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1.
Ariel Mizrahi Yevgeny Berdichevsky Patrick J. Casey Edgar Pick 《The Journal of biological chemistry》2010,285(33):25485-25499
The superoxide-generating NADPH oxidase complex of resting phagocytes includes cytochrome b559, a membrane-associated heterodimer composed of two subunits (Nox2 and p22phox), and four cytosolic proteins (p47phox, p67phox, Rac, and p40phox). 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 b559 and its phospholipid environment. We described the construction of a tripartite chimera (trimera) consisting of strategic domains of p47phox, p67phox, 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-p47phox-p67phox-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 p47phox phox homology domain and, partially, on the Rac1 polybasic domain, but the requirement for Trp193 in p47phox persists. Prenylated GFP-p47phox-p67phox-Rac1 trimera acts as a quintessential single molecule oxidase activator of potential use in high throughput screening of inhibitors. 相似文献
2.
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. 相似文献
3.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2022,1869(9):119276
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. 相似文献
4.
Yuichi Maehara Kei Miyano Satoru Yuzawa Risa Akimoto Ryu Takeya Hideki Sumimoto 《The Journal of biological chemistry》2010,285(41):31435-31445
The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The membrane-integrated protein gp91phox serves as the catalytic core, because it contains a complete electron-transporting apparatus from NADPH to molecular oxygen for superoxide production. Activation of gp91phox requires the cytosolic proteins p67phox, p47phox, and Rac (a small GTPase). p67phox, comprising 526 amino acids, moves upon cell stimulation to the membrane together with p47phox and there interacts with Rac; these processes are prerequisite for gp91phox activation. Here we show that a region of p67phox (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 p67phox to support superoxide production by gp91phox-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 gp91phox-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 p67phox interaction with the gp91phox NADPH-binding domain, although these mutations affect neither the protein integrity nor the Rac binding activity. Thus the extended activation domain of p67phox (amino acids 190–210) containing the D(Y/F)LGK motif plays an essential role in oxidase activation probably by interacting with gp91phox. 相似文献
5.
《生物化学与生物物理学报:生物膜》2023,1865(7):184180
In phagocytes, superoxide anion (O2−), the precursor of reactive oxygen species, is produced by the NADPH oxidase complex to kill pathogens. Phagocyte NADPH oxidase consists of the transmembrane cytochrome b558 (cyt b558) and four cytosolic components: p40phox, p47phox, p67phox, and Rac1/2. The phagocyte activation by stimuli leads to activation of signal transduction pathways. This is followed by the translocation of cytosolic components to the membrane and their association with cyt b558 to form the active enzyme.To investigate the roles of membrane-interacting domains of the cytosolic proteins in the NADPH oxidase complex assembly and activity, we used giant unilamellar phospholipid vesicles (GUV). We also used the neutrophil-like cell line PLB-985 to investigate these roles under physiological conditions. We confirmed that the isolated proteins must be activated to bind to the membrane. We showed that their membrane binding was strengthened by the presence of the other cytosolic partners, with a key role for p47phox. We also used a fused chimera consisting of p47phox(aa 1–286), p67phox(aa 1–212) and Rac1Q61L, as well as mutated versions in the p47phox PX domain and the Rac polybasic region (PB). We showed that these two domains have a crucial role in the trimera membrane-binding and in the trimera assembly to cyt b558. They also have an impact on O2.- production in vitro and in cellulo: the PX domain strongly binding to GUV made of a mix of polar lipids; and the PB region strongly binding to the plasma membrane of neutrophils and resting PLB-985 cells. 相似文献
6.
