Cytosolic Components Of NADPH Oxidase Research Articles

Mycobacterium tuberculosis protein PPE15 (Rv1039c) possesses eukaryote-like SH3 domain that interferes with NADPH Oxidase assembly and Reactive Oxygen Species production

Inhibition of Reactive Oxygen Species (ROS) is one of the strategies that Mycobacterium tuberculosis (Mtb) employs as its defence mechanism. In this study, the role of PPE15 (Rv1039c), a late-stage protein, has been investigated in modulating the cellular ROS. We discovered PPE15 to be a secretory protein that downregulates ROS generation in THP1 macrophages. Our in-silico analysis revealed the presence of a eukaryote-like SH3 (SH3e) domain in PPE15. The predicted SH3e-domain of PPE15 was found to interact with cytosolic components of NADPH Oxidase (NOX), p67phox and p47phox through molecular docking. In-vitro experiments using THP1 macrophages showed a diminished NADP/NADPH ratio, indicating reduced NOX activity. We also observed increased levels of p67phox and p47phox in the cytoplasmic fraction of PPE15 treated macrophages as compared to the plasma membrane fraction. To understand the role of the SH3e-domain in ROS modulation, this domain was deleted from the full-length PPE15 (PPE15-/-SH3). We observed an increase in cellular ROS and NADP/NADPH ratio in response to PPE15-/-SH3 protein. The interaction of PPE15-/-SH3 with p67phox or p47phox was also reduced in the cytoplasm, indicating migration of NOX subunits to the plasma membrane. Additionally, M. smegmatis expressing PPE15 was observed to be resistant to oxidative stress with significant intracellular survival in THP1 macrophages as compared to M. smegmatis expressing PPE15-/-SH3. These observations suggest that the SH3e-domain of PPE15 interferes with ROS generation by sequestering NOX components that inhibit NOX assembly at the cell membrane. Therefore, PPE15 acts like a molecular mimic of SH3-domain carrying eukaryotic proteins that can be employed by Mtb at late stages of infection for its survival. These findings give us new insights about the pathogen evading strategy of Mtb which may help in improving the therapeutics for TB treatment.

Neuroprotective effects of tannic acid in the postischemic brain via direct chelation of Zn2+

ABSTRACT Tannic acid (TA) is a polyphenolic compound that exerts protective effects under pathological conditions. The diverse mechanisms of TA can exert beneficial anti-oxidative, anti-inflammatory, and anti-cancer effects. Herein, we reported that TA affords robust neuroprotection in an animal model of stroke (transient middle cerebral artery occlusion; tMCAO) and exhibits Zn2+-chelating and anti-oxidative effects in primary cortical neurons. Following tMCAO induction, intravenous administration of TA (5 mg/kg) suppressed infarct formation by 32.9 ± 16.2% when compared with tMCAO control animals, improving neurological deficits and motor function. We compared the chelation activity under several ionic conditions and observed that TA showed better Zn2+ chelation than Cu2+. Furthermore, TA markedly decreased lactate dehydrogenase release following acute Zn2+ treatment and subsequently reduced the expression of p67 (a cytosolic component of NADPH oxidase), indicating the potential mechanism underlying TA-mediated Zn2+ chelation and anti-oxidative effects in primary cortical neurons. These findings suggest that anti-Zn2+ toxicity and anti-oxidative effects participate in the TA-mediated neuroprotective effects in the postischemic brain.

Role of l-amino acid oxidase isolated from Calloselasma rhodostoma venom on neutrophil NADPH oxidase complex activation

The action of Cr-LAAO, an l-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, on NADPH oxidase activation in isolated human neutrophil function was investigated. This enzyme has an intrinsic activity of hydrogen peroxide production. Cr-LAAO, in its native form, induces the ROS production in neutrophil and migration of cytosolic NADPH oxidase components p40phox, p47phox and p67phox to the membrane, and Rac, a GTPase protein member, with the involvement of intracellular signaling mediated by phospho PKC-α. In its inactive form, iCr-LAAO does not induce NADPH oxidase activation in neutrophil showing that the intrinsic enzymatic activity does not have a role in this process, suggesting that its primary structure is essential for the cell's stimulation. Accordingly, the data showed for the first time that the Cr-LAAO has a role in NADPH oxidase complex activation triggering relevant proinflammatory events in human neutrophils.

