Taura Masahiko, Miyano Kei, Minakami Reiko, Kamakura Sachiko, Takeya Ryu, Sumimoto Hideki
Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Biochem J. 2009 Apr 15;419(2):329-38. doi: 10.1042/BJ20082028.
The superoxide-producing NADPH oxidase in phagocytes is crucial for host defence; its catalytic core is the membrane-integrated protein gp91phox [also known as Nox2 (NADPH oxidase 2)], which forms a stable heterodimer with p22phox. Activation of the oxidase requires membrane translocation of the three cytosolic proteins p47phox, p67phox and the small GTPase Rac. At the membrane, these proteins assemble with the gp91phox-p22phox heterodimer and induce a conformational change of gp91phox, leading to superoxide production. p47phox translocates to membranes using its two tandemly arranged SH3 domains, which directly interact with p22phox, whereas p67phox is recruited in a p47phox-dependent manner. In the present study, we show that a short region N-terminal to the bis-SH3 domain is required for activation of the phagocyte NADPH oxidase. Alanine substitution for Ile152 in this region, a residue that is completely conserved during evolution, results in a loss of the ability to activate the oxidase; and the replacement of Thr153 also prevents oxidase activation, but to a lesser extent. In addition, the corresponding isoleucine residue (Ile155) of the p47phox homologue Noxo1 (Nox organizer 1) participates in the activation of non-phagocytic oxidases, such as Nox1 and Nox3. The I152A substitution in p47phox, however, does not affect its interaction with p22phox or with p67phox. Consistent with this, a mutant p47phox (I152A), as well as the wild-type protein, is targeted upon cell stimulation to membranes, and membrane recruitment of p67phox and Rac normally occurs in p47phox (I152A)-expressing cells. Thus the Ile152-containing region of p47phox plays a crucial role in oxidase activation, probably by functioning at a process after oxidase assembly.
吞噬细胞中产生超氧化物的NADPH氧化酶对宿主防御至关重要；其催化核心是膜整合蛋白gp91phox [也称为Nox2（NADPH氧化酶2）]，它与p22phox形成稳定的异源二聚体。氧化酶的激活需要三种胞质蛋白p47phox、p67phox和小GTPase Rac向膜的转位。在膜上，这些蛋白与gp91phox-p22phox异源二聚体组装并诱导gp91phox的构象变化，从而导致超氧化物的产生。p47phox利用其两个串联排列的SH3结构域转位到膜上，这两个结构域直接与p22phox相互作用，而p67phox以p47phox依赖的方式被募集。在本研究中，我们表明在双SH3结构域N端的一个短区域是吞噬细胞NADPH氧化酶激活所必需的。该区域中Ile152的丙氨酸替代，这是一个在进化过程中完全保守的残基，导致激活氧化酶的能力丧失；Thr153的替代也阻止氧化酶激活，但程度较小。此外，p47phox同源物Noxo1（Nox组织者1）的相应异亮氨酸残基（Ile155）参与非吞噬性氧化酶如Nox1和Nox3的激活。然而，p47phox中的I152A替代并不影响其与p22phox或p67phox的相互作用。与此一致的是，突变型p47phox（I152A）以及野生型蛋白在细胞刺激后靶向到膜上，并且p67phox和Rac在表达p47phox（I152A）的细胞中正常发生膜募集。因此，p47phox的含Ile152区域可能通过在氧化酶组装后的一个过程中发挥作用，在氧化酶激活中起关键作用。
