Cyclophilin A facilitates translocation of the Clostridium botulinum C2 toxin across membranes of acidified endosomes into the cytosol of mammalian cells
Cyclophilin A facilitates translocation of the Clostridium botulinum C2 toxin across membranes of acidified endosomes into the cytosol of mammalian cells
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DOI：
10.1111/j.1462-5822.2009.01291.x
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发表时间：
2009-05-01
影响因子：
3.4
通讯作者：
Barth, Holger
中科院分区：
文献类型：
--
作者：
Kaiser, Eva;Pust, Sascha;Barth, Holger
二元肉毒梭菌C2毒素由结合/易位组分C2IIa和单独的酶组分C2I组成，所述酶组分C2I在真核细胞中使肌动蛋白单ADP核糖基化。早期内体膜中C2IIa的孔形成促进未折叠的C2I易位到胞质溶胶中。我们早先发现C2I的易位依赖于宿主细胞伴侣热休克蛋白Hsp90的活性。在这里，我们表明，环孢菌素A，它抑制亲环素的肽基脯氨酰顺/反异构酶活性，抑制中毒的细胞与C2毒素，并防止摄取C2I进入胞质溶胶。环孢菌素A阻断了完整细胞和部分纯化的早期内体的跨膜C2I活性的pH依赖性易位。在体外，加入细胞质到C2毒素负载的内涵体诱导易位的C2I活性进入细胞质，这是防止预处理的细胞质与亲环素A的抗体。从C2毒素处理的细胞裂解物的下拉实验显示亲环素A的N-末端结构域的C2I的特异性结合。总之，我们的研究结果表明，亲环素A的易位C2 I跨内体膜在摄取C2毒素到哺乳动物细胞中的重要作用。
The binary Clostridium botulinum C2 toxin consists of the binding/translocation component C2IIa and the separate enzyme component C2I, which mono-ADP-ribosylates actin in eukaryotic cells. Pore formation of C2IIa in early endosomal membranes facilitates translocation of unfolded C2I into the cytosol. We discovered earlier that translocation of C2I depends on the activity of the host cell chaperone heat shock protein Hsp90. Here, we demonstrate that cyclosporin A, which inhibits the peptidyl-prolyl cis/trans isomerase activity of cyclophilins, inhibited intoxication of cells with C2 toxin and prevented uptake of C2I into the cytosol. Cyclosporin A blocked the pH-dependent translocation of C2I activity across membranes of intact cells and of partially purified early endosomes. In vitro, the addition of cytosol to C2 toxin-loaded endosomes induced translocation of C2I activity into the cytosol, which was prevented by pretreatment of the cytosol with an antibody against cyclophilin A. Pull-down experiments with lysates from C2 toxin-treated cells revealed specific binding of cyclophilin A to the N-terminal domain of C2I. In conclusion, our results suggest an essential role of cyclophilin A for translocation of C2I across endosomal membranes during the uptake of C2 toxin into mammalian cells.