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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 investigate if BAG3 is a direct substrate of caspase. METHODS: The human pancreatic cancer cells of SW1990, respectively provided broth treated control group, the proteasome inhibitor MG132 alone or with caspase inhibitors z-YVAD, z-DEVD, z-IETD or z-LEHD joint processing; using Recombinant caspase was used to digest recombinant BAG3 protein in vitro. Western blotting was used to detect the cleavage of BAG3 protein in each group. Results: 2-mol / L z-YVAD caspase-1 inhibitor and 2-mol / L caspase-8 inhibitor z-IETD could be partially inhibited in vitro, and 2mol / L caspase 3 inhibitor z- While caspase 9 inhibitor z-LEHD did not inhibit MG132-induced BAG3 cleavage; in caspase-mediated digestion experiments, recombinant caspases 1, 3 and 8 cleave BAG3, whereas caspase 9 can not cleave BAG3. In addition, the efficiency of cleaving BAG3 by caspase 3 is the highest, cleaving BAG3 with 0.1 mol / L caspase 3, and completely digesting BAG3 with 0.5 mol / L caspase 3. CONCLUSION: BAG3 is a direct substrate for caspase and caspase 3 is the major caspase that performs BAG3 cleavage.