Unsaturated fatty acids stimulate NADPH-dependent superoxide production by cell-free system derived from macrophages

Bromberg, Yael; Pick, Edgar

doi:10.1016/0008-8749(84)90066-2

Cited by 330 publications

(134 citation statements)

References 24 publications

Supporting

Mentioning

126

Contrasting

Order By: Relevance

“…Many reports emphasize the effects of AA on p47 phox . Early studies showed AA or other anionic amphiphiles activate ROS production in cell-free assays of Nox2 activity (Bromberg and Pick, 1984;Curnutte, 1985). It is thought that AA induces conformational changes in p47 phox that may mimic the effects of phosphorylation, resulting in exposure of its binding PX and SH3 domains and translocation of p47 phox and p67 phox (Shiose and Sumimoto, 2000).…”

Section: Discussionmentioning

confidence: 99%

“…However, recent studies in p40 phox -deficient mice or in Fc␥IIA receptor-reconstituted cells indicate p40 phox is an essential component of the Nox2 system (Ellson et al, 2006;Suh et al, 2006). Arachidonic acid (AA) has been used frequently as a activator of Nox2 in cell-free assay systems (Bromberg and Pick, 1984;Curnutte, 1985). Although relatively high concentrations of AA (50 -200 M) are required for activation of Nox2 both in vitro and in vivo, functional roles for cytosolic phospholipase A 2 (cPLA 2 ), which produces AA, in Nox2 activation have been demonstrated at the cellular level (Dana et al, 1998;Zhao et al, 2002;Shmelzer et al, 2003).…”

mentioning

confidence: 99%

See 1 more Smart Citation

A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox

Ueyama

Tatsuno

Kawasaki

et al. 2007

MBoC

101

View full text Add to dashboard Cite

In the phagocytic cell, NADPH oxidase (Nox2) system, cytoplasmic regulators (p47 phox , p67 phox , p40 phox , and Rac) translocate and associate with the membrane-spanning flavocytochrome b 558 , leading to activation of superoxide production. We examined membrane targeting of phox proteins and explored conformational changes in p40 phox that regulate its translocation to membranes upon stimulation. GFP-p40 phox translocates to early endosomes, whereas GFP-p47 phox translocates to the plasma membrane in response to arachidonic acid. In contrast, GFP-p67 phox does not translocate to membranes when expressed alone, but it is dependent on p40 phox and p47 phox for its translocation to early endosomes or the plasma membrane, respectively. Translocation of GFP-p40 phox or GFP-p47 phox to their respective membrane-targeting sites is abolished by mutations in their phox (PX) domains that disrupt their interactions with their cognate phospholipid ligands. Furthermore, GFP-p67 phox translocation to either membrane is abolished by mutations that disrupt its interaction with p40 phox or p47 phox . Finally, we detected a head-to-tail (PX-Phox and Bem1 [PB1] domain) intramolecular interaction within p40 phox in its resting state by deletion mutagenesis, cell localization, and binding experiments, suggesting that its PX domain is inaccessible to interact with phosphatidylinositol 3-phosphate without cell stimulation. Thus, both p40 phox and p47 phox function as diverse p67 phox "carrier proteins" regulated by the unmasking of membrane-targeting domains in distinct mechanisms. INTRODUCTIONIn phagocytic cells, reactive oxygen species (ROS) are produced by NADPH oxidase (Nox2 system). The enzyme is a multiprotein complex assembled from a membrane-spanning flavocytochrome b 558 (composed of gp91 phox [Nox2] and p22 phox ) and four cytoplasmic components (p47 phox , p67 phox , p40 phox , and Rac) (Leto, 1999;Nauseef, 2004;Quinn and Gauss, 2004). In unstimulated phagocytes, the oxidase is dissociated and inactive: the flavocytochrome b 558 is stored on the membranes of intracellular granules (Jesaitis et al., 1990), Rac is maintained in a GDP-bound cytoplasmic complex dimerized with Rho-guanine nucleotide dissociation inhibitor (GDI) , and the other phox proteins associate in a separate ternary cytoplasmic complex (p47 phox -p67 phox -p40 phox ) in a dephosphorylated state (Bolscher et al., 1989;Rotrosen and Leto, 1990;Kuribayashi et al., 2002;Lapouge et al., 2002). During phagocyte activation, intracellular granules containing flavocytochrome b 558 fuse with phagosomes; p47 phox is phosphorylated, thereby inducing conformational changes in p47 phox that promote the interaction of the cytoplasmic complex with the flavocytochrome b 558 ; and Rac dissociates from Rho-GDI and translocates independently to the membrane after exchange of GDP for GTP (Heyworth et al., 1994;Zhao et al., 2003), resulting in generation of superoxide anion by the transfer of electrons from cytoplasmic NADPH to molecular oxygen.Chronic granulomatous disease...

