SapM诱导的自噬体-溶酶体融合阻断依赖于SapM和Rab7的相互作用  

SapM-induced fusion blocking of autophagosome-lysosome is depended on interaction with Rab7

作　　者：胡东[1,2] 王婉[1] 赵润鹏[1] 徐雪维 邢应如[3] 倪升发[3] 徐从景[3] 铁保贤[3] 张荣波[1,2] 吴静[1,2] 

机构地区：[1]安徽理工大学医学检验中心 [2]安徽理工大学医学院医学免疫学与检验教研室 [3]安徽理工大学附属肿瘤医院,安徽淮南232001

出　　处：《细胞与分子免疫学杂志》2016年第9期1178-1182,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81302524;81202294;81571528);安徽省科技攻关项目(1604a0802091)

摘　　要：目的研究Rab7在结核分枝杆菌毒力因子分泌性酸性磷酸酶(SapM)诱导的自噬体-溶酶体融合阻断中的作用。方法采用Rab7小干扰RNA(si Rab7)转染Raw264.7细胞,Western blot法检测P62蛋白水平。m Cherry-SapM转染Raw264.7细胞后免疫荧光检测SapM与Rab7共定位情况,免疫共沉淀分析SapM与Rab7的关系。用3种SapM的突变体SapM△ARCA,SapM△FRED和SapM△CT转染Raw264.7细胞,分析SapM的不同突变体与Rab7的关系。结果 si Rab7处理后,细胞P62水平显著增高;免疫组织化学染色结果显示SapM与Rab7在胞内共定位;免疫共沉淀分析显示SapM与Rab7可相互沉淀;3种不同突变形式的SapM中,只有SapM△CT不能与Rab7相互作用。结论 SapM诱导的自噬体-溶酶体融合阻断依赖于SapM与Rab7的相互作用。Objective To study the role of Rab7 in the blockage of autophagosome-lysosome fusion induced by secretory acid phosphatase （SapM）, a virulence factor of mycobacterium tuberculosis. Methods The Raw264.7 cells were transfected with siRabT, and the P62 was detected using Western blotting. After transfected with mCherry-SapM, the co-localization of SapM and Rab7 in Raw264.7 cells was detected by immunofluorescence cytochemistry and the interaction of SapM with Rab7 was determined by co-immunoprecipitation. SapM mutants including SapM△ARCA, SapM△FRED and SapM△CT were used to transfect Raw264.7 cells, and their associations with Rab7 were analyzed. Results The treatment of siRab7 induced a significant increase of P62 in these cells. Immunofluorescence cytochemistry showed the intracellular co-localization of SapM and Rab7. Co-immunoprecipitation showed that SapM and Rab7 were precipitated by each other. Only SapM^cT failed to interact with Rab7 among the three SapM mutants. Conclusion The inhibition of autophagosome-lysosome fusion induced by SapM is dependent on the interaction between SapM and RabT.

关 键 词：分泌性酸性磷酸酶(SapM) 自噬 Rab7 融合 

分 类 号：R392-33[医药卫生—免疫学] Q932[医药卫生—基础医学]