Reconstitution and characterization of the human neutrophil respiratory burst oxidase using recombinant p47-phox, p67-phox and plasma membrane

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118

gp91 phox (Nox2), the catalytic subunit of the superoxide-generating respiratory burst oxidase, is regulated by subunits p47 phox and p67 phox . Nox1, a homolog of gp91 phox , is regulated by NOXO1 and NOXA1, homologs of p47 phox and p67 phox , respectively. For both Nox1 and gp91 phox , an organizer protein (NOXO1 or p47 phox ) cooperates with an activator protein (NOXA1 or p67 phox ) to regulate the catalytic subunit. Herein, we investigate the subunit regulation of Nox3 compared with that of other Nox enzymes. Nox3, like gp91 phox , was activated by p47 phox plus p67 phox . Whereas gp91 phox activity required the protein kinase C activator phorbol myristate acetate (PMA), Nox3 activity was already high without PMA, but was further stimulated ϳ30% by PMA. gp91 phox was also activated by NOXO1/NOXA1 and required PMA for high activity. gp91 phox regulation required an intact activation domain in the activator protein, as neither p67 phox (V204A) nor NOXA1(V205A) were effective. In contrast, p67 phox (V204A) was effective (along with p47 phox ) in activating Nox3. Unexpectedly, Nox3 was strongly activated by NOXO1 in the absence of NOXA1 or p67 phox . Nox3 activity was regulated by PMA only when p47 phox but not NOXO1 was present, consistent with the phosphorylation-regulated autoinhibitory region in p47 phox but not in NOXO1. Deletion of the autoinhibitory region from p47 phox rendered this subunit highly active in the absence of PMA toward both gp91 phox and Nox3, and high activity required an activator subunit. The unique regulation of Nox3 supports a model in which multiple interactions with regulatory subunits stabilize an active conformation of the catalytic subunit.The Nox family of NAD(P)H oxidases has recently been described (1-10). These enzymes are structural homologs of gp91 phox (a.k.a. Nox2), the catalytic subunit of the phagocyte NADPH oxidase, and are distributed in a variety of non-phagocytic tissues such as colon, kidney, vascular smooth muscle, and brain. Reactive oxygen species generated by these novel enzymes are proposed to function in signal transduction related to cell growth and cancer (1,(11)(12)(13)(14)(15)(16)(17), angiogenesis (18), and in innate immunity (15), e.g. in barrier cells such as epithelium. In addition, reactive oxygen species from these enzymes are proposed to be causally linked to pathological states including atherosclerosis (19), cancer (11,15,20,21), and diabetes (22).The human Nox enzymes are encoded by at least 7 genes. NOX1, NOX3, and NOX4 encode proteins that are similar in size and domain structure to gp91 phox . These consist of a Cterminal flavoprotein domain containing both an FAD-binding site and an NADPH-binding site, and an N-terminal membrane-associated hydrophobic domain consisting of 6 transmembrane ␣ helices that provide binding sites for 2 hemes (3). Nox5 consists of these same domains along with an N-terminal calcium-binding domain (9); this enzyme is dormant when expressed in cells, but is activated by calcium. Duox1 and Duox2 build upon the Nox5 str...

Section: Methodsmentioning

confidence: 99%

Nox3 Regulation by NOXO1, p47 , and p67

Cheng

Ritsick

Lambeth

2004

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118

“…p67 phox and prenylated Rho GTPases were expressed and purified as GST fusion proteins in baculovirus-infected Sf9 cells as previously described (10,26). GST fusion proteins were cleaved with thrombin for use in enzyme assays.…”

Section: Methodsmentioning

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...

“…Cytochrome b 558 was solubilized with cholate and octyl-glucoside and purified using several column chromatographic steps as described previously (30,31). Recombinant proteins p47 phox and p67 phox were produced in insect cells and purified as described by Uhlinger et al (12,32). Protein concentration was determined according to Bradford (33).…”

Section: Methodsmentioning

confidence: 99%

Rac “Insert Region” Is a Novel Effector Region That Is Implicated in the Activation of NADPH Oxidase, but Not PAK65

Freeman

Abo²,

Lambeth

1996

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133

The small GTPase Rac assembles with the cytosolic p47 phox and p67 phox and the membrane-associated flavocytochrome b 558 to form the multicomponent respiratory burst oxidase. Mutation of amino acids in a region of Rac (residues 26 -45), homologous to an effector region in Ras, was previously shown to interfere with Rac binding to the oxidase. Herein we have elucidated an additional region in Rac involved in regulating oxidase activity. Rho family small GTPases contain a 12-amino acid "insert" region (residues 124 -135) that is not present in Ras. Point mutations in and deletion of this region were constructed and used for in vitro studies of the activation of PAK65 and NADPH oxidase. During the respiratory burst, neutrophils and other phagocytic cells reduce molecular oxygen to generate superoxide anion, with subsequent production of secondary products such as hydrogen peroxide and hydroxyl radical, all of which participate in microbial killing. The enzyme that initiates the respiratory burst, the NADPH oxidase, is a multicomponent enzyme that utilizes reducing equivalents from NADPH to reduce oxygen to superoxide (reviewed in Ref.