Eric M. Lewis Susan Sergeant Bill Ledford Natalie Stull Mary C. Dinauer Linda C. McPhail 《The Journal of biological chemistry》2010,285(5):2959-2967
NADPH oxidase comprises both cytosolic and membrane-bound subunits, which, when assembled and activated, initiate the transfer of electrons from NADPH to molecular oxygen to form superoxide. This activity, known as the respiratory burst, is extremely important in the innate immune response as indicated by the disorder chronic granulomatous disease. The regulation of this enzyme complex involves protein-protein and protein-lipid interactions as well as phosphorylation events. Previously, our laboratory demonstrated that the small membrane subunit of the oxidase complex, p22phox, is phosphorylated in neutrophils and that its phosphorylation correlates with NADPH oxidase activity. In this study, we utilized site-directed mutagenesis in a Chinese hamster ovarian cell system to determine the phosphorylation sites within p22phox. We also explored the mechanism by which p22phox phosphorylation affects NADPH oxidase activity. We found that mutation of threonine 147 to alanine inhibited superoxide production in vivo by more than 70%. This mutation also blocked phosphorylation of p22phox in vitro by both protein kinase C-α and -δ. Moreover, this mutation blocked the p22phox-p47phox interaction in intact cells. When phosphorylation was mimicked in vivo through mutation of Thr-147 to an aspartyl residue, NADPH oxidase activity was recovered, and the p22phox-p47phox interaction in the membrane was restored. Maturation of gp91phox was not affected by the alanine mutation, and phosphorylation of the cytosolic component p47phox still occurred. This study directly implicates threonine 147 of p22phox as a critical residue for efficient NADPH oxidase complex formation and resultant enzyme activity. 相似文献
7.
P-Rex1 (phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1) is a Rac-specific guanine nucleotide exchange factor activated by Gβγ subunits and by PtdIns(3,4,5)P3. Recent studies indicate that P-Rex1 plays an important role in signaling downstream of neutrophil chemoattractant receptors. Here we report that heterologous expression of P-Rex1, but not Vav1, reconstitutes formyl peptide receptor 1 (FPR1)-mediated NADPH oxidase activation in the transgenic COSphox cells expressing gp91phox, p22phox, p67phox and p47phox. A successful reconstitution requires the expression of a full-length P-Rex1 with intact DH and PH domains, and is accompanied by P-Rex1 membrane localization as well as Rac1 activation. P-Rex1-dependent superoxide generation in the reconstituted COSphox cells was further enhanced by expression of the novel PKC isoform PKCδ and by overexpression of Akt. Heterologous expression of P-Rex1 in COSphox cells potentiated fMet-Leu-Phe-induced Akt phosphorylation, whereas expression of a constitutively active form of Akt enhanced Rac1 activation. In contrast, a dominant negative Akt mutant reduced the fMet-Leu-Phe stimulated superoxide generation as well as Rac1 activation. These results demonstrate that in COSphox cells, P-Rex1 is a critical component for FPR1-mediated signaling leading to NADPH oxidase activation, and there is a crosstalk between the P-Rex1-Rac pathway and Akt in superoxide generation. 相似文献
8.
Xing Jun Li Wei Tian Natalie D. Stull Sergio Grinstein Simon Atkinson Mary C. Dinauer 《Molecular biology of the cell》2009,20(5):1520-1532
The assembly of cytosolic p47phox and p67phox with flavocytochrome b558 at the membrane is crucial for activating the leukocyte NADPH oxidase that generates superoxide for microbial killing. p47phox and p67phox 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 p67phox 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 p67phox to the phagosomal cup, and, after phagosome internalization, a probe for PI(3)P followed by a YFP-tagged fragment derived from the p47phox PRR. This fragment was recruited in a flavocytochrome b558-dependent, p67phox-specific, and PI(3)P-independent manner. These findings indicate that p47PRR fragment probes the status of the p67phox SH3b domain and suggest that the p47phox/p67phox tail-to-tail interaction is disrupted after oxidase assembly such that the p67phox-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. 相似文献
9.
The NADPH-oxidase of phagocytic cells is a multicomponent enzyme that generates superoxide. It comprises a membrane flavocytochrome b558 and four cytosolic proteins; p67phox, p47phox, p40phox and Rac. The NADPH-binding site of this complex was shown to be located on the flavocytochrome b558. However, a number of studies have suggested the presence of another site on the p67phox subunit which is the key activating component. Using several approaches like tryptophan quenching fluorescence measurement, inhibition by 2′,3′-dialdehyde NADPH, and free/bound NADPH concentration measurements, we demonstrate that no NADPH binds on p67phox, thus definitively solving the controversy on the number and location of the NADPH-binding sites on this complex. 相似文献
10.