Endocytosis is required for exocytosis and priming of respiratory burst activity in human neutrophils.

Neutrophil generation of reactive oxygen species (ROS) is enhanced by exposure to pro-inflammatory agents in a process termed priming. Priming is depending on exocytosis of neutrophil granules and p47phox phosphorylation-dependent translocation of cytosolic NADPH oxidase components. Clathrin-mediated endocytosis was recently reported to be necessary for priming, but the mechanism linking endocytosis to priming was not identified. The present study examined the hypothesis that endocytosis regulates neutrophil priming by controlling granule exocytosis. Clathrin-mediated endocytosis by isolated human neutrophils was inhibited by chlorpromazine, monodansylcadaverine, and sucrose. Exocytosis of granule subsets was measured as release of granule components by ELISA or chemiluminescence. ROS generation was measured as extracellular release of superoxide as reduction of ferrocytochrome c. p38 MAPK activation and p47phox phosphorylation were measured by immunoblot analysis. Statistical analysis was performed using a one-way ANOVA with the Tukey-Kramer multiple-comparison test. Inhibition of endocytosis prevented priming of superoxide release by TNFα and inhibited TNFα stimulation and priming of exocytosis of all four granule subsets. Inhibition of endocytosis did not reduce TNFα-stimulated p38 MAPK activation or p47phox phosphorylation. Inhibition of NADPH oxidase activity blocked TNFα stimulation of secretory vesicle and gelatinase granule exocytosis. Endocytosis is linked to priming of respiratory burst activity through ROS-mediated control of granule exocytosis.

7-Keto-cholesterol and 25-hydroxy-1 cholesterol rapidly enhance ROS production in human neutrophils.

Oxysterols are cholesterol-oxygenated derivatives generated in the organism and also present in foods because of cholesterol oxidation during processing and storage. They are the natural ligands of liver X receptors (LXRs) and are generally recognized as hypocholesterolemic and anti-inflammatory molecules although this latter property is still controversial. Most oxysterol studies have been performed in macrophages, whereas the effects of oxysterols in neutrophils are poorly known. In this study, human neutrophils were exposed to two different oxysterols, 7-keto-cholesterol (7-k-chol) and 25-hydroxy-cholesterol (25-OH-chol), and their possible participation in inflammatory process was evaluated. Human neutrophils were incubated with 7-k-chol and 25-OH-chol, and ROS production, translocation of the NADPH oxidase cytosolic components, hemoxygenase-1 (HO-1) expression and lysozyme secretion were analyzed. An increase in ROS production was observed within a short period of time (minutes) with both molecules. These oxysterols also stimulated the cellular membrane translocation of the NADPH oxidase cytosolic components, p47phox and p67phox. On the other hand, HO-1 expression, a cytoprotector enzyme, is inhibited in human neutrophils upon oxysterols treatment. Moreover, both oxysterols were associated with high lysozyme enzyme secretion at 5 and 18h of incubation. The present paper describes for the first time that two oxysterols (7-k-chol and 25-OH-chol) enhance the ROS production within a short period of time in human neutrophils, stimulate the translocation of the cytosolic components of NADPH oxidase to the cellular membrane and increase lysozyme secretion. These data suggest that both oxysterols are able to activate pro-inflammatory effects in human neutrophils which contrasts with the role assigned to the oxysterols when they act through LXR at long time of incubation.

Arjunolic acid ameliorates reactive oxygen species via inhibition of p47phox-serine phosphorylation and mitochondrial dysfunction

Impaired cardiovascular function during acute myocardial infarction (MI) is partly associated with recruitment of activated polymorphonuclear neutrophils. The protective role of arjunolic acid (AA; 2,3,23-trihydroxy olean-12-en-28-oic acid) is studied in the modulation of neutrophil functions in vitro by measuring the reactive oxygen species (ROS) generation. Neutrophils were isolated from normal and acute MI mice to find out the efficacy of AA in reducing oxidative stress. Stimulation of neutrophils with phorbol-12-myristate-13-acetate (PMA) resulted in an oxidative burst of superoxide anion (O2−) and enhanced release of lysosomal enzymes. The treatment of neutrophils with PMA induced phosphorylation of Ser345 on p47phox, a cytosolic component of NADPH oxidase. Furthermore, we observed activated ERK induced phosphorylation of Ser345 in MI neutrophils. Treatment with AA significantly inhibited the phosphorylation of P47phox and ERK in the stimulated controls and MI neutrophils. Oxidative phosphorylation activities in MI cells were lower than in control, while the glycolysis rates were elevated in MI cells compared to the control. In addition, we observed AA decreased intracellular oxidative stress and reduced the levels of O2− in neutrophils. This study therefore identifies targets for AA in activated neutrophils mediated by the MAPK pathway on p47phox involved in ROS generation.

