Cytosolic Proteins P47phox Research Articles

ROCK2 interacts with p22phox to phosphorylate p47phox and to control NADPH oxidase activation in human monocytes

Monocytes play a key role in innate immunity by eliminating pathogens, releasing high levels of cytokines, and differentiating into several cell types, including macrophages and dendritic cells. Similar to other phagocytes, monocytes produce superoxide anions through the NADPH oxidase complex, which is composed of two membrane proteins (p22phox and gp91phox/NOX2) and four cytosolic proteins (p47phox, p67phox, p40phox and Rac1). The pathways involved in NADPH oxidase activation in monocytes are less known than those in neutrophils. Here, we show that p22phox is associated with Rho-associated coiled-coil kinase2 (ROCK2) in human monocytes but not neutrophils. This interaction occurs between the cytosolic region of p22phox (amino acids 132 to 195) and the coiled-coil region of ROCK2 (amino acids 400 to 967). Interestingly, ROCK2 does not phosphorylate p22phox, p40phox, p67phox, or gp91phox invitro but phosphorylates p47phox on Ser304, Ser315, Ser320 and Ser328. Furthermore, KD025, a selective inhibitor of ROCK2, inhibited reactive oxygen species (ROS) production and p47phox phosphorylation in monocytes. Specific inhibition of ROCK2 expression in THP1-monocytic cell line by siRNA inhibited ROS production. These data show that ROCK2 interacts with p22phox and phosphorylates p47phox, and suggest that p22phox could be a shuttle for ROCK2 to allow p47phox phosphorylation and NADPH oxidase activation in human monocytes.

Consequences of the constitutive NOX2 activity in living cells: Cytosol acidification, apoptosis, and localized lipid peroxidation

The phagocyte NADPH oxidase (NOX2) is a key enzyme of the innate immune system generating superoxide anions (O2•-), precursors of reactive oxygen species. The NOX2 protein complex is composed of six subunits: two membrane proteins (gp91phox and p22phox) forming the catalytic core, three cytosolic proteins (p67phox, p47phox and p40phox) and a small GTPase Rac. The sophisticated activation mechanism of the NADPH oxidase relies on the assembly of cytosolic subunits with the membrane-bound components. A chimeric protein, called ‘Trimera’, composed of the essential domains of the cytosolic proteins p47phox (aa 1–286), p67phox (aa 1–212) and full-length Rac1Q61L, enables a constitutive and robust NOX2 activity in cells without the need of any stimulus. We employed Trimera as a single activating protein of the phagocyte NADPH oxidase in living cells and examined the consequences on the cell physiology of this continuous and long-term NOX activity. We showed that the sustained high level of NOX activity causes acidification of the intracellular pH, triggers apoptosis and leads to local peroxidation of lipids in the membrane. These local damages to the membrane correlate with the strong tendency of the Trimera to clusterize in the plasma membrane observed by FRET-FLIM microscopy.

Impaired p47phox phosphorylation in neutrophils from patients with p67phox-deficient chronic granulomatous disease

AbstractSuperoxide production by the phagocyte reduced NAD phosphate (NADPH) oxidase is essential for innate immunity as shown in chronic granulomatous disease (CGD), an immunodeficiency disease resulting from mutations in 1 of its genes. The NADPH oxidase is composed of 2 membrane proteins (gp91phox/NOX2 and p22phox) and 4 cytosolic proteins (p47phox, p67phox, p40phox, and Rac1/2). The phosphorylation of p47phox is required for NADPH oxidase activation in cells. As p47phox and p67phox can form a tight complex in cells, we hypothesized that p67phox could regulate p47phox phosphorylation. To investigate this hypothesis, we used phospho-specific antibodies against 5 major p47phox-phosphorylated sites (Ser304, Ser315, Ser320, Ser328, and Ser345) and neutrophils from healthy donors and from p67phox−/− CGD patients. Results showed that formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate induced a time- and a concentration-dependent phosphorylation of p47phox on Ser304, Ser315, Ser320, and Ser328 in healthy human neutrophils. Interestingly, in neutrophils and Epstein-Barr virus-transformed B lymphocytes from p67phox−/− CGD patients, phosphorylation of p47phox on serine residues was dramatically reduced. In COSphox cells, the presence of p67phox led to increased phosphorylation of p47phox. In vitro studies showed that recombinant p47phox was phosphorylated on Ser304, Ser315, Ser320, and Ser328 by different PKC isoforms and the addition of recombinant p67phox alone or in combination with p40phox potentiated this process. Thus, p67phox and p40phox are required for optimal p47phox phosphorylation on Ser304, Ser315, Ser320, and Ser328 in intact cells. Therefore, p67phox and p40phox are novel regulators of p47phox-phosphorylation.

