Cytosolic P47phox Research Articles

NADPH oxidase activator p67phox behaves in solution as a multidomain protein with semi-flexible linkers

The NADPH oxidase complex is involved in the destruction of phagocytosed pathogens through the production of reactive oxygen species. This activatable complex consists of a membranous heterodimeric flavocytochrome b, a small G-protein Rac1/Rac2 and cytosolic factors, p47(phox), p67(phox) and p40(phox). p67(phox), due to its modular structure, is the NADPH oxidase component for which global structure information is most scarce despite its mandatory role in activation and its central position in the whole complex organization. Indeed, p67(phox) is the only factor establishing interaction with all others. In this study, we report the SAXS analysis of p67(phox). Our data reveals that p67(phox) behaves as a multidomain protein with semi-flexible linkers. On the one hand, it appears to be a very elongated molecule with its various domains organized as beads on a string. Linkers are predicted to be partially or mainly unstructured and features of our experimental data do point towards inter-domain flexibility. On the other hand, our work also suggests that the protein is not as extended as unstructured linkers could allow, thereby implying the existence of intra-molecular interactions within p67(phox). We suggest that the dual character of p67(phox) conformation in solution is central to ensure the numerous interactions to be accommodated.

Inhibition of the human neutrophil NADPH oxidase by Coxiella burnetii

Coxiella burnetii is an obligate intracellular Gram-negative pathogen. A notable feature of C. burnetii is its ability to replicate within acidic phagolysosomes; however, the mechanisms utilized in evading host defenses are not well defined. Here, we investigated human neutrophil phagocytosis of C. burnetii (Nine Mile, phase II; NMII) and the effect of phagocytosed organisms on neutrophil reactive oxygen species (ROS) production. We found that opsonization with immune serum substantially enhanced phagocytosis of NMII. Human neutrophils phagocytosing opsonized NMII generated very little ROS compared to cells phagocytosing opsonized Staphylococcus aureus, Escherichia coli, or zymosan. However, phagocytosis of NMII did not affect the subsequent ROS response to a soluble agonist, indicating inhibition was localized to the phagolysosome and was not a global effect. Indeed, analysis of NADPH oxidase assembly in neutrophils after phagocytosis showed that translocation of cytosolic NADPH oxidase proteins, p47 phox and p67 phox , to the membrane was absent in cells phagocytosing NMII, as compared to cells phagocytosing S. aureus or activated by phorbol myristate acetate. Thus, phagocytosed NMII is able to disrupt assembly of the human neutrophil NADPH oxidase, which represents a novel virulence mechanism for this organism and appears to be a common mechanism of virulence for many intracellular pathogens.

NADPH oxidase signaling regulates Epithelial Sodium Channel (ENaC) activity

In general, over‐production of superoxide (O2−) in the kidneys leads to serious renal disorders. However, when released in low quantities in a controlled manner, O2− may serve as an important signaling molecule in epithelial cells. Presently, we examine whether sodium transporting epithelial cells can control release of O2− , through regulated NADPH oxidase activity, in order to regulate ENaC in renal epithelia. First, we showed that A6 distal nephron cells express each of the critical NADPH oxidase (NOX) components that catalyze the production of O2− (membrane bound gp91phox and p22phox; cytosolic p67 phox and p47 phox subunits; and small G protein Rac1) using western blot. Since complete activation of NOX is believed to occur following Rac1activation, we then tested whether inhibition or activation of Rac1 influenced ENaC activity. We report that, indeed, 1μM NSC23766 inhibition of Rac1 significantly decreases single channel measurements of ENaC activity from 1.16+.27 to .50+.11 (mean NPo values+SE, n=6, p<0.05). Conversely, 10ng/mL EGF activation of Rac1 significantly increased ENaC NPo values, on average, from .93+.25 to 1.30+.31 in 6 independent observations (p<0.05). HPLC measurements verify that Rac1 inhibition decreases O2− release. Very briefly, we conclude that NADPH‐oxidase induced O2− signaling is a new pathway for ENaC regulation.

