Molecular mechanism for activation of the reactive-oxygen-species-producing phagocyte NADPH oxidase involved in host defense against microbial infections

NADPH氧化酶是一种产生活性氧的酶，在宿主防御微生物感染中起着至关重要的作用。吞噬细胞NADPH氧化酶的催化核心是膜整合细胞色素b558，由gp91^<Phox>和p22^<Phox>组成。氧化酶的激活需要刺激诱导特异性衔接蛋白（p47^<Phox>, p67^<Phox>和p40^<Phox>，每个都含有SH3结构域）和小GTPase Rac的膜移位。最近的研究已经确定了gp91^<Phox>的几种哺乳动物同源物，目前被称为nox家族氧化酶：例如，在结肠上皮细胞中丰富的Noxl可能参与局部宿主防御。在本项目中，我们研究了氧化酶活化的分子机制，得到了以下新结果。(1)我们的生化分析表明，在gp91^<Phox>的激活过程中，p47^<Phox>通过PX结构域介导的与磷酸肌苷的相互作用和5113结构域介导的与p22^<Phox>的结合转运到膜上。我们还确定了p47^<Phox> SH3结构域与p22^<Phox>脯氨酸富区（PRR）的复杂结构。(2)我们发现p67^<Phox>以一种新颖的方式通过c端S113结构域与p47^<Phox>相互作用，并确定了p67^<Phox> SH3结构域和p47^<Phox> PRR之间复合物的核磁共振结构。(3)我们已经证明p4O^<Phox>通过增强p47^<Phox>和p67^<Phox>的膜移位来促进氧化酶的激活，这种作用是通过PB1结构域介导的p67^<Phox>的异源二聚化介导的。(4)我们鉴定并克隆了p47^<Phox>和p67^<Phox>的新同源蛋白，分别命名为Noxol和Noxal，并证明这两种蛋白都是激活Noxl所必需的。(5)我们最近的研究表明，Rac参与了Noxl的激活，这是通过它与Noxal的相互作用介导的。

The NADPH oxidase is an enzyme that produces reactive oxygen species, which play a crucial role in host defense against microbial infections. The catalytic core of the phagocyte NADPH oxidase is membrane-integrated cytochrome b558, comprising gp91^<Phox> and p22^<Phox>. Activation of the oxidase requires stimulus-induced membrane translocation of specific adaptor proteins (p47^<Phox>, p67^<Phox>, and p40^<Phox>, each containing SH3 domain) and the small GTPase Rac. Recent studies have identified several mammalian homologues of gp91^<Phox>, presently known as Nox-family oxidases : e.g., Noxl, abundant in the colon epithelial cells, is likely involved in local host defense. In the present project, we investigated the molecular mechanism for oxidase activation, and obtained the following novel results. (1) Our biochemical analyses have revealed that, in activation of gp91^<Phox>, p47^<Phox> translocates to the membrane via both PX domain-mediated interaction with phosphoinositides and 5113 domain-mediated binding to p22^<Phox>. We have also determined the complex structure of the p47^<Phox> SH3 domains with the p22^<Phox> proline-rich region (PRR). (2) We have shown that p67^<Phox> interacts with p47^<Phox> via the C-terminal S113 domain in a novel fashion, and determined the NMR structure of a complex between the p67^<Phox> SH3 domain and the p47^<Phox> PRR. (3) We have demonstrated that p4O^<Phox> facilitates the oxidase activation by enhancing the membrane translocation of p47^<Phox> and p67^<Phox>, which effect is mediated via the PB1 domain-mediated heterodimeriztion with p67^<Phox>. (4) We have identified and cloned novel homologues of p47^<Phox> and p67^<Phox>, designated Noxol and Noxal, respectively, and shown that both proteins are required for activation of Noxl. (5) Our recent study has shown that Rac participates in Noxl activation, which is mediated via its interaction with Noxal.