结直肠癌细胞β-catenin蛋白脱酰胺化修饰的实验研究

Identification of deamidation modification of β-catenin in colorectal cancer cell lines

摘要:

目的 鉴定结直肠癌细胞β-catenin的脱酰胺化修饰及其相关脱酰胺化酶。方法 采用二维电泳结合脱酰胺化抑制剂6-重氮-5-氧代-L-正亮氨酸（DON）方法鉴定内源性β-catenin蛋白脱酰胺修饰的存在。通过β-catenin蛋白的纯化与质谱分析筛选出β-catenin蛋白候选可能的脱酰胺化酶。利用免疫共沉淀实验检测候选脱酰胺化酶与β-catenin蛋白的相互作用。结果 二维电泳在结直肠癌细胞系HCT116中鉴定出β-catenin蛋白可以发生脱酰胺化修饰。成功构建pEE14.4-β-catenin质粒表达载体，并且在细胞中成功验证其表达；利用蛋白纯化结合质谱分析鉴定出β-catenin蛋白的候选脱酰胺化酶——谷氨酰胺酶复合酶（CAD）；Western blotting再次验证CAD能够与内源性β-catenin蛋白发生相互作用。结论 结直肠癌细胞β-catenin蛋白能够发生脱酰胺化修饰，CAD蛋白可能为介导β-catenin蛋白脱酰胺化修饰的相关酶。

Abstract:

Objective To identify the deamidation modification of β-catenin and related deamidases in colorectal cancer cell lines.Methods The two-dimensional electrophoresis combined with the deamidation inhibitor 6-Diazo-5-oxo-L-norleucine (DON) was applied to determine the presence of deamidation modification of endogenous β-catenin. The candidate deamidases were screened out by β-catenin purification and mass spectrometry analysis. The interaction between the candidate deamidases and β-catenin was detected by co-immunoprecipitation (co-IP).Results Two-dimensional electrophoresis verified that β-catenin protein exhibited deamidation modification in colorectal cancer cell lines HCT116. The pEE14.4-β-catenin plasmid was constructed and expressed in cells successfully. Protein purification combined with mass spectrometry analysis found that CAD was a candidate deamidase, and co-IP further confirmed the interaction between CAD and endogenous β-catenin.Conclusions β-catenin could be modified via deamidation in colorectal cancer cell lines, where CAD represents possibly the key deamidase responsible for this modification.