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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 .
However, the mechanism of inactivation of p5 3 protein has not been clarified until now.For the purpose of revealing the pathogenesis of nasopharyngeal carcinoma (NPC), p5 3 gene mutation is rare, but most of NPC cells have p5 3 overexpression / The mechanism of inactivation of p5 3 protein in pharyngeal carcinoma was investigated. The co-immunoprecipitation method was used to enrich the p5 3 binding protein of nasopharyngeal carcinoma cell lines HNE1 and HNE2 respectively. SDS PAGE was used to evaluate the immunoprecipitation complex After separation, the p5 3-binding protein bands were excised from the gel and resolved by gel in-house electrospray ionization tandem mass spectrometry (LC ESI MS / MS) to obtain the corresponding peptide sequence tags (PSTs) Nine p5 3 binding proteins were identified in pharyngeal carcinoma cell lines, which are GRP 78 and GRP 75, members of HSP70 family, GRP 94 and Lamin A / C, members of HSP90 family, α actinin 4, Ezrin / Cytovillin, DNA replication permitting agent / minichromosome maintenance 3 protein (MCM3), CD98 / 4F2 heavy chain and protein kinase C (PKC), and co-immunoprecipitation and Western blot analysis One of the p5 3-interacting proteins identified by HNE1 cell protein band 3, HSP78, was validated.First, nine p5 3-binding proteins were identified in nasopharyngeal carcinoma cells. To elucidate the aggregation and inactivation of p5 3 protein in NPC, The mechanism provides an important basis and clues.