羧基末端结合蛋白CtBP2与泛素结合酶UBE2Ⅰ的相互作用  

Interaction between C-terminal binding protein 2 and ubiquitin-conjugating enzyme E2Ⅰ

作　　者：叶晓霞[1] 石彦[2] 霍克克[2] 陈东[1] 吴民华[1] 

机构地区：[1]广东医学院基础医学院组织学与胚胎学教研室,广东湛江524023 [2]复旦大学生命科学院遗传研究所,上海200433

出　　处：《第三军医大学学报》2011年第17期1783-1788,共6页Journal of Third Military Medical University

基　　金：广东省卫生厅医学科研基金(B2009192);广东医学院科研基金(B2009010)~~

摘　　要：目的筛选并确定人羧基末端结合蛋白CtBP2的相互作用蛋白。方法用酵母双杂交技术,以人CtBP2为诱饵蛋白筛选人肝cDNA文库,获得与之相互作用的候选蛋白,采用酵母共转化和细胞免疫共沉淀实验确认相互作用的特异性,最后利用荧光蛋白融合表达技术和荧光显微镜观察确认CtBP2与其相互作用蛋白的亚细胞共定位情况。结果酵母双杂交筛选获得了41个与Bd-CtBP2相互作用的阳性克隆,编码10个不同的蛋白,其中有4个克隆编码泛素结合酶UBE2Ⅰ的不同片段。从人肝cDNA文库中扩增获得UBE2Ⅰ的全长编码序列,酵母共转化和细胞免疫共沉淀实验证实CtBP2与UBE2Ⅰ能够特异性结合。亚细胞定位研究表明GFP-CtBP2弥散分布于细胞核,DsRed-UBE2Ⅰ不均匀分布于细胞核,有部分点状聚集。两者共转染时能够呈点状共定位于细胞核。结论人CtBP2能够在细胞核内与UBE2Ⅰ特异性相互作用,是UBE2Ⅰ的靶蛋白。Objective To screen and identify the proteins that can interact with human C-terminal binding protein 2（CtBP2）.Methods Yeast two-hybrid screening technique,with CtBP2 as bait protein,was used to screen out the genes of the candidate CtBP2-interacting proteins from human liver cDNA library.Then,yeast co-transformation assay and co-immunoprecipitation assay were performed to confirm the specificity of interaction.Fluorescent fusion proteins and a fluorescence microscope were used for subcellular co-localization of CtBP2 and its interacting proteins in HeLa cells.Results The yeast two-hybrid screening method yielded 41 positive clones that coded 10 CtBP2-interacting proteins.Among them,4 clones coded different fragments of ubiquitin-conjugating enzyme E2Ⅰ（UBE2Ⅰ）.The full-length coding sequence of UBE2Ⅰ was amplified from human liver cDNA library.The specific interaction between CtBP2 and UBE2Ⅰ was confirmed by the yeast co-transformation assay and co-immunoprecipitation assay.The subcellular localization results showed that：（1） GFP-CtBP2 was dispersed in the nuclei of HeLa cells,（2） DsRed-UBE2Ⅰ was unevenly distributed in the nuclei of HeLa cells with some spotty accumulation,and（3） GFP-CtBP2 and DsRed-UBE2Ⅰ could be co-localized in nuclear speckles when the HeLa cells were transfected with both pEGFP-CtBP2 and pDsRed-UBE2Ⅰ.Conclusion Human CtBP2 can interact with UBE2Ⅰ specifically in the nucleus and is a target protein of UBE2Ⅰ.

关 键 词：羧基末端结合蛋白 酵母双杂交 泛素结合酶UBE2Ⅰ 免疫共沉淀 亚细胞共定位 

分 类 号：R394.33[医药卫生—医学遗传学] R394.3[医药卫生—基础医学]