Regulation of NADPH Oxidase in Vascular Endothelium: The Role of Phospholipases, Protein Kinases, and Cytoskeletal Proteins

The generation of reactive oxygen species (ROS) in the vasculature plays a major role in the genesis of endothelial cell (EC) activation and barrier function. Of the several potential sources of ROS in the vasculature, the endothelial NADPH oxidase family of proteins is a major contributor of ROS associated with lung inflammation, ischemia/reperfusion injury, sepsis, hyperoxia, and ventilator-associated lung injury. The NADPH oxidase in lung ECs has most of the components found in phagocytic oxidase, and recent studies show the expression of several homologues of Nox proteins in vascular cells. Activation of NADPH oxidase of nonphagocytic vascular cells is complex and involves assembly of the cytosolic (p47(phox), p67(phox), and Rac1) and membrane-associated components (Noxes and p22(phox)). Signaling pathways leading to NADPH oxidase activation are not completely defined; however, they do appear to involve the cytoskeleton and posttranslation modification of the components regulated by protein kinases, protein phosphatases, and phospholipases. Furthermore, several key components regulating NADPH oxidase recruitment, assembly, and activation are enriched in lipid microdomains to form a functional signaling platform. Future studies on temporal and spatial localization of Nox isoforms will provide new insights into the role of NADPH oxidase-derived ROS in the pathobiology of lung diseases.

Effect of Seven Tricyclic Diterpenoids from Needles ofTaxus mediavar.Hicksiion Stimulus-Induced Superoxide Generation, Tyrosyl or Serine/Threonine Phosphorylation and Translocation of Cytosolic Compounds to the Cell Membrane in Human Neutrophils

Taxol has been widely used as an anticancer drug for ovarian, breast, lung and prostate cancer. Some kinds of Taxus plants are widely distributed in the Northeast Asia region. We have isolated seven tricyclic diterpenoids, taxinine, taxagifine, 5-O-cinnamoyltaxacin I triacetate, 5-decinnamoyltaxinine J, 5-cinnamoyl-9-acetyltaxicin I, taxacin and taxol from the needles of Taxus media var. Hicksii, and investigated their effects on stimulus-induced superoxide generation and translocation of cytosolic compounds to the cell membrane in human neutrophils. Six tricyclic diterpenoids used in this experiment suppressed the superoxide generation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) and arachidonic acid (AA) in a concentration-dependent manner. Taxinine significantly suppressed the superoxide generation induced by phorbol 12-myristate 13-acetate (PMA). The compounds also suppressed fMLP- and AA-induced tyrosyl or PMA-induced serine/threonine phosphorylation, and translocation of cytosolic compounds, p47 (phox), p67 (phox) and Rac to the cell membrane in parallel with the suppression of the stimulus-induced superoxide generation.

Angiotensin II inhibits the Na+-K+ pump via PKC-dependent activation of NADPH oxidase

The sarcolemmal Na(+)-K(+) pump, pivotal in cardiac myocyte function, is inhibited by angiotensin II (ANG II). Since ANG II activates NADPH oxidase, we tested the hypothesis that NADPH oxidase mediates the pump inhibition. Exposure to 100 nmol/l ANG II increased superoxide-sensitive fluorescence of isolated rabbit ventricular myocytes. The increase was abolished by pegylated superoxide dismutase (SOD), by the NADPH oxidase inhibitor apocynin, and by myristolated inhibitory peptide to epsilon-protein kinase C (epsilonPKC), previously implicated in ANG II-induced Na(+)-K(+) pump inhibition. A role for epsilonPKC was also supported by an ANG II-induced increase in coimmunoprecipitation of epsilonPKC with the receptor for the activated kinase and with the cytosolic p47(phox) subunit of NADPH oxidase. ANG II decreased electrogenic Na(+)-K(+) pump current in voltage-clamped myocytes. The decrease was abolished by SOD, by the gp91ds inhibitory peptide that blocks assembly and activation of NADPH oxidase, and by epsilonPKC inhibitory peptide. Since colocalization should facilitate NADPH oxidase-dependent regulation of the Na(+)-K(+) pump, we examined whether there is physical association between the pump subunits and NADPH oxidase. The alpha(1)-subunit coimmunoprecipitated with caveolin 3 and with membrane-associated p22(phox) and cytosolic p47(phox) NADPH oxidase subunits at baseline. ANG II had no effect on alpha(1)/caveolin 3 or alpha(1)/p22(phox) interaction, but it increased alpha(1)/p47(phox) coimmunoprecipitation. We conclude that ANG II inhibits the Na(+)-K(+) pump via PKC-dependent NADPH oxidase activation.

