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首页> 外文期刊>Biochemistry >Targeted Protein Degradation of Outer Membrane Decaheme Cytochrome MtrC Metal Reductase in Shewanella oneidensis MR-1 Measured Using Biarsenical Probe CrAsH-EDT_2
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Targeted Protein Degradation of Outer Membrane Decaheme Cytochrome MtrC Metal Reductase in Shewanella oneidensis MR-1 Measured Using Biarsenical Probe CrAsH-EDT_2

机译：用双砷探针CrAsH-EDT_2测得的沙瓦氏假单胞菌MR-1中外膜十氢血红素细胞色素MtrC金属还原酶的靶向蛋白质降解

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摘要

Development of efficient microbial biofuel cells requires an ability to exploit interfacial electron transfer reactions to external electron acceptors, such as metal oxides; such reactions occur in the facultative anaerobic Gram-negative bacterium Shewanella oneidensis MR-1 through the catalytic activity of the outer membrane decaheme c-type cytochrome MtrC. Central to the utility of this pathway to synthetic biology is an understanding of cellular mechanisms that maintain optimal MtrC function, cellular localization, and renewal by degradation and resynthesis. In order to monitor trafficking to , the outer membrane, and the environmental sensitivity of MtrC, we have engineered a . tetracysteine tag (i.e., CCPGCC) at its C-terminus that permits labeling by the cell τ impermeable biarsenical fluorophore carboxy-FIAsH (CrAsH) of MtrC at the surface of living Shewanella oneidensis MR-1 cells. In comparison, the cell permeable reagent FlAsH permits labeling of the entire population of MtrC, including proteolytic fragments resulting from incorrect maturation. We demonstrate specific labeling by CrAsH of engineered MtrC (MtrC~*) which is dependent on the presence of a functional type 2 secretion system (T2S), as evidenced by T2S system gspD or gspG deletion mutants which are incapable of CrAsH labeling. Under these latter conditions, MtrC* undergoes proteolytic degradation to form a large 35-38 kDa fragment; this degradation product is also resolved during normal tumover of the CrAsH-labeled MtrC protein. No MtrC protein is released into the medium during turnover, suggesting the presence of cellular turnover systems involving MtrC reuptake and degradation. The mature MtrC localized on the outer membrane is a long-lived protein, with a turnover rate of 0.043 h~(-1) that is insensitive to O_2 concentration. Maturation of MtrC is relatively inefficient, with substantial rates of turnover of the immature protein prior to export to the outer membrane (i.e., 0.028 h~(-1)) that are consistent with the inherent complexity associated with correct heme insertion and acylation of MtrC that occurs in the periplasm prior to its targeting to the outer membrane. These latter results suggest that MtrC protein trafficking to the outer membrane and its subsequent degradation are tightly regulated, which is consistent with cellular processing pathways that target MtrC to extracellular structures and their possible role in promoting electron transfer from Shewanella to extracellular acceptors.
机译：开发有效的微生物生物燃料电池需要具有开发与外部电子受体（例如金属氧化物）的界面电子转移反应的能力。这样的反应通过外膜十聚体c型细胞色素MtrC的催化活性发生在兼性厌氧革兰氏阴性细菌MR-1中。合成生物学途径实用性的核心是对维持最佳MtrC功能，细胞定位以及通过降解和再合成进行更新的细胞机制的理解。为了监控MtrC的运输，外膜和对环境的敏感性，我们设计了MtrC。在其C末端的四半胱氨酸标签（即CCPGCC），允许在活的希瓦氏wan虫（Shewanella oneidensis）MR-1细胞表面通过MtrC的细胞τ不可渗透的双砷荧光团羧基-FIAsH（CrAsH）进行标记。相比之下，细胞可渗透试剂FlAsH可以标记整个MtrC群体，包括由于不正确的成熟而产生的蛋白水解片段。我们证明了由工程化的MtrC（MtrC〜*）的CrAsH进行的特异性标记，这取决于功能性2型分泌系统（T2S）的存在，正如T2S系统的gspD或gspG缺失突变体所证明的那样，这些突变体无法进行CrAsH标记。在后一种条件下，MtrC *进行蛋白水解降解，形成一个35-38 kDa的大片段。在CrAsH标记的MtrC蛋白正常转移过程中，这种降解产物也会被分解。周转期间没有MtrC蛋白释放到培养基中，表明存在涉及MtrC重摄取和降解的细胞周转系统。定位在外膜上的成熟MtrC是一种长寿蛋白，其转换速率为0.043 h〜（-1），对O_2浓度不敏感。 MtrC的成熟效率相对较低，未成熟蛋白质输出到外膜之前的周转率很高（即0.028 h〜（-1）），这与正确的血红素插入和MtrC的酰化作用相关的固有复杂性是一致的在周质靶向外膜之前发生在周质中。这些后面的结果表明，MtrC蛋白运输到外膜及其后续降解受到严格调控，这与将MtrC靶向细胞外结构的细胞加工途径及其在促进电子从希瓦氏菌到细胞外受体转移中的作用相一致。

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