【作者】 许健； 沈俊俊； 蒋菲； 李细胞鹏； 滕达； 于自强； 何金艳； 李霞； 李永清； 吴文学； 李金祥；

【Author】 Jian Xu;Junjun Shen;Fei Jiang;Peng Li;Da Teng;Ziqiang Yu;Jinyan He;Li Xia;Yongqing Li;Wenxue Wu;Jinxiang Li;Laboratory of Rapid Diagnostic Technology for Animal Disease,College of Veterinary Medicine,China Agricultural University;Institute of Animal Husbandry and Veterinary Medicine,Beijing Academy of agricultural and Forestry Sciences;Key Laboratory of Animal Epidemiology and Zoonosis,Ministry of Agriculture,College of Veterinary Medicine,China Agricultural University;Chinese Academy of Agricultural Sciences;

【机构】 中国农业大学动物医学院动物疫病快速诊断实验室； 北京市农林科学院畜牧兽医研究所； 中国农业大学动物医学院农业部动物原虫及流行病学重点实验室； 中国农业科学院；

【摘要】 鸡毒支原体（Mycoplasma gallisepticum）核酸酶样蛋白MGA₀676是重要的核酸酶样蛋白,具有核酸酶活性,可以内化进入宿主细胞诱导鸡细胞凋亡功能。为研究MGA₀676诱导鸡细胞凋亡的分子机制,本研究采用间接免疫荧光及激光共聚焦技术对MGA₀676内化细胞的机理进行研究,并对MGA₀676蛋白的细胞靶蛋白采用puNdown、质谱测序、免疫共沉淀及激光共聚焦技术进行研究,结果发现抑制Caveolin细胞内吞途径,抑制了rMGA₀676蛋白内化DF-1细胞,鸡毒支原体MGA₀676蛋白与鸡DF-1细胞的NEDD8激活酶调节亚基（NEDD8-activating enzyme E1 regulatory subunit,NAE）蛋白相互作用,缺失了MGA₀676蛋白的SNC区域或NAE蛋白的Thif区域均阻止了MGA₀676蛋白与NAE蛋白的相互作用,这表明鸡毒支原体MGA₀676蛋白通过依赖于Caveolin的细胞内吞作用进入细胞,其SNC区域与鸡DF-1细胞的NAE蛋白的Thif区域相互作用。为进一步研究MGA₀676蛋白与宿主NAE蛋白相互作用所引起的生物学效应与细胞凋亡的关系,采用小RNA干扰抑制DF-1细胞内源性的NAE蛋白的表达,证实MGA₀676与其的相互作用对细胞凋亡的影响,并通过检测细胞内NF-κB活性,研究DF-1细胞NF-κB的激活与rMGA₀676诱导细胞凋亡的关系,结果发现,抑制DF-1细胞NAE表达阻止了rMGA₀676蛋白及鸡毒支原体诱导的细胞凋亡,rMGA₀676蛋白可以激活宿主细胞NF-κB信号途径,阻断MGA₀676与NAE相互作用抑制了rMGA₀676蛋白诱导的NF-κB的激活,抑制细胞内源性的NF-κB,抑制了rMGA₀676蛋白诱导的细胞凋亡,研究结果表明鸡毒支原体MGA₀676蛋白通过Caveolin介导的细胞内吞进入细胞,与DF-1细胞NAE蛋白相互作用激活宿主细胞NF-κB信号通路,诱导细胞凋亡。研究结果揭示了MGA₀676蛋白引起的宿主细胞凋亡的分子致病机理,阐明了NAE及NF-κB在鸡毒支原体MGA₀676蛋白在引起细胞凋亡中发挥的作用,为深入探讨鸡毒支原体引起的细胞凋亡的分子致病机理提供了重要的理论依据。更多还原

【Abstract】 MGA₀676 has been characterized as a Mycoplasma gallisepticum nuclease that can induce apoptosis of chicken cells,and Staphylococcal nuclease（SNC） region is essential for its nuclease activity and apoptotic induction.In the present study,we uncovered a yet unknown mechanism of apoptosis induced by MGA₀676.We show that MGA₀676 was internalized by chicken embryo fibroblasts（DF-1）through caveolin-mediated endocytosis.Then,it interacts with NEDD8-activating enzyme E1 regulatory subunit（NAE）as detected by pulldown and immunoprecipitation,and their co-location in the perinuclear and nuclear regions of DF-1 cell was observed by Laser scanning confocal microscopy（LSCM）.By constructing mutants with deleted functional regions,we discovered the interaction between MGA₀676 and NAE was dependent on SNC domain of MGA₀676 and Thif region of NAE.The apoptosis induced by MGA₀676 decreased significantly after NF-κB was inhibited by siRNA or BAY11-7082 or NAE was silenced by siRNA.Levels of NF-κB in the nucleus significantly rose after DF-1 cells were treated with rMGA₀676,but fell down in DF-1 cells treated with MGA₀676^△SNCor in DF-1 cells with knockdown NAE and treated with MGA₀676.LSCM also confirmed that NF-κB lost the ability to translocate to the nucleus in DF-1 cells treated with MGA₀676^△SNCand in rMGA₀676-treated DF-1 cells that had undergone NAE-knockdown.In conclusion,the above results suggest that MGA₀676 was internalized through caveolin-mediated endocytosis,and the interaction between MGA₀676 and NAE dependent on SNC and Thif regions plays a key role on the activation of NF-κB and the apoptosis induced by M.gallisepticum.The study elaborated the MGA₀676 was an important virulence factor in cellular pathology and may play a unique role in the life cycle events of M.gallisepticum.it also illuminated the NAE and NF-κB played a vital role in the process of mechanism of MGA₀676 in the host cells,which would be provided important theoretical basis on the host apoptosis induction after M.gallisecpticum infected.更多还原