A fluorescently tagged C-terminal fragment of p47phox detects NADPH oxidase dynamics during phagocytosis.

The assembly of cytosolic p47(phox) and p67(phox) with flavocytochrome b(558) at the membrane is crucial for activating the leukocyte NADPH oxidase that generates superoxide for microbial killing. p47(phox) and p67(phox) are linked via a high-affinity, tail-to-tail interaction involving a proline-rich region (PRR) and a C-terminal SH3 domain (SH3b), respectively, in their C-termini. This interaction mediates p67(phox) translocation in neutrophils, but is not required for oxidase activity in model systems. Here we examined phagocytosis-induced NADPH oxidase assembly, showing the sequential recruitment of YFP-tagged p67(phox) to the phagosomal cup, and, after phagosome internalization, a probe for PI(3)P followed by a YFP-tagged fragment derived from the p47(phox) PRR. This fragment was recruited in a flavocytochrome b(558)-dependent, p67(phox)-specific, and PI(3)P-independent manner. These findings indicate that p47PRR fragment probes the status of the p67(phox) SH3b domain and suggest that the p47(phox)/p67(phox) tail-to-tail interaction is disrupted after oxidase assembly such that the p67(phox)-SH3b domain becomes accessible. Superoxide generation was sustained within phagosomes, indicating that this change does not correlate with loss of enzyme activity. This study defines a sequence of events during phagocytosis-induced NADPH oxidase assembly and provides experimental evidence that intermolecular interactions within this complex are dynamic and modulated after assembly on phagosomes.

Advanced oxidation protein products induce mesangial cell perturbation through PKC-dependent activation of NADPH oxidase

Mesangial deposition of extracellular matrix (ECM) is a hallmark of several glomerular diseases including diabetic nephropathy. Accumulation of advanced oxidation protein products (AOPPs) has been found in diabetes and chronic kidney disease and linked to mesangial ECM deposition and progressive glomerulosclerosis in these disorders. Although emerging evidence implicates AOPPs as the renal pathogenic factors, the underlying mechanisms have not been investigated. Here, using cultured rat mesangial cells (MCs) as a model, we identify AOPPs as the important mediators for activation of MC NADPH oxidase. Exposure of MCs to AOPPs, through membrane-associated phosphorylation of PKCalpha, induced rapid phosphorylation of cytosolic p47(phox) and its membrane translocation, enhanced interaction of p47(phox) with the membrane components p22(phox) and Nox4, and increased expression of these key regulatory subunits of NADPH oxidase. Challenge with AOPPs triggered cytosolic superoxide generation, resulting in upregulation of fibronectin and collagen IV genes and proteins and overexpression of TGF-beta1 via a PKC-NADPH oxidase-dependent pathway, as these downstream events were blocked by the inhibitors of PKC, inhibitors of NADPH oxidase, or the cytosolic superoxide scavenger. These data provide new information for understanding the molecular basis underlying AOPP-induced MC perturbation and might be a central step toward development of new interventions.

FcγR-stimulated activation of the NADPH oxidase: phosphoinositide-binding protein p40phox regulates NADPH oxidase activity after enzyme assembly on the phagosome

AbstractThe phagocyte NADPH oxidase generates superoxide for microbial killing, and includes a membrane-bound flavocytochrome b558 and cytosolic p67phox, p47phox, and p40phox subunits that undergo membrane translocation upon cellular activation. The function of p40phox, which binds p67phox in resting cells, is incompletely understood. Recent studies showed that phagocytosis-induced superoxide production is stimulated by p40phox and its binding to phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide enriched in membranes of internalized phagosomes. To better define the role of p40phox in FcγR-induced oxidase activation, we used immunofluorescence and real-time imaging of FcγR-induced phagocytosis. YFP-tagged p67phox and p40phox translocated to granulocyte phagosomes before phagosome internalization and accumulation of a probe for PI3P. p67phox and p47phox accumulation on nascent and internalized phagosomes did not require p40phox or PI3 kinase activity, although superoxide production before and after phagosome sealing was decreased by mutation of the p40phox PI3P-binding domain or wortmannin. Translocation of p40phox to nascent phagosomes required binding to p67phox but not PI3P, although the loss of PI3P binding reduced p40phox retention after phagosome internalization. We conclude that p40phox functions primarily to regulate FcγR-induced NADPH oxidase activity rather than assembly, and stimulates superoxide production via a PI3P signal that increases after phagosome internalization.

