摘要
目的明确人肺泡Ⅱ型上皮细胞(AECⅡ)中CCAAT增强子结合蛋白α(C/EBPα)能否发生小泛素相关修饰物(SUMO)修饰,并进一步鉴定其修饰位点。方法采用免疫荧光双标法检测C/EBPα和SUMO1在人AECⅡ中的表达情况。免疫共沉淀(Co-IP)检测C/EBPα和SUMO1在AECⅡ中的相互作用,明确人AECⅡ中C/EBPα能否发生SUMO化修饰。利用SUMOsp软件预测人C/EBPα的SUMO化修饰位点为C/EBPα上第161位赖氨酸(即K161),构建野生型GFP-C/EBPα质粒和突变型GFP-K161R质粒并转染AECⅡ,采用Co-IP鉴定C/EBPα的SUMO化位点。结果免疫荧光双标发现AECⅡ中SUMO1和C/EBPα存在共定位现象,且主要定位于细胞核中; C/EBPα和SUMO1的Co-IP结果出现C/EBPα-SUMO条带,提示C/EBPα能与SUMO1相互作用。野生型GFP-C/EBPα转染的AECⅡ中,可检测到C/EBPα-SUMO特异性条带,而突变型GFP-C/EBPα转染的AECⅡ中,不能检测到C/EBPα-SUMO特异性条带,提示C/EBPα的SUMO化修饰位点为C/EBPα第161位赖氨酸。结论人AECⅡ中C/EBPα能发生SUMO化修饰且其修饰位点为C/EBPα第161位赖氨酸。
Objective To clarify the interaction between CCAAT enhancer-binding protein a ( C/EBPa) and small ubiquitin-related modification (SUMO) in human alveolar type Ⅱ epithelial cells (AEC Ⅱ), and further identify its modification sites. Methods The expression of C/EBPa and SUMO1 in human AEC Ⅱ was detected by immunofluorescence double labeling. Co-IP was used to detect the interaction of C/EBPα and SUMO1 in AEC Ⅱ. The SUMO site of C/EBPa was predicted to be the 161st lysine (K161) by the SUMOsp software. The wild-type GFP-C/EBPα plasmids and mutant GFP-K161R plasmids were constructed and transfected into AECⅡ. The SUMO site of C/EBPα was identified by Co-IP. Results Immunofluorescence double staining found that SUMO1 and C/EBPα were co-located in the nucleus. C/EBPa-SUMO band could be marked by Co-IP, which suggested that C/EBPα could interact with SUMO1. When AEC Ⅱ was transfected by wild-type GFP-C/EBPa plasmids. C/EBPα-SUMOl band could be detected by immunoprecipitation (IP), but could not be detected when transfected by mutant GFP-C/EBPα plasmids. These suggested that the SUMO site of C/EBPα was the 161st lysine. Conclusion C/EBPa can be modified by SUMO1 and the site of its modification is the 161st lysine in human AEC Ⅱ.
作者
郝晓波
卢红艳
朱玥
万峰云
万雪晴
HAO Xiaobo;LU Hongyan;ZHU Yue;WAN Fengyun;WAN Xueqing(Department of Pediatrics, Affiliated Hospital of Jiangsu University, Zhenjiang 212001, China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2019年第2期158-161,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(81741052)
江苏省自然科学基金(BK20161356)
