鼻咽癌细胞中p53相互作用蛋白质的分离和鉴定  

Separation and Identification of p53 Interacting Proteins From Human Nasopharyngeal Carcinoma Cells

作　　者：胡巍[1] 肖志强[1] 陈主初[1] 李建玲[1] 张鹏飞[1] 冯雪萍[1] 易红[1] 余艳辉[2] 唐新科[3] 刘清萍[3] 梁宋平[3] 

机构地区：[1]中南大学湘雅医院医学实验研究中心 [2]中南大学湘雅医学院肿瘤研究所,长沙410078 [3]湖南师范大学生命科学学院,长沙410081

出　　处：《生物化学与生物物理进展》2004年第7期628-634,共7页Progress In Biochemistry and Biophysics

基　　金：国家重点基础研究发展规划项目(973)(2001CB5102);国家自然科学基金资助项目(30000028;30240056;30370642);教育部跨世纪优秀人才培养计划基金资助项目(教技函[2002]48);湖南省科技重点科研项目(02SSY20011);湖南省卫生厅重点科研项目(Z0204)~~

摘　　要：鼻咽癌中p5 3基因突变罕见 ,但绝大部分鼻咽癌中存在p5 3蛋白过表达 /聚集且功能失活 .然而 ,到目前为止p5 3蛋白失活的机制仍然不清楚 .为揭示鼻咽癌中p5 3蛋白功能失活的机制 ,采用免疫共沉淀技术分别富集鼻咽癌细胞系HNE1和HNE2的p5 3结合蛋白 ,SDS 聚丙烯酰胺凝胶电泳 (SDS PAGE)对免疫沉淀复合物进行分离 ,从胶中切取p5 3结合蛋白条带 ,胶内酶解后进行电喷雾串联质谱 (LC ESI MS/MS)分析 ,得到相应的肽序列标签 (peptidesequencetags ,PST) ,通过搜索数据库在鼻咽癌细胞系中鉴定了 9个p5 3结合蛋白 .分别是热休克蛋白 70 (HSP70 )家族成员GRP 78和GRP 75、HSP90家族成员GRP 94、核纤层蛋白A/C (LaminA/C)、α actinin 4、Ezrin/Cytovillin、DNA复制准许因子 /MCM3蛋白 (DNAreplicationlicensingfactor/minichromosomemaintenance 3protein ,MCM3)、CD98/ 4F2heavychain和蛋白激酶C (PKC) .并用免疫共沉淀和蛋白质印迹分析技术对HNE1细胞蛋白条带 3鉴定的p5 3相互作用蛋白之一HSP78进行了验证 .首次在鼻咽癌细胞中鉴定了 9个p5 3结合蛋白 ,为阐明鼻咽癌中p5 3蛋白聚集及失活的机制提供了重要依据和线索 .Although p53 gene mutation is a rare event in human nasopharyngeal carcinoma (NPC overexpressed/accumulated p53 protein is dysfunction in most of NPC. However, till now the mechanism of p53 protein inactivation remains unclear. In order to elucidate the mechanisms of p53 inactivation, p53 interacting proteins in the human NPC HNE1 and HNE2 cells were separeted and identified. p53 binding proteins were recovered by anti-p53 antibody immunoprecipitation with the total proteins from NPC HNE1 and HNE2 cells respectively. The recovered protein complexes were subjected to SDS-PAGE. The 5 separated protein bands were cut from the gel, in-gel digested and analyzed by LC-ESI-MS/MS. Then proteins were identified by peptide sequence tags (PST) and database searching, and were confirmed by immunoprecipitation and Western blot analysis. The results show that nine p53 binding proteins were identified, which were GRP-78 and GRP-75 of HSP 70 family members, GRP-94 of HSP 90 family members, laminA/C, Alpha-actinin 4, Ezrin/Cytovillin, DNA replication licensing factor/MCM3 protein, CD98/4F2 heavy chain and protein kinase C. It can be concluded that this study first time identified nine p53 binding proteins in NPC, which provide important clues to elucidate the mechanism of p53 overexpression and inactivation in NPC.

关 键 词：P53 鼻咽癌 免疫共沉淀 液相色谱-电喷雾串联质谱 相互作用蛋白 蛋白质印迹 

分 类 号：R739.62[医药卫生—肿瘤]