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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2012, Vol. 32 Issue (09): 9-14    
研究报告     
丝氨酸蛋白酶HtrA2/Omi可参与调节线粒体中的适配蛋白p66Shc
刘姝1,2, 姜长安1,2
1. 四川大学华西第二医院 西部妇幼医学研究院 华西发育与干细胞研究所 成都 610041;
2. 四川大学华西医院 生物治疗国家重点实验室 成都 610041
Serine Protease Omi/HtrA2 Regulates Adaptor Protein p66Shc in Mitochondria
LIU Shu1,2, JIANG Chang-an 1,2
1. West China Development and Stem Cell Institute, West China Institute of Women and Children's Health, West China Second University Hospital, Chengdu 610041, China;
2. State Key Laboratory of Biotherapy, West China Hospital, Chengdu 610041, China
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摘要: 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。
关键词: HtrA2/Omip66Shc线粒体神经退行性疾病    
Abstract: Objective: Both accumulation of p66Shc in mitochondria and loss of Omi/HtrA2 lead to mitochondrial dysfunction, which triggers apoptosis. To study the regulation of HtrA2 to the mitochondrial pool of p66Shc. Methods: The p66Shc and mature HtrA2 cDNA were cloned into eukaryote expression vector, then the constructs were cotransfected into HEK293T cell line, analyze p66Shc by Western blot; Purify proteins by pET System, then conduct in vitro cleavage assay, SDS-PAGE and Coomassie brilliant blue staining; analyze mitochondrial p66Shc protein level in mammalian cells overexpressed full length HtrA2 or knocked down HtrA2; Analyze p66Shc protein level in mitochondria isolated from mnd2 mouse brain. Results: p66Shc was cleaved by HtrA2 in vivo and in vitro; in mammalian cells, overexpressing full length HtrA2 down regulate mitochondrial p66Shc, and knocking down HtrA2 up regulate mitochondrial p66Shc; In mnd2 mouse brain, the mitochondrial p66Shc protein level was higher than that of the wild type mouse(P<0.05). Conclusion: Omi/HtrA2 cleaves p66Shc, and regulates the protein level of mitochondrial p66Shc,suggesting a possible new mechanism of how Omi/HtrA2 protects neuronal cells.
Key words: HtrA2/Omi    p66Shc    Mitochondria    Neurodegenerative disorder
收稿日期: 2012-04-17 出版日期: 2012-09-25
ZTFLH:  Q78  
基金资助: 国家自然科学基金资助项目(30871032)
通讯作者: 姜长安     E-mail: cjcareer@gmail.com
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引用本文:

刘姝, 姜长安. 丝氨酸蛋白酶HtrA2/Omi可参与调节线粒体中的适配蛋白p66Shc[J]. 中国生物工程杂志, 2012, 32(09): 9-14.

LIU Shu, JIANG Chang-an. Serine Protease Omi/HtrA2 Regulates Adaptor Protein p66Shc in Mitochondria. China Biotechnology, 2012, 32(09): 9-14.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/        https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I09/9

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