文章摘要
张亚萍,李建涛,施芃曦,阙玲琍,李跃华.Pellino1 SUMO修饰位点突变对TRAF6介导的NF⁃κB信号转导的影响[J].南京医科大学学报,2018,(7):863~868
Pellino1 SUMO修饰位点突变对TRAF6介导的NF⁃κB信号转导的影响
Effects of Pellino1 SUMO modification site mutation on TRAF6 mediated NF⁃κB signal transduction
投稿时间:2018-03-19  
DOI:10.7655/NYDXBNS20180701
中文关键词: Pellino1  SUMO修饰  TRAF6  NF⁃κB
英文关键词: Pellino1  SUMOylation  TRAF6  NF⁃κB
基金项目:国家自然科学基金(81470418,81270292)
作者单位
张亚萍 南京医科大学病理生理学系江苏 南京 211166 
李建涛 南京医科大学病理生理学系江苏 南京 211166 
施芃曦 南京医科大学病理生理学系江苏 南京 211166 
阙玲琍 南京医科大学病理生理学系江苏 南京 211166 
李跃华 南京医科大学病理生理学系江苏 南京 211166 
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中文摘要:
      目的:研究Pellino1蛋白翻译后修饰——小泛素相关修饰物(small ubiquitin?related modifier,SUMO)修饰对肿瘤坏死因子受体相关分子?6(TNF receptor associated factor 6,TRAF6)介导的核因子κB(nuclear factor κB,NF?κB)信号转导的影响。方法:构建Pellino1 SUMO修饰位点突变的质粒,与TRAF6、泛素(ubiquitin,Ub)质粒共转染至HEK?293细胞中,荧光显微镜观察转染效率,免疫共沉淀的方法检测突变型Pellino1与TRAF6的相互结合作用;同时,检测Pellino1和TRAF6的泛素化修饰水平。采用脂多糖(lipopolysaccharide,LPS)诱导炎症反应,分提胞浆胞核蛋白,Western blot检测核因子κB的抑制蛋白α(inhibitor of NF?κBα,IκBα)的磷酸化和NF?κB P65的核转位,分析Pellino1蛋白SUMO修饰突变对TRAF6介导的NF?κB信号通路的影响。结果:与野生型Pellino1相比,SUMO修饰突变型Pellino1不仅增加Pellino1的自身泛素化修饰水平,而且可增加其与TRAF6的相互结合作用,并增强TRAF6的泛素化修饰;LPS刺激可增加IκBα的磷酸化和NF?κB P65的核转位,转染使Pellino1 SUMO修饰突变高表达可明显增强LPS诱导的NF?κB信号激活。结论:Pellino1 SUMO修饰突变,可通过增加Pellino1的自身泛素化修饰及其与TRAF6的结合,增强TRAF6的泛素化,最终促进TRAF6介导的NF?κB信号通路的激活。
英文摘要:
      Objective:To study the effects of Pellino1’s post?translational modification—small ubiquitin?related modifier(SUMO)modification of Pellino1 in the nuclear factor κB(NF?κB)signaling pathway mediated by the TNF receptor associated factor6(TRAF6). Methods:We constructed the plasmid of the SUMO modification sites mutantation of Pellino1,then cotransfected with TRAF6 plasmid and ubiquitin(Ub)plasmid into HEK?293 cells. The efficiency of plasmid transfection was observed by fluorescence microscopy. We detected the combination of mutant Pellino1 with TRAF6 by co?immunoprecipitation,and monitored the ubiquitination level of Pellino1 and TRAF6. Lipopolysaccharide(LPS)was used to induce inflammatory response,and the cytoplasmic nucleocapsid was extracted. Western blot was used to detect the phosphorylation of inhibitor of NF?κBα(IκBα)and the nuclear translocation of NF?κB P65. The effect of Pellino1 SUMO modification on TRAF6 mediated NF?κB signaling pathway was analyzed. Results:Compared with wild type Pellino1,mutant of Pellino1 SUMOylation ylation not only increased ubiquitination of Pellino1 itself,but also the combination with TRAF6,and the ubiquitination modification of TRAF6 were also increased. LPS stimulation significantly increased the phosphorylation of inhibitor of NF?κBα(IkBα)and NF?κB P65 nuclear translocation. High expression of mutant of Pellino1 SUMO after transfection significantly enhanced the LPS induced NF?κB signal activation. Conclusion:Mutant of Pellino1 SUMOylation can promote TRAF6 mediated activation of NF?κB signaling pathway by increasing the ubiquitination of Pellino1 and binding with TRAF6 and the ubiquitination level of TRAF6.
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