show abstract

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox

Ueyama

Tatsuno

Kawasaki

et al. 2007

MBoC

101

View full text Add to dashboard Cite

show abstract

“…Preparation of Phagocyte Membrane Vesicles-Phagocyte membranes were prepared from guinea pig peritoneal macrophages, as described (7). The membranes were solubilized by 40 mM n-octyl-␤-Dglucopyranoside, and membrane vesicles were prepared by extensive dialysis against detergent-free buffer, as described (18).…”

Section: Methodsmentioning

confidence: 99%

“…This hypothesis also implies that p47 phox and Rac serve either as "carriers" for p67 phox from the cytosol to the membrane or, following their own translocation, as membrane "anchors" for the correct positioning of p67 phox in the assembled complex. Oxidase assembly can be elicited in vitro in a cell-free system consisting of phagocyte membranes and the cytosolic components p47 phox , p67 phox , and Rac, exposed to an anionic amphiphile (7,8). The role of the amphiphile is to induce a conformational change in p47 phox , resulting in its binding to p22 phox and the passive translocation of p67 phox , by virtue of its affinity for p47 phox , to the vicinity of gp91 phox .…”

mentioning

confidence: 99%

The Guanine Nucleotide Exchange Factor Trio Activates the Phagocyte NADPH Oxidase in the Absence of GDP to GTP Exchange on Rac

Sigal¹,

Gorzalczany²,

Sarfstein³

et al. 2003

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The superoxide-generating NADPH oxidase complex of phagocytes consists of a membrane-associated flavocytochrome b 559 and four cytosolic components as follows: p47 phox , p67 phox , p40 phox , and the small GTPase Rac (1 or 2). Activation of the oxidase is the result of assembly of the cytosolic components with cytochrome b 559 and can be mimicked in vitro by mixtures of membrane and cytosolic components exposed to an anionic amphiphile, serving as activator. We reported that prenylation of Rac1 endows it with the ability to support oxidase activation in conjunction with p67 phox but in the absence of amphiphile and p47 phox . We now show the following 6 points. 1) The Rac guanine nucleotide exchange factor Trio markedly potentiates oxidase activation by prenylated Rac1-GDP. 2) This occurs in the absence of exogenous GTP or any other source of GTP generation, demonstrating that the effect of Trio does not involve GDP to GTP exchange on Rac1. 3) Trio does not potentiate oxidase activation by prenylated Rac1-GTP, by nonprenylated Rac1-GDP in the presence or absence of amphiphile, and by a prenylated [p67 phox -Rac1] chimera in GDP-bound form. 4) Rac1 mutants defective in the ability to bind Trio or to respond to Trio by nucleotide exchange fail to respond to Trio by enhanced oxidase activation. 5) A Trio mutant with conserved Rac1-binding ability but lacking nucleotide exchange activity fails to enhance oxidase activation. 6) The effect of Trio is mimicked by displacement of Mg 2؉ from Rac1-GDP. These results reveal the existence of a novel mechanism of Rac activation by a guanine nucleotide exchange factor and suggest that the induction by Trio of a conformational change in Rac1, in the absence of nucleotide exchange, is sufficient for enhancing its effector function. . 4). The identity of the cytosolic component(s) responsible for causing the conformational change in gp91 phox is controversial. The principal candidate is p67 phox , based on the identification of an "activation domain" in p67 phox , consisting of residues 199 -210 (5), and on direct evidence of binding of p67 phox to gp91 phox , an interaction enhanced by Rac1 (6). This hypothesis also implies that p47 phox and Rac serve either as "carriers" for p67 phox from the cytosol to the membrane or, following their own translocation, as membrane "anchors" for the correct positioning of p67 phox in the assembled complex. Oxidase assembly can be elicited in vitro in a cell-free system consisting of phagocyte membranes and the cytosolic components p47 phox , p67 phox , and Rac, exposed to an anionic amphiphile (7,8). The role of the amphiphile is to induce a conformational change in p47 phox , resulting in its binding to p22 phox and the passive translocation of p67 phox , by virtue of its affinity for p47 phox , to the vicinity of gp91 phox . The in vivo equivalent of amphiphile action is the phosphorylation of critical serines in p47 phox , a process representing the starting point of a "p47 phoxinitiated" pathway of oxidase activation.Under certain condi...

