scholarly journals Characterization the binding of cytosolic phospholipase A2 alpha and NOX2 NADPH oxidase in mouse macrophages

2021 ◽  
Author(s):  
Yulia Solomonov ◽  
Nurit Hadad ◽  
Rachel Levy
Keyword(s):  
Amino Acids ◽  
Nadph Oxidase ◽  
Plasma Membranes ◽  
Superoxide Production ◽  
C2 Domain ◽  
Cytosolic Phospholipase ◽  
Px Domain ◽  
Mouse Macrophages ◽  
Nadph Oxidase Activation

Abstract Background: Previous studies have demonstrated that Cytosolic phospholipase A2a (cPLA2a) is absolutely required for NOX2 NADPH oxidase activation in human and mouse phagocytes. Moreover, upon stimulation, cPLA2a translocates to the plasma membranes of by binding to the assembled oxidase, forming a complex between its C2 domain and the PX domain of the oxidase cytosolic factor, p47phox in human phagocytes. Intravenous administration of an antisense against cPLA2a that significantly inhibited its expression in mouse peritoneal neutrophil and macrophages also inhibited superoxide production, in contrast to cPLA2a knockout mice that showed normal superoxide production. The aim of the present study was to determine whether there is a binding between cPLA2a-C2 domain and p47phox-PX in mouse macrophages, to further support the role of cPLA2a in oxidase regulation also in mouse phagocytes. Methods and Results: A significant binding of mouse GST-p47phox-PX domain fusion protein and cPLA2a in stimulated mouse phagocyte membranes was demonstrated by pull down experiments, although lower than that detected by human p47phox-PX domain. Substituting the amino acids Phe98, Asn99 and Gly100 to Cys98 Ser99 and Thr100 in mouse p47phox-PX domain (that are present in human p47phox-PX domain) caused strong binding that was similar to that detected by the human p47phox-PX domain. Conclusions: the binding between cPLA2a-C2 and p47phox-PX domains exist in mouse macrophages and is not unique to human phagocytes. The binding between the two proteins is lower in the mice probably due to the absence of amino acids Cys98 Ser99 and Thr100 in p47phox-PX domain that facilitate the binding to cPLA2a.

The Phosphoinositide-Binding Protein p40phox Regulates NADPH Oxidase Activation Rather Than Assembly during FcγIIA Receptor-Induced Phagocytosis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 678-678 ◽  
Author(s):  
Wei Tian ◽  
Xing Jun Li ◽  
Natalie D. Stull ◽  
Chang-Il Suh ◽  
Sergio Grinstein ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Superoxide Production ◽  
Null Mouse ◽  
Target Sequence ◽  
Resting Cells ◽  
Nadph Oxidase Activity ◽  
Px Domain ◽  
Significant Difference ◽  
Nadph Oxidase Activation