Apocynin, NADPH Oxidase, and Vascular Cells

Apocynin (4-hydroxy-3-methoxyacetophenone), isolated from the traditional medicinal plant Picrorhiza kurroa , is a naturally occurring methoxy-substituted catechol, experimentally used as an inhibitor of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase.1 Activated neutrophil NADPH oxidase, a multisubunit complex composed of membrane-associated gp91phox (Nox2) and p22phox and cytosolic subunits, p47phox, p67phox, and p40phox, produces superoxide anion, a precursor of microbicidal reactive oxygen species (ROS), thereby playing a crucial role in host defense. Apocynin inhibits the release of superoxide anion by NADPH oxidase by blocking migration of p47phox to the membrane, critically involved in initiating assembly of the functional NADPH oxidase complex.1 The underlying chemistry of apocynin’s actions has been elucidated in phagocytic cells.2 Apocynin needs to be activated (oxidized) for it to inhibit the oxidase. In the presence of H2O2 and myeloperoxidase (MPO), abundant in neutrophils, apocynin is oxidized and has as products dimer and trimer derivatives resulting in diapocynin formation, the metabolically active compound of apocynin. The reactivity of apocynin radical with thiol compounds is possibly the mechanism involved in the inhibitory effect of apocynin on the NADPH oxidase complex.2,3 In the early 1990s, with the discovery that cardiovascular cells possess functionally active NADPH oxidase critically involved in ROS generation and oxidative stress in the vascular wall, many investigators extended neutrophil findings and used apocynin as a pharmacological agent to specifically inhibit the vascular cell oxidase. Convincing data from numerous in vitro studies demonstrated that, in endothelial cells, vascular smooth muscle cells, and adventitial fibroblasts, apocynin blocks NADPH oxidase activity and superoxide anion generation.4,5 In vivo studies, in which mice and …

Les Nox/Duox : une nouvelle famille de NADPH oxydases

Reactive oxygen species (ROS) generated by the NADPH oxidases are conventionally thought to be cytotoxic and mutagenic and at high levels induce an oxidative stress response. The phagocyte NADPH oxidase catalyzes the NADPH-dependent reduction of molecular oxygen to generate superoxide O2-., which can dismute to generate ROS species. Together, these ROS participate in host defence by killing or damaging invading microbes. Flavocytochrome b558 is the catalytic core of the phagocyte NADPH oxidase and consists of a large glycoprotein gp91phox or Nox-2 and a small protein p22phox. The other components of the NADPH oxidase are cytosolic proteins, namely p67phox, p47phox, p40phox and Rac. A defect in any of the genes encoding gp91phox, p22phox, p67phox or p47phox results in chronic granulomatous disease, a genetic disorder characterized by severe and recurrent infections. Evidence is rapidly accumulating that low level of ROS were produced by NADPH oxidase homologs in non-phagocytic cells. To date, six human homologs (Nox-1, Nox-3, Nox-4, Nox-5, Duox-1 and Duox-2) have been recently identified in a variety of non-phagocytic cells. The identification of Nox-1 was quickly followed by the cloning of Nox-3, Nox-4, and Nox-5. In parallel, two very large members of the Nox family were discovered, namely Duox-1 and Duox-2, initially also referred to as thyroid oxidases. The physiological functions of Nox-dependent ROS generation are in progress and still require detailed characterization. Activation mechanisms and tissue distribution of the different members of the Nox family are very different, suggesting distinct physiological functions. Nox family enzymes are likely to be involved in a variety of physiological events including cell proliferation, host defence, differentiation, apoptosis, senescence and activation of growth-related signaling pathways. An increase and a decrease in the function of Nox enzymes can contribute to a wide range of pathological processes.

