胱天蛋白酶3介导BAG3蛋白剪切的研究

  目的 解析胱天蛋白酶（caspase）3介导BCL2结合抗凋亡基因3（BAG3）酶切的具体作用靶点，明确caspase 3裂解对BAG3抗凋亡作用的影响。方法 采用生物信息学方法筛选BAG3蛋白序列中潜在胱天蛋白酶caspase酶切位点；采用定点突变法构建潜在胱天蛋白酶caspase酶切位点突变体；体外翻译野生型和突变体BAG3蛋白；利用重组胱天蛋白酶caspase对野生型或突变型重组BAG3蛋白进行体外剪切实验；构建BAG3裂解片段的真核表达载体，突变型或片段BAG3表达载体转染SW1990细胞；利用Western blot检测各组细胞中BAG3蛋白的裂解情况；利用流式细胞仪（FCM）检测细胞凋亡率。结果 在BAG3蛋白序列中存在K344EVD347和L515EAD518 2个潜在胱天蛋白酶caspase酶切位点；胱天蛋白酶caspase3可有效剪切D518A突变型BAG3，而不能剪切D347A突变型BAG3；D518A突变型和野生型BAG3同等程度抑制MG132诱导的细胞凋亡，D347A突变型BAG3对细胞凋亡的保护作用高于D518A突变型或野生型BAG3，而N末端和C末端剪切片段BAG3对MG132诱导的细胞凋亡无作用。结论 胱天蛋白酶caspase3主要在D347位点裂解BAG3蛋白；BAG3在D347位点的剪切使其丧失了原有的抗凋亡作用。

Investigation of Cleavage of BAG3 Mediated by Caspase 3

Objective To investigate the cleavage site of BAG3 and the function of BAG3 cleavage mediated by caspase 3.Methods Potential cleavage sites in BAG3 protein were predicted by bioinformatic methods;expression vectors containing cleavage site mutation were constructed using site-directed mutagenesis system.Wild-type(WT) or mutated(D347A or D518A) BAG3 were in vitro translated,and cleaved by caspase 3 in vitro;SW1990 cells were transfected with WT,D347A,D518A N-terminal or C-terminal fragments of BAG3;cleavage of BAG3 and apoptotic cells were analyzed using Western blot and flow cytometry(FCM),respectively.Results Two potential caspase cleavage sites(K344EVD347 and L515EAD518) were predicted in the BAG3 protein;D347A BAG3 were not cleaved by caspase 3,whereas D518A were cleaved by caspase 3 efficiently in vitro.D518A and WT BAG3 inhibited MG132-induced apoptosis with similar extent.D347A BAG3 had more potent effect on protecting cells from MG132 as compared with WT BAG3;on the other hand,N-terminal or C-terminal fragment of BAG3 had no effect on MG132-induced apoptosis.Conclusion BAG3 is mainly cleaved at D347 site by caspase 3,and the cleavage of BAG3 by caspase 3 compromises its antiapoptotic effect.