Gilda Karimi Chantal Houée Levin Marie Claire Dagher Laura Baciou Tania Bizouarn 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
The 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.Methods
A 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.Results
The 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 significance
The 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. 相似文献11.
12.
Rumi Matono Kei Miyano Takuya Kiyohara Hideki Sumimoto 《The Journal of biological chemistry》2014,289(36):24874-24884
The phagocyte NADPH oxidase Nox2, heterodimerized with p22phox 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 p47phox and GDP/GTP exchange on the small GTPase Rac. These proteins, in an active form, bind to their respective targets, p22phox and p67phox, 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 p47phox 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 p47phox 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 p67phox, AA induces the direct interaction of Rac-GTP-bound p67phox with the C-terminal cytosolic region of Nox2. p67phox-Rac-Nox2 assembly and superoxide production are both abrogated by alanine substitution for Tyr-198, Leu-199, and Val-204 in the p67phox activation domain that localizes the C-terminal to the Rac-binding domain. Thus the “third” switch (AA-inducible interaction of p67phox·Rac-GTP with Nox2) is required to be turned on at the same time for Nox2 activation. 相似文献
13.
《Free radical research》2013,47(11-12):1366-1378
AbstractThe NADPH oxidase (NOX) family of enzymes oxidase catalyzes the transport of electrons from NADPH to molecular oxygen and generates O2??, which is rapidly converted into H2O2. We aimed to identify in hepatocytes the protein NOX complex responsible for H2O2 synthesis after α1-adrenoceptor (α1-AR) stimulation, its activation mechanism, and to explore H2O2 as a potential modulator of hepatic metabolic routes, gluconeogenesis, and ureagenesis, stimulated by the ARs. The dormant NOX2 complex present in hepatocyte plasma membrane (HPM) contains gp91phox, p22phox, p40phox, p47phox, p67phox and Rac 1 proteins. In HPM incubated with NADPH and guanosine triphosphate (GTP), α1-AR-mediated H2O2 synthesis required all of these proteins except for p40phox. A functional link between α1-AR and NOX was identified as the Gα13 protein. Alpha1-AR stimulation in hepatocytes promotes Rac1-GTP generation, a necessary step for H2O2 synthesis. Negative cross talk between α1-/β-ARs for H2O2 synthesis was observed in HPM. In addition, negative cross talk of α1-AR via H2O2 to β-AR-mediated stimulation was recorded in hepatocyte gluconeogenesis and ureagenesis, probably involving aquaporine activity. Based on previous work we suggest that H2O2, generated after NOX2 activation by α1-AR lightening in hepatocytes, reacts with cAMP-dependent protein kinase A (PKA) subunits to form an oxidized PKA, insensitive to cAMP activation that prevented any rise in the rate of gluconeogenesis and ureagenesis. 相似文献
14.
Yuichi Maehara Kei Miyano Hideki Sumimoto 《Biochemical and biophysical research communications》2009,379(2):589-31445
The membrane-bound NADPH oxidase in phagocytes, gp91phox (a.k.a. Nox2), produces superoxide, a precursor of microbicidal oxidants, thereby playing a crucial role in host defense. Activation of gp91phox/Nox2 requires assembly with the cytosolic proteins p67phox and p47phox, each containing two SH3 domains. Although the C-terminal SH3 domain of p67phox is responsible for binding to p47phox, little is known about the role for the first (N-terminal) SH3 domain [SH3(N)]. Here we show that truncation of p67phox-SH3(N), but not substitution of arginine for the invariant residue Trp-277 in SH3(N), results in an impaired activation of gp91phox/Nox2. The impairment is overcome by higher expression of an SH3(N)-defective p67phox in cells, suggesting that SH3(N) primarily increases the affinity of p67phox for the oxidase complex. On the other hand, p67phox-SH3(N) is not involved in activation of Nox1 and Nox3, closely-related homologues of gp91phox/Nox2. Thus p67phox-SH3(N) specifically functions in gp91phox/Nox2 activation probably via facilitating oxidase assembly. 相似文献
15.
