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[1]袁韵,林之蔚,郭加加,等.细胞膜结合的雌激素受体的特异性标记[J].应用与环境生物学报,2014,20(02):227-232.[doi:10.3724/SP.J.1145.2014.00227]
 YUAN Yun,LIN Zhiwei,GUO Jiajia,et al.Specific labeling of the cell membrane-bound estrogen receptor[J].Chinese Journal of Applied & Environmental Biology,2014,20(02):227-232.[doi:10.3724/SP.J.1145.2014.00227]
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细胞膜结合的雌激素受体的特异性标记()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
20卷
期数:
2014年02期
页码:
227-232
栏目:
研究论文
出版日期:
2014-04-25

文章信息/Info

Title:
Specific labeling of the cell membrane-bound estrogen receptor
作者:
袁韵林之蔚郭加加王飞袁小红
1西南科技大学生命学院 绵阳 621000 2中国科学院成都生物研究所 成都 610041 3成都理工大学 成都 610059
Author(s):
YUAN Yun LIN Zhiwei GUO Jiajia WANG Fei YUAN Xiaohong
1College of Life Sciences, Southwest University of Science and Technology, Mianyang 621000, China 2Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China 3Chengdu University of Technology, Chengdu 610059, China
关键词:
雌激素受体磷酸泛酰巯基乙胺基转移酶标记辅酶A
Keywords:
estrogen receptor phosphopantetheinyl transferase labeling coenzyme A
分类号:
Q291 : Q78
DOI:
10.3724/SP.J.1145.2014.00227
文献标志码:
A
摘要:
细胞膜结合的雌激素受体(Cell membrane-bound estrogen receptor, cmER)介导了雌激素的非基因组作用,参与了乳腺癌等诸多疾病的发生,但是作用机制研究受限于能够有效区分cmER和细胞质内ER(Estrogen receptor)的标记手段. 为了实现cmER的特异性标记,首先在体外分别表达含有A1、S6和ybbR三个不同短肽的谷胱甘肽S转移酶融合蛋白,比较磷酸泛酰巯基乙胺基转移酶(Phosphopantetheinyl transferase, PPTase)-AcpS和Sfp对其的识别选择性. 选取识别效率较高和特异性较好的A1短肽,将其构建到雌激素受体ERα的C端(ERα-A1),结果表明该偶联并不影响ERα的蛋白质表达及其在雌激素作用下的转录. 进一步研究表明,来源于大肠杆菌的PPTase-AcpS可以以辅酶A-生物素(Coenzyme A-biotin)为底物,将生物素特异性标记到ERα-A1. 这些结果为进一步研究雌激素及cmER介导的非基因组调控机制提供了新的方法.
Abstract:
Cell membrane-bound estrogen receptors (cmERs) mediate the non-genomic effects of estrogen, which is involved in the occurrence of breast cancer and many other estrogen-related diseases. However, the mechanism study is hindered by the lack of a specific labeling method which can efficiently differentiate between cmERs and cytoplasmic ERs. The purpose of this study was to develop the specific labeling of the cmER. We expressed and purified glutathione S-transferase fusion proteins which contained three different specific peptides A1, S6 and ybbR. Then we compared the labeling specificity and efficiency of the proteins by two different phosphopantetheinyl transferase (PPTase): AcpS and Sfp. A1 peptide, which was shown with higher labeling efficiency, was constructed into the C-terminus of the estrogen receptor α (ERα-A1) and then the ERα-A1 expression, its genomic and nongenomic function and labeling of biotin by AcpS were measured in cells. The results showed that coupling of A1 tag did not affect the ER protein expression or its activity. AcpS could specifically label biotin to cmERα-A1with the coenzyme A-biotin as a substrate. These results provide a new method for the study of cmER-mediated estrogen nongenomic signal transduction.

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备注/Memo

备注/Memo:
国家自然科学基金项目(30900769)资助
更新日期/Last Update: 2014-05-04