Premium
Assembly of the phagocyte NADPH oxidase complex: chimeric constructs derived from the cytosolic components as tools for exploring structure‐function relationships
Author(s) -
Mizrahi Ariel,
Berdichevsky Yevgeny,
Ugolev Yelena,
MolshanskiMor Shahar,
Nakash Yael,
Dahan Iris,
Alloul Nathalie,
Gorzalczany Yara,
Sarfstein Rive,
Hirshberg Miriam,
Pick Edgar
Publication year - 2006
Publication title -
journal of leukocyte biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.819
H-Index - 191
eISSN - 1938-3673
pISSN - 0741-5400
DOI - 10.1189/jlb.1005553
Subject(s) - nadph oxidase , nicotinamide adenine dinucleotide phosphate , biology , biochemistry , oxidase test , p22phox , rac gtp binding proteins , rac1 , biophysics , superoxide , förster resonance energy transfer , conformational change , gtpase , cytosol , microbiology and biotechnology , enzyme , signal transduction , fluorescence , physics , quantum mechanics
Phagocytes generate superoxide (O 2 .− ) 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 O 2 .− , is flavocytochrome b 559 , located in the membrane and consisting of gp91 phox and p22 phox subunits. NADPH oxidase activation is initiated by the translocation to the membrane of the cytosolic components p47 phox , p67 phox , and the GTPase Rac. Cytochrome b 559 is converted to an active form by the interaction of gp91 phox with p67 phox , leading to a conformational change in gp91 phox 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 p67 phox (1–212)‐Rac1 (1–192) is a potent activator in a cell‐free system, also containing membrane p47 phox and an anionic amphiphile. Chimeras behave like bona fide GTPases and can be prenylated, and prenylated (p67 phox ‐Rac1) chimeras activate the oxidase in the absence of p47 phox 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 p67 phox and from p67 phox to gp91 phox .