Thiol-based regulation of protein modifications in a host-pathogen setting

在优先计划SPP 1710的第一个资助期内，我们研究了暴露于我们免疫系统细胞的细菌蛋白质中巯基的氧化还原状态。我们认识到，次氯酸（HOCl）是影响被吞噬细菌中蛋白质巯基氧化还原状态的主要反应物种。这些中性粒细胞吞噬溶酶体中HOCl的释放导致吞噬细菌中细胞巯基-二硫键氧化还原状态的完全破坏。在本资助期内，我们建议阐明吞噬溶酶体中HOCl和其他反应物质的释放如何影响免疫细胞本身。我们建议使用roGFP 2为基础的氧化还原探针，我们已经成功地建立了确定吞噬细菌的氧化还原状态，研究反应物种在宿主细胞中的分布。此外，我们打算研究，在吞噬细胞中的蛋白质硫醇的反应性物种的影响，以揭示潜在的氧化还原调节机制。在我们项目的第三部分，我们想研究硫氧还蛋白参与去除蛋白质中的N-氯化。赖氨酸和精氨酸侧链的N-氯化是由HOCl引起的翻译后修饰，并且，在第一个资助期间，我们发现它可以被硫氧还蛋白还原，将这种HOCl诱导的氧化蛋白质修饰与细胞的巯基氧化还原系统联系起来。在嗜酸性粒细胞样细胞系PLB-985中表达基于roGFP 2的探针，以实时监测相关细胞区室响应于暴露于调理的E. coli细胞。监测细菌作用下嗜人大肠杆菌样细胞株PLB-985不同区室巯基氧化还原蛋白质组状态的变化.阐明硫氧还蛋白依赖NADPH去除蛋白质中N-氯修饰的机制。

During the first funding period of the priority program SPP1710, our group has studied the redox state of thiol groups in the proteins of bacteria exposed to cells in our immune system. We recognized that hypochlorous acid (HOCl) is the major reactive species influencig the thiol redox state of proteins in phagocytized bacteria. The release of HOCl in the phagolysosome of these neutrophils leads to the complete breakdown of the cellular thiol-disulfide redox state in the phagocytized bacteria.In this funding period, we propose to elucidate how the release of HOCl and other reactive species in the phagolysosome affects the immune cells themselves. We propose to study the distribution of reactive species in the host cell using the roGFP2-based redox probes that we already successfully established to determine the redox state of phagocytized bacteria. Additionally, we intend to study, which protein thiols in the phagocytic cell are affected by the reactive species, in order to uncover potential redox regulation mechanisms. In a third part of our project we want to study the involvement of thioredoxin in the removal of N-chlorinations from proteins. N-chlorination of lysine and arginine sidechains is a post-translational modification caused by HOCl, and, during the first funding period, we found out that it can be reduced by thioredoxin, linking this HOCl-induced oxidative protein modification to the thiol-redox system of the cell.To test our hypotheses, we plan to:1. Express roGFP2-based probes in the neutrophil-like cell line PLB-985 to real-time monitor redox changes in relevant cellular compartments in response to the exposition to opsonized E. coli cells.2. Monitor changes in the thiol redox proteome state of different compartments of the neutrophil-like cell line PLB-985 when exposed to bacteria.3. Elucidate the mechanism of the NADPH-dependent removal of N-Chlorination modifications in proteins by thioredoxin.