title: Tripartite chimeras comprising functional domains derived from the cytosolic NADPH oxidase components p47phox, p67phox, and Rac1 elicit activator-independent superoxide production by phagocyte membranes: an essential role for anionic membrane phospholipids.
abstract: The superoxide-generating NADPH oxidase is converted to an active state by the assembly of a membrane-localized cytochrome b(559) with three cytosolic components: p47(phox), p67(phox), and GTPase Rac1 or Rac2. Assembly involves two sets of protein-protein interactions: among cytosolic components and among cytosolic components and cytochrome b(559) within its lipid habitat. We circumvented the need for interactions among cytosolic components by constructing a recombinant tripartite chimera (trimera) consisting of the Phox homology (PX) and Src homology 3 (SH3) domains of p47(phox), the tetratricopeptide repeat and activation domains of p67(phox), and full-length Rac1. Upon addition to phagocyte membrane, the trimera was capable of oxidase activation in vitro in the presence of an anionic amphiphile. The trimera had a higher affinity (lower EC(50)) for and formed a more stable complex (longer half-life) with cytochrome b(559) compared with the combined individual components, full-length or truncated. Supplementation of membrane with anionic but not neutral phospholipids made activation by the trimera amphiphile-independent. Mutagenesis, truncations, and domain replacements revealed that oxidase activation by the trimera was dependent on the following interactions: 1) interaction with anionic membrane phospholipids via the poly-basic stretch at the C terminus of the Rac1 segment; 2) interaction with p22(phox) via Trp(193) in the N-terminal SH3 domain of the p47(phox) segment, supplementing the electrostatic attraction; and 3) an intrachimeric bond among the p67(phox) and Rac1 segments complementary to their physical fusion. The PX domain of the p47(phox) segment and the insert domain of the Rac1 segment made only minor contributions to oxidase assembly.
authors.affiliation: Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research and the Ela Kodesz Institute of Host Defense against Infectious Diseases, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.
authors.forename: Yevgeny
authors.initials: Y
authors.lastname: Berdichevsky
authors.forename: Ariel
authors.initials: A
authors.lastname: Mizrahi
authors.forename: Yelena
authors.initials: Y
authors.lastname: Ugolev
authors.forename: Shahar
authors.initials: S
authors.lastname: Molshanski-Mor
authors.forename: Edgar
authors.initials: E
authors.lastname: Pick
mesh_terms: D000818:Animals; D003600:Cytosol / Q000201:enzymology*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006168:Guinea Pigs; D046911:Macromolecular Substances / Q000378:metabolism; D008563:Membrane Lipids / Q000502:physiology; D050506:Mutant Chimeric Proteins / Q000302:isolation & purification / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism*; D010586:Phagocytes / Q000502:physiology*; D010743:Phospholipids / Q000502:physiology*; D010750:Phosphoproteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
chemical_list: D046911:Macromolecular Substances; D008563:Membrane Lipids; D050506:Mutant Chimeric Proteins; D010743:Phospholipids; D010750:Phosphoproteins; C107953:RAC1 protein, human; D011994:Recombinant Proteins; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; D006165:Guanylyl Imidodiphosphate; D019255:NADPH Oxidases; D020830:rac1 GTP-Binding Protein