目的 研究E46K突变体α-突触核蛋白(α-synuclein, α-syn)对线粒体自噬的影响。方法 构建稳定表达E46K突变体α-syn的PC12细胞株,检测细胞内活性氧含量和细胞凋亡的发生,并通过western blot和免疫共沉淀方法检测线粒体自噬相关蛋白Parkin、PINK1的细胞内定位和相互作用情况。结果 本研究成功构建稳定表达E46K突变体α-syn的细胞株。在线粒体解偶联剂CCCP作用下,过表达E46K突变体α-syn可导致细胞内活性氧含量和细胞凋亡显著增加,并抑制线粒体自噬蛋白Parkin由细胞浆向线粒体募集,导致Parkin与PINK1相互作用减少。结论 过表达E46K突变体α-syn可能通过抑制线粒体自噬发生促进帕金森病(PD)的发生和发展。
Abstract
OBJECTIVE To explore the effects of E46K mutant α-synuclein (α-syn) on mitophagy. METHODS Stable PC12 cells lines expressing E46K mutant α-syn were constructed. The levels of reactive oxygen species (ROS) and apoptosis rate were detected by flow cytometry, and the levels and location of mitophagy-related proteins such as PINK1 and Parkin were evaluated by Western blot and co-immunoprecipitation assays. RESULTS Overexpression of E46K mutant α-syn could significantly increase ROS levels and apoptosis rate under CCCP treatment. Otherwise, expressing E46K mutant α-syn inhibited translocation of Parkin from cytoplasm to mitochondrial and interaction with PINK1. CONCLUSIONS Overexpression of E46K mutant α-syn may promote PD development by inhibiting mitophagy.
关键词
α-突触核蛋白 /
E46K突变体 /
线粒体自噬
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Key words
α-synuclein /
E46K mutant /
mitophagy
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中图分类号：
R966
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基金
国家自然科学基金项目资助(82003973);北京市优秀人才培养资助青年骨干个人项目资助(2018000032600G402)
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