Myristoylation of Novel Protein Kinase C Beta II Peptide Inhibitor is Required for the Attenuation of Phorbol 12‐myristate 13‐acetate‐induced Superoxide Release in Isolated Rat Polymorphonuclear Leukocytes

Polymorphonuclear leukocyte (PMN) superoxide (SO) production by NADPH oxidase (NOX‐2) activation contributes to myocardial ischemia‐reperfusion (I/R) injury. Protein kinase C beta‐II (PKC βII) is a principal mediator of NOX‐2 activation via phosphorylation of NOX‐2 cytosolic protein p47phox. Phorbol 12‐myristate 13‐acetate (PMA) is a known broad‐spectrum PKC agonist that induces PMN SO release. In prior studies, selective PKCβII inhibition with myristoylated PKCβII peptide inhibitor (N‐myr‐SLNPEWNET; myr‐PKCβII‐) attenuated PMA‐induced PMN SO release and myocardial I/R injury in a dose‐dependent manner. However, the role of myristoylation mediating the inhibitory effects of myr‐PKCβII‐ on PMN SO release needs to be determined. Therefore, we aim to confirm the role of PKCβII by using myristoylated PKCβII peptide activator (N‐myr‐SVEIWD; myr‐PKCβII+) and myr‐PKCβII‐ that influence PKCβII translocation. Whereas, myr‐scrambled PKCβII‐ (N‐myr‐WNPESLNTE; myr‐PKCβII‐scram) is a control for myristoylation. We hypothesize that myr‐PKCβII+ should augment, myr‐PKCβII‐should attenuate, and myr‐PKCβII‐scram should have no effect on PMA‐induced PMN SO release compared to non‐treated and unconjugated peptide controls. PMNs (5×106) isolated from male Sprague‐Dawley rats (~400g) were incubated for 15 min at 37°C in the presence/absence of SO dismutase (SOD; 10μg/mL, positive control), unconjugated PKCβII+/− (20 μM), myr‐PKCβII+/− (20 μM), or myr‐PKCβII‐scram (20 μM). SO release was evaluated by the absorbance change (at 550 nm) via ferricytochrome c reduction after PMA stimulation (100 nM) for 390 sec. Data were analyzed by ANOVA using Bonferroni‐Dunn post‐hoc analysis. Myr‐PKCβII‐ significantly attenuated PMA‐induced PMN SO release (0.29±0.02; n=36; p<0.05) when compared to myr‐PKCβII+ (0.42±0.03; n=29), myr‐PKCβII‐scram (0.53±0.05; n=10), and non‐treated controls (0.41±0.02; n=55). Unconjugated PKCβII+ (0.41±0.04; n=16) and PKCβII‐(0.40±0.04; n=28) were similar to non‐treated controls. SOD (n=8) significantly reduced SO release by 94±7% compared to all groups (p<0.01). Cell viability determined by 0.2% trypan blue exclusion was similar in all groups, 94±2%. Unexpectedly, myr‐PKCβII‐scram rather than myr‐PKCβII+ PMNs exhibited the highest PMA‐induced PMN SO release but was not significantly different from untreated controls. Additional experiments will determine whether myr‐PKCβII‐scram significantly enhances SO release. Results suggest myr‐ conjugation improved myr‐PKCβII‐ delivery compared to unconjugated PKCβII‐ but does not contribute to the inhibitory effects of PMA‐induced PMN SO release. Therefore, myr‐PKCβII‐ may be an effective therapeutic intervention to limit inflammation‐induced I/R injury.Support or Funding InformationThis research was supported by the Division of Research, Department of Biomedical Sciences, and the Center for Chronic Disorders of Aging at Philadelphia College of Osteopathic Medicine. Current license is supported by Young Therapeutics, LLC.

