Changing the Conformation State of Cytochrome b 558 Initiates NADPH Oxidase Activation

Abstract

The phagocyte respiratory burst depends on the NADPH reduction of molecular oxygen and generation of superoxide anion O 2 . upon activation of the cell with soluble (chemotactic factors) or particulate (bacteria or fungi) stimuli (1). The O 2 . -generating NADPH oxidase (EC 1.6.99.6) is a heterogeneous complex compartmentalized in resting cells, between plasma membrane, cytosol, and the cytoskeleton, which assembles at the plasma membrane level once activated. Cytochrome b 558 is the membrane redox core of the oxidase complex, which, after activation, transfers electrons from NADPH to molecular oxygen. It is constituted of gp91 phox (the ␤-catalytic subunit) and p22 phox (␣-subunit), which is necessary in phagocytes to stabilize the 1/1 (␣/␤) heterodimer (2). Three soluble factors, p67 phox , p47 phox , and p40 phox , participate in the activation process, as does the monomeric G protein Rac1/2. Another monomeric G protein, Rap1A, has been identified in the membrane and proposed as a possible regulation factor of the oxidase complex (3, 4). In phagocytes, O 2 . and derivatives are synthesized in large amounts and used as bactericidal tools, but uncontrolled production is also injurious to tissues and triggers inflammatory reactions (5, 6). Phagocyte NADPH oxidase activity is transitory; it is submitted to a strict up-and down-regulation process. Up to now, oxidase activation has been investigated, but the molecular support of down-regulation has not. In fact, the role of a membrane-associated GTPase-activating protein has been described and related to the deactivation of NADPH oxidase (7,8). Recent observations would support an allosteric regulation of NADPH oxidase activity where cytochrome b 558 is the catalytic subunit. In the oxidase assembly, p67 phox is the limiting factor (9 -11). The binding of p67 phox to cytochrome b 558 mediates the transition from an inactive to an active conformation of cytochrome b 558 (11). In the process, p47 phox and Rac1/2 are introduced as positive effectors, whereas the p40 phox function remains undetermined. In chronic granulomatous disease (CGD) 1 , there is no oxidase activity (12).All the components of the phagocyte NADPH oxidase complex are expressed in Epstein-Barr virus-immortalized B lymphocytes. However, in these cells, the activity of stimulated oxidase is 100 times inferior to that of human neutrophils (13). Both neutrophils and EBV-B cells are qualitatively similar in * This work was supported by grants from the Ministère de l'Enseignement supérieur de la Recherche et Technologie, Paris, the Région Rhône Alpes, programme Mobilité Internationale Rhône-Alpes 2001, the Groupement des Entreprises Françaises dans la lutte contre le Cancer, délégation de Grenoble, the Fondation pour la Recherche Médicale, Isère, the Direction Régionale de la Recherche Clinique, and the Association Française, la Ligue Nationale contre le Cancer. We acknowledge the financial support of Mission Physique et Chimie du Vivant (CNRS) and la Fondation pour la Recherche Médicale for atomic force...

Myeloid-Related Protein-14 Is a p38 MAPK Substrate in Human Neutrophils

“…Phosphorylation has been implicated in translocation of the MRP-14/ MRP-8 heterodimer to membranes and cytoskeleton in monocytes (30). MRP-14 was also reported to participate in the initiation of NADPH oxidase activity in human neutrophils (31,32). The present study showed that p38 MAPK phosphorylates MRP-14 on Thr 113 .…”

“…The MRP-14/MRP-8 complex was reported to undergo phosphorylation-dependent translocation to the plasma membrane and cytoskeleton in monocytes after cell stimulation (30). The MRP-14/MRP-8 complexes were also found to bind arachidonic acid (38,39) and to participate in the activation of NADPH oxidase (31,32). MRP-14/MRP-8 increased the affinity of p67 phox for cytochrome b 558 , and oxidase activity was enhanced (32).…”

“…The MRP-14/MRP-8 complexes were also found to bind arachidonic acid (38,39) and to participate in the activation of NADPH oxidase (31,32). MRP-14/MRP-8 increased the affinity of p67 phox for cytochrome b 558 , and oxidase activity was enhanced (32). Targeted disruption of the MRP-14 gene in mice does not produce consistent phenotypic changes (52,53), although Manitz et al (53) reported impaired in vitro chemotaxis and up-regulation of CD11b.…”

“…MRP-14 is a known phosphoprotein that binds Ca 2ϩ (24 -30) and arachidonic acid (38,39). MRP-14 also acts as a binding partner for the cytosolic factors of the NADPH oxidase (31,32). As these findings suggest that MRP-14 may mediate some p38 MAPK-dependent neutrophil responses, additional experiments were performed to confirm that MRP-14 is a true substrate of p38 MAPK.…”

Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity

“…The capacity of electron transfer in vitro was evaluated using an adapted protocol previously described [17]. Briefly, 10 pmol of purified proteins was incubated for 5 min at 4°C in PBS buffer containing 10 lM FAD (with or without 300 lg of cytosol isolated from HEK293E cells) and 100 lM INT (iodonitro tetrazolium chloride) or 100 lM cytochrome c. The reaction was initiated by the addition of 150 lM NADPH, and the reduction of these compounds was followed during 30 min (at 500 nm for INT, e 500nm = 11 mM À1 cm À1 or at 550 nm for cytochrome c, e 550nm = 21.1 mM À1 cm À1 ).…”