scholarly journals Activation of the superoxide-producing phagocyte NADPH oxidase requires co-operation between the tandem SH3 domains of p47phox in recognition of a polyproline type II helix and an adjacent α-helix of p22phox

2006 ◽  
Vol 396 (1) ◽  
pp. 183-192 ◽  
Author(s):  
Ikuo Nobuhisa ◽  
Ryu Takeya ◽  
Kenji Ogura ◽  
Noriko Ueno ◽  
Daisuke Kohda ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nadph Oxidase ◽  
Regulatory Protein ◽  
Sh3 Domain ◽  
Alanine Scanning Mutagenesis ◽  
Catalytic Core ◽  
Intact Cells ◽  
Sh3 Domains ◽  
Phagocyte Nadph Oxidase ◽  
Phagocyte Oxidase

Activation of the superoxide-producing phagocyte NADPH oxidase, crucial for host defence, requires an SH3 (Src homology 3)-domain-mediated interaction of the regulatory protein p47phox with p22phox, a subunit of the oxidase catalytic core flavocytochrome b558. Although previous analysis of a crystal structure has demonstrated that the tandem SH3 domains of p47phox sandwich a short PRR (proline-rich region) of p22phox (amino acids 151–160), containing a polyproline II helix, it has remained unknown whether this model is indeed functional in activation of the oxidase. In the present paper we show that the co-operativity between the two SH3 domains of p47phox, as expected from the model, is required for oxidase activation. Deletion of the linker between the p47phox SH3 domains results not only in a defective binding to p22phox but also in a loss of the activity to support superoxide production. The present analysis using alanine-scanning mutagenesis identifies Pro152, Pro156 and Arg158 in the p22phox PRR as residues indispensable for the interaction with p47phox. Pro152 and Pro156 are recognized by the N-terminal SH3 domain, whereas Arg158 contacts with the C-terminal SH3 domain. Amino acid substitution for any of the three residues in the p22phox PRR abrogates the superoxide-producing activity of the oxidase reconstituted in intact cells. The bis-SH3-mediated interaction of p47phox with p22phox thus functions to activate the phagocyte oxidase. Furthermore, we provide evidence that a region C-terminal to the PRR of p22phox (amino acids 161–164), adopting an α-helical conformation, participates in full activation of the phagocyte oxidase by fortifying the association with the p47phox SH3 domains.

A region N-terminal to the tandem SH3 domain of p47phox plays a crucial role in the activation of the phagocyte NADPH oxidase

2009 ◽  
Vol 419 (2) ◽  
pp. 329-338 ◽  
Author(s):  
Masahiko Taura ◽  
Kei Miyano ◽  
Reiko Minakami ◽  
Sachiko Kamakura ◽  
Ryu Takeya ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Crucial Role ◽  
Sh3 Domain ◽  
Small Gtpase ◽  
Dependent Manner ◽  
Wild Type ◽  
Catalytic Core ◽  
Membrane Recruitment ◽  
Phagocyte Nadph Oxidase ◽  
Isoleucine Residue

The superoxide-producing NADPH oxidase in phagocytes is crucial for host defence; its catalytic core is the membrane-integrated protein gp91phox [also known as Nox2 (NADPH oxidase 2)], which forms a stable heterodimer with p22phox. Activation of the oxidase requires membrane translocation of the three cytosolic proteins p47phox, p67phox and the small GTPase Rac. At the membrane, these proteins assemble with the gp91phox–p22phox heterodimer and induce a conformational change of gp91phox, leading to superoxide production. p47phox translocates to membranes using its two tandemly arranged SH3 domains, which directly interact with p22phox, whereas p67phox is recruited in a p47phox-dependent manner. In the present study, we show that a short region N-terminal to the bis-SH3 domain is required for activation of the phagocyte NADPH oxidase. Alanine substitution for Ile152 in this region, a residue that is completely conserved during evolution, results in a loss of the ability to activate the oxidase; and the replacement of Thr153 also prevents oxidase activation, but to a lesser extent. In addition, the corresponding isoleucine residue (Ile155) of the p47phox homologue Noxo1 (Nox organizer 1) participates in the activation of non-phagocytic oxidases, such as Nox1 and Nox3. The I152A substitution in p47phox, however, does not affect its interaction with p22phox or with p67phox. Consistent with this, a mutant p47phox (I152A), as well as the wild-type protein, is targeted upon cell stimulation to membranes, and membrane recruitment of p67phox and Rac normally occurs in p47phox (I152A)-expressing cells. Thus the Ile152-containing region of p47phox plays a crucial role in oxidase activation, probably by functioning at a process after oxidase assembly.

