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.
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, $6 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 a（ERa-A1） and then the ERa-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 cmER^- Alwith the coenzyme A-biotin as a substrate. These results provide a new method for the study of cmER-mediated estrogen nongenomic signal transduction.
出处
《应用与环境生物学报》
CAS
CSCD
北大核心
2014年第2期227-232,共6页
Chinese Journal of Applied and Environmental Biology
基金
国家自然科学基金项目(30900769)资助~~