Activation of apocynin-sensitive NADPH oxidase (Nox2) activity in INS-1 832/13 cells under glucotoxic conditions

Several lines of recent evidence provided compelling evidence to suggest increased generation of reactive oxygen species (ROS) as causal for mitochondrial dysregulation and apoptosis in islet β-cells exposed to noxious stimuli including high glucose, lipids and proinflammatory cytokines. Studies along these lines are also suggestive of a significant contributory role for NADPH oxidase in the generation of ROS under the above conditions. We have recently reported a marked increase in the expression and activation of cytosolic components of NADPH oxidase (p47phox, Rac1) in cell culture models of glucotoxicity and in islets from T2DM animals (Zucker Diabetic Fatty rat) and humans. In this communication, we provide further evidence indicating significant activation of NADPH activity (~2-fold) in INS-1 832/13 cells exposed to chronic hyperglycemic conditions (20 mM; 48 h). We also report marked attenuation of this activity, by apocynin, a selective inhibitor of phagocyte-like NADPH oxidase (Nox2) activity. Together, our findings implicate Nox2 as a source for ROS generation in β-cells exposed to glucotoxic conditions.

Adipocyte expression of PU.1 transcription factor causes insulin resistance through upregulation of inflammatory cytokine gene expression and ROS production

We have reported previously that ETS family transcription factor PU.1 is expressed in mature adipocytes of white adipose tissue. PU.1 expression is increased greatly in mouse models of genetic or diet-induced obesity. Here, we show that PU.1 expression is increased only in visceral but not subcutaneous adipose tissues of obese mice, and the adipocytes are responsible for this increase in PU.1 expression. To further address PU.1's physiological function in mature adipocytes, PU.1 was knocked down in 3T3-L1 cells using retroviral-mediated expression of PU.1-targeting shRNA. Consistent with previous findings that PU.1 regulates its target genes, such as NADPH oxidase subunits and proinflammatory cytokines in myeloid cells, the mRNA levels of proinflammatory cytokines (TNFα, IL-1β, and IL-6) and cytosolic components of NADPH oxidase (p47phox and p40phox) were downregulated significantly in PU.1-silenced adipocytes. NADPH oxidase is a main source for reactive oxygen species (ROS) generation. Indeed, silencing PU.1 suppressed NADPH oxidase activity and attenuated ROS in basal or hydrogen peroxide-treated adipocytes. Silencing PU.1 in adipocytes suppressed JNK1 activation and IRS-1 phosphorylation at Ser(307). Consequently, PU.1 knockdown improved insulin signaling and increased glucose uptake in basal and insulin-stimulated conditions. Furthermore, knocking down PU.1 suppressed basal lipolysis but activated stimulated lipolysis. Collectively, these findings indicate that obesity induces PU.1 expression in adipocytes to upregulate the production of ROS and proinflammatory cytokines, both of which lead to JNK1 activation, insulin resistance, and dysregulation of lipolysis. Therefore, PU.1 might be a mediator for obesity-induced adipose inflammation and insulin resistance.

Stable accumulation of p67phox at the phagosomal membrane and ROS production within the phagosome

Production of ROS by the leukocyte NADPH oxidase is essential for the destruction of pathogenic bacteria inside phagosomes. The enzyme is a complex of cytosolic and membranous subunits that need to assemble upon activation. Biochemical data suggest that the complex is renewed continuously during activity. Furthermore, it is generally assumed that complex assembly and activity occur in parallel. However, information about the oxidase assembly in individual phagosomes in live cells is scarce. We studied the dynamic behavior of the crucial cytosolic NADPH oxidase component p67(phox) during phagocytosis by videomicroscopy. p67(phox) is involved in the regulation of electron flow from NADPH to oxygen, leading to superoxide radical formation inside the phagosome. p67(phox)-citrine, expressed in myeloid PLB-985 cells, accumulated at the phagosomal membrane during phagocytosis of yeast particles. Using photobleaching techniques (FRAP, FLIP), we demonstrated that p67(phox)-citrine diffused freely in this phagosomal membrane, but the phagosomal pool of p67(phox)-citrine did not exchange with the cytosolic pool. This result suggests that once assembled in the NADPH oxidase complex, p67(phox) is stable in this complex. Furthermore, the time of the presence of p67(phox)-citrine at the phagosome increased substantially in the presence of complement in the opsonizing serum compared with decomplemented serum. PI(3)P also accumulated around phagosomes for twice as long in the presence of complement. The presence of p67(phox)-citrine was correlated with the duration of phagosomal ROS production in different opsonization conditions. These data support the critical role of p67(phox) for ROS production on the level of individual phagosomes.

