Tripartite chimeras comprising functional domains derived from the cytosolic NADPH oxidase components p47phox, p67phox, and Rac1 elicit activator-independent superoxide production by phagocyte membranes -: An essential role for anionic membrane phospholipids
Tripartite chimeras comprising functional domains derived from the cytosolic NADPH oxidase components p47phox, p67phox, and Rac1 elicit activator-independent superoxide production by phagocyte membranes -: An essential role for anionic membrane phospholipids
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DOI：
10.1074/jbc.m701497200
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发表时间：
2007-07-27
影响因子：
4.8
通讯作者：
Pick, Edgar
中科院分区：
文献类型：
--
作者：
Berdichevsky, Yevgeny;Mizrahi, Ariel;Pick, Edgar
产生超氧化物的NADPH氧化酶通过膜定位的细胞色素B（559）与三种胞质组分p47（phox）、p67（phox）和GT β Rac 1或Rac 2的组装转化为活性状态。组装涉及两组蛋白质-蛋白质相互作用：胞质组分之间和胞质组分与其脂质栖息地内的细胞色素b559之间。我们规避了胞质组分之间的相互作用的需要，通过构建一个重组三方嵌合体（三聚体）组成的Phox同源性（PX）和Src同源性3（SH 3）结构域的p47（phox），tetratricopeptide重复和激活结构域的p67（phox），和全长Rac 1。加入吞噬细胞膜后，在阴离子两亲物存在的情况下，三聚体能够在体外激活氧化酶。与组合的单个组分（全长或截短的）相比，三聚体对细胞色素b559具有更高的亲和力（较低的EC 50），并与细胞色素b559形成更稳定的复合物（较长的半衰期）。补充膜与阴离子，而不是中性磷脂的三聚体两亲独立的激活。突变、截短和结构域置换表明，三聚体对氧化酶的激活依赖于以下相互作用：1）通过Rac 1片段C端的多元拉伸与阴离子膜磷脂相互作用，2）通过p47（phox）片段N端SH 3结构域中的Trp（193）与p22（phox）相互作用，补充静电吸引;和3）p67（phox）和Rac 1片段之间的嵌合内键，与它们的物理融合互补。PX域的p47（phox）段和插入域的Rac 1段的氧化酶组装只有很小的贡献。
The superoxide-generating NADPH oxidase is converted to an active state by the assembly of a membrane-localized cytochrome b(559) with three cytosolic components: p47(phox), p67(phox), and GTPase Rac1 or Rac2. Assembly involves two sets of protein-protein interactions: among cytosolic components and among cytosolic components and cytochrome b559 within its lipid habitat. We circumvented the need for interactions among cytosolic components by constructing a recombinant tripartite chimera (trimera) consisting of the Phox homology (PX) and Src homology 3 (SH3) domains of p47(phox), the tetratricopeptide repeat and activation domains of p67(phox), and full-length Rac1. Upon addition to phagocyte membrane, the trimera was capable of oxidase activation in vitro in the presence of an anionic amphiphile. The trimera had a higher affinity (lower EC50) for and formed a more stable complex (longer half-life) with cytochrome b559 compared with the combined individual components, full-length or truncated. Supplementation of membrane with anionic but not neutral phospholipids made activation by the trimera amphiphile-independent. Mutagenesis, truncations, and domain replacements revealed that oxidase activation by the trimera was dependent on the following interactions: 1) interaction with anionic membrane phospholipids via the polybasic stretch at the C terminus of the Rac1 segment; 2) interaction with p22(phox) via Trp(193) in the N-terminal SH3 domain of the p47(phox) segment, supplementing the electrostatic attraction; and 3) an intrachimeric bond among the p67(phox) and Rac1 segments complementary to their physical fusion. The PX domain of the p47(phox) segment and the insert domain of the Rac1 segment made only minor contributions to oxidase assembly.