Daniel N. Meijles Lampson M. Fan Brendan J. Howlin Jian-Mei Li 《The Journal of biological chemistry》2014,289(33):22759-22770
Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47phox is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47phox phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47phox protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47phox is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22phox binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47phox−/− coronary microvascular cells. Compared with wild-type p47phox cDNA transfected cells, the single mutation of S379A completely blocked p47phox membrane translocation, binding to p22phox and endothelial O2⨪ production in response to acute stimulation of PKC. p47phox C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47phox conformational changes and NADPH oxidase-dependent superoxide production by cells. 相似文献
16.
The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the reduction of oxygen to O–
2 at the expense of NADPH. During activation, the cytosolic oxidase components p47phox and p67phox, each containing two Src homology 3 (SH3) domains, migrate to the plasma membrane. p47phox and p67phox associate with cytochrome b558, a membrane-integrated flavohemoprotein, to assemble the active oxidase. Oxidase activation can be mimicked in a cell-free system using an anionic amphiphile, such as sodium dodecyl sulfate or arachidonic acid, as an activating agent. Activators of the oxidase in vitro cause exposure of the SH3 domains of p47phox, which has probably been masked by the C-terminal region of this protein in a resting state. We show here that the fluorescence exhibited by the covalently labeled N,N-di-methyl-N(iodoacetyl)-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethyleneamine (IANBD) was increased when N-terminal-truncated p47phox-(SH3)2-C was treated with anionic amphiphiles. This finding was similar to the results obtained with the full-length p47phox. However, the fluorescence of C-terminal-truncated p47phox-N-(SH3)2 and that of both C-terminal and N-terminal truncated p47phox-(SH3)2 were not altered by the activators. These results indicate that the C-terminal region of p47phox is a primary target of the conformational change during the activation of NADPH oxidase. 相似文献
17.
AngII (angiotensin II)-induced excessive ROS (reactive oxygen species) generation and proliferation of VSMCs (vascular smooth muscle cells) is a critical contributor to the pathogenesis of atherosclerosis. PGC-1α [PPARγ (peroxisome-proliferator-activated receptor γ) co-activator-1α] is involved in the regulation of ROS generation, VSMC proliferation and energy metabolism. The aim of the present study was to investigate whether PGC-1α mediates AngII-induced ROS generation and VSMC hyperplasia. Our results showed that the protein content of PGC-1α was negatively correlated with an increase in cell proliferation and migration induced by AngII. Overexpression of PGC-1α inhibited AngII-induced proliferation and migration, ROS generation and NADPH oxidase activity in VSMCs. Conversely, Ad-shPGC-1α (adenovirus-mediated PGC-1α-specific shRNA) led to the opposite effects. Furthermore, the stimulatory effect of Ad-shPGC-1α on VSMC proliferation was significantly attenuated by antioxidant and NADPH oxidase inhibitors. Analysis of several key subunits of NADPH oxidase (Rac1, p22phox, p40phox, p47phox and p67phox) and mitochondrial ROS revealed that these mechanisms were not responsible for the observed effects of PGC-1α. However, we found that overexpression of PGC-1α promoted NOX1 degradation through the proteasome degradation pathway under AngII stimulation and consequently attenuated NOX1 (NADPH oxidase 1) expression. These alterations underlie the inhibitory effect of PGC-1α on NADPH oxidase activity. Our data support a critical role for PGC-1α in the regulation of proliferation and migration of VSMCs, and provide a useful strategy to protect vessels against atherosclerosis. 相似文献
18.
