DNA损伤修复蛋白PARP1聚ADP-核糖基化有丝分裂蛋白BUB3调控HeLa细胞的有丝分裂  

DNA damage repair protein PARP1 poly(ADP-ribosyl)ates mitotic protein BUB3 regulates mitosis in HeLa cells

作　　者：杨雪 徐波 YANG Xue;XU Bo(Department of Biochemistry and Molecular Biology,National Clinical Research Center for Cancer,Key Laboratory of Breast Cancer Prevention and Therapy of Ministry of Education,Cancer Institute and Hospital,Tianjin Medical University,Tianjin,300060;Institute of Inelligent Oncology,Chongqing Key Laboratory of Intelligent Oncology for Breast Cancer,Chongqing University Cancer Hospital,Chongqing,400030;Schoo of Medicine,Chongqing University,Chongqing,400040,China)

机构地区：[1]天津医科大学肿瘤医院肿瘤研究所生物化学与分子生物学研究室,国家肿瘤临床医学研究中心,乳腺癌防治教育部重点实验室,天津300060 [2]重庆大学附属肿瘤医院智能肿瘤学研究中心,乳腺癌智能诊疗重庆市重点实验室,重庆400030 [3]重庆大学医学院,重庆400040

出　　处：《陆军军医大学学报》2023年第16期1682-1692,共11页Journal of Army Medical University

基　　金：国家自然科学基金面上项目(81974464,82272754)。

摘　　要：目的探讨DNA损伤修复蛋白聚ADP-核糖聚合酶1[poly(ADP-ribose)polymerase 1,PARP1]对HeLa细胞有丝分裂的影响及其分子机制。方法使用流式细胞术、细胞免疫荧光、活细胞成像检测PARP1对HeLa细胞有丝分裂进程的影响。采用染色体核型分析、细胞免疫荧光检测PARP1对HeLa细胞染色体稳定性的影响。使用蛋白免疫印迹和免疫共沉淀检测HeLa细胞有丝分裂期PARP1的蛋白表达及酶活性变化。使用蛋白质组质谱分析方法鉴定与PARP1在有丝分裂期具有特异性相互作用的蛋白并进行免疫共沉淀及聚ADP-核糖基化实验验证。结果流式细胞术和细胞免疫荧光结果显示,敲低PARP1或使用奥拉帕尼抑制PARP1的酶活性后,HeLa细胞对诺考达唑诱导的有丝分裂阻滞反应显著下降(P<0.05)。活细胞成像结果显示,敲低PARP1后HeLa细胞的平均有丝分裂时间缩短(P<0.01)。染色体核型分析和免疫荧光结果显示,敲低PARP1后非整倍体细胞和多极纺锤体细胞的比例明显增加(P<0.05)。蛋白免疫印迹结果显示有丝分裂期PARP1的蛋白表达量无明显变化,免疫共沉淀实验结果显示其酶活性显著增加。蛋白质组质谱鉴定和免疫共沉淀结果显示,PARP1与有丝分裂检查点复合体(mitotic checkpoint complex,MCC)的主要组成蛋白BUB3在有丝分裂期具有特异性相互作用,并且BUB3可以发生聚ADP-核糖基化修饰。结论DNA损伤修复蛋白PARP1可能通过上调其酶活性并聚ADP-核糖基化MCC蛋白BUB3以调控HeLa细胞有丝分裂的正常进行并维持其染色体稳定性。Objective To investigate the role and molecular mechanism of poly(ADP-ribose)polymerase 1(PARP1)in mitosis of HeLa cells.Methods Flow cytometry,immunofluorescent assay and live cell imaging were used to detect the effect of PARP1 on mitosis of HeLa cells.Chromosome karyotype analysis and immunofluorescent assay were used to detect the effect of PARP1 on chromosome stability.Western blotting and co-immunoprecipitation assay were used to analyze the protein expression and enzyme activity of PARP1 in HeLa cells during mitosis.Proteome mass spectrometry was used to identify proteins that interacted specifically with PARP1 in mitosis,which was verified by co-immunoprecipitation and poly(ADP-ribosyl)ation assay.Results Flow cytometry and immunofluorescence staining showed that the response of HeLa cells to nocodazole-induced mitotic arrest was signifcantly decreased after genetice manipulat ion(knockdown)or pharmacological inhibition(olaparib)of PARP1(P<0.05).The results of live cell imaging showed the mean mitotie time of HeLa cells was shortened after the knockdown of PARP1(P<0.01).Chromosome karyotype analysis and immunofluorescence staining showed that the proportion of aneuploid and multipolar spindle cells was increased significantly after the knockdown of PARP1(P<0.05).Western blotting showed no significant difference in the protein level of PARP1 during the process of mitosis,while the co-immunoprecipitation assay showed the enzyme activity was increased significantly.Proteome mass spectrometry and co-immunoprecipitation assay showed that PARP1 specifically interacted with BUB3,a major component of the mitotic checkpoint complex(MCC),and BUB3 is poly(ADP-ribosyl)ated in mitosis.Conclusion DNA damage repair protein PARP1 may regulate the mitosis of HeLa cells and maintain its chromosome stability by upregul ating enzyme activity and poly(ADP-ibosyl)ating mitotic BUB3.

关 键 词：DNA损伤修复 有丝分裂 聚ADP-核糖聚合酶1 PARP抑制剂 

分 类 号：R341[医药卫生—基础医学] R394.3R737.33