Source Pml-nb - Related Proteins In Cells Ttrap Study On Cytological Features

The gene delivery mediated by phage PhiC31imtegrase is believed to be safer, compared with the traditional gene-therapy method.In order to elucidate its recombination mechanism and potential risk while applied in mammalian cells, it is essential to understand the interaction between PhiC31and cellular proteins.Here we constructed and employed plasmid pLexA-ΦC31 as bait to sceen the human fetal brain cDNA library and established TTRAP as a novel cellular protein associated withΦC31.The subsequent yeast-mating assay and Co-immunoprecipitation further verified this interaction and revealed that N-terminus of TTRAP and C-terminus ofΦC31 integrase was responsible for binding respectively. The integration efficiency ofΦC31 could be upregulated while endogenous TTRAP was knocked-down by siRNAs;In Hela cells transient expression ofΦC31 could inhibit the transcriptional activity of NFκB stimulated by IL-1,in a dose-dependent manner.Moreover,TTRAP has been identified as a novel PML-NB associated protein for its particular intracellular localization. Just as several other NB-associated components, TTRAP could also interact with p53.The binding region located within p53 was exactly its DNA-binding domain, which suggested that TTRAP could possibly play a role in regulating the transcriptional activity of p53.In H1299 cells transiently expression of TTRAP could obviously enhance the expression of reporter gene whose promoter constituted a p53-responsive element. In our research we have identified TTRAP as a transcriptional co-activator of p53.The long-term expression of TTRAP could effectively inhibit the colony formation of tumor cells.As the transient expression of TTRAP could neither induce apoptosis nor cell-cycle arrest, we proposed that this phenomenon was related with the phosphodiesterase activity of TTRAP. We constructed two TTRAP mutants (E152A,D262A whose enzyme activity have been impaired)and repeated the colony formation assay. It is found that these mutants could hardly function as the wild-type TTRAP and this suggested that the phosphodiesterase activity of TTRAP could play a significant role both in maintaining genomic stability and repressing the colony formation of tumor cells.