APOCYNIN, AN INHIBITOR OF THE NADPH OXIDASE COMPLEX

Apocynin is a phenolic compound isolated from the plant Picrorhiza kurroa Royle ex Benth. Such a compound has been extensively investigated for its therapeutic potential in diseases involving inflammatory processes or oxidative stress due to its ability to inhibit the NADPH oxidase multienzyme complex. This complex consists of two transmembrane proteins (Nox2 and p22phox) and four cytosolic regulatory proteins (p67phox, p47phox, p40phox, and GTPase-Rac) and their activation occurs after the stimulation of phagocytic cells by the mediation of the enzyme myeloperoxidase (MPO). NADPH oxidase is the only enzyme complex that is intended for the production of superoxide anion that is precursor of highly oxidizing substances classified as reactive oxygen species (ROS). NADPH oxidase is an enzyme complex that produces superoxide anion from molecular oxygen. Ta the same time, the superoxide anion is a precursor to reactive oxygen species (ROS) catalyzed by enzymes.These oxidative species, when in excess, can induce burst, causing irreparable tissue damage. They can act by modifying the redox state of DNA, protein or lipid molecules, playing a central role in the development of chronic pathologies and various health complications. One can cite vascular problems, hyperglycemia, diabetes, hypertension, Alzheimer's disease, and cancer, among others. Apocynin, previously activated by MPO, blocks the enzyme complex and prevents the formation of these oxidative species. Therefore, the central biological function of compound is to modulate the action of NADPH oxidase, promoting a positive effect in the prevention/remediation of inflammatory diseases.

NADPH oxidase activation in neutrophils: Role of the phosphorylation of its subunits.

Neutrophils are key cells of innate immunity and during inflammation. Upon activation, they produce large amounts of superoxide anion (O2 -. ) and ensuing reactive oxygen species (ROS) to kill phagocytized microbes. The enzyme responsible for O2 -. production is called the phagocyte NADPH oxidase. This is a multicomponent enzyme system that becomes active after assembly of four cytosolic proteins (p47phox , p67phox , p40phox and Rac2) with the transmembrane proteins (p22phox and gp91phox , which form the cytochrome b558 ). gp91phox represents the catalytic subunit of the NADPH oxidase and is also called NOX2. NADPH oxidase-derived ROS are essential for microbial killing and innate immunity; however, excessive ROS production induces tissue injury and prolonged inflammatory reactions that contribute to inflammatory diseases. Thus, NADPH oxidase activation must be tightly regulated in time and space to limit ROS production. NADPH oxidase activation is regulated by several processes such as phosphorylation of its components, exchange of GDP/GTP on Rac2 and binding of p47phox and p40phox to phospholipids. This review aims to provide new insights into the role of the phosphorylation of the NADPH oxidase components, that is gp91phox , p22phox , p47phox , p67phox and p40phox , in the activation of this enzyme.

Conversion of NOX2 into a constitutive enzyme in vitro and in living cells, after its binding with a chimera of the regulatory subunits

During the phagocytosis of pathogens by phagocyte cells, the NADPH oxidase complex is activated to produce superoxide anion, a precursor of microbial oxidants. The activated NADPH oxidase complex from phagocytes consists in two transmembrane proteins (Nox2 and p22phox) and four cytosolic proteins (p40phox, p47phox, p67phox and Rac1-2). In the resting state of the cells, these proteins are dispersed in the cytosol, the membrane of granules and the plasma membrane. In order to synchronize the assembly of the cytosolic subunits on the membrane components of the oxidase, a fusion of the cytosolic proteins p47phox, p67phox and Rac1 named trimera was constructed. The trimera investigated in this paper is composed of the p47phox segment 1–286, the p67phox segment 1–212 and the mutated Rac1(Q61L). We demonstrate that the complex trimera-cyt b558 is functionally comparable to the one containing the separated subunits. Each of the subunits p47phox, p67phox and Rac1Q61L has kept its own activating property. The trimera is produced in an activated conformation as seen by circular dichroism. However, the presence of amphiphile is still necessary in a cell-free system to trigger superoxide anion production. The COS7gp91-p22 cells expressing the trimera produce continuously superoxide anion at high rate. This constitutive activity in cells can be of particular interest for understanding the NADPH oxidase functioning independently of signaling pathways.