Effect of Five Triterpenoid Compounds from the Buds of Aralia elata on Stimulus-Induced Superoxide Generation, Tyrosyl Phosphorylation and Translocation of Cytosolic Compounds to the Cell Membrane in Human Neutrophils

The buds of Aralia elata (Miq.) Seem (Japanese angelica tree) have long been used as a tonic, antiarthritic and antidiabetic agent in China and Japan. We have isolated five triterpenoids, congmuyanosides A, C, D, echinocystic acid and 3-O-[beta-D-glucopyranosyl(1-->2)-beta-D-glucopyranosyl]-hederagenin from the buds of Aralia elata , and investigated their effects on stimulus-induced superoxide generation in human neutrophils. Congmuyanoside A, echinocystic acid and 3-O-[beta-D-glucopyranosyl(1-->2)-beta-D-glucopyranosyl]-hederagenin suppressed the superoxide generation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) in a concentration-dependent manner. Congmuyanosides C, D and echinocystic acid significantly suppressed the superoxide generation induced by phorbol 12-myristate 13-acetate (PMA) and arachidonic acid (AA). The compounds also suppressed fMLP- and AA-induced tyrosyl or PMA-induced serine/threonine phosphorylation and translocation of cytosolic compounds, p47 (phox), p67 (phox) and Rac to the cell membrane.

Effect of Agkistroden blomhoffi (mamushi) on superoxide generation in human neutrophils

Background The purpose of this study is to investigate the effect of Agkistroden blomhoffi (mamushi) aqueous extract on stimulus-induced superoxide generation in human neutrophils. Methods Superoxide generation was assayed by measuring the reduction of ferricytochrome C ( cyt c) using a dual-beam spectrophotometer. The phosphorylation of neutrophil proteins and the translocation of cytosolic compounds to the cell membrane were detected using specific monoclonal antibodies. Results Mamushi aqueous extract markedly suppressed fMLP-induced superoxide generation in a concentration-dependent manner. fMLP-induced translocation of cytosolic compounds p47 phox, p67 phox and Rac to the cell membrane was suppressed in parallel to the suppression of superoxide generation. The fractions with MW below 10 kDa dose-dependently suppressed tyrosyl phosphorylation of 45.0, 77.6 and 99.5 kDa proteins in fMLP-treated human neutrophils; the inhibition of tyrosyl phosphorylation was in parallel to that of the fMLP-induced translocation of p47 phox to the cell membrane and superoxide generation. Conclusions Mamushi aqueous extract markedly suppressed fMLP-induced superoxide generation in human neutrophils and also suppressed the translocation of cytosolic compounds to the cell membrane and tyrosyl phosphorylation of proteins. The efficacious components are the fractions with MW below 10 kDa. O-phosphoserylethanolamine, a new dipeptide isolated from mamushi, also suppressed fMLP-induced superoxide generation in human neutrophils in a dose-dependent manner.

Effects of p47phox C Terminus Phosphorylations on Binding Interactions with p40phox and p67phox: STRUCTURAL AND FUNCTIONAL COMPARISON OF p40phox and p67phox SH3 DOMAINS

The neutrophil NADPH oxidase produces superoxide anions in response to infection. This reaction is activated by association of cytosolic factors, p47phox and p67phox, and a small G protein Rac with the membranous flavocytochrome b558. Another cytosolic factor, p40phox, is associated to the complex and is reported to play regulatory roles. Initiation of the NADPH oxidase activation cascade has been reported as consecutive to phosphorylation on serines 359/370 and 379 of the p47phox C terminus. These serines surround a polyproline motif that can interact with the Src homology 3 (SH3) module of p40phox (SH3p40) or the C-terminal SH3 of p67phox (C-SH3p67). The latter one presents a higher affinity in the resting state for p47phox. A change in SH3 binding preference following phosphorylation has been postulated earlier. Here we report the crystal structures of SH3p40 alone or in complex with a 12-residue proline-rich region of p47phox at 1.46 angstrom resolution. Using intrinsic tryptophan fluorescence measurements, we compared the affinity of the strict polyproline motif and the whole C terminus peptide with both SH3p40 and C-SH3p67. These data reveal that SH3p40 can interact with a consensus polyproline motif but also with a noncanonical motif of the p47phox C terminus. The electrostatic surfaces of both SH3 are very different, and therefore the binding preference for C-SH3p67 can be attributed to the polyproline motif recognition and particularly to the Arg-368p47 binding mode. The noncanonical motif contributes equally to interaction with both SH3. The influence of serine phosphorylation on residues 359/370 and 379 on the affinity for both SH3 domains has been checked. We conclude that contrarily to previous suggestions, phosphorylation of Ser-359/370 does not modify the SH3 binding affinity for both SH3, whereas phosphorylation of Ser-379 has a destabilizing effect on both interactions. Other mechanisms than a phosphorylation induced switch between the two SH3 must therefore take place for NADPH oxidase activation cascade to start.