Peter V. Usatyuk Irina A. Gorshkova Donghong He Yutong Zhao Satish K. Kalari Joe G. N. Garcia Viswanathan Natarajan 《The Journal of biological chemistry》2009,284(22):15339-15352
Phosphatidic acid generated by the activation of phospholipase D (PLD)
functions as a second messenger and plays a vital role in cell signaling. Here
we demonstrate that PLD-dependent generation of phosphatidic acid is critical
for Rac1/IQGAP1 signal transduction, translocation of
p47phox to cell periphery, and ROS production. Exposure of
[32P]orthophosphate-labeled human pulmonary artery endothelial
cells (HPAECs) to hyperoxia (95% O2 and 5% CO2) in the
presence of 0.05% 1-butanol, but not tertiary-butanol, stimulated PLD as
evidenced by accumulation of [32P]phosphatidylbutanol. Infection of
HPAECs with adenoviral constructs of PLD1 and PLD2 wild-type potentiated
hyperoxia-induced PLD activation and accumulation of
/reactive oxygen species
(ROS). Conversely, overexpression of catalytically inactive mutants of PLD
(hPLD1-K898R or mPLD2-K758R) or down-regulation of expression of PLD with PLD1
or PLD2 siRNA did not augment hyperoxia-induced
[32P]phosphatidylbutanol accumulation and ROS generation. Hyperoxia
caused rapid activation and redistribution of Rac1, and IQGAP1 to cell
periphery, and down-regulation of Rac1, and IQGAP1 attenuated
hyperoxia-induced tyrosine phosphorylation of Src and cortactin and ROS
generation. Further, hyperoxia-mediated redistribution of Rac1, and IQGAP1 to
membrane ruffles, was attenuated by PLD1 or PLD2 small interference RNA,
suggesting that PLD is upstream of the Rac1/IQGAP1 signaling cascade. Finally,
small interference RNA for PLD1 or PLD2 attenuated hyperoxia-induced cortactin
tyrosine phosphorylation and abolished Src, cortactin, and
p47phox redistribution to cell periphery. These results
demonstrate a role of PLD in hyperoxia-mediated IQGAP1 activation through Rac1
in tyrosine phosphorylation of Src and cortactin, as well as in
p47phox translocation and ROS formation in human lung
endothelial cells.Phagocytic cells of the immune system (neutrophils, eosinophils, monocytes,
and macrophages) generate superoxide
()2
instrumental in the killing of invading pathogens solely by NADPH oxidase
(1-3).
Deficiency of results in
the genetically inherited disorder chronic granulomatous disease, a condition
in which the affected individuals are susceptible to infection
(4). Phagocytic NADPH oxidase
is activated when cytosolic p47phox,
p67phox, and Rac2 translocate to the phagosomes and plasma
membrane and form a complex with integral membrane cytochrome
b558, which, in turn, is a Nox2
(gp91phox)/p22phox heterodimer
(5,
6). Assembly of phagocytic
NADPH oxidase is initiated by two signals. The first is the phosphorylation of
multiple serine and tyrosine residues in the p47phox
domain, which leads to unmasking of p47phox SH3 domains
that bind to a proline-rich target in the C terminus of
p22phox
(7-10).
The interaction between p47phox and
p22phox seems to be an essential requirement for the
translocation of other cytosolic components of the oxidase. The second signal
is the binding of GTP to Rac2, which leads to the dissociation of Rac from
Rho-GDI and binding to p67phox, followed by translocation
of p67phox/GTP-Rac2 to the membrane
(11). Nonphagocytic cells
express predominantly Rac1, Tiam1 (a GEF involved in Rac1 activation), Nox1-5,
and most of the other cytosolic phagocytic oxidase components
(12); however, the oxidative
output of non-phagocytes is much smaller compared with the phagocytes. A
recent study indicates that IQGAP1, an effector of Rac1, may link Nox2 to
actin, thereby enhancing ROS production and contributing to cell motility in
ECs (13). The one or more
mechanisms responsible for differences in the oxidative burst between the
phagocytic and non-phagocytic cells are yet to be defined.We have demonstrated previously that hyperoxia activates lung endothelial
NADPH oxidase, which in part is mediated by ERK, p38 MAPK
(14,
15), and Src
(16), and hyperoxia-induced
p47phox tyrosine phosphorylation and translocation to cell
periphery is dependent on Src
(16). Further, tyrosine
phosphorylation of cortactin mediated by Src is essential for
hyperoxia-induced p47phox translocation and
/ROS generation in HPAECs
(17). In addition to Src,
phosphatidic acid (PA) or diacylglycerol also stimulated phosphorylation of
p47phox and p22phox in neutrophils
both in vivo and in vitro
(18-20).
