Bissonnette Sarah A, Glazier Christina M, Stewart Mary Q, Brown Glenn E, Ellson Chris D, Yaffe Michael B
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
J Biol Chem. 2008 Jan 25;283(4):2108-19. doi: 10.1074/jbc.M706639200. Epub 2007 Nov 20.
In response to bacterial infection, the neutrophil NADPH oxidase assembles on phagolysosomes to catalyze the transfer of electrons from NADPH to oxygen, forming superoxide and downstream reactive oxygen species (ROS). The active oxidase is composed of a membrane-bound cytochrome together with three cytosolic phox proteins, p40(phox), p47(phox), and p67(phox), and the small GTPase Rac2, and is regulated through a process involving protein kinase C, MAPK, and phosphatidylinositol 3-kinase. The role of p40(phox) remains less well defined than those of p47(phox) and p67(phox). We investigated the biological role of p40(phox) in differentiated PLB-985 neutrophils, and we show that depletion of endogenous p40(phox) using lentiviral short hairpin RNA reduces ROS production and impairs bacterial killing under conditions where p67(phox) levels remain constant. Biochemical studies using a cytosol-reconstituted permeabilized human neutrophil cores system that recapitulates intracellular oxidase activation revealed that depletion of p40(phox) reduces both the maximal rate and total amount of ROS produced without altering the K(M) value of the oxidase for NADPH. Using a series of mutants, p47PX-p40(phox) chimeras, and deletion constructs, we found that the p40(phox) PX domain has phosphatidylinositol 3-phosphate (PtdIns(3)P)-dependent and -independent functions. Translocation of p67(phox) requires the PX domain but not 3-phosphoinositide binding. Activation of the oxidase by p40(phox), however, requires both PtdIns(3)P binding and an Src homology 3 (SH3) domain competent to bind to poly-Pro ligands. Mutations that disrupt the closed auto-inhibited form of full-length p40(phox) can increase oxidase activity approximately 2.5-fold above that of wild-type p40(phox) but maintain the requirement for PX and SH3 domain function. We present a model where p40(phox) translocates p67(phox) to the region of the cytochrome and subsequently switches the oxidase to an activated state dependent upon PtdIns(3)P and SH3 domain engagement.
作为对细菌感染的反应，中性粒细胞NADPH氧化酶在吞噬溶酶体上组装，催化电子从NADPH转移到氧，形成超氧化物和下游活性氧（ROS）。活性氧化酶由一个膜结合细胞色素以及三种胞质phox蛋白p40(phox)、p47(phox)和p67(phox)，以及小GTP酶Rac2组成，并通过涉及蛋白激酶C、丝裂原活化蛋白激酶和磷脂酰肌醇3激酶的过程进行调节。与p47(phox)和p67(phox)相比，p40(phox)的作用仍不太明确。我们研究了p40(phox)在分化的PLB - 985中性粒细胞中的生物学作用，结果表明，在p67(phox)水平保持恒定的条件下，使用慢病毒短发夹RNA耗尽内源性p40(phox)会减少ROS的产生并损害细菌杀伤能力。使用一种模拟细胞内氧化酶激活的胞质重构通透人中性粒细胞核心系统进行的生化研究表明，耗尽p40(phox)会降低ROS产生的最大速率和总量，而不会改变氧化酶对NADPH的米氏常数（K(M)）。通过一系列突变体研究、p47PX - p40(phox)嵌合体研究和缺失构建体研究，我们发现p40(phox)的PX结构域具有依赖磷脂酰肌醇3 - 磷酸（PtdIns(3)P）和不依赖PtdIns(3)P的功能。p67(phox)的易位需要PX结构域，但不需要3 - 磷酸肌醇结合。然而，p40(phox)对氧化酶的激活既需要PtdIns(3)P结合，也需要一个能够与多聚脯氨酸配体结合的Src同源3（SH3）结构域。破坏全长p40(phox)的封闭自抑制形式的突变可使氧化酶活性比野生型p40(phox)提高约2.5倍，但仍保持对PX和SH3结构域功能的需求。我们提出了一个模型，其中p40(phox)将p67(phox)转运到细胞色素区域，随后将氧化酶转换为依赖PtdIns(3)P和SH3结构域结合的激活状态。