The NADPH oxidase cytosolic component p67phox is constitutively phosphorylated in human neutrophils: Regulation by a protein tyrosine kinase, MEK1/2 and phosphatases 1/2A

Neutrophils play a key role in host defense and inflammation through the production of superoxide anion and other reactive oxygen species (ROS) by the enzyme complex NADPH oxidase. The cytosolic NADPH oxidase component, p67phox, has been shown to be phosphorylated in human neutrophils but the pathways involved in this process are largely unknown. In this study, we show that p67phox is constitutively phosphorylated in resting human neutrophils and that neutrophil stimulation with PMA further enhanced this phosphorylation. Inhibition of the constitutively active serine/threonine phosphatases type 1 and type 2A (PP1/2A) by calyculin A resulted in the enhancement of p67phox phosphorylation. Constitutive and calyculin A-induced phosphorylation of p67phox was completely inhibited by the protein tyrosine kinase inhibitor genistein and partially inhibited by the MEK1/2 inhibitor PD98059, but was unaffected by GF109203X, wortmannin and SB203580, inhibitors of PKC, PI3K and p38MAP kinase, respectively. Two-dimensional phosphopeptide mapping revealed that constitutive and calyculin A-induced p67phox phosphorylation occurred on the same major sites. Interestingly, calyculin A enhanced formyl-Met-Leu-Phe (fMLP)-induced superoxide production, while genistein inhibited this process. Taken together, these results suggest that (i) p67phox undergoes a continual cycle of phosphorylation/dephosphorylation in resting cells; (ii) p67phox phosphorylation is controlled by MEK1/2 and an upstream tyrosine kinase; (iii) PP1/2A directly or indirectly antagonize this process. Thus, these pathways could play a role in regulating ROS production by human neutrophils at inflammatory sites.

Angiotensin II induces human astrocyte senescence through reactive oxygen species production

Angiotensin II (Ang II)-induced astrocyte senescence may be involved in cerebral ischemic injury and age-associated neurodegenerative disease. This study was conducted to determine the roles of reactive oxygen species production in Ang II-induced cellular senescence in cultured human astrocytes. Human astrocytes were stimulated with Ang II either with or without an angiotensin type 1 receptor blocker, CV11974, or an antioxidant, tempol. Application of Ang II to human astrocytes resulted in a concentration-dependent increase in staining for dihydroethidium. Ang II (100 nM for 30 min) increased the translocation of two cytosolic components of NADPH oxidase, p47phox and p67phox, to the cell membrane and formation of the complex of p47phox, p67phox and p22phox. Ang II concentration-dependently induced an increase in β-galactosidase staining. Pretreatment with CV11974 (100 nM) or tempol (3 mM) abolished Ang II-induced astrocyte β-galactosidase staining. Moreover, Ang II significantly upregulated p16 mRNA expression, which was inhibited by pretreatment with CV11974 or tempol. These findings indicate that superoxide production contributes to Ang II-induced astrocyte senescence.

Fluoxetine prevents LPS-induced degeneration of nigral dopaminergic neurons by inhibiting microglia-mediated oxidative stress

