噬菌体ФC31整合酶互作细胞蛋白的鉴定与分析

【作者】 徐冠兰；

【导师】 薛京伦；

【作者基本信息】 复旦大学 ， 遗传学， 2008， 博士

【摘要】 噬菌体ФC31整合酶介导的基因转运相对于逆转录病毒载体往往被视为是一种更为安全的方法,因为该整合酶可以将目的基因插入到哺乳动物基因组的有限位点上。为了弄清ФC31整合酶在哺乳动物细胞中的整合机制以及可能存在的风险,研究ФC31整合酶和细胞蛋白的相互作用则是必要的。以pLexA-ФC31为诱饵,用酵母双杂交的方法对ФC31整合酶的互作蛋白进行了筛选。从10~6个阳性克隆中挑选了61个进行测序,结果鉴定到了11个可能的ФC31整合酶互作蛋白,分别是DAXX,TTRAP,SP100,RIP,BRD7,ZNF403,RPL14,UBA2,TARDBP,SEC61G和PRKDC。其中DAXX出现的频率最高(51/61),它和ФC31整合酶的相互作用又使用免疫共沉淀的方法得以确认。缺失突变分析表明DAXX的Fas结合域负责和ФC31整合酶的相互作用。而利用ФC31整合酶的肽段阵列和HEK293细胞裂解液之间的杂交表明ФC31整合酶C端的一个四氨基酸肽段,即451RFGK454,负责和DAXX的相互作用。这四个氨基酸对ФC31整合酶的活性也是必需的,因为缺失这四个氨基酸以后几乎丧失了全部的整合活性。DAXX和ФC31整合酶的功能上的相互作用也得以证实,因为在沉默细胞内源DAXX的情况下,ФC31整合酶介导的整合反应效率有显著的提高。另外,我们也对ФC31整合酶和TTRAP的相互作用进行了深入的研究。首先利用免疫共沉淀的方法进一步确认了二者的相互结合。突变分析表明TTRAP的N端和ФC31整合酶的C端分别负责彼此的结合。在TTRAP沉默的情况下,ФC31整合酶介导整合反应的效率也有明显的提高。有趣的是,类似于DAXX,TTRAP被证实是一个新的PML核体相关蛋白,因为我们发现TTRAP可以和PML共定位,其转录和表达可以受IFN-γ的上调。另外,我们利用酵母配对实验发现了TTRAP可以和PML以及DAXX相互作用。因此,DAXX和TTRAP作为PML核体相关蛋白,可能是作为宿主细胞的防御机制来抑制ФC31整合酶的活性。我们接着对TTRAP的功能进行了初步的分析。首先我们鉴定到TTRAP也可以和P53,CDC20,SP100,噬菌体ФBT1整合酶以及HIV整合酶相互作用,其次TTRAP可能是P53转录的调节因子,对P53转录活性有促进作用。另外,我们表达和纯化了GST-TTRAP的融合蛋白并对其酶活性进行分析,结果我们并没有发现TTRAP具有生物信息学所推测的磷酸二酯酶活性。我们也初步分析了ФC31整合酶对细胞信号通路的影响,结果表明ФC31整合酶对TNFαNIL-1激活的NFκB活性具有显著的抑制作用,然而这个过程是否通过DAXX或TTRAP起作用的还有待证实。更多还原

【Abstract】 PhageφC31 integrase-mediated gene delivery is believed to be safer than using retroviral vectors since,the protein confines its insertion of the target gene to a limited number of sites in mammalian genomes.To understand the mechanism involved inφC31 integrase-mediated integration and potential risk of the integrase,it is essential to study interactions between integrase and cellular proteins.Using pLexA-φC31 as bait,we employed yeast two hybridization assay to screen theφC31 integrase interacting proteins.61 positives were isolated from independent clones and analyzed, the result suggested that 11 potentialφC31 integrase-interacting proteins were identified,known as DAXX,TTRAP,SP100,RIP,BRD7,ZNF403,RPL14,UBA2, TARDBP,SEC61G and PRKDC.DAXX was identified to interact withφC31 integrase by the highest frequency(51/61),which was also confirmed by co-immunoprecipitation assay.Deletion analysis revealed that the Fas-binding domain of DAXX is also the region forφC31 integrase binding.Hybridization between aφC31 integrase peptide array and an HEK293 cell extract revealed that a tetramer, 451RFGK454,in the C-terminus ofφC31 integrase is responsible for the interaction with DAXX.This tetramer is also necessary forφC31 integrase activity as removal of this tetramer resulted in a complete loss of integration activity.The functional interaction between DAXX andφC31 integrase was also investigated.Knocking down endogenous DAXX resulted in significantly increased integration efficiency. Furthermore,the interaction between TTRAP andφC31 integrase was further studied. At first,the interaction between TTRAP andφC31 integrase was confirmed by co-immunoprecipitation assay.And mutation analysis revealed that N-terminus of TTRAP and C-terminus ofφC31 integrase was responsible for binding.TheφC31 integrase-mediated integration efficiency was also significantly improved when endogenous TTRAP was silenced.Interestingly,like DAXX,TTRAP was also demonstrated to be associated PML nuclear bodies,since we found that TTRAP could be co-localized with PML and induced by IFN-γ.In addition,interaction between TTRAP and PML/DAXX was also confirmed by yeast-mating assay.Hence,given PML NBs-associated proteins,DAXX and TTRAP could play a role as host defense mechanism to inhibitφC31 integrase activity.Subsequently,we performed some preliminary functional analysis of TTRAP.It was identified that TTRAP could interact with P53,CDC20,SP100,phageφBT1 integrase and HIV integrase.TTRAP might also serve as an activator of P53 transcription.Furthermore.GST-TTRAP was expressed and purified,whose enzyme activity was also analyzed.However,we didn’t identify the phosphodiesterase activity as predicted by bioinformatics analysis.At last, the effect ofφC31 integrase on cellular signaling was also investigated.The result suggested thatφC31 integrase could inhibit the NFκB activity-stimulated by TNFαand IL-1.However,it will be under further investigation whether the phenotype was correlated with DAXX or TTRAP.更多还原