Souabni Hajer, Ezzine Aymen, Bizouarn Tania, Baciou Laura
Laboratoire de Chimie Physique, UMR 8000 CNRS Université Paris Sud, Université Paris Saclay, 91405, Orsay Cedex, France.
Methods Mol Biol. 2017;1635:27-43. doi: 10.1007/978-1-4939-7151-0_2.
Activation of phagocyte cells from an innate immune system is associated with a massive consumption of molecular oxygen to generate highly reactive oxygen species (ROS) as microbial weapons. This is achieved by a multiprotein complex, the so-called NADPH oxidase. The activity of phagocyte NADPH oxidase relies on an assembly of more than five proteins, among them the membrane heterodimer named flavocytochrome b (Cytb ), constituted by the tight association of the gp91 (also named Nox2) and p22 proteins. The Cytb is the membrane catalytic core of the NADPH oxidase complex, through which the reducing equivalent provided by NADPH is transferred via the associated prosthetic groups (one flavin and two hemes) to reduce dioxygen into superoxide anion. The other major proteins (p47, p67, p40, Rac) requisite for the complex activity are cytosolic proteins. Thus, the NADPH oxidase functioning relies on a synergic multi-partner assembly that in vivo can be hardly studied at the molecular level due to the cell complexity. Thus, a cell-free assay method has been developed to study the NADPH oxidase activity that allows measuring and eventually quantifying the ROS generation based on optical techniques following reduction of cytochrome c. This setup is a valuable tool for the identification of protein interactions, of crucial components and additives for a functional enzyme. Recently, this method was improved by the engineering and the production of a complete recombinant NADPH oxidase complex using the combination of purified proteins expressed in bacterial and yeast host cells. The reconstitution into artificial membrane leads to a fully controllable system that permits fine functional studies.
来自先天免疫系统的吞噬细胞的激活与大量消耗分子氧以产生活性很强的氧物种（ROS）作为微生物武器有关。这是通过一种多蛋白复合物，即所谓的NADPH氧化酶来实现的。吞噬细胞NADPH氧化酶的活性依赖于五种以上蛋白质的组装，其中包括名为黄素细胞色素b（Cytb）的膜异二聚体，它由gp91（也称为Nox2）和p22蛋白紧密结合构成。Cytb是NADPH氧化酶复合物的膜催化核心，通过它，NADPH提供的还原当量经由相关的辅基（一个黄素和两个血红素）转移，将双氧还原为超氧阴离子。复合物活性所需的其他主要蛋白质（p47、p67、p40、Rac）是胞质蛋白。因此，NADPH氧化酶的功能依赖于一种协同的多伙伴组装，由于细胞的复杂性，在体内很难在分子水平上进行研究。因此，已经开发了一种无细胞测定方法来研究NADPH氧化酶活性，该方法允许基于细胞色素c还原后的光学技术测量并最终定量ROS的产生。这种设置是鉴定蛋白质相互作用、功能性酶的关键成分和添加剂的有价值工具。最近，通过工程技术和使用在细菌和酵母宿主细胞中表达的纯化蛋白质的组合生产完整的重组NADPH氧化酶复合物，对该方法进行了改进。重组到人工膜中产生了一个完全可控的系统，允许进行精细的功能研究。