PA is generated in mammalian cells via de novo biosynthesis or
hydrolysis of membrane phospholipids catalyzed by phospholipase D (PLD)
(21-25).
Activation of polymorphonuclear leukocytes with formyl-Met-Leu-Phe enhanced
the oxidative burst that correlated with PA accumulation, and inclusion of
short-chain primary alcohols attenuated the NADPH oxidase mediated
/ROS generation,
suggesting a potential role for PLD in the regulation of NADPH oxidase
(12,
26,
27). However, the downstream
targets of PLD that signal NADPH oxidase activation have not been fully
characterized.Here, we identify for the first time that activation of IQGAP1 by Rac1 is
downstream of PLD in hyperoxia-induced ROS generation. In addition, we show
that activation of Rac1/IQGAP1 by PLD also regulates Src-dependent tyrosine
phosphorylation of cortactin and p47phox translocation to
cell periphery. Thus, our results define a novel molecular mechanism for
hyperoxia-induced NADPH oxidase activation by PLD/PA-mediated
p47phox membrane translocation via
Rac1/IQGAP1/Src/cortactin signaling cascade. 相似文献
19.
Summary Plant cells respond to a variety of external signals with the production of reactive-oxygen species. The enzyme system generating these reactive-oxygen species is believed to be an NADPH oxidase located in the plasma membrane and sharing similarities with the NADPH oxidase from mammalian macrophages. Antibodies directed against individual subunits (p22
phox
, p47
phox
, p67
phox
) of the human NADPH oxidase cross-react with soybean proteins of a similar size and subcellular location. An extensive expression screening of a soybean cDNA-library with the anti-human NADPH oxidase antibodies gave a single class of cDNA-clones for each antibody. However, the sequence analysis of these clones clearly demonstrates that the different antibodies recognise proteins which are unrelated to the expected oxidase subunits. The anti-p22
phox
antibody recognised a microsomal protein with no significant homology to any known protein in the database. One anti-p47
phox
antibody cross-reacted with the UDP-glucose dehydrogenase and another antibody bound to the chaperon peptidyl prolyl-cis-trans isomerase, both soluble cytosolic proteins. The anti-p67
phox
antibody detected the soluble enzyme acetohydroxy acid reductoisomerase. Chromatography of soybean protein extracts on an ion-exchange column (MonoQ, FPLC) gave a perfect comigration of the enzyme activity with the antibody signal, thus confirming these unexpected results by independent biochemical experiments.Abbreviations AARI
acetohydroxy acid reductoisomerase
- DPI
diphenylene iodonium
- GST
glutathione-S-transferase
- phox NADPH
oxidase of phagocytes
- ROS
reactive-oxygen species 相似文献
20.
Julien Marcoux Petr Man Mathieu Castellan Corinne Vivès Eric Forest Franck Fieschi 《FEBS letters》2009,583(4):835-20244
The neutrophil NADPH oxidase is an enzymatic complex involved in innate immunity. Phosphorylation of p47phox promotes its translocation with p67phox and p40phox, followed by membrane interaction and assembly with flavocytochrome b558 into a functional complex. To characterise p47phox conformational changes during activation, we used wild-type and the S303/304/328E triple mutant mimicking the phosphorylated state. Hydrogen/deuterium exchange and limited proteolysis coupled to mass spectrometry were used to discriminate between the various structural models. An increase in deuteration confirmed that p47phox adopts an open and more flexible conformation after activation. Limited proteolysis correlated this change with increased auto-inhibitory region (AIR) accessibility. These results establish a structural link between the AIR release and the exposure of the Phox homology (PX) domain. 相似文献