An SH3 domain-mediated interaction between the phagocyte NADPH oxidase factors p40 phox and p47 phox

FEBS Letters ◽  
1996 ◽  
Vol 385 (3) ◽  
pp. 229-232 ◽  
Author(s):  
Takashi Ito ◽  
Rika Nakamura ◽  
Hideki Sumimoto ◽  
Koichiro Takeshige ◽  
Yoshiyuki Sakaki
Keyword(s):  
Nadph Oxidase ◽  
Sh3 Domain ◽  
Phagocyte Nadph Oxidase

scholarly journals Properties of phagocyte NADPH oxidase p47-phox mutants with unmasked SH3 (Src homology 3) domains: full reconstitution of oxidase activity in a semi-recombinant cell-free system lacking arachidonic acid

2003 ◽  
Vol 373 (1) ◽  
pp. 221-229 ◽  
Author(s):  
Guihong PENG ◽  
Jin HUANG ◽  
Mellonie BOYD ◽  
Michael E. KLEINBERG
Keyword(s):  
Arachidonic Acid ◽  
Nadph Oxidase ◽  
Sh3 Domain ◽  
Free System ◽  
Cell Free System ◽  
Src Homology 3 ◽  
Rich Domain ◽  
Phagocyte Nadph Oxidase ◽  
Px Domain ◽  
Src Homology

In an early step in the assembly of the phagocyte NADPH oxidase, p47-phox translocates from the cytosol to the membrane, mediated by engagement of the N-termini of two p47-phox Src homology 3 (SH3) domains with a proline-rich region (PRR) in the p22-phox subunit of cytochrome b558. In response to phagocyte activation, several serine residues in a C-terminal arginine/lysine-rich domain of p47-phox are phosphorylated, leading to changes in the conformation of p47-phox and exposure of its N-terminal SH3 domain that is normally masked by internal association with the arginine/lysine-rich domain. We report that triple alanine substitutions at Asp-217, Glu-218 and Glu-223 in a short sequence that links the tandem p47-phox SH3 domains unmasked the N-terminal SH3 domain, similar to the effects of aspartic acid substitutions at Ser-310 and Ser-328 in the arginine/lysine-rich region. Recombinant p47-phox proteins with mutations in either the linker region or the arginine/lysine-rich domain were active in the absence of arachidonic acid stimulation in a cell-free NADPH oxidase system consisting of recombinant p67-phox, Rac1–guanosine 5′-[γ-thio]triphosphate and neutrophil membranes. Supplementing neutrophil membranes with phosphoinositides or other negatively charged phospholipids markedly enhanced cell-free superoxide generation by these p47-phox mutants in the absence of arachidonic acid, to levels equivalent to those generated by wild-type p47-phox following arachidonic acid activation. This enhancement may be related to recruitment to the membrane of p47-phox mediated by a novel secondary phox homology (PX) domain binding site that broadly recognizes phospholipids. No specific enhancement by specific phosphorylated phosphatidylinositols was found to suggest a dominant role for the p47-phox primary PX domain binding site. Truncated p47-phox S310D S328D lacking the C-terminal PRR was inactive in the cell-free system without arachidonic acid, but was fully active with arachidonic acid. This suggests that activation of NADPH oxidase in an arachidonate-free cell-free system requires association of the p47-phox C-terminal PRR with the p67-phox C-terminal SH3 domain.

