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

蛋白质二硫键（SS）形成、重排和氧化还原控制的机制理解是真核细胞生物学中的一个严峻挑战，因为SS蛋白不仅存在于内质网（ER）和线粒体膜间隙中，而且存在于胞质溶胶和几乎所有其他隔室中。另一方面，SS键断裂也发生在这些隔室中，不仅用于损伤修复，而且用于细胞内信号传导。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/细胞因子，仍然知之甚少，特别是在低pH值区室中控制蛋白硫醇的机制，如内吞和晚期分泌途径。我们关于病毒A/B毒素（K28）及其杀死真核靶细胞的策略的数据显示，连接A/B二聚体中的两个毒素亚基的单个SS键的最关键的还原裂解必须仅发生在胞质溶胶中，并且必须在毒素逆行运输期间被阻止。这预测了细胞控制系统，特别是在低pH区室，如内体和高尔基体，感测和调节SS-键合蛋白质中的分子间/分子内巯基转换。同样地，K28从ER到胞质溶胶的逆易位涉及PDI和Hsp 40共分子伴侣的氧化还原依赖性分子伴侣活性，以使A/B毒素进入易位能力构象，而不破坏其SS-键合结构，但仍允许ER离开。特别是调节早期内体中和通过分泌途径的逆行毒素转运期间的SS键转换。在这里，我们打算使用K28作为模型来研究胞内SS键的形成/重排的内吞和分泌途径内的调节，了解蛋白质氧化还原开关控制在真核细胞中的基本原则。通过解剖毒素免疫的遗传筛选，我们打算确定尚未未知的细胞因子控制时空蛋白质巯基开关在细胞内pH值变化的条件下，在各个舱室。由于在体外，A-毒素从二聚体的释放在pH >6.0时自发发生，我们想了解这是如何在低pH区室如内体和高尔基体中被阻止的。在中性pH的体外条件下，K28还形成SS键合的寡聚体，其导致体内毒性的完全丧失;我们想了解在自然杀伤情况下，特别是在ER腔中，如何防止这种情况（在pH 7.2下），其中K28应甚至具有更强的形成无活性寡聚物的倾向;同时，ER中A亚单位的释放是细胞毒性的，必须加以阻止以避免自杀。通过使用K28作为工具，这些机制的分析将促进我们对A/B毒素作用的理解，并确定控制氧化还原开关的细胞因子。

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.