-substrates and inhibitors ok QR2 studied by electrochemistry allowed to classify them according to their capacity to be reduced. The enzymatic activity of the protein, either purified or intracellular, was followed by various methodologies (electron paramagnetic resonance, UV-visible and fluorescence spectroscopy, U(H)PLC-MS, confocal fluorescence microscopy). Production of superoxide radical is observed in the presence of cell lines overexpressing or not QR1 and QR2. Quinones are reduced enzymatically to form hydroquinones via the activity of quinone reductase (QR1 and QR2) and semiquinone via the activity of one electron reductases (e.g. CytP540 reductase). Reoxidation of these products is responsible for a greater or lesser production of the superoxide radical, according to the initial structure of the quinone and the affinity for different reductases. Menadione causes a higher production of cellular superoxide in the absence of QR1 and QR2. These analyzes have also shown that, like its counterpart QR1, QR2 is capable of reducing ortho-quinones including catecholquinones (aminochrome, dopachrome, adrenochrome) known for their neuronal toxicity

La médecine personnalisée appliquée à l'efficacité et à la sécurité des médicaments

LIMOGES-BU Médecine pharmacie (870852108) / SudocLYON1-BU Santé (693882101) / SudocSudocFranceF

Redox cycling of QR2 substrates studied through physic-chemical approaches

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Joint Myocardial Motion and Contraction Phase Estimation from Cine MRI using Variational Data Assimilation.

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Oxidative stress and neurodegeneration: The possible contribution of quinone reductase 2

International audienceThere is increasing evidence that oxidative stress is involved in the etiology and pathogenesis of neurodegenerative disorders. Overproduction of reactive oxygen species (ROS) is due in part to the reactivity of catecholamines, such as dopamine, adrenaline, and noradrenaline. These molecules are rapidly converted, chemically or enzymatically, into catechol-quinone and then into highly deleterious semiquinone radicals after 1-electron reduction in cells. Notably, the overexpression of dihydronicotinamide riboside:quinone oxidoreductase (QR2) in Chinese hamster ovary (CHO) cells increases the production of ROS, mainly superoxide radicals, when it is exposed to exogenous catechol-quinones (e.g. dopachrome, aminochrome, and adrenochrome). Here we used electron paramagnetic resonance analysis to demonstrate that the phenomenon observed in CHO cells is also seen in human leukemic cells (K562 cells) that naturally express QR2. Moreover, by manipulating the level of QR2 in neuronal cells, including immortalized neuroblast cells and ex vivo neurons isolated from QR2 knockout animals, we showed that there is a direct relationship between QR2-mediated quinone reduction and ROS overproduction. Supporting this result, the withdraw of the QR2 co-factor (BNAH) or the addition of the specific QR2 inhibitor S29434 suppressed oxidative stress. Taken together, these data suggest that the overexpression of QR2 in brain cells in the presence of catechol quinones might lead to ROS-induced cell death via the rapid conversion of superoxide radicals into hydrogen peroxide and then into highly reactive hydroxyl radicals. Thus, QR2 may be implicated in the early stages of neurodegenerative disorders

Oxidative stress-induced inflammation in susceptible airways by anthropogenic aerosol

Ambient air pollution is one of the leading five health risks worldwide. One of the most harmful air pollutants is particulate matter (PM), which has different physical characteristics (particle size and number, surface area and morphology) and a highly complex and variable chemical composition. Our goal was first to comparatively assess the effects of exposure to PM regarding cytotoxicity, release of pro-inflammatory mediators and gene expression in human bronchial epithelia (HBE) reflecting normal and compromised health status. Second, we aimed at evaluating the impact of various PM components from anthropogenic and biogenic sources on the cellular responses. Air-liquid interface (ALI) cultures of fully differentiated HBE derived from normal and cystic fibrosis (CF) donor lungs were exposed at the apical cell surface to water-soluble PM filter extracts for 4 h. The particle dose deposited on cells was 0.9–2.5 and 8.8–25.4 μg per cm2 of cell culture area for low and high PM doses, respectively. Both normal and CF HBE show a clear dose-response relationship with increasing cytotoxicity at higher PM concentrations. The concurrently enhanced release of pro-inflammatory mediators at higher PM exposure levels links cytotoxicity to inflammatory processes. Further, the PM exposure deregulates genes involved in oxidative stress and inflammatory pathways leading to an imbalance of the antioxidant system. Moreover, we identify compromised defense against PM in CF epithelia promoting exacerbation and aggravation of disease. We also demonstrate that the adverse health outcome induced by PM exposure in normal and particularly in susceptible bronchial epithelia is magnified by anthropogenic PM components. Thus, including health-relevant PM components in regulatory guidelines will result in substantial human health benefits and improve protection of the vulnerable population

