Assembly of the phagocyte NADPH oxidase complex: Chimeric constructs derived from the cytosolic components as tools for exploring structure-function relationships

Ariel Mizrahi, Yevgeny Berdichevsky, Yelena Ugolev, Shahar Molshanski-Mor, Yael Nakash, Iris Dahan, Nathalie Alloul, Yara Gorzalczany, Rive Sarfstein, Miriam Hirshberg, Edgar Pick*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)881-895
Number of pages15
JournalJournal of Leukocyte Biology
Volume79
Issue number5
DOIs
StatePublished - May 2006

Keywords

  • GTP/GDP
  • Oxygen radicals
  • Rac
  • Respiratory burst
  • Small GTPases
  • Superoxide
  • p47
  • p67

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