Molecular mechanism for activation of the phagocyte NADPH oxidase involued in host defense

参与宿主防御的吞噬细胞NADPH氧化酶的酶核心是细胞膜上的细胞色素b_(558)。该酶的激活需要刺激诱导含有胞浆蛋白的SH3结构域的膜移位，即p47；lt；Phox&gt；，p67；lt；Phox&gt；和p40^&lt；Phox&gt；与细胞色素b_(558)相互作用。(1)确定了Px/Pb2结构域的三维结构，这是我们在p47^&lt；Phox&gt；和p40^&lt；Phox&gt；的N末端发现的一个新模块。此外，我们还发现Px/Pb2结构域具有与磷脂酰肌醇结合的活性，并且P47的Px结构域对于该蛋白的膜转位和NADPH氧化酶的激活是必不可少的。此外，我们还阐明了p47；lt；Phox&gt；px结构域的脂结合活性受其SH3结构域的分子内相互作用的负调控。(2)我们发现p40^；lt；Phox&gt；通过一种新的蛋白质-蛋白质相互作用与p67^&lt；Phox&gt；在静止吞噬细胞中结合：p67^&lt；Phox&gt；的PB1结构域识别并结合p40^&lt；phx&gt；的PC基序。我们还阐明了这种模块化相互作用也发生在发芽酵母中的信号蛋白Bem1p和CDc24p中，并在极性建立中发挥了关键作用，并确定了Bem1p的PB1结构域的三维结构。虽然p40；；Phox&gt；的作用尚不清楚，但我们的研究表明，p40；Phox&gt；通过与p67；；lt；Phox&gt；通过PB1-PC相互作用，促进了p67；lt；Phox&gt；和p47；；

The enzymatic core of the phagocyte NADPH oxidase, that participates in host defense, is cytochrome b_<558> in the membrane. Activation of this enzyme requires stimulus-induced membrane translocation of SH3 domain containing cytosolic proteins, namely p47^<phox>, p67^<phox>, and p40^<phox> to interact with cytochrome b_<558>. We studied mechanism for the oxidase activation obtained the following results.(1) We determined the three-dimensional structure of the PX/PB2 domain a novel module that we had found in the N-termini of p47^<phox> and p40^<phox>. Furthermore, we found that the PX/PB2 domain has an activity to bind to phosphoinositides, and that the PX domain of p47^<phox> is essential for membrane translocation of this protein and activation of the NADPH oxidase. In addition, we also clarified that the lipid-binding activity of the p47^<phox> PX domain is negatively regulated by intramolecular interaction with its SH3 domains.(2) We found that p40^<phox> associates with p67^<phox> in resting phagocytes via a novel protein-protein interaction : the PB1 domain of p67^<phox> recognizes and binds to the PC motif of p40^<phox>. We also clarified that this modular interaction also occurs in Bem1p and Cdc24p, signaling proteins in the budding yeast, and plays a crucial role in polarity establishment, and determined the three-dimensional structure of the PB1 domain of Bem1p. Although the role of p40^<phox> had remained unknown, our study revealed that p40^<phox> enhances membrane translocation of p67^<phox> and P47^<phox> via the PB1-PC interaction with p67^<phox>, thereby positively regulating activation of the phagocyte NADPH oxidase.