Lipopolysaccharide (LPS)-induced microglial activation causes degeneration of nigral dopaminergic (DA) neurons. Here, we examined whether fluoxetine prevents LPS-induced degeneration of DA in the rat substantia nigra (SN) in vivo. Seven days after LPS injection into the SN, immunostaining for tyrosine hydroxylase (TH) revealed a significant loss of nigral DA neurons. Parallel activation of microglia (visualized by OX-42 and ED1 immunohistochemistry), production of reactive oxygen species (ROS) (assessed by hydroethidine histochemistry), and degeneration of nigral DA neurons were also observed in the SN. Western blot analyses and double-label immunohistochemistry showed an increase in the expression of inducible nitric oxide synthase (iNOS) within activated microglia. LPS also induced translocation of p67 phox, the cytosolic component of NADPH oxidase, to the membrane of SN microglia, indicating activation of NADPH oxidase. The LPS-induced loss of nigral DA neurons was partially inhibited by fluoxetine, and the observed neuroprotective effects were associated with fluoxetine-mediated suppression of microglial NADPH oxidase activation and iNOS upregulation, and decreased ROS generation and oxidative stress. These results suggest that fluoxetine and analogs thereof may be beneficial for the treatment of neurodegenerative diseases, such as PD, that are associated with microglia-derived oxidative damage.

Subcellular localisation of the p40phox component of NADPH oxidase involves direct interactions between the Phox homology domain and F-actin

Cytosolic components of the NADPH oxidase interact with the actin cytoskeleton. These interactions are thought to be important for the activation of this enzyme system but they are poorly characterised at the molecular level. Here we have explored the interaction between the actin cytoskeleton and p40phox, one of the cytosolic components of NADPH oxidase. Full length p40phox expressed in COS cells co-localised with F-actin in a peripheral lamellar compartment. The co-localisation was lost after deletion of the Phox homology (PX) domain and the PX domain in isolation (p40PX) showed the same F-actin co-localisation as the full length protein. PX domains are known lipid-binding modules however, a mutant p40PX which did not bind lipids still co-localised with F-actin suggesting that lipid-independent interactions underlie the localisation. Affinity chromatography identified actin as a binding partner for p40PX in neutrophil extracts. Pure actin interacted with both p40phox and with p40PX suggesting it is a direct interaction. Disruption of the actin cytoskeleton with cytochalasin D resulted in actin rearrangement and concomitantly the localisation of full length p40phox proteins and that of p40PX changed. Thus p40PX is a dual F-actin/lipid-binding module and F-actin interactions with the PX domain dictate at least in part the intracellular localisation of the cytosolic p40phox subunit of the NADPH oxidase.

Prothrombin kringle-2-induced oxidative stress contributes to the death of cortical neurons in vivo and in vitro: Role of microglial NADPH oxidase

In the present study, we examine whether prothrombin kringle-2 (pKr-2), a domain of prothrombin distinct from thrombin and a potent microglial activator induces reactive oxygen species (ROS) generation through stimulation of microglial NADPH oxidase activity, and whether this phenomenon contributes to oxidative damage and consequent neurodegeneration. Intracortical injection of pKr-2 caused significant loss of cortical neurons in vivo after seven days, as evident from Nissl staining and immunohistochemical analysis using the neuronal-specific nuclear protein (NeuN) antibody. In parallel, pKr-2-activated microglia and ROS production were observed in rat cortex displaying degeneration of cortical neurons. Reverse transcription-PCR at various time points after pKr-2 administration disclosed early and transient expression of inducible nitric oxide synthase (iNOS) and proinflammatory cytokines, such as interleukin 1β (IL-1β). Co-localization of iNOS, IL-1β, and TNF-α within microglia was evident with double-label immunohistochemistry. Additionally, pKr-2 induced upregulation of cytosolic components of NADPH oxidase (p67 phox), translocation of cytosolic p67 phox protein to the membrane, and p67 phox expression in microglia in the cortex in vivo, signifying NADPH oxidase activation. The pKr-2-induced oxidation of proteins and loss of cortical neurons were partially inhibited by DPI, an NADPH oxidase inhibitor, and trolox, an antioxidant. Consistent with our hypothesis, following treatment with pKr-2 in vitro, neurotoxicity was detected exclusively in co-cultures of cortical neurons and microglia, but not in microglia-free neuron-enriched cortical cultures, indicating that microglia are required for pKr-2 neurotoxicity. Our results strongly suggest that pKr-2 as an endogenous compound participates in cortical neuron death through microglial NADPH oxidase-mediated oxidative stress.