Synaptic localization of a functional NADPH oxidase in the mouse hippocampus

Superoxide has been shown to be critical for hippocampal long-term potentiation (LTP) and hippocampus-dependent memory function. A possible source for the generation of superoxide during these processes is NADPH oxidase. The active oxidase consists of two membrane proteins, gp91 phox and p22 phox , and four cytosolic proteins, p40 phox , p47 phox , p67 phox , and Rac. Upon stimulation, the cytosolic proteins translocate to the membrane to form a complex with the membrane components, which results in production of superoxide. Here, we determined the presence, localization, and functionality of a NADPH oxidase in mouse hippocampus by examining the NADPH oxidase proteins as well as the production of superoxide. All of the NADPH oxidase proteins were present in hippocampal homogenates and enriched in synaptoneurosome preparations. Immunocytochemical analysis of cultured hippocampal neurons indicated that all NADPH oxidase proteins were localized in neuronal cell bodies as well as dendrites. Furthermore, double labeling analysis using antibodies to p67 phox and the presynaptic marker synaptophysin suggest a close association of the NADPH oxidase subunits with synaptic sites. Finally, stimulation of hippocampal slices with phorbol esters triggered translocation of the cytoplasmic NADPH oxidase proteins to the membrane and an increase in superoxide production that was blocked by inhibitors of NADPH oxidase. Taken together, our data suggest that NADPH oxidase is present in mouse hippocampus and might be the source of superoxide production required for LTP and memory function.

Rac1-NADPH oxidase-regulated generation of reactive oxygen species mediates glutamate-induced apoptosis in SH-SY5Y human neuroblastoma cells

Reactive oxygen species (ROS) are known to play an important role in glutamate-induced neuronal cell death. In the present study, we examined whether NADPH oxidase serves as a source of ROS production and plays a role in glutamate-induced cell death in SH-SY5Y human neuroblastoma cells. Stimulation of the cells with glutamate (100 mM) induced apoptotic cell death and increase in the level of ROS, and these effects of glutamate were significantly suppressed by the inhibitors of the NADPH oxidase, diphenylene iodonium, apocynin, and neopterine. In addition, RT-PCR revealed that SH-SY5Y cells expressed mRNA of gp91phox, p22phox and cytosolic p47phox, p67phox and p40phox, the components of the plasma membrane NADPH oxidase. Treatment with glutamate also resulted in activation and translocation of Rac1 to the plasma membrane. Moreover, the expression of Rac1N17, a dominant negative mutant of Rac1, significantly blocked the glutamate-induced ROS generation and cell death. Collectively, these results suggest that the plasma membrane-bound NADPH oxidase complex may play an essential role in the glutamate-induced apoptotic cell death through increased production of ROS.

Effect of sulfur amino acids on stimulus-induced superoxide generation and translocation of p47 phox and p67 phox to cell membrane in human neutrophils and the scavenging of free radical

