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目的:确定α-Synuclein蛋白与线粒体相互作用的功能结构域,并检测该结构域对线粒体功能的影响。方法:构建重组融合蛋白质粒pCMV-myc/α-Syn-WT、pCMV-myc/α-Syn-N和pCMV-myc/α-Syn-△N,转染人胚肾HEK293T细胞,通过免疫共沉淀明确α-Synuclein蛋白与线粒体相互作用的功能结构域。使用表达α-Synuclein蛋白的病毒上清感染小鼠多巴胺能神经细胞MN9D,通过免疫荧光、流式细胞术检测线粒体膜电位及Cytochrome c释放。结果:成功构建了pCMV-myc/α-Syn-WT、pCMV-myc/α-Syn-N和pCMV-myc/α-Syn-△N融合蛋白质粒,免疫共沉淀明确α-Synuclein N-端为其功能结构域,JC-1染色发现N-端使线粒体膜电位降低,流式细胞术证实N-端使Cytochrome c释放明显增加。结论:α-Synuclein N-端是其与线粒体相互作用的功能结构域,N-端降低线粒体功能。
OBJECTIVE: To determine the functional domain of α-Synuclein interacting with mitochondria and to examine the effect of this domain on mitochondrial function. Methods: Recombinant fusion protein pCMV-myc / α-Syn-WT, pCMV-myc / α-Syn-N and pCMV-myc / α-Syn- △ N were constructed and transfected into human embryonic kidney HEK293T cells. Identify the functional domains of α-Synuclein interacting with mitochondria. The mouse dopaminergic neuron MN9D was infected with virus supernatant expressing α-Synuclein protein and mitochondrial membrane potential and release of Cytochrome c were detected by immunofluorescence and flow cytometry. RESULTS: Plasmid pCMV-myc / α-Syn-WT, pCMV-myc / α-Syn-N and pCMV-myc / α-Syn- △ N were successfully constructed and co- Its functional domain, JC-1 staining found that the N-terminal mitochondrial membrane potential decreased, flow cytometry confirmed N-terminal Cytochrome c release increased significantly. CONCLUSION: N-terminal α-Synuclein is a functional domain of its interaction with mitochondria, N-terminal decreases mitochondrial function.