The adaptor protein p40phox as a positive regulator of the superoxide-producing phagocyte oxidase.

Activation of the superoxide-producing phagocyte NADPH oxidase, crucial in host defense, requires the cytosolic proteins p67phox and p47phox. They translocate to the membrane upon cell stimulation and activate flavocytochrome b558, the membrane-integrated catalytic core of this enzyme system. The activators p67phox and p47phox form a ternary complex together with p40phox, an adaptor protein with unknown function, comprising the PX/PB2, SH3 and PC motif- containing domains: p40phox associates with p67phox via binding of the p40phox PC motif to the p67phox PB1 domain, while p47phox directly interacts with p67phox but not with p40phox. Here we show that p40phox enhances membrane translocation of p67phox and p47phox in stimulated cells, which leads to facilitated production of superoxide. The enhancement cannot be elicited by a mutant p40phox carrying the D289A substitution in PC or a p67phox with the K355A substitution in PB1, each being defective in binding to its respective partner. Thus p40phox participates in activation of the phagocyte oxidase by regulating membrane recruitment of p67phox and p47phox via the PB1–PC interaction with p67phox.

A time course of NADPH-oxidase up-regulation and endothelial nitric oxide synthase activation in the hippocampus following neurotrauma

Nicotinamide adenine dinucleotide phosphate oxidase (NADPH-oxidase; NOX) is a complex enzyme responsible for increased levels of reactive oxygen species (ROS), superoxide (O2•−). NOX-derived O2•− is a key player in oxidative stress and inflammation-mediated multiple secondary injury cascades (SIC) following traumatic brain injury (TBI). The O2•− reacts with nitric oxide (NO), produces various reactive nitrogen species (RNS), and contributes to apoptotic cell death. Following a unilateral cortical contusion, young adult rats were killed at various times postinjury (1, 3, 6, 12, 24, 48, 72, and 96h). Fresh tissue from the hippocampus was analyzed for NOX activity, and level of O2•−. In addition we evaluated the translocation of cytosolic NOX proteins (p67Phox, p47Phox, and p40Phox) to the membrane, along with total NO and the activation (phosphorylation) of endothelial nitric oxide synthase (p-eNOS). Results show that both enzymes and levels of O2•− and NO have time-dependent injury effects in the hippocampus. Translocation of cytosolic NOX proteins into membrane, NOX activity, and O2•− were also increased in a time-dependent fashion. Both NOX activity and O2•− were increased at 6h. Levels of p-eNOS increased within 1h, with significant elevation of NO at 12h post-TBI. Levels of NO failed to show a significant association with p-eNOS, but did associate with O2•−. NOX up-regulation strongly associated with both the levels of O2•− and the total NO. The initial 12h post-TBI are very important as a possible window of opportunity to interrupt SIC. It may be important to selectively target the translocation of cytosolic subunits for the modulation of NOX function.

Assembly of phagocyte NADPH oxidase: A concerted binding process?

BackgroundThe phagocyte NADPH-oxidase is a multicomponent enzyme that generates superoxide anions. It comprises a membrane redox component flavocytochrome b558 and four cytosolic proteins (p67phox, p47phox, p40phox and Rac) that must assemble to produce an active system. In this work we focused on the spatio-temporal control of the activation process of phagocyte NADPH oxidase. MethodsA wide range of techniques including fast kinetics with a stopped-flow apparatus and various combinations of the activating factors was used to test the order of assembly and the role of the p47phox–p67phox complex. ResultsThe data presented here are consistent with the absence of a catalytic role of the p47phox–p67phox interacting state and support the idea of independent binding sites for the cytosolic proteins on the flavocytochrome b558 allowing random binding order. However, the formation of the active complex appears to involve a synergistic process of binding of the activated cytosolic subunits to cytochrome b558. All partners should be in the vicinity for optimal assembly, a delay or the absence of one of the partners in this process seems to lead to a decrease in the efficiency of the catalytic core. Conclusion and general significanceThe activation and assembly of the NADPH oxidase components have to be achieved simultaneously for the formation of an efficient and optimal enzyme complex. This mechanism appears to be incompatible with continuous fast exchanges of the cytosolic proteins during the production of superoxide ion in the phagosome.

