Assembly and Activation of the Phagocyte NADPH Oxidase

The superoxide-generating NADPH oxidase complex of phagocytes consists of a membranal heterodimeric flavocytochrome (cytochrome b 559 ), composed of gp91 phox and p22 phox subunits, and four cytosolic proteins, p47phox , p67 phox , p40 phox , and the small GTPase Rac (1 or 2). All redox stations involved in electron transport from NADPH to oxygen are located in gp91 phox . NADPH oxidase activation is the consequence of assembly of cytochrome b 559 with cytosolic proteins, a process reproducible in a cell-free system, consisting of phagocyte membranes, and recombinant cytosolic components, activated by an anionic amphiphile. p22phox is believed to act as a linker between the cytosolic components and gp91phox . We applied "peptide walking" to mapping of domains in p22 phox participating in NADPH oxidase assembly. Ninety one synthetic overlapping pentadecapeptides, spanning the p22 phox sequence, were tested for the ability to inhibit NADPH oxidase activation in the cell-free system and to bind individual cytosolic NADPH oxidase components. We conclude the following. 1) The p22 phox subunit of cytochrome b 559 serves as an anchor for both p47 phox and p67 phox . 2) p47 phox binds not only to the proline-rich region, located at residues 151-160 in the cytosolic C terminus of p22 phox , but also to a domain (residues 51-63) located on a loop exposed to the cytosol. 3) p67 phox shares with p47 phox the ability to bind to the proline-rich region (residues 151-160) and also binds to two additional domains, in the cytosolic loop (residues 81-91) and at the start of the cytosolic tail (residues 111-115). 4) The binding affinity of p67 phox for p22 phox peptides is lower than that of p47 phox . 5) Binding of both p47 phox and p67 phox to proline-rich p22 phox peptides occurs in the absence of an anionic amphiphile. A revised membrane topology model of p22 phox is proposed, the core of which is the presence of a functionally important cytosolic loop (residues 51-91).

contrasting

confidence: 96%

contrasting

confidence: 93%

Mapping of Functional Domains in the p22 Subunit of Flavocytochrome b 559 Participating in the Assembly of the NADPH Oxidase Complex by “Peptide Walking”

Dahan¹,

Issaeva²,

Gorzalczany³

et al. 2002

“…The polymerase chain reaction product was subcloned into pGEX-4T (Amersham Pharmacia Biotech), and subjected to DNA sequencing for the confirmation of precise construction. The GST fusion protein was expressed in E. coli BL21 cells and purified by glutathione-Sepharose-4B (18,20,21).…”

Section: Methodsmentioning

confidence: 99%

Phosphoinositide 3-Kinase-dependent and -independent Activation of the Small GTPase Rac2 in Human Neutrophils

Akasaki

Koga

Sumimoto

1999

Self Cite

158

145

The small GTPase Rac participates in various cellular events such as cytoskeletal reorganization. It has remained, however, largely unknown about intracellular signaling pathways for Rac activation because of the lack of a simple and reliable assay to estimate the activation. Here we describe a novel method to detect the GTP-bound, active Rac in cells by pulling it down with the Rac-binding domain of the protein kinase PAK. Experiments using this method reveal that stimulation of human neutrophils with the G i -coupled receptor agonists N-formyl-methionyl-leucyl-phenylalanine (fMLP) and leukotriene B 4 (LTB 4 ) leads to a rapid and transient increase in the GTP-bound state of Rac2, whereas phorbol myristate acetate (PMA) causes a slow but more sustained activation of Rac2. Pretreatment of cells with pertussis toxin results in defective activation of Rac2 in response to fMLP and LTB 4 , indicating that coupling of the receptors to G i plays a crucial role in the activation. Furthermore, the phosphoinositide 3-kinase (PI3K) inhibitors wortmannin and LY294002 block Rac2 activation elicited by the receptor agonists, but not that by PMA. Thus the G i -coupled receptors likely mediate Rac2 activation via PI3K, whereas PMA activates Rac2 in a PI3K-independent manner.

“…Some controversy exists for the NADPH binding site, which is thought to be positioned in Nox2 (33), whereas others suggest localization in p67phox (34). p22phox is the binding partner of p47phox (35), the subunits required for oxidase assembly. Phosphorylation of p22phox in vitro has been demonstrated in the course of activation (36) but certainly is not central for oxidase activation.…”

Section: Fig 8 Co-immunoprecipitation Of Endogenous P22phox Bymentioning

confidence: 99%

Direct Interaction of the Novel Nox Proteins with p22phox Is Required for the Formation of a Functionally Active NADPH Oxidase

Ambasta

Kumar

Griendling

et al. 2004

477

376

Nox1 and Nox4, homologues of the leukocyte NADPH oxidase subunit Nox2 (gp91phox) mediate superoxide anion formation in various cell types. However, their interactions with other components of the NADPH oxidase are poorly defined. We determined whether a direct interaction of Nox1 and Nox4 with the p22phox subunit of the NADPH oxidase occurs. Using confocal microscopy, co-localization of p22phox with Nox1, Nox2, and Nox4 was observed in transiently transfected vascular smooth muscle cells (VSMC) and HEK293 cells. Plasmids coding for fluorescent fusion proteins of p22phox and the Nox proteins with cyan-and yellowfluorescent protein (cfp and yfp, respectively) were constructed and expressed in VSMC and HEK293 cells. The cfp-tagged p22phox expression level increased upon cotransfection with Nox1 or Nox4. Protein-protein interaction between the fluorescent fusion proteins of p22phox and the Nox partners was observed using the fluorescence resonance energy transfer technique. Immunoprecipitation of native Nox1 from human VSMC revealed co-precipitation of p22phox. Immunoprecipitation from transfected HEK293 cells revealed co-precipitation of native p22phox with yfp-tagged Nox1, Nox2, and Nox4. Following mutation of a histidine (corresponding to the position 115 in human Nox2) to leucine, this interaction was abolished. Transfection of rat p22phox (but not Noxo1 and Noxa1) increased the radical generation in cells expressing Nox4. We provide evidence that p22phox directly interacts with Nox1 and Nox4, to form an superoxide-generating NADPH oxidase and demonstrate that mutation of the potential heme binding site in the Nox proteins disrupts the complex formation of Nox1 and Nox4 with p22phox.