【作者】 熊舸；

【导师】 肖波；

【作者基本信息】 中南大学 ， 神经病学， 2008， 博士

【摘要】 目的:应用酵母双杂交系统和免疫荧光技术研究野生型和变异型LRR(Il122V)主域在体外与parkin分子之间是否存在相互作用,为逐个解析LRRK2蛋白其他主域的功能,全面探讨LRRK2蛋白在帕金森病发病机制中作用的后续研究奠定基础。方法:通过分子克隆技术构建诱饵pGBDU-LRR质粒,转染进入酵母菌成为PJ69-4α/pGBDU-LRR菌株,应用酵母双杂交系统在猎物书架(prey of bookshelf)中筛选出可能与野生型LRRK2蛋白的LRR主域存在相互作用的蛋白。随后分别转染含FLAG标记的PRK5-parkin质粒(对照组),HA标记的野生型pCMV-LRR质粒+PRK5-parkin质粒(实验组1,parkin+LRR(W)),HA标记的变异型pCMV-LRR质粒+PRK5-parkin质粒(实验组2,parkin+LRR(M)),采用Hoechst染色试剂盒检测细胞凋亡,western blot检测parkin蛋白和野生型/变异型LRR主域的表达,免疫荧光技术进一步观察野生型/变异型LRR主域在细胞内是否共定位,免疫沉淀技术证实LRR主域在体外与parkin分子之间是否存在相互作用。结果:我们通过酵母双杂交系统从猎物书架中筛选出parkin分子可能与野生型LRR主域之间存在相互作用,通过共同转染野生型/变异型pCMV-HA-LRR质粒和PRK5-FLAG-parkin质粒进入HEK293细胞,western blot检测到parkin蛋白和野生型/变异型LRR主域在HEK293细胞成功表达,免疫荧光技术观察到parkin在细胞胞浆中表达(绿色荧光),野生型LRR亦在细胞胞浆中表达(红色荧光),融合后,可见parkin与野生型LRR共同转染的HEK293细胞胞浆呈橙色,说明两者之间在细胞内共定位,可能存在相互作用;然而变异型LRR在HEK293细胞的胞核中形成包涵体,融合后核内的包涵体仍然为红色,说明parkin与变异型LRR在体外表达时细胞内定位不同,两者之间没有相互作用。免疫沉淀实验进一步证实parkin分子与野生型LRR主域之间存在相互作用。Hoechst染色发现细胞凋亡比率较对照组、实验组1明显升高,免疫荧光也表明实验组2的细胞核内有类似于路易体的包涵体形成,所以我们建立了帕金森病的细胞模型。结论:野生型LRRK2蛋白的LRR主域在体外表达时与parkin位于细胞内同一亚结构,两者之间存在相互作用。突变的LRR主域(Il122V)与parkin之间的相互作用缺失。变异型LRRK2蛋白之LRR主域与parkin在体外共同表达时可以形成类似路易体样的细胞核内包涵体。更多还原

【Abstract】 Objectives: After screening the prey of bookshelf to get the candidates that interact with the LRR domain of LRRK2 by yeast two hybrid system, the candidate molecule was further identified by immunofluorescence staining, which may offer a way to dissect the functions of other domains of LRRK2 protein in vitro to investigate the potential role of LRRK2 in the pathogenesm of Parkinson’s disease.Methods: The bait plasmid pGBDU-LRR was constructed by molecular cloning and transformed into yeast to acquired the strain PJ69-4a/pGBDU-LRR. Then the yeast two hybrid system was set up to investigate which prey plasmid (alpha-synuclein,parkin,UCHLl and DJ1)interacted with the bait. There were three groups of HEK293 cells transfected with different combinations of FLAG or HA tagged plasmids persectively: the control (parkin only), the experiment group 1(parkin+wild type LRR) and the experiment group 2 (parkin+mutant LRR). Hoechst staining was done to investigate the cell apoptosis. Immunofluorescent staining was done to show if there is a colocalization of LRR domain and parkin. The western blot was to test whether wild type or mutant LRR domain can coexpress with parkin in HEK293 cells. The immunoprecipitation were performed to further show whether there is an interaction between parkin and LRR(wild type or mutant).Results: It was indicated that protein parkin may interact with wild type LRR domain by yeast two hybrid. Western blot showed that LRR and parkin were co-expressed in HEK293 cells and immunoprecipitation proved that there was an interaction between LRR domain (wild type) and parkin. The immunofluorscence also found out that parkin (green) and wild type LRR domain (Red) were both expressed in cytoplasm and the merged image was orange. It was indicated that LRR domain (wild type) and parkin were colocalized and there was an interaction between parkin and wild type LRR in vitro. However, the mutant LRR domain was expressed in nuclei as aggregates and the merged image was not orange. So mutant LRR domain and parkin were not colocalized and there was no interaction between parkin and mutant LRR. The Hoechst staining showed that the experiment group 2 had a higher rate of cell apoptosis than that of experiment group 1 and control. Moreover, immunostaining also found that there were intranuclear aggregates in experiment group 2 that were similar to Lewy’s bodies in patients of Parkinson’s disease. Therefore, we had set up the cellular model of Parkinson’s disease.Conclusions: The wild type LRR domain colocalized with parkin in the same subcellular compartment and there is an interaction between parkin and wild type LRR, but no evidence of interaction between parkin and mutant LRR domain. The mutant LRR domain can form a intranuclear aggregates that is similar to the structure of Lewy body which is the hallmark of Parkinson’s disease.更多还原