The p67 Activation Domain Regulates Electron Flow from NADPH to Flavin in Flavocytochromeb 558

124

147

NADPH oxidase activation involves the assembly of membrane-localized cytochrome b 559 with the cytosolic components p47 phox , p67 phox , and the small GTPase Rac. Assembly is mimicked by a cell-free system consisting of membranes and cytosolic components, activated by an anionic amphiphile. We reported that a chimeric construct, consisting of residues 1-212 of p67 phox and fulllength Rac1, activates the oxidase in vitro in an amphiphile-dependent manner, and when prenylated, in the absence of amphiphile and p47 phox . We subjected chimera p67 phox -(1-212)-Rac1 to mutational analysis and found that: 1) replacement of a single basic residue at the C terminus of the Rac1 moiety by glutamine is sufficient for loss of activity by the non-prenylated chimera; replacement of all six basic residues by glutamines is required for loss of activity by the prenylated chimera. 2) A V204A mutation in the activation domain of the p67 phox moiety leads to a reduction in activity. 3) Mutating residues, known to participate in the interaction between free p67 phox and Rac1, in the p67 phox -(R102E) or Rac1 (A27K, G30S) moieties of the chimera, leads to a marked decrease in activity, indicating a requirement for intrachimeric bonds, in addition to the engineered fusion. 4) Chimeras, inactive because of mutations A27K or G30S in the Rac1 moiety, are reactivated by supplementation with exogenous Rac1-GTP but not with exogenous p67 phox . This demonstrates that Rac has a dual role in the assembly of NADPH oxidase. One is to tether p67 phox to the membrane; the other is to induce an "activating" conformational change in p67 phox . . production, is probably the consequence of a conformational change in gp91 phox , caused by the interaction of cytochrome b 559 with one or several of the cytosolic oxidase components (8). Establishing contact with cytochrome b 559 requires the translocation to the plasma membrane of p47 phox , p67 phox , and Rac, a process known as oxidase assembly (reviewed in Refs. 9 and 10). Stimulus-elicited activation of the oxidase in intact cells can be mimicked in a cell-free system, in which phagocyte membranes or purified and relipidated cytochrome b 559 are mixed with the cytosolic components p47 phox , p67 phox , and Rac, in the presence of an anionic amphiphile, serving as an activator (11-15). The amphiphile acts by causing a conformational change in p47 phox (16) and, possibly, also by the induction of structural changes in cytochrome b 559 (17).The impairment of O 2 . production in the p47 phox -deficient form of chronic granulomatous disease shows that p47 phox is * This work was supported by the Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research, the Ela Kodesz Institute of Host Defense against Infectious Diseases, Israel Science Foundation Grant 428/01, the Roberts-Guthman Chair in Immunopharmacology (to E. P.), and National Institutes of Health Grant GM46372 (to C. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefor...

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confidence: 60%

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confidence: 98%

Dual Role of Rac in the Assembly of NADPH Oxidase, Tethering to the Membrane and Activation of p67

Sarfstein

Gorzalczany

Mizrahi

et al. 2004

124

147

“…The Rac Switch I domain in its GTP-bound form interacts with the tetratricopeptide repeat of p67 phox (36), a direct activator of the NADPH oxidase (43). The crystal structure of the Rac-p67 phox complex revealed that Ala 27 and Gly 30 of Rac are crucial to the stability of this assembly (36).…”

Section: Discussionmentioning

confidence: 99%

Antagonistic Cross-talk between Rac and Cdc42 GTPases Regulates Generation of Reactive Oxygen Species

Diebold

Fowler

et al. 2004

Cross-talk between Rho GTPase family members (Rho, Rac, and Cdc42) plays important roles in modulating and coordinating downstream cellular responses resulting from Rho GTPase signaling. The NADPH oxidase of phagocytes and nonphagocytic cells is a Rac GTPaseregulated system that generates reactive oxygen species (ROS) for the purposes of innate immunity and intracellular signaling. We recently demonstrated that NADPH oxidase activation involves sequential interactions between Rac and the flavocytochrome b 558 and p67 phox oxidase components to regulate electron transfer from NADPH to molecular oxygen. Here we identify an antagonistic interaction between Rac and the closely related GTPase Cdc42 at the level of flavocytochrome b 558 that regulates the formation of ROS. Cdc42 is unable to stimulate ROS formation by NADPH oxidase, but Cdc42, like Rac1 and Rac2, was able to specifically bind to flavocytochrome b 558 in vitro. Cdc42 acted as a competitive inhibitor of Rac1-and Rac2-mediated ROS formation in a recombinant cell-free oxidase system. Inhibition was dependent on the Cdc42 insert domain but not the Switch I region. Transient expression of Cdc42Q61L inhibited ROS formation induced by constitutively active Rac1 in an NADPH oxidase-expressing Cos7 cell line. Inhibition of Cdc42 activity by transduction of the Cdc42-binding domain of Wiscott-Aldrich syndrome protein into human neutrophils resulted in an enhanced fMetLeuPhe-induced oxidative response, consistent with inhibitory cross-talk between Rac and Cdc42 in activated neutrophils. We propose here a novel antagonism between Rac and Cdc42 GTPases at the level of the Nox proteins that modulates the generation of ROS used for host defense, cell signaling, and transformation.The process by which cells produce reactive oxygen species (ROS) 1 has gained much interest because of the diverse functions attributed to this class of molecules. In nonphagocytic cells, oxidants affect a variety of cellular processes, including transcription factor activation, proliferation, transformation, and apoptosis. In neutrophils and other phagocytes, oxidants play an important role in cellular innate immune responses. A critical component of the bactericidal activity of phagocytes is the NADPH oxidase, also referred to as "phox" (phagocytic oxidase) (1-3), which generates superoxide anion and, subsequently, a number of other ROS. The phagocyte NADPH oxidase is a multiprotein system whose activity is regulated by the RhoGTPase Rac2 in human cells (4 -6). Electrons are transferred from NADPH to molecular oxygen through the action of an integral membrane flavocytochrome b 558 (cyt b 558 ), composed of subunits gp91 phox and p22 phox . In addition to Rac2, the activity of the NADPH oxidase is regulated by the cytosolic components p47 phox , p67 phox , and p40 phox , which exist as a heterotrimeric complex in the cytosol of unstimulated neutrophils (7). In a separate cytosolic complex are Rac2 (or Rac1 in certain species) and GDP dissociation inhibitor (8). When neutrophils are activate...

“…An allosteric regulation mechanism was suggested in which p47 phox behaves as a positive effector that increases the affinity of p67 phox for cytochrome b 558 (53,54,11). In vivo, phosphorylated p47 phox serves as an adapter protein bringing p67 phox into proximity with flavocytochrome b 558 (55).…”

Section: Discussionmentioning

confidence: 99%

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

Berthier

Paclet

Lerouge

et al. 2003

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...