鼻咽癌下调新基因 NOR1相互作用蛋白的筛选和鉴定

Screen and Identification of The Protein-protein Interactors of NOR1, a Novel Gene Down-regulated in Nasopharyngeal Carcinoma

NOR1基因是新的鼻咽癌相关基因,该基因在鼻咽癌细胞系HNE1和鼻咽癌组织中表达下调.在鼻咽癌细胞HNE1中恢复NOR1基因表达抑制了鼻咽癌细胞的生长和增殖能力.为了探讨NOR1基因的生物学功能,以NOR1基因为诱饵运用酵母双杂交技术在人胎脑文库中筛选其交互作用蛋白,挑选阳性克隆,进行DNA序列分析和同源检索,阳性克隆编码7个不同的蛋白质,其中一个阳性克隆编码线粒体ATP合成酶亚基OSCP蛋白.瞬时转染pCMV-myc-NOR1质粒进入鼻咽癌5-8F细胞,通过密度梯度离心法分离线粒体蛋白,Western blot检测表明myc-NOR1蛋白分布于线粒体与胞浆.免疫荧光检测表明在鼻咽正常上皮细胞NP69中内源性NOR1蛋白与线粒体存在明显共定位.随后采用特异性酵母双杂交、免疫荧光共定位、免疫共沉淀技术证实了NOR1与OSCP在线粒体内存在交互作用.提示,NOR1是一个新的线粒体蛋白,可能通过结合OSCP蛋白调控细胞能量代谢,为深入探讨其功能提供了重要线索.

NOR1 is a novel nasopharyngeal carcinoma（NPC） associated gene. It is significantly down-regulated in NPC cell line HNE1 and spontaneous NPC biopsies. Over-expression of NOR1 in HNE1 cell line is effective to inhibit HNE1 cell growth and proliferation. To establish a clearer picture of what the functions of NOR1 might be and to identify cellular proteins that might bind to NOR1, a yeast two-hybrid screen was performed to search for proteins that interact with NOR1. 10 positive clones which encoded for 7 polypeptides were successfully isolated. Among these 7 candidate interaction proteins, one candidate was mitochondria ATP synthase subunit OSCP. NPC cell line 5-8F cells were transfected with pCMV-myc-NOR1 plasmids, then the mitochondrial proteins and cytoplasmic protein were isolated by cellular subftration and subjected to Western blot assay. The data showed that myc-NOR1 protein distributes in mitochondrion and cytoplasm. Immunofluoresence assay also showed the endogenous NOR1 protein co-localized with mitochondrion in human normal nasopharyngeal epithelial cells NP69, which indicate that NORI is a novel mitochondrial protein. The interaction between NOR1 and OSCP was confirmed by specific yeast two-hybrid assay, immunofluoresence co-localization and co-immunoprecipitation assay. These primary work suggests NOR1 may be involved in energe metabolism.