show abstract

“…Since the inception of the cell-free oxidase assay (21)(22)(23), several theories have been put forth to explain oxidase activation by anionic amphiphiles (e.g., arachidonate, SDS, or LDS). There is evidence for direct interactions with the cytosolic subunits (24,25) that induce conformational changes, fostering the notion that certain Src homology 3 binding domains in both p47 phox and p67 phox remain inaccessible until exposed to anionic amphiphile.…”

Section: Translocation Assaymentioning

confidence: 99%

Functional Epitope on Human Neutrophil Flavocytochrome b558

Burritt

Foubert

Baniulis

et al. 2003

The Journal of Immunology

View full text Add to dashboard Cite

mAb NL7 was raised against purified flavocytochrome b558, important in host defense and inflammation. NL7 recognized the gp91phox flavocytochrome b558 subunit by immunoblot and bound to permeabilized neutrophils and neutrophil membranes. Epitope mapping by phage display analysis indicated that NL7 binds the 498EKDVITGLK506 region of gp91phox. In a cell-free assay, NL7 inhibited in vitro activation of the NADPH oxidase in a concentration-dependent manner, and had marginal effects on the oxidase substrate Michaelis constant (Km). mAb NL7 did not inhibit translocation of p47phox, p67phox, or Rac to the plasma membrane, and bound its epitope on gp91phox independently of cytosolic factor translocation. However, after assembly of the NADPH oxidase complex, mAb NL7 bound the epitope but did not inhibit the generation of superoxide. Three-dimensional modeling of the C-terminal domain of gp91phox on a corn nitrate reductase template suggests close proximity of the NL7 epitope to the proposed NADPH binding site, but significant separation from the proposed p47phox binding sites. We conclude that the 498EKDVITGLK506 segment resides on the cytosolic surface of gp91phox and represents a region important for oxidase function, but not substrate or cytosolic component binding.

show abstract

Unsaturated fatty acids stimulate NADPH-dependent superoxide production by cell-free system derived from macrophages

Cited by 330 publications

References 24 publications

A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox

A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox

The Guanine Nucleotide Exchange Factor Trio Activates the Phagocyte NADPH Oxidase in the Absence of GDP to GTP Exchange on Rac

Functional Epitope on Human Neutrophil Flavocytochrome b558

Contact Info

Product

Resources

About

Unsaturated fatty acids stimulate NADPH-dependent superoxide production by cell-free system derived from macrophages

Cited by 330 publications

References 24 publications

A Regulated Adaptor Function of p40phox: Distinct p67phoxMembrane Targeting by p40phoxand by p47phox

A Regulated Adaptor Function of p40phox: Distinct p67phoxMembrane Targeting by p40phoxand by p47phox

The Guanine Nucleotide Exchange Factor Trio Activates the Phagocyte NADPH Oxidase in the Absence of GDP to GTP Exchange on Rac

Functional Epitope on Human Neutrophil Flavocytochrome b558

Contact Info

Product

Resources

About

A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox

A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox