鼻咽癌细胞中p53相互作用蛋白质的分离和鉴定

鼻咽癌中p53基因突变罕见，但绝大部分鼻咽癌中存在p53蛋白过表达/聚集且功能失活.然而，到目前为止p53蛋白失活的机制仍然不清楚.为揭示鼻咽癌中p53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p53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p53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（peptide sequence tags, PST）,通过搜索数据库在鼻咽癌细胞系中鉴定了9个p53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 （Lamin A/C）、α-actinin 4、Ezrin/Cytovillin、DNA复制准许因子/MCM3蛋白(DNA replication licensing factor/minichromosome maintenance 3 protein, MCM3)、CD98/4F2 heavy chain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p53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p53结合蛋白，为阐明鼻咽癌中p53蛋白聚集及失活的机制提供了重要依据和线索.

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

Although p53 gene mutation is a rare event in human nasopharyngeal carcinoma (NPC), over-expressed/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 over-expression and inactivation in NPC.