含MPN结构域蛋白（MPND）通过上调乙型肝炎病毒X蛋白水平影响其功能MPN Domain-Containing Protein(MPND) Increases Hepatitis B Virus X Protein Levels and Promotes Its Biological Functions
张翼;吴琼;施佳健;张璐;林旭;陈婉南;
摘要(Abstract)：
乙型肝炎病毒X蛋白（Hepatitis B virus X protein,HBx）在生成后即被快速降解，但目前影响HBx稳定性的机制仍未完全阐明。课题组前期通过酵母双杂交筛选与HBx具有相互作用的去泛素化酶（Deubiqutinases,DUBs），含MPN结构域蛋白（MPN domain containing protein,MPND）为筛选获得的蛋白之一。本研究首先采用免疫共沉淀和激光共聚焦实验验证HBx与MPND的相互作用，并通过酵母双杂交实验进一步分析两者相互作用的区域。Western blot检测过表达MPND的肝癌细胞中HBx蛋白水平的变化。以蛋白合成抑制剂环己酮亚胺（Cycloheximide,CHX）或蛋白酶体抑制剂MG132处理细胞，检测MPND对HBx蛋白半衰期及降解途径的影响。通过泛素化实验分析MPND对HBx泛素化水平的影响。采用双荧光素酶报告基因检测实验和克隆形成实验检测MPND对HBx生物学功能的影响。结果显示，MPND与HBx在肝癌细胞内存在相互作用，且HBx的26～50和81～120位氨基酸与MPND的272～362位氨基酸介导HBx与MPND的结合；过表达MPND的肝癌细胞中，HBx蛋白水平显著增高，HBx的半衰期明显延长，且蛋白酶体抑制剂处理后，MPND对HBx蛋白降解的抑制效果更为显著，但HBx泛素化水平没有变化；共表达MPND与HBx的肝癌细胞中，MPND通过上调HBx蛋白水平，进而促进了HBx对NF-κB启动子的反式激活作用，且进一步抑制克隆形成。本研究表明，MPND通过泛素非依赖-蛋白酶体途径抑制HBx降解，从而增加HBx蛋白水平，调控HBx功能，可能参与HBV致病机制，以此蛋白相互作用为靶标，将为HBV感染相关肝脏疾病的治疗提供有益思路。
关键词(KeyWords)： 乙型肝炎病毒X蛋白;去泛素化酶;蛋白酶体;蛋白降解
基金项目(Foundation): 福建省财政厅项目（项目号：2019B027）,题目：去泛素化酶通过调控乙型肝炎病毒X蛋白水平影响肝细胞及病毒复制;; 国家自然科学基金项目（项目号：81672031）,题目：BRG1相关因子155对乙型肝炎病毒X蛋白翻译后水平的调控及意义;; 福建省自然科学基金项目（项目号：2019J01299）,题目：乙型肝炎病毒剪接特异性蛋白HBDSP通过诱导上皮间质转化促进肝癌细胞侵袭转移机制研究;; 福建医科大学启航基金项目（项目号：2018QH2005）,题目：泛素特异性肽酶USP22对乙型肝炎病毒X蛋白的影响及机制~~
作者(Authors): 张翼;吴琼;施佳健;张璐;林旭;陈婉南;
DOI: 10.13242/j.cnki.bingduxuebao.004217
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