The Phage §¶c31 Integrase Interaction The Identification And Analysis Of Cell Protein

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.