Background Various cystathionine metabolites are in the urine of the patients with cystathioninuria. Among these metabolites, cystathionine ketimine significantly enhanced N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide generation in parallel with tyrosyl phosphorylation of 45 kDa protein in human neutrophils. Methods We investigated the effect of various sulfur amino acids on fMLP-, phorbol-12-myristate-13-acetate (PMA)- and arachidonic acid (AA)-induced superoxide generation in human neutrophils. In addition, the effects of these sulfur amino acids on the membrane translocation of cytosolic compounds p47 phox and p67 phox and on the scavenging of superoxide anions were investigated. Results When the cells were preincubated with various sulfur amino acids, fMLP-induced superoxide generation was enhanced by d,l-homocysteine and d,l-homocysteine-thiolactone but was inhibited by other sulfur amino acids in a concentration-dependent manner. The AA-induced superoxide was enhanced by l-cysteine, N-acetyl- l-cysteine and d,l-homocysteine. The strength of enhancing effect was: l-cysteine≫ N-acetyl- l-cysteine> d,l-homocysteine. On the other hand, the superoxide generation was weakly inhibited by l-cystathionine. The superoxide generation induced by PMA was weakly inhibited by l-cysteine, N-acetyl- l-cysteine and l-cystathionine. Homocysteine and d,l-homocysteine-thiolactone had no effect. In addition, d,l-homocysteine also enhanced translocation to the cell membrane of cytosolic compounds p47 phox and p67 phox. Conversely, l-cystathionine and N-acetyl- l-cysteine inhibited the translocation to membrane of p47 phox and p67 phox in a concentration-dependent manner. N-acetyl- l-cysteine and l-cysteine revealed scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. The sulfur amino acids tested also indicated radical scavenging activity on superoxide anion generated by phenazine methoxysulfate (PMS)–NADH system. Conclusion d,l-homocysteine and d,l-homocysteine-thiolactone enhanced fMLP-induced superoxide generation by the increment of translocation to membrane of p47 phox and p67 phox. l-cystathionine and N-acetyl- l-cysteine suppressed fMLP- and PMA-induced superoxide generation by the inhibition of translocation to membrane of p47 phox and p67 phox. N-acetyl- l-cysteine also had scavenging activity against DPPH radicals and superoxide anion.

Localization of NADPH oxidase subunits in neonatal sympathetic neurons

Reactive oxygen species (ROS) trigger programmed cell death in neonatal sympathetic neurons that have been deprived of nerve growth factor (NGF), however, the source of these oxygen intermediates has not been established. Using laser scanning confocal microscopy (LSCM), the intracellular distribution of the subunits of the ROS-generating enzyme NADPH oxidase was examined in sympathetic neurons of the superior cervical ganglion (SCG). Optical sectioning using LSCM showed that gp91-phox and p22-phox co-localize in neurons at the cell membrane, while the p47-phox and p67-phox subunits are found uniformly distributed in the cytoplasm of neurons maintained in the presence of NGF. Within 4 h after NGF deprivation, both the p47-phox and p67-phox subunits exhibit punctate staining in the cytoplasm and at the membrane. Furthermore, a sub-population of the cytosolic p47-phox appeared to co-localize with the membrane-bound gp91-phox in NGF-deprived neurons. These data provide support for the presence of NADPH oxidase in sympathetic neurons and suggest that this enzyme may become activated following the withdrawal of NGF.

Arachidonic Acid Rescues Defects on PMA-Elicited NADPH Oxidase Activity in Rac2-Deficient Neutrophils.

Rac2 is a hematopoietic-specific Rho-GTPase implicated as an important constituent of the NADPH oxidase complex. We previously showed that Rac2 plays a stimulus-specific role in regulating NADPH oxidase activation and other functional responses in neutrophils [Kim and Dinauer, JI 166, 2001]. Here we investigate the effect of arachidonic acid (AA) on Rac2-regulated NADPH oxidase activity. Superoxide production in rac2-/- neutrophils was significantly lower (~4-fold) than that of wild-type when stimulated with PMA or AA alone. However, exogenously added AA (10 μM) fully restored the defect in PMA-elicited NADPH oxidase activity in rac2−/ − neutrophils, while having no effect on FMLP-elicited superoxide production. Impaired PMA- or AA-induced F-actin polymerization in rac2−/ − neutrophils was also not restored by co-stimulation with PMA and AA. Taken together, these observations suggest that there are agonist- and pathway-specific differences in the underlying basis of functional defects in rac2−/ − neutrophils. To further investigate possible mechanisms of AA-mediated rescue of PMA-stimulated NADPH oxidase activation in rac2−/ − neutrophils, we measured protein expression and activity of cytosolic phospholipase A2 (cPLA2) and protein kinase C (PKC). The expression of cPLA2 and PMA-stimulated release of AA was similar between wild-type and rac2−/ − neutrophils, suggesting that defects in AA production by PMA-stimulated rac2−/ − neutrophils do not account for the effect of exogenous AA on oxidase activity. The neutrophil expression of PKC isoforms (α, β, δ, ζ) was also similar between genotypes. The cytosolic p47phox and p67phox components of NADPH oxidase were translocated to the plasma membrane upon stimulation with PMA in both genotypes, and no additional translocation in either wild-type or rac2−/ − neutrophils was detected upon co-stimulation with AA. The level of activated Rac1-GTP was similar between genotypes following stimulation, and was not increased by co-stimulation with PMA and AA. These studies indicate that the addition of exogenous AA reconstitutes PMA-elicited superoxide production in rac2−/ − neutrophils independent of the effects on translocation of p47phox and p67phox and activation of Rac1 GTPase. We hypothesize that the effect of AA is exerted through conformational changes of the assembled NADPH oxidase.

