宫颈癌细胞放射损伤后IER5蛋白表达及其相互作用蛋白的筛选和验证

目的 了解IER5蛋白经放射损伤后的表达变化，并筛选IER5蛋白在放射损伤后可能参与DNA损伤修复通路中的相互作用蛋白。方法 HeLa细胞经4 Gy γ射线照射后在不同时间点收集细胞，提取总蛋白质和细胞核蛋白行Western blot检验；构建3×Flag标签融合表达的IER5蛋白表达载体，转染人肾上皮细胞293T并照射，收集细胞行免疫沉淀富集IER5的结合蛋白，十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离免疫共沉淀复合物，考马斯亮蓝染色观察差异条带并切胶酶解后行质谱分析，对可能相互作用蛋白结果进行Western blot验证。结果 IER5蛋白照后4 h表达逐渐增加，12 h至高峰，持续至48 h；细胞核内IER5蛋白表达也有增加趋势；质谱分析数据表明，照射组检测出374个蛋白，对照组检测出256个蛋白，两组比较差异蛋白41个，照射组中包括与DNA结合、代谢、损伤修复相关的10个蛋白；其中，与DNA损伤修复密切相关的1型聚ADP核糖基化聚合酶（PARP1）得到Western blot验证。结论 IER5是放射损伤相关蛋白，其可能参与DNA损伤修复通路。

Preliminary screening of novel IER5 interacting proteins after ionizing irradiation

Objective To investigate the expression of radiation-induced IER5 protein and screen its potential interaction proteins that may participate in DNA repair process. Methods HeLa cells were irradiated with 4 Gy ionizing radiation. IER5 protein expression in whole cell lysate and in nuclear fraction were detected by Western blot at different timepoints after irradiation. 3×Flag-IER5 pCMV plasmid was constrcuted and the Flag tagged-IER5 expression was verified by Western blot. 293T cells were transfected with 3×Flag-IER5 pCMV plasmid. After irradiation the cells were collected and proteins were extracted. The IER5 interaction proteins were purified using immunoprecipitation and separated by 12% SDS-polyacrylamide gel electrophoresis. Then the binding proteins were cut from the gel and analyzed by Mass spectrometry. Results The expression of IER5 protein began to increase 4 hour post-irradiation and its peak level was observed at 12 hour post-irradiation, and it lasted until 48 hour after irradiation. The expression level of IER5 protein in whole cell lysate and nuclear fraction were both increased. With the mass spectrometry analysis, a total of 347 proteins and 256 proteins were identified in irradiated and non-irradiated groups, respectively. Fourty one differential proteins were obtained, where 10 proteins were associated with DNA metabolic process and DNA rapair in the irradiated group and the poly(ADP-ribose) polymerase 1 (PARP1) protein was further confirmed by Western blot. Conclusions IER5 protein is an DNA damage related protein, and it may participate in DNA repair process.