MDM2与端粒保护蛋白1的相互作用及其功能

Interaction and Function Between MDM2 and POT1

目的:探究鼠双微体2同源体(MDM2)与端粒保护蛋白1(POT1)在细胞水平是否有相互作用,及其是否发挥E3泛素化连接酶的功能。方法:首先,用蛋白酶体抑制剂MG132处理稳定表达Flag-POT1的HeLa细胞,Western印迹检测Flag-POT1的表达情况;其次,在HeLa细胞中转入外源的Myc-MDM2和Flag-POT1质粒,48 h后收集细胞,通过免疫共沉淀方法验证Myc-MDM2和Flag-POT1是否具有相互作用;再次,在稳定表达Flag-POT1的HeLa细胞中转入Myc-MDM2或MDM2 siRNA,48 h后收集细胞,Western印迹检测Flag-POT1的表达水平。结果:MG132处理后,Flag-POT1的表达量明显升高且有拖尾现象,免疫共沉淀显示Myc-MDM2和Flag-POT1具有相互作用,但无论转入Myc-MDM2还是MDM2 siRNA,Flag-POT1的表达水平没有明显变化。结论:POT1通过泛素化途径降解,MDM2与POT1具有相互作用,但MDM2不是POT1主要的E3泛素化连接酶。

Objective： To investigate whether there was interaction between mouse double minute 2 homolog （MDM2） and protection of telomeres（POT1）, and whether POT1 was degradated by MDM2 as an E3 ubiquitin li-gase. Methods： Firstly, HeLa cells with overexpression of Flag-POT1 were treated with proteasome inhibitor MG132, then Flag-POT1 level was detected by Western blot. Secondly, HeLa cells transfected with Myc-MDM2 and Flag-POT1 plasmids were incubated for 48 h before harvest, then MDM2 and POT1 interaction was identified by immunoprecipitation. Thirdly, Myc-MDM2 and MDM2 siRNA were transfected into HeLa cells with overexpres-sion of Flag-POT1, POT1 level were detected by Western blot. Results： The expression level of Flag-POT1 in-creased after treatment with MG132, immunoprecipitation test demonstrated the interaction between POT1 and MDM2, there was no obvious expression change of Flag-POT1 with or without Myc-MDM2 or MDM2 siRNA. Con-clusion： POT1 was degradated through the ubiquitin pathways, there was interaction between MDM2 and POT1, and MDM2 was not an E3 ubiquitin ligase for POT1.