Protein Network of the Pseudomonas aeruginosa Denitrification Apparatus
Protein Network of the Pseudomonas aeruginosa Denitrification Apparatus
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DOI：
10.1128/jb.00055-16
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发表时间：
2016-05-01
影响因子：
3.2
通讯作者：
Jahn, Dieter
中科院分区：
文献类型：
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作者：
Borrero-de Acuna, Jose Manuel;Rohde, Manfred;Jahn, Dieter
使用多组分的膜相关蛋白复合体进行氧化磷酸化是细胞产生能量的最有效方式。在这里，我们系统地研究了致病菌铜绿假单胞菌反硝化装置的多种蛋白质-蛋白质相互作用。在反硝化过程中，硝酸盐(NAR)、亚硝酸盐(NIR)、一氧化氮(NOR)和一氧化二氮(NOS)还原酶催化N3-->；NO2-->；NO->；N2O->；N-2的反应级联反应。遗传学实验表明，一氧化氮还原酶NorBC和调节蛋白NOSR是反硝化蛋白网络的核心。我们利用膜相互作用组学结合电子显微镜共定位研究来阐明相应的蛋白质-蛋白质相互作用。完整的膜蛋白NORC、NorB和NOSR形成核心组装平台，通过其成熟因子NirF结合硝酸还原酶NarGHI和周质亚硝酸盐还原酶NIRS。胞质周围的一氧化二氮还原酶NOSZ通过NOSR连接。硝酸盐转运蛋白NarK2、硝酸盐调控系统NarXL、多种亚硝酸盐还原酶成熟蛋白、NirEJMNQ和Nos装配脂蛋白NosFL也被发现连接。许多与能量产生相关的蛋白质，包括电子供体脱氢酶、完整的ATP合成酶、几乎所有的三羧酸(TCA)循环酶和SEC蛋白质运输系统，以及许多其他蛋白质，都与反硝化蛋白质相互作用。这个推测的硝酸盐呼吸系统可能只是连接细胞质、内膜和周质蛋白的广泛的细胞质膜锚定蛋白网络的一部分，以介导发生在细胞质和外部环境之间的屏障/界面上的关键活动。这些络合物相互作用的分子基础还知之甚少。我们使用膜相互作用组学和电子显微镜来确定所涉及的蛋白质-蛋白质相互作用。以研究较多的铜绿假单胞菌反硝化酶复合体为模型。反硝化是整个氮循环的关键步骤之一，为细菌提供了氧气呼吸的有效替代。这个过程允许细菌形成生物膜，生物膜创造低氧栖息地，这是感染机制的关键。我们的结果为呼吸的分子基础提供了新的见解，并为这种病原体的感染策略打开了新的窗口。
Oxidative phosphorylation using multiple-component, membrane-associated protein complexes is the most effective way for a cell to generate energy. Here, we systematically investigated the multiple protein-protein interactions of the denitrification apparatus of the pathogenic bacterium Pseudomonas aeruginosa. During denitrification, nitrate (Nar), nitrite (Nir), nitric oxide (Nor), and nitrous oxide (Nos) reductases catalyze the reaction cascade of NO3- -> NO2- -> NO -> N2O -> N-2. Genetic experiments suggested that the nitric oxide reductase NorBC and the regulatory protein NosR are the nucleus of the denitrification protein network. We utilized membrane interactomics in combination with electron microscopy colocalization studies to elucidate the corresponding protein-protein interactions. The integral membrane proteins NorC, NorB, and NosR form the core assembly platform that binds the nitrate reductase NarGHI and the periplasmic nitrite reductase NirS via its maturation factor NirF. The periplasmic nitrous oxide reductase NosZ is linked via NosR. The nitrate transporter NarK2, the nitrate regulatory system NarXL, various nitrite reductase maturation proteins, NirEJMNQ, and the Nos assembly lipoproteins NosFL were also found to be attached. A number of proteins associated with energy generation, including electron-donating dehydrogenases, the complete ATP synthase, almost all enzymes of the tricarboxylic acid (TCA) cycle, and the Sec system of protein transport, among many other proteins, were found to interact with the denitrification proteins. This deduced nitrate respirasome is presumably only one part of an extensive cytoplasmic membrane-anchored protein network connecting cytoplasmic, inner membrane, and periplasmic proteins to mediate key activities occurring at the barrier/interface between the cytoplasm and the external environment.IMPORTANCEThe processes of cellular energy generation are catalyzed by large multiprotein enzyme complexes. The molecular basis for the interaction of these complexes is poorly understood. We employed membrane interactomics and electron microscopy to determine the protein-protein interactions involved. The well-investigated enzyme complexes of denitrification of the pathogenic bacterium Pseudomonas aeruginosa served as a model. Denitrification is one essential step of the universal N cycle and provides the bacterium with an effective alternative to oxygen respiration. This process allows the bacterium to form biofilms, which create low-oxygen habitats and which are a key in the infection mechanism. Our results provide new insights into the molecular basis of respiration, as well as opening a new window into the infection strategies of this pathogen.