Abstract Many critical features of the organization and regulation of the phagocyte NADPH oxidase, a complex multi-subunit enzyme that generates superoxide for microbial killing, remain poorly defined. The active enzyme includes a membrane-bound flavocytochrome b along with p47phox, p67phox, p40phox, and Rac-GTP that are present in the cytosol of resting cells. p67phox is linked by high affinity interactions with both p47phox and p40phox, which appear to translocate as a trimeric complex upon cellular activation. The p47phox subunit acts as an adaptor to promote translocation by docking at a proline-rich target sequence on the flavocytochrome, and p67phox is a Rac-GTP effector containing a domain that activates electron transport. In contrast, the function of p40phox, which is not required for high level oxidase activity in cell free systems, is poorly understood. Recently, our group showed that p40phox plays key role in the activation of superoxide production during phagocytosis of IgG-opsonized targets in COSphoxFcγR cells. This model cell line contains stable transgenes for the flavocytochrome, p47phox, p67phox, and the FcγIIA receptor, without or with an additional transgene for p40phox. p40phox-dependent coupling of FcγR-mediated phagocytosis to superoxide production required an intact p40phox PX domain, which binds to phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide generated by class III PI3 kinases in phagosome membranes (Suh et al J Exp Med 203, 1915Suh et al J Exp Med 203, 2006). Furthermore, a newly developed p40phox-null mouse exhibits reduced neutrophil NADPH oxidase activity in response to selected agonists, including IgG-opsonized targets (Ellson et al J Exp Med 203, 1927Ellson et al J Exp Med 203, 2006). In the current study, we investigated whether p40phox is required for translocation of p67phox during phagocytosis. We generated COSphoxFcγR cells expressing YFP-tagged p67phox from a stable transgene instead of untagged p67phox. Following incubation with IgG-opsonized sheep red blood cells (IgG-RBC), p67phox was detected on phagosome membranes at both early stages of phagosome cup formation and after closure, independent of whether or not p40phox was also co-expressed. However, NADPH oxidase activity was not detected in IgG-RBC phagosomes in COSphoxFcγR-p67phox-YFP cells unless p40phox was present. PMA-activated superoxide production was independent of p40phox, and Western blotting indicated there was no significant difference in expression of the other oxidase subunits in COSphoxFcγR-p67phox-YFP cells without or with the p40phox transgene. Further studies in PLB-985 granulocytes expressing stable transgenes for either YFP-tagged p67phox or p40phox showed that the PI3K inhibitor wortmannin inhibited phagosome NADPH oxidase activity and translocation of p40phox, but localization of p67phox to phagosomes was unaffected. These results indicate that although p40phox positively regulates NADPH oxidase activation during phagocytosis, recruitment of p67phox to the phagosome is independent of p40phox. Taken together, these data suggest that the PX domain of p40phox acts as a PI3P-dependent switch to activate the membrane-assembled NADPH oxidase complex.

scholarly journals Cytosolic Phospholipase A2α Is Targeted to the p47phox-PX Domain of the Assembled NADPH Oxidase via a Novel Binding Site in Its C2 Domain

2008 ◽  
Vol 283 (46) ◽  
pp. 31898-31908 ◽  
Author(s):  
Zeev Shmelzer ◽  
Maria Karter ◽  
Miriam Eisenstein ◽  
Thomas L. Leto ◽  
Nurit Hadad ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Binding Site ◽  
C2 Domain ◽  
Cytosolic Phospholipase ◽  
Px Domain ◽  
Cytosolic Phospholipase A2α

scholarly journals Olfactomedin 4 contributes to hydrogen peroxide-induced NADPH oxidase activation and apoptosis in mouse neutrophils

2018 ◽  
Vol 315 (4) ◽  
pp. C494-C501 ◽  
Author(s):  
Wenli Liu ◽  
Yueqin Liu ◽  
Hongzhen Li ◽  
Griffin P. Rodgers
Keyword(s):  
Hydrogen Peroxide ◽  
Nadph Oxidase ◽  
Molecular Mechanisms ◽  
Oxidase Inhibitor ◽  
Superoxide Production ◽  
Wild Type ◽  
Escherichia Coli Bacteria ◽  
Induced Apoptosis ◽  
Nadph Oxidase Activation ◽  
Deficient Mice