The cytosolic 5’-nucleotidase cN-II lowers the adaptability to glucose deprivation in human breast cancer cells

International audienceThe cytosolic 5'-nucleotidase cN-II is a highly conserved enzyme implicated in nucleotide metabolism. Based on recent observations suggesting additional roles not directly associated to its enzymatic activity, we studied human cancer cell models with basal or decreased cN-II expression. We developed cancer cells with stable inhibition of cN-II expression by transfection of shRNA-coding plasmids, and studied their biology. We show that human breast cancer cells MDA-MB-231 with decreased cN-II expression better adapt to the disappearance of glucose in growth medium under normoxic conditions than cells with a baseline expression level. This is associated with enhanced in vivo growth and a lower content of ROS in cells cultivated in absence of glucose due to more efficient mechanisms of elimination of ROS. Conversely, cells with low cN-II expression are more sensitive to glucose deprivation in hypoxic conditions. Overall, our results show that cN-II regulates the cellular response to glucose deprivation through a mechanism related to ROS metabolism and defence

Piceatannol and resveratrol share inhibitory effects on hydrogen peroxide release, monoamine oxidase and lipogenic activities in adipose tissue, but differ in their antilipolytic properties

International audiencePiceatannol is a hydroxylated derivative of resveratrol. While both dietary polyphenols coexist in edible plants and fruits, and share equivalent concentrations in several wines, the influence of piceatannol on adiposity has been less studied than that of resveratrol. Though resveratrol is now recognized to limit fat deposition in various obesity models, the benefit of its dietary supplementation remains under debate regarding human obesity treatment or prevention. The research for more potent resveratrol analogs is therefore still undergoing. This prompted us to compare various effects of piceatannol and resveratrol directly on human adipose tissue (hAT). Hydrogen peroxide release was measured by Amplex Red-based fluorescence in subcutaneous hAT samples from obese patients. Interactions of stilbenes with human amine oxidases and quinone reductase were assessed by radiometric methods, computational docking and electron paramagnetic resonance. Influences on lipogenic and lipolytic activities were compared in mouse adipocytes. Resveratrol and piceatannol inhibited monoamine oxidase (MAO) with respective IC50 of 18.5 and 133.7 μM, but not semicarbazide-sensitive amine oxidase (SSAO) in hAT. For both stilbenes, the docking scores were better for MAO than for SSAO. Piceatannol and resveratrol similarly hampered hydrogen peroxide detection in assays with and without hAT, while they shared pro-oxidant activities when incubated with purified quinone reductase. They exhibited similar dose-dependent inhibition of adipocyte lipogenic activity. Only piceatannol inhibited basal and stimulated lipolysis when incubated at a dose ≥100 μM. Thus, piceatannol exerted on fat cells dose-dependent effects similar to those of resveratrol, except for a stronger antilipolytic action. In this regard, piceatannol should be useful in limiting the lipotoxicity related to obesity when ingested or administered alone - or might hamper the fat mobilization induced by resveratrol when simultaneously administered with it

Role of Quinone Reductase 2 in the Antimalarial Properties of Indolone-Type Derivatives

Indolone-N-oxides have antiplasmodial properties against Plasmodium falciparum at the erythrocytic stage, with IC50 values in the nanomolar range. The mechanism of action of indolone derivatives involves the production of free radicals, which follows their bioreduction by an unknown mechanism. In this study, we hypothesized that human quinone reductase 2 (hQR2), known to act as a flavin redox switch upon binding to the broadly used antimalarial chloroquine, could be involved in the activity of the redox-active indolone derivatives. Therefore, we investigated the role of hQR2 in the reduction of indolone derivatives. We analyzed the interaction between hQR2 and several indolone-type derivatives by examining enzymatic kinetics, the substrate/protein complex structure with X-ray diffraction analysis, and the production of free radicals with electron paramagnetic resonance. The reduction of each compound in cells overexpressing hQR2 was compared to its reduction in naïve cells. This process could be inhibited by the specific hQR2 inhibitor, S29434. These results confirmed that the anti-malarial activity of indolone-type derivatives was linked to their ability to serve as hQR2 substrates and not as hQR2 inhibitors as reported for chloroquine, leading to the possibility that substrate of hQR2 could be considered as a new avenue for the design of new antimalarial compounds