Régulation de l'activité NADPH oxydase phagocytaire -Mécanismes moléculaires de la super-activité oxydase du cytochrome b558 D-loopNox4-Nox2

The phagocytic NADPH oxidase, responsible for superoxide anions production, becomes activated after assembly of cytosolic proteins with cytochrome b558, the transmembrane redox core of the enzyme composed of Nox2 and p22phox. We have previously observed that replacement of the D-loop of Nox2 with D-loop of Nox4 (mutant D-loopNox4-Nox2) induced a NADPH oxidase super-activity. The present work was to elucidate molecular mechanisms responsible for the overproduction of superoxide anions by the D-loopNox4-Nox2 mutant. The super-mutant exhibits an ex vivo oxidase activity which is 2 to 8 times higher than that of the WT-Nox2 PLB-985 cells, after stimulation by soluble and particulate agonists. The highest NADPH oxidase activity is obtained after ionomycin and fMLF chemotactic peptide activation, including signalling pathways with an increase of Ca2+i. This overproduction is also observed in vitro in a cell free system using only purified proteins of the oxidase complex, and activated by phosphatidic acid. Ex vivo oxidase activity of the mutant demonstrated an enhanced sensitivity to calcium influx. The mutated cytochrome b558 is less dependent on phosphorylation events ERK1/2-dependent occurring during activation with fMLF. Then, the super-oxidase activity of the D-loopNox4-Nox2 mutant could become from a conformational change of Nox2 improving the NADPH oxidase activated state. Moreover, superoxide anions overproduction by the mutant improves its killing activity against attenuated strain of P. aeruginosa PAO1. At last, we have found that the bactericidal process of this microorganism by phagocytes consists in two distinct and interdependent phases.La NADPH-oxydase phagocytaire, responsable de la production d'anions superoxydes microbicides, résulte de l'assemblage des protéines cytosoliques avec le cytochrome b558 membranaire redox formé de Nox2 et p22phox. Nous avons mis en évidence précédemment que le remplacement de la boucle D de Nox2 par celle de Nox4 (mutant D-loopNox4-Nox2) était à l'origine d'une " super-activité " oxydase. Le présent travail a consisté à élucider les mécanismes moléculaires à l'origine de la super-activité oxydase de ce mutant. Le mutant présente une activité oxydase ex vivo 2 à 8 fois supérieure à celle de cellules PLB-985 WT-Nox2, en réponse aux stimuli solubles et particulaires. Cette suractivité est plus importante en réponse à l'ionomycine et au facteur chimiotactique fMLF, dont les voies d'activation impliquent une augmentation du taux de Ca2+i. Cette suractivité a également été mise en évidence dans un système simplifié in vitro contenant uniquement les protéines purifiées du complexe oxydase et activé par l'acide phosphatidique. L'activité oxydase du mutant ex vivo présente une sensibilité accrue à un influx de calcium. Le cytochrome b558 muté est moins sensible aux événements de phosphorylation dépendant d'ERK1/2 durant l'activation par le fMLF. Ainsi, la suractivité du mutant D-loopNox4-Nox2 pourrait provenir d'une modification de la conformation de Nox2 mutée, favorisant l'état activé du complexe oxydase. De plus, la super-activité du mutant améliore son pouvoir bactéricide vis-à-vis de la souche atténuée P. aeruginosa PAO1. Enfin, nous avons pu mettre en évidence l'existence de deux phases distinctes et interdépendantes dans le processus de bactéricidie de ce microorganisme

Characterization of superoxide overproduction by the D-Loop(Nox4)-Nox2 cytochrome b(558) in phagocytes-Differential sensitivity to calcium and phosphorylation events.

International audienceNADPH oxidase is a crucial element of phagocytes involved in microbicidal mechanisms. It becomes active when membrane-bound cytochrome b(558), the redox core, is assembled with cytosolic p47(phox), p67(phox), p40(phox), and rac proteins to produce superoxide, the precursor for generation of toxic reactive oxygen species. In a previous study, we demonstrated that the potential second intracellular loop of Nox2 was essential to maintaining NADPH oxidase activity by controlling electron transfer from FAD to O(2). Moreover, replacement of this loop by the Nox4-D-loop (D-loop(Nox4)-Nox2) in PLB-985 cells induced superoxide overproduction. In the present investigation, we demonstrated that both soluble and particulate stimuli were able to induce this superoxide overproduction. Superoxide overproduction was also observed after phosphatidic acid activation in a purified cell-free-system assay. The highest oxidase activity was obtained after ionomycin and fMLF stimulation. In addition, enhanced sensitivity to Ca(2+) influx was shown by thapsigargin, EDTA, or BTP2 treatment before fMLF activation. Mutated cytochrome b(558) was less dependent on phosphorylation triggered by ERK1/2 during fMLF or PMA stimulation and by PI3K during OpZ stimulation. The superoxide overproduction of the D-loop(Nox4)-Nox2 mutant may come from a change of responsiveness to intracellular Ca(2+) level and to phosphorylation events during oxidase activation. Finally the D-loop(Nox4)-Nox2-PLB-985 cells were more effective against an attenuated strain of Pseudomonas aeruginosa compared to WT-Nox2 cells. The killing mechanism was biphasic, an early step of ROS production that was directly bactericidal, and a second oxidase-independent step related to the amount of ROS produced in the first step