Analysis of a p67phoxΔ401 Mutant Identified in Chronic Granulomatous Disease: Deletion of C-Terminal SH3 Binding Site for p47phox Still Supports NADPH Oxidase Activity in Cosphox Cells.

The phagocyte NADPH oxidase plays an essential role in host defense through the production of reactive oxygen species. Oxidase activation occurs via assembly of cytosolic p47phox, p67phox, p40phox and Rac with the membrane flavocytochrome b558. This work presents the functional analysis of a mutant p67phox identified in two unrelated patients affected with chronic granulomatous disease (CGD), a 5 nucleotide deletion in exon 13 that generates a premature stop codon at amino acid position 401. The p67phox-Δ401 (p67-d401) protein, which lacks the wild type (WT) C-terminal residues 398–526, has a truncated PB1 domain while the C-SH3 domain is absent. In neutrophils and EBV-B lymphocytes derived from a patient with this mutant, the protein was not detected and RNA appears to be unstable (Patino et al. 1999). This mutant was transiently expressed in COS7 cells that also expressed gp91phox, p22phox, and p47phox (COS91-22-47 cells) or the first two of these (COS91-22), from stable transgenes. The p67-d401 protein was expressed at lower levels (~38%) compared to p67- WT when the same amount of plasmid DNA was used for transfection, which was consistently observed over a three fold range of plasmid DNA; this suggests instability of the mRNA or protein. When normalized for protein expression, NADPH oxidase activity in cells expressing p67-d401 was similar to those expressing p67-WT in response to PMA and 3 times better in response to arachidonic acid (AA). Co-expression of p47phox was necessary for p67-d401 to support O2- production in response to AA and PMA. Co-expression of p40phox did not affect either expression levels of p67-WT or p67-d401 or NADPH oxidase activity. Very little p67-WT was detected in membranes of transfected resting COS91-22-47 cells, with some membrane translocation observed following stimulation with PMA or AA, as expected. Interestingly, a significant amount of p67-d401 was detected in membranes and very little in the cytosol of resting COS91-22-47 cells transfected with this mutant, similar to C-terminal truncation mutants studied by de Mendez and colleagues (de Mendez et al 1994, de Mendez et al 1996). Upon activation with AA or PMA, a ~1.5 fold increase in p67-d401 and a ~ 1.6 fold increase in p47phox associated with the membrane fraction was observed in COS91-22-47 cells transfected with p67-d401. Finally, transiently transfected p67-d401 co-immunoprecipitated with p47phox both in resting and stimulated COS91-22-47 cells. This indicates that the carboxyl terminal SH3 domain of p67phox is not the only site of interaction between p67phox and p47phox, and suggests that a second binding site exists. In conclusion, the COSphox system represents a transfectable whole cell model for functional analysis of mutant phox proteins with reduced stability in neutrophils. It is likely that over expression of the p67-d401 cDNA in this system permits detectable expression of recombinant p67-d401. The unusual properties of the p67-d401 mutant, with spontaneous membrane translocation as well as ability to interact with p47phox in the absence of the C-terminal p67phox SH3 domain may provide further insight into the multiplicity of interactions between each of the oxidase subunits and their relation with the Rac and the cytochrome b558.