人肝中硒蛋白K相互作用蛋白的筛选与验证

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

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.

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.