ERβ相互作用蛋白HPIP的分离及其在ERβ信号途径中的作用

Isolation of an ERβ interaction protein HPIP and its role in ERβ singnaling

目的以雌激素受体β(ERβ)AF2结构域为诱饵,采用酵母双杂交方法,从人类乳腺cDNA文库筛选的蛋白HPIP,进一步验证其与ERβ相互作用的功能。方法分别用细胞免疫荧光分析和酵母双杂交的方法筛选蛋白的细胞内定位及结合结构域的鉴定,转染293T胚胎肾细胞,利用荧光素酶和β-半乳糖苷酶活性测定,检测HPIP对ER转录活性的影响。结果 HPIP的编码基因主要分布于细胞质中,HPIP与ERβAF2结构域相互作用,而不与ERβAF1和DBD相互作用,HPIP能降低ER的转录活性,而且这种抑制作用在激素存在时更显著,但是雌激素受体抑制剂存在时,HPIP反而增强ERβ的转录活性。结论 HPIP可能通过影响ERβ信号途径在乳腺癌发生、发展中起重要作用。

Objective To generate the HPIP and to test the function of its interaction with ERβ on the bait of structure field of ERβ AF2 and filtering proteins combined with ERβ in the cDNA library of human mammary glands using the method of yeast two-hybrid method.Methods The methods of fluorescence analysis of cellular immunity and yeast two-hybrid were used respectively in filtering intracellular positioning and identifying the binding domain of proteins.The transfections of HEK293T cell,the determination of activity with luciferase and β-galactosidase enzyme,and the examinations on the influence of HPIP to ERβ transcription activity were conducted.Results The coding genes of HPIP were mainly located in cytoplasm.HPIP interacts with the structure field of ERβ AF2 instead of ERβ AF1 and DBD.HPIP was with the function of reducing the transcription activity of ER.This inhibition was more significant with the existence of hormone.However,HPIP increased the transcription activity of ERβ when the inhibitors of estrogen receptors exist.Conclusion HPIP may be essential in breast cancer grouth and progression through ERβ singnaling.