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目的:p66Shc在线粒体内积累和HtrA2/Omi的功能缺陷都能导致线粒体损伤,诱导细胞凋亡。探讨在线粒体中HtrA2对p66Shc的调控作用。方法:构建p66Shc和成熟型HtrA2的真核表达质粒,共转染HEK293T细胞,免疫印迹法(Western blot)检测p66Shc蛋白;构建原核表达质粒,大肠杆菌纯化蛋白,体外切割实验,SDS-PAGE分离后考马斯亮蓝染色检测;提取HtrA2功能缺陷小鼠(mnd2)大脑组织的线粒体,检测线粒体内p66Shc的蛋白水平。结果:细胞实验和体外实验证明HtrA2可以切割p66Shc,且在mnd2小鼠大脑中,线粒体内p66Shc的蛋白水平明显升高(P<0.05)。结论:p66Shc是HtrA2的直接底物,且HtrA2参与调节线粒体中p66Shc的蛋白水平,揭示了HtrA2发挥神经保护功能新的可能机制。
PURPOSE: The in vivo mitochondrial accumulation of p66Shc and the functional defects of HtrA2 / Omi all lead to mitochondrial damage and apoptosis. To investigate the regulatory effect of HtrA2 on p66Shc in mitochondria. Methods: Eukaryotic expression plasmid p66Shc and mature HtrA2 were constructed and transfected into HEK293T cells. The p66Shc protein was detected by Western blot. The prokaryotic expression plasmid, E. coli purified protein, in vitro cleavage experiment and SDS-PAGE separation Coomassie brilliant blue staining; mitochondria were extracted from brain tissue of HtrA2-deficient mice (mnd2) and the protein level of p66Shc in mitochondria was detected. Results: Both cell and in vitro experiments showed that HtrA2 can cleave p66Shc, and the mitochondrial p66Shc protein level was significantly increased in mnd2 mouse brain (P <0.05). Conclusion: p66Shc is the direct substrate of HtrA2, and HtrA2 is involved in the regulation of p66Shc protein level in mitochondria, revealing a new possible mechanism of HtrA2 playing a neuroprotective role.