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Role of peptidyl-prolyl cis/trans-isomerases (PPlases) during uptake of binary bacterial toxins into the cytosol of mammalian cells

肽基脯氨酰顺/反异构酶（PPlases）在二元细菌毒素摄取到哺乳动物细胞胞浆中的过程中的作用

基本信息

批准号：
110265127
负责人：
Professor Dr. Holger Barth
金额：
--
依托单位：
Institut für Experimentelle und Klinische Pharmakologie,Toxikologie und Naturheilkunde
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2008
资助国家：
德国
起止时间：
2007-12-31 至 2015-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/110265127?language=en
关键词：
Role peptidyl prolyl cis trans

项目摘要

Many bacterial toxins, such as diphtheria toxin, enter mammalian cells by receptor-mediated endocytosis and then deliver an enzymatic active moiety from endosomes into the cytosol, where it ADP-ribosylates specific cellular substrate molecules. This results in cell damage and the typical clinical symptoms of several important infectious diseases. The mechanisms of how the enzyme moieties translocate across endosomal membranes are not understood for most toxins and therefore of scientific and medical interest. We investigate how the enzyme components of binary actin-ADP-ribosylating toxins of Clostridium botulinum (C2 toxin), C. perfringens (Iota toxin) und C. difficile (CDT) translocate from endosomal vesicles into the cytosol. Here, a separate transport component forms pores in the membranes of acidified endosomes which serve as translocation channels for the respective unfolded enzyme components to reach the cytosol. We have discovered that besides the chaperone Hsp90, the activity of peptidyl-prolyl cis/trans isomerases (PPIases) is crucial for membrane translocation of the enzyme components. Moreover, we demonstrated for the first time that the enzyme component of C2 toxin (C2I), interacts with cyclophilins (CyPA, CyP-40) and FK506 binding proteins (FKBP 51/52) in vitro and in living cells. Cyclosporine A, an inhibitor of CyPs, as well as FK506, an inhibitor of FKBPs prevented translocation of the enzyme components from acidified endosomes into the cytosol and thereby protected cultured cells from intoxication by the binary actin-ADP-ribosylating toxins, but also from intoxication with diphtheria toxin. Prompted by these findings, we will investigate whether the discovered PPIase/Hsp90-dpendent translocation is specific for ADP-ribosylating toxins and focus on the mechanism by which PPIases and Hsp90 interact with ADP-ribosyltransferases. Most importantly, we will include the isolated recombinant ADP-ribosyltransferase domains of binary actin-ADP-ribosylating toxins in our ongoing studies in addition to their complete enzyme components. We will establish a novel approach to exploit the transport component of Anthrax toxins for delivery of the isolated ADP-ribosyltransferase domains into cells and to use specific inhibitors and antibodies to elucidate the role of CyPs, FKBPs and Hsp90 during membrane translocation of ADP-ribosyltransferases in more detail. Furthermore, we will investigate the role of CyPs and FKBPs during cellular uptake of diphtheria toxin. The suggested approaches will provide significant new knowledge on intracellular membrane transport of bacterial toxins. Prompted by these findings, we will screen novel non-immunosuppresive PPIase-inhibitors to develop and characterize substances that prevent uptake of bacterial ADP-ribosylating toxins into human cells. Such compounds could lead to alternative therapeutic strategies against toxin-producing (antibiotics-resistant) bacteria.

许多细菌毒素，如白喉毒素，通过受体介导的内吞作用进入哺乳动物细胞，然后将内切体中的酶活性部分传递到胞浆中，在胞浆中ADP-核糖化特定的细胞底物分子。这会导致细胞损伤和几种重要传染病的典型临床症状。对于大多数毒素来说，酶部分如何跨内膜转运的机制尚不清楚，因此引起了科学和医学上的兴趣。我们研究了肉毒梭菌(C2毒素)、产气荚膜梭菌(IOTA毒素)和艰难梭菌(CDT)二元肌动蛋白-ADP-核糖化毒素(CDT)的酶组分是如何从内体小泡转移到胞浆中的。在这里，一个单独的转运组分在酸化的内体体膜上形成孔，作为各个未折叠的酶组分到达胞浆的转运通道。我们发现，除了分子伴侣Hsp90外，肽基-脯氨酰顺式/反式异构酶(PPIase)的活性对酶组分的膜转位至关重要。此外，我们还首次证明了C2毒素的酶组分(C2I)在体外和活细胞中与亲环素(CyPA，CYP-40)和FK506结合蛋白(FKBP 51/52)相互作用。环孢素A是一种细胞色素P450的抑制剂，FK506是一种FKBPs的抑制剂，可以防止酶组分从酸化的内吞体移入胞浆，从而保护培养细胞免受肌动蛋白-ADP-核糖化毒素的毒害，也保护培养细胞免受白喉毒素的毒害。在这些发现的推动下，我们将调查已发现的PPIase/Hsp90依赖的易位是否是ADP核糖化毒素所特有的，并重点研究PPIase和Hsp90与ADP核糖基转移酶相互作用的机制。最重要的是，我们将在我们正在进行的研究中包括分离的二元肌动蛋白-ADP-核糖化毒素的重组ADP-核糖转移酶结构域，以及它们的完整酶组分。我们将建立一种新的方法来利用炭疽毒素的运输成分将分离的ADP-核糖基转移酶结构域传递到细胞内，并使用特定的抑制剂和抗体来更详细地阐明CyPS、FKBPs和Hsp90在ADP-核糖基转移酶膜转位中的作用。此外，我们还将研究Cyps和FKBPs在细胞摄取白喉毒素过程中的作用。建议的方法将为细菌毒素的细胞内膜运输提供重要的新知识。在这些发现的推动下，我们将筛选新的非免疫抑制PPIase抑制剂，以开发和表征防止细菌ADP-核糖化毒素摄取到人类细胞的物质。这些化合物可能会导致针对产生毒素(抗药性)细菌的替代治疗策略。