Neutrophils increase production of reactive oxygen species, including superoxide, hydrogen peroxide (H2O2), and hydroxyl radical, to destroy invading microorganisms under pathological conditions. Conversely, oxidative stress conditions, such as the presence of H2O2, induce neutrophil apoptosis, which helps to remove neutrophils after inflammation. However, the detailed molecular mechanisms that are involved in the latter process have not been elucidated. In this study, we investigated the potential role of olfactomedin 4 (Olfm4) in H2O2-induced superoxide production and apoptosis in mouse neutrophils. We have demonstrated that Olfm4 is not required for maximal-dosage PMA- and Escherichia coli bacteria-induced superoxide production, but Olfm4 contributes to suboptimal-dosage PMA- and H2O2-induced superoxide production. Using an NADPH oxidase inhibitor and gp91phox-deficient mouse neutrophils, we found that NAPDH oxidase was required for PMA-stimulated superoxide production and that Olfm4 mediated H2O2-induced superoxide production through NADPH oxidase, in mouse neutrophils. We have shown that neutrophils from Olfm4-deficient mice exhibited reduced H2O2-induced apoptosis compared with neutrophils from wild-type mice. We also demonstrated that neutrophils from Olfm4-deficient mice exhibited reduced H2O2-stimulated mitochondrial damage and membrane permeability, and as well as reduced caspase-3 and caspase-9 activity, compared with neutrophils from wild-type mice. Moreover, the cytoplasmic translocation of the proapoptotic mitochondrial proteins Omi/HtrA2 and Smac/DIABLO in response to H2O2was reduced in neutrophils from Olfm4-deficient mice compared with neutrophils from wild-type mice. Our study demonstrates that Olfm4 contributes to H2O2-induced NADPH oxidase activation and apoptosis in mouse neutrophils. Olfactomedin 4 might prove to be a potential target for future studies on inflammatory neutrophil biology and for inflammatory disease treatment.

Quercetin inhibits vascular superoxide production induced by endothelin-1: Role of NADPH oxidase, uncoupled eNOS and PKC

2009 ◽  
Vol 202 (1) ◽  
pp. 58-67 ◽  
Author(s):  
Miguel Romero ◽  
Rosario Jiménez ◽  
Manuel Sánchez ◽  
Rocío López-Sepúlveda ◽  
Maria José Zarzuelo ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Superoxide Production ◽  
Endothelin 1

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

Blood ◽  
2008 ◽  
Vol 112 (9) ◽  
pp. 3867-3877 ◽  
Author(s):  
Wei Tian ◽  
Xing Jun Li ◽  
Natalie D. Stull ◽  
Wenyu Ming ◽  
Chang-Il Suh ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Superoxide Production ◽  
Resting Cells ◽  
Cellular Activation ◽  
Nadph Oxidase Activity ◽  
Phagocyte Nadph Oxidase ◽  
Before And After ◽  
Membrane Bound

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.

scholarly journals Restoration of high-level transport activity by human reduced folate carrier/ThTr1 thiamine transporter chimaeras: role of the transmembrane domain 6/7 linker region in reduced folate carrier function

2003 ◽  
Vol 369 (1) ◽  
pp. 31-37 ◽  
Author(s):  
Xiang Y. LIU ◽  
Teah L. WITT ◽  
Larry H. MATHERLY
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Plasma Membranes ◽  
Transmembrane Domain ◽  
K562 Cells ◽  
Reduced Folate Carrier ◽  
Wild Type ◽  
Linker Region ◽  
Conserved Amino Acids

The reduced folate carrier (RFC; SLC19A1) is closely related to the thiamine transporter, SLC19A2 (ThTr1). Hydropathy models for these homologous transporters predict up to 12 transmembrane domains (TMDs), with internally oriented N- and C-termini and a large central loop between TMDs 6 and 7. The homologies are localized mostly in the TMDs. However, there is little similarity in their N- and C-terminal domains and the central peptide linkers connecting putative TMDs 1—6 and TMDs 7—12. To explore the functional role of the 61-amino acid central linker in the human RFC (hRFC), we introduced deletions of 49 and 60 amino acids into this region, differing by the presence of a stretch of 11 highly conserved amino acids between the human and rodent RFCs (positions 204—214). An additional hRFC construct was prepared in which only the 11 conserved amino acids were deleted. The resulting hRFCD215—R263Δ, hRFCK204—R263Δ and hRFCK204—R214Δ proteins were transfected into transport-impaired K562 cells. The deletion constructs were all expressed in plasma membranes; however, they were completely inactive for methotrexate and (6S)5-formyl tetrahydrofolate transport. Insertion of non-homologous 73- and 84-amino acid fragments from the structurally analogous ThTr1 linker region into position 204 of hRFCK204—R263Δ restored low levels of transport (16—21% of the wild type). Insertion of the ThTr1 linkers into hRFCD215—R263Δ at position 215 restored 60—80% of wild-type levels of transport. Collectively, our results suggest that the role of the hRFC linker peptide is to provide the proper spatial orientation between the two halves of the hRFC protein for optimal function, and that this is largely independent of amino acid sequence. Our results also demonstrate a critical transport role for the stretch of 11 conserved amino acids starting at position 204 of hRFC.