Detection of Non-ΔGTNCF-1Mutations in Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) is a rare inherited disorder caused by mutations in the subunits of the NADPH oxidase complex, leaving phagocytes unable to produce superoxide and thereby unable to kill invading microorganisms. A subgroup of CGD patients (approximately 20%) is reported to have mutations in NCF-1 encoding p47-phox, which is part of the cytosolic component of NADPH oxidase. More than 94% of these patients share the same mutation, a 2 bp GT deletion in the GTGT dinucleotide repeat in the start of exon 2. The presence of two pseudogenes more than 98% homologous to the functional NCF-1 has complicated the identification of other mutations in the gene. The aim of this study was to find a general technique for detection of non-GT deletion mutations in the coding region of NCF-1. A technique involving GeneScan analysis followed by amplification of cDNA with intact dinucleotide repeat was set up and used to identify a novel mutation in exon 7 of NCF-1 in a patient with autosomal recessive CGD, explaining the disease by changing a UGG codon to a premature UGA STOP codon. The method is generally applicable for the detection of NCF-1 mutations in patients with suspected CGD.

Proteomic analysis of Legionella-containing phagosomes isolated from Dictyostelium

Legionella pneumophila, the agent of Legionnaires’ disease, replicates intracellularly within specialized phagosomes of human macrophages and amoebae. In this study, we have developed a protocol for the isolation of Legionella-containing phagosomes from Dictyostelium discoideum. Cell fractionation, two-dimensional gel electrophoresis and MALDI-TOF MS combined with genomic data identified 157 phagosome host proteins. In addition to proteins with an evident role in phagosome maturation, we identified proteins for which a function remains to be elucidated. Possible interactions of coronin with cytosolic NADPH oxidase components and protein kinase C inhibitors which together may lead to an inhibition of phagosomal superoxide generation are discussed. Comparative proteomics of phagosomes containing highly virulent L. pneumophila Corby versus less virulent L. hackeliae revealed distinctive protein expression patterns, e.g., an abundance of RhoGDI in L. hackeliae degrading phagosomes versus little RhoGDI in L. pneumophila Corby replicative phagosomes. We present a kinetic dissection of phagosome maturation including the complex alterations of the phagosome protein composition. A reference flow chart suggests so far unrecognized consequences of infection for host cell physiology, actin degradation on phagosomes, and a putative cysteine proteinase inhibitor interference with lysosomal enzyme sorting and activation processes.

Regulation of the phagocyte NADPH oxidase activity: phosphorylation of gp91phox/NOX2 by protein kinase C enhances its diaphorase activity and binding to Rac2, p67phox, and p47phox

Neutrophils generate microbicidal oxidants through activation of a multicomponent enzyme called NADPH oxidase. During activation, the cytosolic NADPH oxidase components (p47(phox), p67(phox), p40(phox), and Rac2) translocate to the membranes, where they associate with flavocytochrome b(558), which is composed of gp91(phox)/NOX2 and p22(phox), to form the active system. During neutrophil stimulation, p47(phox), p67(phox), p40(phox), and p22(phox) are phosphorylated; however, the phosphorylation of gp91(phox)/NOX2 and its potential role have not been defined. In this study, we show that gp91(phox) is phosphorylated in stimulated neutrophils. The gp91(phox) phosphoprotein is absent in neutrophils from chronic granulomatous disease patients deficient in gp91(phox), which confirms that this phosphoprotein is gp91(phox). The protein kinase C inhibitor GF109203X inhibited phorbol 12-myristate 13-acetate-induced phosphorylation of gp91(phox), and protein kinase C (PKC) phosphorylated the recombinant gp91(phox)- cytosolic carboxy-terminal flavoprotein domain. Two-dimensional tryptic peptide mapping analysis showed that PKC phosphorylated the gp91(phox)-cytosolic tail on the same peptides that were phosphorylated on gp91(phox) in intact cells. In addition, PKC phosphorylation increased diaphorase activity of the gp91(phox) flavoprotein cytosolic domain and its binding to Rac2, p67(phox), and p47(phox). These results demonstrate that gp91(phox) is phosphorylated in human neutrophils by PKC to enhance its catalytic activity and assembly of the complex. Phosphorylation of gp91(phox)/NOX2 is a novel mechanism of NADPH oxidase regulation.