Arachidonic acid induces direct interaction of the p67(phox)-Rac complex with the phagocyte oxidase Nox2, leading to superoxide production.

The phagocyte NADPH oxidase Nox2, heterodimerized with p22(phox) in the membrane, is dormant in resting cells but becomes activated upon cell stimulation to produce superoxide, a precursor of microbicidal oxidants. Nox2 activation requires two switches to be turned on simultaneously: a conformational change of the cytosolic protein p47(phox) and GDP/GTP exchange on the small GTPase Rac. These proteins, in an active form, bind to their respective targets, p22(phox) and p67(phox), leading to productive oxidase assembly at the membrane. Although arachidonic acid (AA) efficiently activates Nox2 both in vivo and in vitro, the mechanism has not been fully understood, except that AA induces p47(phox) conformational change. Here we show that AA elicits GDP-to-GTP exchange on Rac at the cellular level, consistent with its role as a potent Nox2 activator. However, even when constitutively active forms of p47(phox) and Rac1 are both expressed in HeLa cells, superoxide production by Nox2 is scarcely induced in the absence of AA. These active proteins also fail to effectively activate Nox2 in a cell-free reconstituted system without AA. Without affecting Rac-GTP binding to p67(phox), AA induces the direct interaction of Rac-GTP-bound p67(phox) with the C-terminal cytosolic region of Nox2. p67(phox)-Rac-Nox2 assembly and superoxide production are both abrogated by alanine substitution for Tyr-198, Leu-199, and Val-204 in the p67(phox) activation domain that localizes the C-terminal to the Rac-binding domain. Thus the "third" switch (AA-inducible interaction of p67(phox)·Rac-GTP with Nox2) is required to be turned on at the same time for Nox2 activation.

Trans Arachidonic acid isomers inhibit NADPH-oxidase activity by direct interaction with enzyme components

NADPH-oxidase is an enzyme that represents, when activated, the major source of non-mitochondrial reactive oxygen species. In phagocytes, this production is an indispensable event for the destruction of engulfed pathogens. The functional NADPH-oxidase complex consists of a catalytic membrane flavocytochrome b (Cytb558) and four cytosolic proteins p47phox, p67phox, Rac and p40phox. The NADPH-oxidase activity is finely regulated spatially and temporally by cellular signaling events that trigger the translocation of the cytosolic subunits to its membrane partner involving post-translational modifications and activation by second messengers such as arachidonic acid (AA). Arachidonic acid in its natural cis-poly unsaturated form (C20:4) has been described to be an efficient activator of the enzyme in vivo and in vitro. In this work, we examined in a cell-free system whether a change of the natural cis geometry to the trans configuration, which could occur either by diet or be produced by the action of free radicals, may have consequences on the functioning of NADPH-oxidase. We showed the inability of mono-trans AA isomers to activate the NADPH-oxidase complex and demonstrated the inhibitory effect on the cis-AA-induced NADPH oxidase activation. The inhibition is mediated by a direct effect of the mono-trans AA which targets both the membrane fraction containing the cytb558 and the cytosolic p67phox. Our results suggest that the loss of the natural geometric feature (cis-AA) induces substantial structural modifications of p67phox that prevent its translocation to the complex.