scholarly journals A new genetic subgroup of chronic granulomatous disease with autosomal recessive mutations in p40phox and selective defects in neutrophil NADPH oxidase activity

Blood ◽  
2009 ◽  
Vol 114 (15) ◽  
pp. 3309-3315 ◽  
Author(s):  
Juan D. Matute ◽  
Andres A. Arias ◽  
Nicola A. M. Wright ◽  
Iwona Wrobel ◽  
Christopher C. M. Waterhouse ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Oxidase Activity ◽  
Autosomal Recessive ◽  
Premature Stop Codon ◽  
Superoxide Production ◽  
Human Neutrophils ◽  
Granulomatous Disease ◽  
Recessive Mutations ◽  
Px Domain

Abstract Chronic granulomatous disease (CGD), an immunodeficiency with recurrent pyogenic infections and granulomatous inflammation, results from loss of phagocyte superoxide production by recessive mutations in any 1 of 4 genes encoding subunits of the phagocyte NADPH oxidase. These include gp91phox and p22phox, which form the membrane-integrated flavocytochrome b, and cytosolic subunits p47phox and p67phox. A fifth subunit, p40phox, plays an important role in phagocytosis-induced superoxide production via a phox homology (PX) domain that binds to phosphatidylinositol 3-phosphate (PtdIns(3)P). We report the first case of autosomal recessive mutations in NCF4, the gene encoding p40phox, in a boy who presented with granulomatous colitis. His neutrophils showed a substantial defect in intracellular superoxide production during phagocytosis, whereas extracellular release of superoxide elicited by phorbol ester or formyl-methionyl-leucyl-phenylalanine (fMLF) was unaffected. Genetic analysis of NCF4 showed compound heterozygosity for a frameshift mutation with premature stop codon and a missense mutation predicting a R105Q substitution in the PX domain. Parents and a sibling were healthy heterozygous carriers. p40phoxR105Q lacked binding to PtdIns(3)P and failed to reconstitute phagocytosis-induced oxidase activity in p40phox-deficient granulocytes, with premature loss of p40phoxR105Q from phagosomes. Thus, p40phox binding to PtdIns(3)P is essential for phagocytosis-induced oxidant production in human neutrophils and its absence can be associated with disease.

scholarly journals PI(3)P-p40 phox binding regulates NADPH oxidase activation in mouse macrophages and magnitude of inflammatory responses in vivo

2016 ◽  
Vol 101 (2) ◽  
pp. 449-457 ◽  
Author(s):  
Juhi Bagaitkar ◽  
Emilia A. Barbu ◽  
Lizet J. Perez-Zapata ◽  
Anthony Austin ◽  
Guangming Huang ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Inflammatory Responses ◽  
Mouse Macrophages ◽  
Nadph Oxidase Activation

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

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2386-2386
Author(s):  
Chaekyun Kim ◽  
Mary C. Dinauer
Keyword(s):  
Arachidonic Acid ◽  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Actin Polymerization ◽  
Rho Gtpase ◽  
Superoxide Production ◽  
Wild Type ◽  
Functional Responses ◽  
Nadph Oxidase Activity ◽  
Nadph Oxidase Activation

Abstract 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.

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