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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氧化酶激活的分子机制

基本信息

批准号：
12470028
负责人：
SUMIMOTO Hideki
金额：
$ 9.28万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

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.

参与宿主防御的吞噬细胞NADPH氧化酶的酶核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质蛋白的SH3结构域的膜移位，即p47^<phox>, p67^<phox>和p40^<phox>与细胞色素b_<558>相互作用。对氧化酶的活化机理进行了研究，得到了以下结果。(1)我们在p47^<phox>和p40^<phox>的n端发现了新的模块PX/PB2结构域的三维结构。此外，我们发现PX/PB2结构域具有与磷酸肌苷结合的活性，并且p47^<phox>的PX结构域对于该蛋白的膜易位和NADPH氧化酶的激活至关重要。此外，我们还澄清了p47^<phox> PX结构域的脂质结合活性受到与其SH3结构域的分子内相互作用的负调控。(2)我们发现p40^<phox>在静止的吞噬细胞中通过一种新的蛋白-蛋白相互作用与p67^<phox>结合：p67^<phox>的PB1结构域识别并结合p40^<phox>的PC基序。我们还明确了这种模块化相互作用也发生在出芽酵母中的信号蛋白Bem1p和Cdc24p中，并在极性建立中起着至关重要的作用，并确定了Bem1p的PB1结构域的三维结构。尽管p40^<phox>的作用尚不清楚，但我们的研究表明，p40^<phox>通过PB1-PC与p67^<phox>的相互作用，增强了p67^<phox>和P47^<phox>的膜移位，从而积极调节吞噬细胞NADPH氧化酶的激活。