Cannabinoids prevent lipopolysaccharide-induced neurodegeneration in the rat substantia nigra in vivo through inhibition of microglial activation and NADPH oxidase

We investigated the effects of synthetic cannabinoids, WIN55,212-2 and HU210, on LPS-injected rat substantia nigra in vivo. Intranigral injection of LPS resulted in a significant loss of nigral dopaminergic (DA) neurons, as determined by Nissl staining and TH immunohistochemistry. LPS-induced neurotoxicity was accompanied by microglial activation, as demonstrated by OX-42 immunohistochemistry. In parallel, Western blot analysis, ELISA assay and hydroethidine histochemistry revealed activation of NADPH oxidase, as demonstrated by increased translocation of the cytosolic proteins p47phox and p67phox, generation of reactive oxygen species (ROS) and increased level of proinflammatory cytokines (TNF-α and IL-1β), where degeneration of nigral DA neurons was evident. Interestingly, WIN55,212-2 and HU210 increased the survival of nigral DA neurons at 7days post-LPS treatment. Consistent with these results, cannabinoids inhibited activation of NADPH oxidase, ROS production and production of proinflammatory cytokines in the rat SN. The present data suggest that cannabinoids may be beneficial for the treatment of neurodegenerative diseases, such as PD, that are associated with microglial activation.

Relative contributions of mitochondria and NADPH oxidase to deoxycorticosterone acetate-salt hypertension in mice

We assessed the relative contribution of the mitochondrial respiratory chain and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase to deoxycorticosterone acetate (DOCA)-salt hypertension in mice. The daily mean arterial pressure was monitored by radiotelemetry in DOCA-salt-treated mice given vehicle or the mitochondrial respiratory chain complex I inhibitor rotenone. This treatment produced remarkable attenuation of DOCA-salt hypertension. Similar results were obtained with other inhibitors of mitochondrial function, including 5-hydroxydecanoate (specific for mitochondrial potassium-ATP channels), benzylguanidine (complexes I and III), and the cell-permeable manganese tetrakis (4-benzoic acid) porphyrin (a mimic of mitochondrial superoxide dismutase). In parallel with the blood pressure-lowering effect of rotenone, the DOCA-salt-induced increases in urinary 8-isoprostane excretion and in reactive oxygen species production of isolated kidney mitochondria were both significantly attenuated. Conversely, the DOCA-salt-induced reduction of urinary nitrate/nitrite excretion was significantly elevated. Following DOCA-salt treatment, mice deficient in NADPH oxidase subunits gp91(phox) or p47(phox) exhibited a partial attenuation of the hypertensive response at early but not later time points. Thus, the mitochondrial respiratory chain is a major source of oxidative stress in DOCA-salt hypertension, whereas NADPH oxidase may have a relatively minor role during the early stage of hypertension.

NADPH-oxidase activation and cognition in Alzheimer disease progression

Superoxide production via NADPH-oxidase (NOX) has been shown to play a role in a variety of neurological disorders, including Alzheimer disease (AD). To improve our understanding of the NOX system and cognitive impairment, we studied the various protein components of the phagocytic isoform (gp91phox, or NOX2) in the frontal and temporal cortex of age- and postmortem-matched samples. Individuals underwent antemortem cognitive testing and postmortem histopathologic assessment to determine disease progression and assignment to one of the following groups: no cognitive impairment (NCI), preclinical AD, mild cognitive impairment (MCI), early AD, and mild-to-moderate AD. Biochemical methods were used to determine overall NOX activity as well as levels of the various subunits (gp91phox, p67phox, p47phox, p40phox, and p22phox). Overall enzyme activity was significantly elevated in the MCI cohort in both cortical regions compared to the NCI cohort. This activity level remained elevated in the AD groups. Only the NOX cytosolic subunit proteins (p67phox, p47phox, and p40phox ) were significantly elevated with disease progression; the membrane-bound subunits (gp91phox and p22phox) remained stable. In addition, there was a robust correlation between NOX activity and the individual's cognitive status such that as the enzyme activity increased, cognitive performance decreased. Collectively, these data show that upregulated NADPH-oxidase in frontal and temporal cortex suggests that increases in NOX-associated redox pathways might participate in early pathogenesis and contribute to AD progression.

