高等学校化学学报

高等学校化学学报 ›› 2012, Vol. 33 ›› Issue (02): 313.doi: 10.3969/j.issn.0251-0790.2012.02.018

• 生物化学 • 上一篇    下一篇

人肝中硒蛋白K相互作用蛋白的筛选与验证

梁雪莹1, 刘琼1, 陈平1,2, 霍克克3, 胡田勇1, 倪嘉缵1,2   

  1. 1. 深圳大学生命科学学院, 深圳 518060;
    2. 中国科学院长春应用化学研究所, 长春 130022;
    3. 上海复旦大学生命科学学院, 上海 200433
  • 收稿日期:2011-01-21 出版日期:2012-02-10 发布日期:2012-01-13
  • 通讯作者: 倪嘉缵, 男, 研究员, 中国科学院院士, 主要从事硒蛋白以及稀土生物化学等研究. E-mail: jzni@szu.edu.cn 刘 琼, 女, 博士, 教授, 主要从事硒的生物化学和分子生物学研究. E-mail: liuqiong@szu.edu.cn E-mail:jzni@szu.edu.cn;liuqiong@szu.edu.cn
  • 基金资助:

    国家自然科学基金(批准号: 31070731, 30901182)和广东省自然科学基金(批准号: 10151806001000023)资助.

Screen and Verification of Interactive Protein of Selenoprotein K in Human Liver

LIANG Xue-Ying1, LIU Qiong1, CHEN Ping1,2, HUO Ke-Ke3, HU Tian-Yong1, NI Jia-Zuan1,2   

  1. 1. College of Life Sciences, Shenzhen University, Shenzhen 518060, China;
    2. Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China;
    3. College of Life Sciences, Fudan University, Shanghai 200433, China
  • Received:2011-01-21 Online:2012-02-10 Published:2012-01-13
  • Contact: Jiazuan Ni E-mail:jzni@szu.edu.cn;liuqiong@szu.edu.cn

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被引次数

8

摘要： 以硒蛋白K(SelK)突变体为"诱饵", 采用酵母双杂交系统对人肝cDNA文库进行筛选, 得到一个与SelK相互作用的蛋白──环腺苷酸应答元件结合蛋白3(CREB3). 将SelK与CREB3共同转染酵母细胞, 验证了SelK与CREB3的相互作用; 并采用受体漂白、敏化发射和荧光寿命3种荧光共振能量转移方法进一步验证了二者间的相互作用, 发现其不受SelK中硒代半胱氨酸(Sec)的影响. 推测SelK可能通过其Sec之前的区域与CREB3发生作用, 参与CREB3介导的内质网相关降解过程, 影响相关癌症的转移和发展.

关键词: 硒蛋白K, 环腺苷酸应答元件结合蛋白3, 酵母双杂交, 荧光共振能量转移, 蛋白质相互作用

Abstract: Selenium, an essential trace element for human health, plays important roles in maintaining redox balance in vivo. Selenium deficiency is associated with many diseases, such as cancer, neurodegenerative diseases and cardiovascular diseases. The biological function of selenium in vivo is mainly exerted through selenoproteins. Selenoprotein K(SelK) is a newly discovered selenoprotein with unknown biological function and molecular mechanism. In this work, human SelK gene was cloned, site-directedly mutated and inserted into the "bait" plasmid to screen the human liver cDNA library using the yeast two-hybrid system. An interactive protein, homo sapiens cAMP responsive element binding protein 3(CREB3), was screened out and analyzed. SelK' and CREB3 genes were then co-transformed into yeast cells to verify the protein interaction, followed by the co-transfection into HEK293T cells to further verify the interaction via three methods of fluorescence resonance energy transfer technique, including the receptor photobleaching, sensitized emission and fluorescence lifetime. The results show that SelK interacte with CREB3 independent of its selenocysteine residue. By interacting with CREB3, SelK may participate in CREB3-mediated endoplasmic reticulum-asso-ciated degradation and make impact on cancer migration and development.

Key words: Selenoprotein K(SelK), Homo sapiens cAMP responsive element binding protein 3(CREB3), Yeast two hybridization, Fluorescence resonance energy transfer(FRET), Protein-protein interaction

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