TY - JOUR
T1 - Assembly of the phagocyte NADPH oxidase complex
T2 - Chimeric constructs derived from the cytosolic components as tools for exploring structure-function relationships
AU - Mizrahi, Ariel
AU - Berdichevsky, Yevgeny
AU - Ugolev, Yelena
AU - Molshanski-Mor, Shahar
AU - Nakash, Yael
AU - Dahan, Iris
AU - Alloul, Nathalie
AU - Gorzalczany, Yara
AU - Sarfstein, Rive
AU - Hirshberg, Miriam
AU - Pick, Edgar
PY - 2006/5
Y1 - 2006/5
N2 - Phagocytes generate superoxide (O2.-) by an enzyme complex known as reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Its catalytic component, responsible for the NADPH-driven reduction of oxygen to O2.-, is flavocytochrome b559, located in the membrane and consisting of gp91phox and p22 phox subunits. NADPH oxidase activation is initiated by the translocation to the membrane of the cytosolic components p47phox, p67phox, and the GTPase Rac. Cytochrome b559 is converted to an active form by the interaction of gp91phox with p67 phox, leading to a conformational change in gp91phox and the induction of electron flow. We designed a new family of NADPH oxidase activators, represented by chimeras comprising various segments of p67 phox and Rac1. The prototype chimera p67phox (1-212)-Rac1 (1-192) is a potent activator in a cell-free system, also containing membrane p47phox and an anionic amphiphile. Chimeras behave like bona fide GTPases and can be prenylated, and prenylated (p67phox-Rac1) chimeras activate the oxidase in the absence of p47phox and amphiphile. Experiments involving truncations, mutagenesis, and supplementation with Rac1 demonstrated that the presence of intrachimeric bonds between the p67 phox and Rac1 moieties is an absolute requirement for the ability to activate the oxidase. The presence or absence of intrachimeric bonds has a major impact on the conformation of the chimeras, as demonstrated by fluorescence resonance energy transfer, small angle X-ray scattering, and gel filtration. Based on this, a "propagated wave" model of NADPH oxidase activation is proposed in which a conformational change initiated in Rac is propagated to p67phox and from p67phox to gp91phox.
AB - Phagocytes generate superoxide (O2.-) by an enzyme complex known as reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Its catalytic component, responsible for the NADPH-driven reduction of oxygen to O2.-, is flavocytochrome b559, located in the membrane and consisting of gp91phox and p22 phox subunits. NADPH oxidase activation is initiated by the translocation to the membrane of the cytosolic components p47phox, p67phox, and the GTPase Rac. Cytochrome b559 is converted to an active form by the interaction of gp91phox with p67 phox, leading to a conformational change in gp91phox and the induction of electron flow. We designed a new family of NADPH oxidase activators, represented by chimeras comprising various segments of p67 phox and Rac1. The prototype chimera p67phox (1-212)-Rac1 (1-192) is a potent activator in a cell-free system, also containing membrane p47phox and an anionic amphiphile. Chimeras behave like bona fide GTPases and can be prenylated, and prenylated (p67phox-Rac1) chimeras activate the oxidase in the absence of p47phox and amphiphile. Experiments involving truncations, mutagenesis, and supplementation with Rac1 demonstrated that the presence of intrachimeric bonds between the p67 phox and Rac1 moieties is an absolute requirement for the ability to activate the oxidase. The presence or absence of intrachimeric bonds has a major impact on the conformation of the chimeras, as demonstrated by fluorescence resonance energy transfer, small angle X-ray scattering, and gel filtration. Based on this, a "propagated wave" model of NADPH oxidase activation is proposed in which a conformational change initiated in Rac is propagated to p67phox and from p67phox to gp91phox.
KW - GTP/GDP
KW - Oxygen radicals
KW - Rac
KW - Respiratory burst
KW - Small GTPases
KW - Superoxide
KW - p47
KW - p67
UR - http://www.scopus.com/inward/record.url?scp=33745303143&partnerID=8YFLogxK
U2 - 10.1189/jlb.1005553
DO - 10.1189/jlb.1005553
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AN - SCOPUS:33745303143
SN - 0741-5400
VL - 79
SP - 881
EP - 895
JO - Journal of Leukocyte Biology
JF - Journal of Leukocyte Biology
IS - 5
ER -