Coupling of 6‐phosphogluconate dehydrogenase with NADPH oxidase in neutrophils: Nox2 activity regulation by NADPH availability

It is well known that activation of the phagocyte NADPH oxidase requires the association of cytosolic proteins (p67-phox, p47-phox, p40-phox, and Rac) with the membrane cytochrome b(558), leading to its conformation change. Recently, the phagocyte NADPH oxidase complex was isolated in a constitutively active form. In this complex, 6-phosphogluconate dehydrogenase (6PGDH), an enzyme involved in the production of intracellular NADPH, was identified. This protein was absent from the oxidase complex isolated from B lymphocytes, suggesting a specific interaction with the neutrophil NADPH oxidase. To clarify the implication of 6PGDH in the NADPH oxidase activity, a siRNA approach was conducted in neutrophil-like PLB985 cells. NADPH oxidase activity of siRNA-transfected cells was shown to be decreased. Similar results were obtained in vitro, after reconstitution of oxidase activity with subcellular fractions isolated from siRNA-transfected cells. Interestingly, the Michaelis constant (K(m)) of Nox2 for NADPH increases in 6PGDH-depleted cells. Moreover, 6PGDH coimmunoprecipitated with oxidase cytosolic factors from cytosol of stimulated cells. Data suggested that the affinity of Nox2 for NADPH is increased in the presence of 6PGDH on cell stimulation. The present work proposes a new way of NADPH oxidase activity regulation by modulating Nox2 affinity for NADPH.

A Conserved Region between the TPR and Activation Domains of p67phox Participates in Activation of the Phagocyte NADPH Oxidase

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The membrane-integrated protein gp91(phox) serves as the catalytic core, because it contains a complete electron-transporting apparatus from NADPH to molecular oxygen for superoxide production. Activation of gp91(phox) requires the cytosolic proteins p67(phox), p47(phox), and Rac (a small GTPase). p67(phox), comprising 526 amino acids, moves upon cell stimulation to the membrane together with p47(phox) and there interacts with Rac; these processes are prerequisite for gp91(phox) activation. Here we show that a region of p67(phox) (amino acids 190-200) C-terminal to the Rac-binding domain is evolutionarily well conserved and participates in oxidase activation at a later stage in conjunction with an activation domain. Alanine substitution for Tyr-198, Leu-199, or Val-204 abrogates the ability of p67(phox) to support superoxide production by gp91(phox)-based oxidase as well as its related oxidases Nox1 and Nox3; the activation also involves other invariant residues such as Leu-193, Asp-197, and Gly-200. Intriguingly, replacement of Gln-192 by alanine or that of Tyr-198 by phenylalanine or tryptophan rather enhances superoxide production by gp91(phox)-based oxidase, suggesting a tuning role for these residues. Furthermore, the Y198A/V204A or L199A/V204A substitution leads to not only a complete loss of the activity of the reconstituted oxidase system but also a significant decrease in p67(phox) interaction with the gp91(phox) NADPH-binding domain, although these mutations affect neither the protein integrity nor the Rac binding activity. Thus the extended activation domain of p67(phox) (amino acids 190-210) containing the D(Y/F)LGK motif plays an essential role in oxidase activation probably by interacting with gp91(phox).

A Prenylated p47phox-p67phox-Rac1 Chimera Is a Quintessential NADPH Oxidase Activator: MEMBRANE ASSOCIATION AND FUNCTIONAL CAPACITY

The superoxide-generating NADPH oxidase complex of resting phagocytes includes cytochrome b(559), a membrane-associated heterodimer composed of two subunits (Nox2 and p22(phox)), and four cytosolic proteins (p47(phox), p67(phox), Rac, and p40(phox)). Upon stimulation, the cytosolic components translocate to the membrane, as the result of a series of interactions among the cytosolic components and among the cytosolic components and cytochrome b(559) and its phospholipid environment. We described the construction of a tripartite chimera (trimera) consisting of strategic domains of p47(phox), p67(phox), and Rac1, in which interactions among cytosolic components were replaced by fusion (Berdichevsky, Y., Mizrahi, A., Ugolev, Y., Molshanski-Mor, S., and Pick, E. (2007) J. Biol. Chem. 282, 22122-22139). We now fused green fluorescent protein (GFP) to the N terminus of the trimera and found the following. 1) The GFP-p47(phox)-p67(phox)-Rac1 trimera activates the oxidase in amphiphile-dependent and -independent (anionic phospholipid-enriched membrane) cell-free systems. 2) Geranylgeranylation of the GFP-trimera makes it a potent oxidase activator in unmodified (native) membranes and in the absence of amphiphile. 3) Prenylated GFP-trimera binds spontaneously to native membranes (as assessed by gel filtration and in-line fluorometry), forming a tight complex capable of NADPH-dependent, activator-independent superoxide production at rates similar to those measured in canonical cell-free systems. 4) Prenylation of the GFP-trimera supersedes completely the dependence of oxidase activation on the p47(phox) phox homology domain and, partially, on the Rac1 polybasic domain, but the requirement for Trp(193) in p47(phox) persists. Prenylated GFP-p47(phox)-p67(phox)-Rac1 trimera acts as a quintessential single molecule oxidase activator of potential use in high throughput screening of inhibitors.

