胱天蛋白酶酶解BAG3蛋白的体外研究

Investigation of BAG3 cleavage mediated by caspases in vitro

目的:探讨BAG3是否为胱天蛋白酶的直接作用底物。方法:选取人胰腺癌细胞SW1990,分别设培养液处理空白对照组、蛋白酶体抑制剂MG132单独或与胱天蛋白酶抑制剂z-YVAD、z-DEVD、z-IETD或z-LEHD联合处理;利用重组胱天蛋白酶对重组BAG3蛋白进行体外酶解;蛋白质印迹法检测各组细胞中BAG3蛋白的裂解情况。结果:在体外,2mol/L胱天蛋白酶1抑制剂z-YVAD和2mol/L胱天蛋白酶8抑制剂z-IETD可以部分抑制,2mol/L胱天蛋白酶3抑制剂z-DEVD可以完全抑制,而胱天蛋白酶9抑制剂z-LEHD不能抑制MG132诱导的BAG3裂解;在胱天蛋白酶的体外酶切实验中,重组胱天蛋白酶1、3和8可以裂解BAG3,而胱天蛋白酶9不能剪切BAG3。另外,胱天蛋白酶3裂解BAG3的效率最高,0.1mol/L胱天蛋白酶3即可以对BAG3进行剪切,0.5mol/L胱天蛋白酶3可以完全酶切BAG3。结论:BAG3是胱天蛋白酶的直接作用底物,胱天蛋白酶3是执行BAG3裂解的主要胱天蛋白酶。

OBJECTIVE：To clarify whether caspases are directly responsible for the cleavage of BAG3. METHODS： Pancreatic cancer SW1990 cells were treated with vehicle, proteasome inhibitor MG132 alone or combination with z-YVAD,z-DEVD,z-IETD or z-LEHD; Recombinant BAG3 was incubated with different active caspases; Cleavage of BAG3 was analyzed using Western blot. RESULTS： In vitro, 2 mol/L caspase inhibitor z-YVAD and z-IETD partially, z-DEVD completely blocked BAG3 cleavage induced by MG132,respectively; z-LEHD had no effect on MG132-mediated cleavage of BAG3. Caspase 1,3 and 8 cleaved BAG3 ,respectively,and caspase 9 couldn＇t cleave BAG3. In addition,caspase 3 was the most effective enzyme responsible for BAG3 cleavage in vitro. And 0.1 mol/L caspase 3 could cleave BAG3,and 0.5 mol/L caspase 3 could cleave BAG3. CONCLUSION： BAG3 is a direct substrate of caspases and caspase 3 is the major caspase responsible for BAG3 cleavage.