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 ofβ-catenin in colorectal cancer cell lines

作　　者：向源楚 廖雨洁 陈杰[1] 沈绎伦 李虎 彭健[1] Xiang Yuan-chu;Liao Yu-jie;Chen Jie;Shen Yi-Lun;Li Hu;Peng Jian(Department of Geriatric Surgery,Xiangya Hospital,Central South University,Changsha,Hunan 410008,China;School of Life Sciences,Central South University,Changsha,Hunan 410013,China)

机构地区：[1]中南大学湘雅医院老年医学老年外科,湖南长沙410008 [2]中南大学生命科学学院,湖南长沙410013

出　　处：《中国现代医学杂志》2023年第14期33-38,共6页China Journal of Modern Medicine

基　　金：湖南省自然科学基金(No:2023JJ30869);长沙市科技计划项目(No:kq2014227)。

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。Objective To identify the deamidation modification ofβ-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 endogenousβ-catenin.The candidate deamidases were screened out byβ-catenin purification and mass spectrometry analysis.The interaction between the candidate deamidases andβ-catenin was detected by co-immunoprecipitation(co-IP).Results Two-dimensional electrophoresis verified thatβ-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 endogenousβ-catenin.Conclusionsβ-catenin could be modified via deamidation in colorectal cancer cell lines,where CAD represents possibly the key deamidase responsible for this modification.

关 键 词：结直肠癌 Β-CATENIN 脱酰胺化 谷氨酰胺酶复合酶 

分 类 号：R735.3[医药卫生—肿瘤]