Mutations in the PX−SH3A Linker of p47phox Decouple PI(3,4)P2 Binding from NADPH Oxidase Activation

NADPH oxidase is essential in the human innate immune response. p47 (phox), a cytosolic NADPH oxidase component, plays a regulatory role in the activation of NADPH oxidase. Our manipulation of p47 (phox) by mutation and amino acid deletion shows that the linker region between the PX and N-terminal SH3 domain plays a role in blocking the binding of the phosphoinositide 3,4-bisphosphate [PI(3,4)P2], a lipid second messenger generated upon neutrophil activation. Replacement of linker residues 151-158 with glycine alters NMR-measured spin lattice relaxation rates and sedimentation velocity compared to those of the wild-type protein, suggesting that the PX domain is released from its autoinhibited conformation. Liposome binding and surface plasmon resonance experiments confirm this result, showing that this mutant has a similar binding affinity for the isolated PX domain toward PI(3,4)P2. However, an in vitro NADPH oxidase activity assay reveals that this glycine mutant of the full-length protein greatly reduced NADPH oxidase activity upon activation even though it displayed PI(3,4)P2 binding activity comparable to that of the isolated PX domain. Our results highlight an active role of the PX-SH3 linker region in maintaining p47 (phox) in its fully autoinhibited form and demonstrate that binding of p47 (phox) to membrane phospholipids is mechanistically distinct from NADPH oxidase activation.

Possible involvement of 12-lipoxygenase activation in glucose-deprivation/reload-treated neurons

The aim of this study was to clarify whether 12-lipoxygenase (12-LOX) activation was involved in reactive oxygen species (ROS) generation, extensive poly(ADP-ribose) polymerase (PARP) activation and neuronal death induced by glucose-deprivation, followed by glucose-reload (GD/R). The decrease of neuronal viability and accumulation of poly(ADP-ribose) induced by GD/R were prevented 3-aminobenzamide, a representative PARP inhibitor, demonstrating this treatment protocol caused the same oxidative stress with the previously reported one. The PARP activation, ROS generation and decrease of neuron viability induced by GD/R treatment were almost completely abolished by an extracellular zinc chelator, CaEDTA. p47(phox), a cytosolic component of NADPH oxidase was translocated the membrane fraction by GD/R, indicating its activation, but it did not generate detectable ROS. Surprisingly, pharmacological inhibition of NADPH oxidase with apocynin and AEBSF further decreased the decreased neuron viability induced by GD/R. On the other hand, AA861, a 12-LOX inhibitor, prevented ROS generation and decrease of neuron viability caused by GD/R. Interestingly, an antioxidant, N-acetyl-l-cysteine rescued the neurons from GD/R-induced oxidative stress, implying effectiveness of antioxidant administration. These findings suggested that activation of 12-LOX, but not NADPH oxidase, following to zinc release might play an important role in ROS generation and decrease of viability in GD/R-treated neurons.

Tripartite Chimeras Comprising Functional Domains Derived from the Cytosolic NADPH Oxidase Components p47phox, p67phox, and Rac1 Elicit Activator-independent Superoxide Production by Phagocyte Membranes: AN ESSENTIAL ROLE FOR ANIONIC MEMBRANE PHOSPHOLIPIDS

The superoxide-generating NADPH oxidase is converted to an active state by the assembly of a membrane-localized cytochrome b(559) with three cytosolic components: p47(phox), p67(phox), and GTPase Rac1 or Rac2. Assembly involves two sets of protein-protein interactions: among cytosolic components and among cytosolic components and cytochrome b(559) within its lipid habitat. We circumvented the need for interactions among cytosolic components by constructing a recombinant tripartite chimera (trimera) consisting of the Phox homology (PX) and Src homology 3 (SH3) domains of p47(phox), the tetratricopeptide repeat and activation domains of p67(phox), and full-length Rac1. Upon addition to phagocyte membrane, the trimera was capable of oxidase activation in vitro in the presence of an anionic amphiphile. The trimera had a higher affinity (lower EC(50)) for and formed a more stable complex (longer half-life) with cytochrome b(559) compared with the combined individual components, full-length or truncated. Supplementation of membrane with anionic but not neutral phospholipids made activation by the trimera amphiphile-independent. Mutagenesis, truncations, and domain replacements revealed that oxidase activation by the trimera was dependent on the following interactions: 1) interaction with anionic membrane phospholipids via the poly-basic stretch at the C terminus of the Rac1 segment; 2) interaction with p22(phox) via Trp(193) in the N-terminal SH3 domain of the p47(phox) segment, supplementing the electrostatic attraction; and 3) an intrachimeric bond among the p67(phox) and Rac1 segments complementary to their physical fusion. The PX domain of the p47(phox) segment and the insert domain of the Rac1 segment made only minor contributions to oxidase assembly.