通过配体文库研究GIPC2 PDZ配体结合特点及其相互作用蛋白

Characterization of the binding property and the identification of the interaction proteins of GIPC2 PDZ domain by validation screening of PDZ ligand library

目的通过验证性筛选配体文库,获得GIPC2 PDZ结构域的配体结合特点,进而找到GIPC2的相互作用蛋白。方法 1)利用酵母双杂交的方法从已有的PDZ配体库中寻找与GIPC2的PDZ结构域配体结合特性;2)根据GIPC2的亚细胞定位和肿瘤相关功能再结合PDZ结构域结合序列的共同特征在蛋白质数据库中预测GIPC2的天然潜在配体;3)将天然潜在配体的C末端序列依次与GIPC2 PDZ结构域或GIPC2全长进行验证反应,从而得到阳性蛋白。结果 1)GIPC2 PDZ结构域的配体结合特性是C末端最后4个氨基酸为-X-S/T-X-V/L/I,是Ⅰ类PDZ配体;2)综合GIPC2的生物学特征和GIPC2 PDZ结构域的配体结合特点,在蛋白质数据库中预测得到47个天然潜在配体;3)将天然潜在配体的C末端序列克隆至酵母双杂交系统进行验证,最后得到10个确定的阳性蛋白。结论获得10个GIPC2相互作用蛋白。

Objective To characterize the binding property of GIPC2 PDZ domain by validation screening of PDZ ligand library and identification of the interaction proteins of GIPC2. Methods 1） The binding properties of GIPC2 PDZ domain were characterized by validation screening of the PDZ ligand library with yeast two-hybrid approach,2） According to the subcellular localization and function in the development of tumor,combined with the PDZ binding properties,the candidate GIPC2 binding ligands were predicted. 3） The candidate ligands were validated with GIPC2 PDZ domain and GIPC2. Results 1） The binding property of GIPC2 PDZ domain is that the last four amino acid residues of C termini are-X-S / T-X-V / L / I and belonging a type I PDZ ligand. 2） Integrated the biological characterization of GIPC2 and the PDZ binding properties,47 candidate GIPC2 binding ligands were predicted. 3）10 of 47 candidate ligands were validated to interact with GIPC2 PDZ domain and GIPC2 using yeast two hybrid.Conclusions Ten interaction proteins of GIPC2 are identified.