scholarly journals Functional modules and expression of mouse p40 phox and p67phox, SH3-domain-containing proteins involved in the phagocyte NADPH oxidase complex

1998 ◽  
Vol 251 (3) ◽  
pp. 573-582 ◽  
Author(s):  
Kazuhito Mizuki ◽  
Kenji Kadomatsu ◽  
Kenichiro Hata ◽  
Takashi Ito ◽  
Qi-Wen Fan ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Sh3 Domain ◽  
Functional Modules ◽  
Phagocyte Nadph Oxidase ◽  
Nadph Oxidase Complex

A region C-terminal to the proline-rich core of p47phox regulates activation of the phagocyte NADPH oxidase by interacting with the C-terminal SH3 domain of p67phox

2005 ◽  
Vol 444 (2) ◽  
pp. 185-194 ◽  
Author(s):  
Kazuhito Mizuki ◽  
Ryu Takeya ◽  
Futoshi Kuribayashi ◽  
Ikuo Nobuhisa ◽  
Daisuke Kohda ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Sh3 Domain ◽  
Phagocyte Nadph Oxidase

The insert region of the Rac GTPases is dispensable for activation of superoxide-producing NADPH oxidases

2009 ◽  
Vol 422 (2) ◽  
pp. 373-382 ◽  
Author(s):  
Kei Miyano ◽  
Hirofumi Koga ◽  
Reiko Minakami ◽  
Hideki Sumimoto
Keyword(s):  
Amino Acids ◽  
Nadph Oxidase ◽  
Essential Role ◽  
Cellular Level ◽  
Nadph Oxidases ◽  
Catalytic Core ◽  
Rac Gtpases ◽  
Nadph Oxidase 1 ◽  
Nox Family ◽  
Insert Region

Rac1 and Rac2, which belong to the Rho subfamily of Ras-related GTPases, play an essential role in activation of gp91phox/Nox2 (cytochrome b-245, β polypeptide; also known as Cybb), the catalytic core of the superoxide-producing NADPH oxidase in phagocytes. Rac1 also contributes to activation of the non-phagocytic oxidases Nox1 (NADPH oxidase 1) and Nox3 (NADPH oxidase 3), each related closely to gp91phox/Nox2. It has remained controversial whether the insert region of Rac (amino acids 123–135), unique to the Rho subfamily proteins, is involved in gp91phox/Nox2 activation. In the present study we show that removal of the insert region from Rac1 neither affects activation of gp91phox/Nox2, which is reconstituted under cell-free and whole-cell conditions, nor blocks its localization to phagosomes during ingestion of IgG-coated beads by macrophage-like RAW264.7 cells. The insert region of Rac2 is also dispensable for gp91phox/Nox2 activation at the cellular level. Although Rac2, as well as Rac1, is capable of enhancing superoxide production by Nox1 and Nox3, the enhancements by the two GTPases are both independent of the insert region. We also demonstrate that Rac3, a third member of the Rac family in mammals, has an ability to activate the three oxidases and that the activation does not require the insert region. Thus the insert region of the Rac GTPases does not participate in regulation of the Nox family NADPH oxidases.

scholarly journals Consequences of the constitutive NOX2 activity in living cells: cytosol acidification, apoptosis, and localized lipid peroxidation

2021 ◽  
Author(s):  
Hana Valenta ◽  
Sophie Dupré-Crochet ◽  
Tania Bizouarn ◽  
Laura Baciou ◽  
Oliver Nüsse ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Nadph Oxidase ◽  
Living Cells ◽  
Small Gtpase ◽  
Activation Mechanism ◽  
Cell Physiology ◽  
List Type ◽  
Catalytic Core ◽  
Cytosolic Proteins ◽  
Phagocyte Nadph Oxidase

ABSTRACTThe 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.HighlightsTrimera is a tool to trigger a continuous ROS production in living cellsContinuous NOX2 activity causes cytosol acidification and apoptosisROS overproduction leads to localized oxidation of the membrane lipidsTrimera tends to clusterize in the plasma membrane of COSNOX and COS-7 cells

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