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P47phox Phox homology domain regulates plasma membrane but not phagosome neutrophil NADPH oxidase activation.

ABSTRACT: The assembly of cytosolic subunits p47(phox), p67(phox), and p40(phox) with flavocytochrome b(558) at the membrane is required for activating the neutrophil NADPH oxidase that generates superoxide for microbial killing. The p47(phox) subunit plays a critical role in oxidase assembly. Recent studies showed that the p47(phox) Phox homology (PX) domain mediates phosphoinositide binding in vitro and regulates phorbol ester-induced NADPH oxidase activity in a K562 myeloid cell model. Because the importance of the p47(phox) PX domain in neutrophils is unclear, we investigated its role using p47(phox) knock-out (KO) mouse neutrophils to express human p47(phox) and derivatives harboring R90A mutations in the PX domain that result in loss of phosphoinositide binding. Human p47(phox) proteins were expressed at levels similar to endogenous murine p47(phox), with the exception of a chronic granulomatous disease-associated R42Q mutant that was poorly expressed, and wild type human p47(phox) rescued p47(phox) KO mouse neutrophil NADPH oxidase activity. Plasma membrane NAPDH oxidase activity was reduced in neutrophils expressing p47(phox) with Arg(90) substitutions, with substantial effects on responses to either phorbol ester or formyl-Met-Leu-Phe and more modest effects to particulate stimuli. In contrast, p47(phox) Arg(90) mutants supported normal levels of intracellular NADPH oxidase activity during phagocytosis of a variety of particles and were recruited to phagosome membranes. This study defines a differential and agonist-dependent role of the p47(phox) PX domain for neutrophil NADPH oxidase activation.

SUBMITTER: Li XJ

PROVIDER: S-EPMC2966130 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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p47phox Phox homology domain regulates plasma membrane but not phagosome neutrophil NADPH oxidase activation.

Li Xing Jun XJ Marchal Christophe C CC Stull Natalie D ND Stahelin Robert V RV Dinauer Mary C MC

The Journal of biological chemistry 20100905 45

The assembly of cytosolic subunits p47(phox), p67(phox), and p40(phox) with flavocytochrome b(558) at the membrane is required for activating the neutrophil NADPH oxidase that generates superoxide for microbial killing. The p47(phox) subunit plays a critical role in oxidase assembly. Recent studies showed that the p47(phox) Phox homology (PX) domain mediates phosphoinositide binding in vitro and regulates phorbol ester-induced NADPH oxidase activity in a K562 myeloid cell model. Because the impo ...[more]

PMID: 20817944

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Project description:The reactive oxygen-generating NADPH oxidases (Noxes) function in a variety of biological roles, and can be broadly classified into those that are regulated by subunit interactions and those that are regulated by calcium. The prototypical subunit-regulated Nox, Nox2, is the membrane-associated catalytic subunit of the phagocyte NADPH-oxidase. Nox2 forms a heterodimer with the integral membrane protein, p22phox, and this heterodimer binds to the regulatory subunits p47phox, p67phox, p40phox and the small GTPase Rac, triggering superoxide generation. Nox-organizer protein 1 (NOXO1) and Nox-activator 1 (NOXA1), respective homologs of p47phox and p67phox, together with p22phox and Rac, activate Nox1, a non-phagocytic homolog of Nox2. NOXO1 and p22phox also regulate Nox3, whereas Nox4 requires only p22phox. In this study, we have assembled and analyzed amino acid sequences of Nox regulatory subunit orthologs from vertebrates, a urochordate, an echinoderm, a mollusc, a cnidarian, a choanoflagellate, fungi and a slime mold amoeba to investigate the evolutionary history of these subunits.Ancestral p47phox, p67phox, and p22phox genes are broadly seen in the metazoa, except for the ecdysozoans. The choanoflagellate Monosiga brevicollis, the unicellular organism that is the closest relatives of multicellular animals, encodes early prototypes of p22phox, p47phox as well as the earliest known Nox2-like ancestor of the Nox1-3 subfamily. p67phox- and p47phox-like genes are seen in the sea urchin Strongylocentrotus purpuratus and the limpet Lottia gigantea that also possess Nox2-like co-orthologs of vertebrate Nox1-3. Duplication of primordial p47phox and p67phox genes occurred in vertebrates, with the duplicated branches evolving into NOXO1 and NOXA1. Analysis of characteristic domains of regulatory subunits suggests a novel view of the evolution of Nox: in fish, p40phox participated in regulating both Nox1 and Nox2, but after the appearance of mammals, Nox1 (but not Nox2) became independent of p40phox. In the fish Oryzias latipes, a NOXO1 ortholog retains an autoinhibitory region that is characteristic of mammalian p47phox, and this was subsequently lost from NOXO1 in later vertebrates. Detailed amino acid sequence comparisons identified both putative key residues conserved in characteristic domains and previously unidentified conserved regions. Also, candidate organizer/activator proteins in fungi and amoeba are identified and hypothetical activation models are suggested.This is the first report to provide the comprehensive view of the molecular evolution of regulatory subunits for Nox enzymes. This approach provides clues for understanding the evolution of biochemical and physiological functions for regulatory-subunit-dependent Nox enzymes.

Dataset Information

P47phox Phox homology domain regulates plasma membrane but not phagosome neutrophil NADPH oxidase activation.

Publications

p47phox Phox homology domain regulates plasma membrane but not phagosome neutrophil NADPH oxidase activation.

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