Arachidonic Acid Induces Direct Interaction of the p67phox-Rac Complex with the Phagocyte Oxidase Nox2, Leading to Superoxide Production*
Arachidonic Acid Induces Direct Interaction of the p67phox-Rac Complex with the Phagocyte Oxidase Nox2, Leading to Superoxide Production*
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DOI：
10.1074/jbc.m114.581785
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发表时间：
2014-07
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影响因子：
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通讯作者：
R. Matono;K. Miyano;Takuya Kiyohara;H. Sumimoto
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文献类型：
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作者：
R. Matono;K. Miyano;Takuya Kiyohara;H. Sumimoto
背景：花生四烯酸(AA)激活吞噬细胞氧化酶NOX2的机制尚不清楚，除了AA诱导的NOX2激活剂p67Phox的伴侣p47Phox的构象变化外。结果：AA还可促进RAC-GTP的形成和NOX2与p67Phox·RAC-GTP复合体的相互作用。结论：AA对NOX2的组装有多步调控作用。意义：p67Phox-NOX2相互作用是一个新的调控步骤。吞噬细胞NADPH氧化酶NOX2与膜上的p22Phox异源二聚，在静息细胞中处于休眠状态，但在细胞刺激下被激活，产生超氧化物，这是杀菌氧化剂的前体。NOX2的激活需要两个开关同时开启：胞浆蛋白p47Phox的构象变化和小GTP酶Rac上的GDP/GTP交换。这些蛋白质以活性形式与它们各自的靶标p22Phox和p67Phox结合，导致在细胞膜上进行高效的氧化酶组装。虽然花生四烯酸(AA)在体内和体外都能有效地激活NOX2，但其机制还不完全清楚，除了引起p47Phox构象的变化。在这里，我们证明了AA在细胞水平上诱导RAC上GDP到GTP的交换，这与它作为一个有效的NOX2激活剂的作用是一致的。然而，即使在HeLa细胞中同时表达了结构性活性形式的p47Phox和rac1，在没有AA的情况下，NOX2也几乎不能诱导超氧化物的产生。在没有AA的无细胞重组系统中，这些活性蛋白也无法有效地激活NOX2。在不影响RAC-GTP与p67Phox结合的情况下，AA诱导RAC-GTP结合的p67Phox与NOX2的C-末端胞液区域直接相互作用。P67Phox-RAC-NOX2组装和超氧化物的产生都被p67Phox激活结构域中Tyr-198、Leu-199和Val-204的丙氨酸取代所废除，该激活结构域将C-末端定位到RAC结合结构域。因此，为了激活NOX2，需要同时开启第三个开关(AA诱导的p67Phox·rac-GTP与NOX2的相互作用)。
Background: The mechanism whereby arachidonic acid (AA) activates the phagocyte oxidase Nox2 is not well understood, except for the AA-induced conformational change of p47phox, a partner of the Nox2 activator p67phox. Results: AA also triggers Rac-GTP formation and Nox2 interaction with the p67phox·Rac-GTP complex. Conclusion: AA regulates Nox2 assembly at multiple steps. Significance: p67phox-Nox2 interaction is a novel regulatory step. The phagocyte NADPH oxidase Nox2, heterodimerized with p22phox in the membrane, is dormant in resting cells but becomes activated upon cell stimulation to produce superoxide, a precursor of microbicidal oxidants. Nox2 activation requires two switches to be turned on simultaneously: a conformational change of the cytosolic protein p47phox and GDP/GTP exchange on the small GTPase Rac. These proteins, in an active form, bind to their respective targets, p22phox and p67phox, leading to productive oxidase assembly at the membrane. Although arachidonic acid (AA) efficiently activates Nox2 both in vivo and in vitro, the mechanism has not been fully understood, except that AA induces p47phox conformational change. Here we show that AA elicits GDP-to-GTP exchange on Rac at the cellular level, consistent with its role as a potent Nox2 activator. However, even when constitutively active forms of p47phox and Rac1 are both expressed in HeLa cells, superoxide production by Nox2 is scarcely induced in the absence of AA. These active proteins also fail to effectively activate Nox2 in a cell-free reconstituted system without AA. Without affecting Rac-GTP binding to p67phox, AA induces the direct interaction of Rac-GTP-bound p67phox with the C-terminal cytosolic region of Nox2. p67phox-Rac-Nox2 assembly and superoxide production are both abrogated by alanine substitution for Tyr-198, Leu-199, and Val-204 in the p67phox activation domain that localizes the C-terminal to the Rac-binding domain. Thus the “third” switch (AA-inducible interaction of p67phox·Rac-GTP with Nox2) is required to be turned on at the same time for Nox2 activation.