DVC1在氮芥致人支气管上皮细胞DNA-蛋白交联损伤修复中的作用  

Role of DVC1 in repair of nitrogen mustard-induced DNA protein crosslink in human bronchial epithelial cells

作　　者：程晋 余文珮 叶枫[1] 董训虎 赵远鹏[1] 但国蓉[1] 邹仲敏[1] CHENG Jin;YU Wenpei;YE Feng;DONG Xunhu;ZHAO Yuanpeng;DAN Guorong;ZOU Zhongmin(State Key Laboratory of Trauma and Chemical Poisoning,Department of Chemical Defense,Faculty of Military Preventive Medicine,Army Medical University(Third Military Medical University),Chongqing,400038,China)

机构地区：[1]陆军军医大学(第三军医大学)军事预防医学系防化医学教研室,创伤与化学中毒全国重点实验室,重庆400038

出　　处：《陆军军医大学学报》2023年第21期2214-2221,共8页Journal of Army Medical University

基　　金：陆军军医大学青年培育项目(2018XQN03);军队医学科技青年培育计划(20QNPY001)。

摘　　要：目的探讨蛋白水解酶DVC1在氮芥诱导人支气管上皮细胞DNA-蛋白交联损伤修复中的作用。方法利用siRNA干预DVC1表达,50μmol/L氮芥染毒细胞后,采用Western blot检测DNA损伤修复蛋白的表达;荧光酶标仪测定1~24 h DNA-蛋白交联总量;Slot blot检测DNA与O 6-甲基鸟嘌呤-DNA甲基转移酶(O^(6)-methylguanine-DNA methyltransferase,MGMT)的共价交联;免疫沉淀检测MGMT泛素化水平。构建GST-DVC1融合蛋白,GST下拉实验检测DVC1与MGMT、泛素的结合。结果si-DVC1干预后,DNA-蛋白交联总量升高。氮芥降低交联蛋白MGMT原型的水平,但增强其泛素化水平;DVC1可结合泛素,si-DVC1可进一步增强MGMT的泛素化及DNA-MGMT的交联量,与泛素结合酶抑制剂NSC697923和蛋白酶体抑制剂MG132引起的交联结果类似。si-DVC1引起DNA损伤修复蛋白Rad51表达降低、Ku70表达升高。结论DNA-蛋白交联的清除依赖于交联蛋白的泛素化;DVC1通过与泛素相互作用,促进氮芥诱导的DNA-蛋白交联的清除,并维持DNA以同源重组方式修复损伤的能力。Objective To investigate the effect of DNA damage protein targeting VCP(DVC1)in DNA protein crosslink repair induced by nitrogen mustard in human bronchial epithelial cells(16HBE).Methods DVC1 siRNA was used to interfere its expression in 16HBE.Then the cells were exposed to 50μmol/L nitrogen mustard,and the expression levels of DNA damage repair proteins were detected by Western blotting.The total content of DNA-protein crosslink was determined by fluorescence microplate analyzer from 1 to 24 h.The crosslink content of DNA with O^(6)-methylguanine-DNA methyltransferase(MGMT),a methyltransferase,was detected by Slot blotting.The level of ubiquitinated MGMT was detected by immunoprecipitation.After GST-DVC1 fusion protein was constructed,GST pull-down was used to detect the binding of DVC1 with MGMT and ubquitin.Results After si-DVC1 intervention,the total content of DNA-protein crosslink was increased,and exposure to nitrogen mustard decreased the level of prototype of MGMT,but increased the level of ubiquitinated MGMT.DVC1 could bind to ubquitin,and si-DVC1 intervention further enhanced the ubiquitination of MGMT,as well as the crosslink between MGMT and DNA,which was similar with NSC697923(an E2 ubiquitin-conjugating inhibitor)and MG132(a proteasome inhibitor)treatment.The expression of Rad51 and Ku70,2 DNA repair proteins,were increased and decreased respectively by si-DVC1 interference.Conclusion Clearance of DNA-protein crossslink is depend on ubiquitination of MGMT.DVC1 promotes the clearance of nitrogen mustard induced DNA-protein crossslink by interacting with ubiquitin,and maintain the ability of homologous recombination repair.

关 键 词：氮芥 DVC1 DNA蛋白交联 O 6-甲基鸟嘌呤-DNA甲基转移酶 人支气管上皮细胞 

分 类 号：R394.3[医药卫生—医学遗传学] R977.3[医药卫生—基础医学] R994.7