Huelsenbeck Johannes, Dreger Stefanie C, Gerhard Ralf, Fritz Gerhard, Just Ingo, Genth Harald
Institut für Toxikologie, Medizinische Hochschule Hannover, D-30625 Hannover, Germany.
Biochemistry. 2007 Apr 24;46(16):4923-31. doi: 10.1021/bi602465z. Epub 2007 Mar 31.
ADP-ribosylation of Rho(A,B,C) by the family of exoenzyme C3-like transferases induces reorganization of the actin cytoskeleton based on inactivation of RhoA. No data are available on the role of RhoB in C3-treated cells. In murine fibroblasts treated with the cell-permeable exoenzyme C3 from Clostridium limosum (C3), an increase in the level of RhoB was observed. This upregulation of RhoB was based on transcriptional activation, as it was responsive to inhibition by actinomycin D and accompanied by activation of the rhoB promoter. Upregulation of RhoB was not observed in cells treated with either the actin ADP-ribosylating C2 toxin from Clostridium botulinum or latrunculin B, suggesting that inactivation of Rho but not actin reorganization was required for the upregulation of RhoB. This notion was confirmed, as the Rho/Rac/Cdc42-glucosylating toxin B from Clostridium difficile (TcdB) but not the Rac/R-Ras-glucosylating variant toxin B from C. difficile strain 1470 serotype F (TcdBF) induced a strong upregulation of RhoB. Upregulation of RhoB was further observed in response to the Rac/(H-,K-,N-,R-)Ras-glucosylating lethal toxin from Clostridium sordellii. The level of active, GTP-bound RhoB was increased in TcdB-treated cells compared to untreated cells (as determined by Rhotekin pull-down assay). In contrast, no active RhoB was found in C3-treated cells. RhoB-GTP was required for the TcdB-induced apoptosis (cytotoxic effect), as this effect was responsive to inhibition by C3. In conclusion, RhoB was upregulated by Rho-/Ras-inactivating toxins, as a consequence of the inactivation of either Rho(A,B,C) or (H-,K-,N-)Ras. In TcdB-treated cells, RhoB escaped its inactivation and was required for the cytotoxic effect.
外切酶C3样转移酶家族对Rho（A、B、C）进行的ADP核糖基化作用，基于RhoA的失活诱导肌动蛋白细胞骨架的重组。目前尚无关于RhoB在C3处理细胞中作用的数据。在用来自破伤风梭菌的细胞可渗透外切酶C3（C3）处理的小鼠成纤维细胞中，观察到RhoB水平升高。RhoB的这种上调基于转录激活，因为它对放线菌素D的抑制有反应，并伴随着rhoB启动子的激活。在用来自肉毒杆菌的肌动蛋白ADP核糖基化C2毒素或拉春库林B处理的细胞中未观察到RhoB的上调，这表明RhoB的上调需要Rho失活而非肌动蛋白重组。这一观点得到了证实，因为来自艰难梭菌的Rho/Rac/Cdc42-葡糖基化毒素B（TcdB）而非来自艰难梭菌1470血清型F菌株（TcdBF）的Rac/R-Ras-葡糖基化变体毒素B诱导了RhoB的强烈上调。进一步观察到，对来自索氏梭菌的Rac/（H-、K-、N-、R-）Ras-葡糖基化致死毒素有反应时，RhoB也会上调。与未处理细胞相比，TcdB处理细胞中活性GTP结合型RhoB的水平升高（通过Rhotekin下拉试验测定）。相反，在C3处理的细胞中未发现活性RhoB。RhoB-GTP是TcdB诱导凋亡（细胞毒性作用）所必需的，因为这种作用对C3的抑制有反应。总之，由于Rho（A、B、C）或（H-、K-、N-）Ras失活，RhoB被Rho-/Ras-失活毒素上调。在TcdB处理的细胞中，RhoB逃避了失活，并且是细胞毒性作用所必需的。
