Role of the phospholipid binding sites,PX of p47phox and PB region of Rac1, in the formation of the phagocyte NADPH oxidase complex NOX2 |
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| Institution: | 1. Université Paris Saclay, Institut de Chimie Physique UMR 8000, CNRS, 91405 Orsay Cedex, France;2. Laboratory of Applied Biotechnology (LBA3B), AZM Center for Research in Biotechnology and its Applications, Doctoral School for Sciences and Technology, Lebanese University, Tripoli 1300, Lebanon;3. Univ. Bordeaux, Bordeaux INP, CNRS, ISM, UMR 5255, F-33402 Talence, France;4. Univ. Bordeaux, CNRS, Bordeaux INP, CBMN, UMR 5248, F-33600 Pessac, France;1. Department of Chemistry and Biochemistry, Stephenson Life Sciences Research Center, University of Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, United States of America;2. Department of Biology, University of Central Oklahoma, 100 North University Drive, Edmond, OK 73034, United States of America;1. Membrane Biochemistry and Biophysics, Department of Chemistry, Faculty of Science, Utrecht University, Utrecht, Netherlands;2. Zhejiang Provincial Key Laboratory of Food Microbiotechnology Research of China, the Zhejiang Gongshang University of China, Hangzhou, China;3. Antimicrobial Pharmacodynamics and Therapeutics, Department of Pharmacology and Therapeutics, Institute of Systems, Molecular & Integrative Biology, University of Liverpool, William Henry Duncan Building, 6 West Derby St, Liverpool L7 8TX, UK;4. Antimicrobial Drug Discovery and Development, Department of Chemistry, The Robert Robinson Laboratories, University of Liverpool, L69 3BX Liverpool, UK |
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| Abstract: | In phagocytes, superoxide anion (O2 ?), the precursor of reactive oxygen species, is produced by the NADPH oxidase complex to kill pathogens. Phagocyte NADPH oxidase consists of the transmembrane cytochrome b558 (cyt b558) and four cytosolic components: p40phox, p47phox, p67phox, and Rac1/2. The phagocyte activation by stimuli leads to activation of signal transduction pathways. This is followed by the translocation of cytosolic components to the membrane and their association with cyt b558 to form the active enzyme.To investigate the roles of membrane-interacting domains of the cytosolic proteins in the NADPH oxidase complex assembly and activity, we used giant unilamellar phospholipid vesicles (GUV). We also used the neutrophil-like cell line PLB-985 to investigate these roles under physiological conditions. We confirmed that the isolated proteins must be activated to bind to the membrane. We showed that their membrane binding was strengthened by the presence of the other cytosolic partners, with a key role for p47phox. We also used a fused chimera consisting of p47phox(aa 1–286), p67phox(aa 1–212) and Rac1Q61L, as well as mutated versions in the p47phox PX domain and the Rac polybasic region (PB). We showed that these two domains have a crucial role in the trimera membrane-binding and in the trimera assembly to cyt b558. They also have an impact on O2.- production in vitro and in cellulo: the PX domain strongly binding to GUV made of a mix of polar lipids; and the PB region strongly binding to the plasma membrane of neutrophils and resting PLB-985 cells. |
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