Thiol-switch control in A/B toxin cell entry and compartmental trafficking

蛋白质二硫键(SS)的形成、重排和氧化还原调控的机理研究是真核细胞生物学中的一项艰巨挑战，因为SS蛋白质不仅存在于内质网和线粒体膜间隙，而且存在于胞浆和几乎所有其他隔室。另一方面，SS键的断裂也发生在这些隔室中，不仅是为了损伤修复，也是为了细胞内的信号传递。然而，硫醇开关控制的潜在机制，包括电子供体和受体，底物识别和遗传/细胞因素，特别是控制低pH区蛋白质硫醇的机制，如内吞和晚期分泌途径，仍然知之甚少。我们关于一种病毒A/B毒素(K28)及其杀死真核靶细胞的策略的数据表明，连接A/B二聚体中两个毒素亚单位的单个SS键的最关键的还原断裂必须只发生在细胞质中，并且在逆行毒素运输过程中必须防止。这预示着一种细胞控制系统，特别是在低pH区，如内小体和高尔基体，来感知和调节SS结合蛋白中分子间/分子内的硫醇开关。同样，K28从内质网逆向易位到胞浆中，涉及PDI和Hsp40辅助伴侣的氧化还原依赖的伴侣活性，使A/B毒素进入有易位能力的构象，而不破坏其SS键结构，但仍然允许ER退出。保护毒素免疫同样需要一个细胞控制系统，特别是调节早期内体中的SS键切换和毒素通过分泌途径逆行运输期间。在这里，我们打算以K28为模型，研究细胞内SS键形成/重排在细胞内和分泌途径中的调节，以了解真核细胞中蛋白质氧化还原开关控制的基本原理。通过解剖遗传筛选中的毒素免疫，我们打算确定在细胞内pH变化的条件下，控制不同隔室中时空蛋白硫醇转换的未知细胞因素。由于在体外，A-毒素从二聚体自发释放在pH&gt；6.0时，我们想了解如何在低pH值的隔间，如内小体和高尔基体中防止这种情况发生。在中性的体外pH条件下，K28也形成SS结合的寡聚体，导致体内毒性完全丧失；我们想了解如何在自然杀伤的情况下防止这种情况，特别是在内质网管腔(在pH 7.2)中，K28甚至应该有更强的倾向形成不活跃的低聚体；同时，A-亚基在内质网中释放是细胞毒性的，必须防止以避免自杀。通过使用K28作为工具，分析这些机制将有助于我们理解A/B毒素的作用，并识别控制氧化还原开关的细胞因素。

Mechanistic understanding of protein disulfide bond (SS) formation, rearrangement and redox control is a demanding challenge in eukaryotic cell biology as SS proteins are not only present in the endoplasmic reticulum (ER) and the mitochondrial intermembrane space, but also in the cytosol and almost all other compartments. On the other hand, SS bond cleavage also occurs in these compartments, not only for damage repair but also for intracellular signaling. However, the underlying mechanisms of thiol switch control, including electron donors and acceptors, substrate recognition and genetic/cellular factors, are still poorly understood, in particular mechanisms controlling protein thiols in low pH compartments such as the endocytic and the late secretory pathway. Our data on a viral A/B toxin (K28) and its strategy to kill eukaryotic target cells revealed that the most critical reductive cleavage of the single SS bond connecting both toxin subunits in the A/B dimer must only take place in the cytosol and has to be prevented during retrograde toxin traffic. This predicts a cellular control system, in particular in low pH compartments such as endosomes and Golgi, to sense and regulate inter-/intramolecular thiol switching in SS-bonded proteins. Likewise, K28 retrotranslocation from the ER into the cytosol involves redox-dependent chaperon activities of PDI and Hsp40 cochaperones to bring the A/B toxin into a translocation competent conformation without disrupting its SS-bonded structure but still allowing ER exit.Protecting toxin immunity likewise requires a cellular control system, in particular to regulate SS bond switching in the early endosome and during retrograde toxin transport through the secretory pathway. Here we intend to use K28 as model to study the regulation of intracellular SS bond formation/rearrangement within the endocytic and secretory pathway to understand the underlying principles of protein redox switch control in eukaryotic cells. By dissecting toxin immunity in a genetic screen, we intend to identify yet unknown cellular factors controlling spatiotemporal protein thiol switching in various compartments under conditions of intracellular pH changes. Since in vitro, A-toxin release from the dimer occurs spontaneously at pH >6.0, we would like to understand how this is prevented in low pH compartments such as endosomes and Golgi. Under in vitro conditions of neutral pH, K28 also forms SS-bonded oligomers that cause total loss of in vivo toxicity; we would like to understand how this is being prevented in the natural situation of killing, in particular in the ER lumen (at pH 7.2) where K28 should even have a stronger tendency to form inactive oligomers; at the same time, A-subunit release in the ER is cytotoxic and must be prevented to avoid suicide. By using K28 as tool, analysis of these mechanisms will foster our understanding of A/B toxin action and identify cellular factors controlling redox switching.