Dixit Garima, Mikoryak Carole, Hayslett Tyler, Bhat Amritha, Draper Rockford K
The Molecular and Cell Biology Department, FO31, The University of Texas at Dallas, Richardson, TX 75083-0688, USA.
Exp Biol Med (Maywood). 2008 Feb;233(2):163-75. doi: 10.3181/0705-RM-132.
Cholera toxin (CT) contains one A chain and five B chains. The A chain is an enzyme that covalently modifies a trimeric G protein in the cytoplasm, resulting in the overproduction of cAMP. The B chain binds the glycosphingolipid G(M1), the cell surface receptor for CT, which initiates receptor-mediated endocytosis of the toxin. After endocytosis, CT enters the endoplasmic reticulum (ER) via retrograde vesicular traffic where the A chain retro-translocates through the ER membrane to reach the cytoplasm. The retro-translocation mechanism is poorly understood, but may involve proteins of the ER stress response, including the ER associated degradation (ERAD) pathway. We report here that treating cells with CT or CTB quickly up-regulates the levels of BiP, Derlin-1, and Derlin-2, known participants in the ER stress response and ERAD. CT did not induce calnexin, another known responder to ER stress, indicating that the CT-mediated induction of ER proteins is selective in this time frame. These data suggest that CT may promote retro-translocation of the A chain to the cytoplasm by rapidly up-regulating a set of ER proteins involved in the retro-translocation process. In support of this idea, a variety of conditions that induced BiP, Derlin-1, and Derlin-2 sensitized cells to CT and conditions that inhibited their induction de-sensitized cells to CT. Moreover, specifically suppressing Derlin-1 with siRNA protected cells from CT. In addition, Derlin-1 co-immunoprecipitated with CTA or CTB from CT-treated cells using anti-CTA or anti-CTB antibodies. Altogether, the results are consistent with the hypothesis that the B chain of CT up-regulates ER proteins that may assist in the retro-translocation of the A chain across the ER membrane.
霍乱毒素（CT）由一条A链和五条B链组成。A链是一种酶，可共价修饰细胞质中的三聚体G蛋白，导致cAMP过度产生。B链与糖鞘脂G(M1)结合，G(M1)是CT的细胞表面受体，可启动毒素的受体介导的内吞作用。内吞作用后，CT通过逆行囊泡运输进入内质网（ER），在此A链通过内质网膜逆向转运到达细胞质。逆向转运机制尚不清楚，但可能涉及内质网应激反应的蛋白质，包括内质网相关降解（ERAD）途径。我们在此报告，用CT或CTB处理细胞可迅速上调BiP、Derlin-1和Derlin-2的水平，这些都是内质网应激反应和ERAD中的已知参与者。CT未诱导钙连蛋白，钙连蛋白是另一种已知的内质网应激反应蛋白，这表明CT介导的内质网蛋白诱导在这个时间框架内具有选择性。这些数据表明，CT可能通过快速上调一组参与逆向转运过程的内质网蛋白来促进A链向细胞质的逆向转运。支持这一观点的是，多种诱导BiP、Derlin-1和Derlin-2的条件使细胞对CT敏感，而抑制它们诱导的条件则使细胞对CT脱敏。此外，用siRNA特异性抑制Derlin-1可保护细胞免受CT的影响。此外，使用抗CTA或抗CTB抗体从CT处理的细胞中，Derlin-1与CTA或CTB共免疫沉淀。总之，这些结果与以下假设一致：CT的B链上调内质网蛋白，这些蛋白可能有助于A链穿过内质网膜的逆向转运。