Ketamine reduces inducible superoxide generation in human neutrophilsin vitroby modulating the p38 mitogen-activated protein kinase (MAPK)-mediated pathway

Many cellular stresses and inflammatory stimuli can activate p38 mitogen-activated protein kinase (MAPK), a serine/threonine kinase in the MAPK family. The different stimuli act via different receptors or signalling pathways to induce phosphorylation of the cytosolic protein p47(phox), one subunit of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Formyl-methionyl-leucyl-phenylalanine (fMLP) has been shown to induce the p38 MAPK phosphorylation during the respiratory burst in human neutrophils. Here, we show that treatment with S(+)-ketamine or R(-)-ketamine at different concentrations (50, 100, 200, 400 microM) reduced fMLP-induced superoxide anion generation and p47(phox) phosphorylation in neutrophils in a concentration-dependent manner (y = -0.093x + 93.35 for S(+)-ketamine and y = -0.0982x + 95.603 for R(-)-ketamine, respectively). While treatment with 50 microM ketamine inhibited fMLP-induced superoxide generation by 10%, treatment with 400 microM S(+)-ketamine and R(-)-ketamine reduced fMLP-induced superoxide generation to 60.5 +/- 8.3% and 60.0 +/- 8.5%, respectively, compared with that in neutrophils treated with fMLP alone. Furthermore, treatment with ketamine down-regulated both fMLP-induced p47(phox) and isoproterenol-induced p38 MAPK phosphorylation and superoxide production. Interestingly, treatment with SB203580, the p38 MAPK inhibitor, also mitigated fMLP-induced superoxide anion generation and p38 MAPK and p47(phox) phosphorylation as well as apoptosis in a concentration-dependent fashion in neutrophils. Therefore, ketamine racemes inhibited fMLP-induced superoxide anion generation and p47(phox) phosphorylation by modulating fMLP-mediated p38 MAPK activation in neutrophils.

Structure of the N-terminal Regulatory Domain of a Plant NADPH Oxidase and Its Functional Implications

Plant NADPH oxidases (Rboh, for respiratory burst oxidase homolog) produce reactive oxygen species that are key regulators of various cellular events including plant innate immunity. Rbohs possess a highly conserved cytoplasmic N-terminal region containing two EF-hand motifs that regulate Rboh activity. Rice (Oryza sativa) RbohB (OsRbohB) is regulated by the direct binding of a small GTPase (Rac1) to this regulatory region as well as by Ca(2+) binding to the EF-hands. Here, we present the atomic structure of the N-terminal region of OsRbohB. The structure reveals that OsRbohB forms a unique dimer stabilized by swapping the EF-hand motifs. We identified two additional EF-hand-like motifs that were not predicted from sequence data so far. These EF-hand-like motifs together with the swapped EF-hands form a structure similar to that found in calcineurin B. We observed conformational changes mediated by Ca(2+) binding to only one EF-hand. Structure-based in vitro pulldown assays and NMR titration experiments defined the OsRac1 binding interface within the coiled-coil region created by swapping the EF-hands. In addition, we demonstrate a direct intramolecular interaction between the N and C terminus, and that the complete N-terminal cytoplasmic region is required for this interaction. The structural features and intramolecular interactions characterized here might be common elements shared by Rbohs that contribute to the regulation of reactive oxygen species production.