PML-NBs Protein Intracellular Defense Mechanism To Study And Clone A New Bax Isomer

Our work firstly focused on the function of PML-NBs protein as a cellular defense system inhibiting site-specific integrase and virus. ΦBT1integrase and adenovirus were used as models of anti-integration and anti-virus respectively.The bacterial phage ΦBT1integrase is a promising tool due to its site-specific transgene character. It enriches the site-specific transgenic tools and provides the possibility for multiple site-specific transgenic manipulations. In our study, we identified that a tetrapeptide433RFAL436in ΦBT1integrase was responsible for interaction between ΦBT1and Daxx. We also demonstrated that over-expression of Daxx could reduce ΦBT1mediated recombination rate in293T cells by using ΦBT1report system. It is the first time to identify a cellular protein interacting with ΦBT1integrase and inhibiting its recombination efficiency. This result might be useful for improving the ΦBT1integrase mediated transgene methods and directing the selection of the target cells for ΦBT1integrase.E1B55K, an adenovirus early protein, was reported to interact with p53and mediate p53degradation. Inactivation of p53facilitates adenovirus DNA replication. Previous report suggested that adeno-associated virus (AAV) could reduce the transforming potential of adenovirus by rescuing p53from E1B55K mediated degradation. However, the details were not clear yet. In our study, we found that a Rep78-p53interaction could be detected in co-IP assay. Following assay revealed that Rep78could inhibit ElB55K-mediated p53cytoplasm recruitment and relieve p53from degradation. Rep68could not bind to p53and had more impaired effection. However, Rep78could not release E1B55K from p53-E1B55K complex. Correspondingly, Rep78could not relieve the transcriptional inactivation of p53induced by E1B55K. Moreover, the distribution of p53sequestered by Rep78in the nuclear changed to a distinct puncta manner similar to a typical PML-NBs localization, indicating that PML-NBs might be involved in the whole process. Our results discovered a new possible mechanism how Rep78and PML-NBs inhibit adenovirus.Proteins of the Bcl-2family are key regulators of programmed cell death and characterized by up to four Bcl-2homology domains (BH1-BH4). The Bcl-2family consists of three major subclasses of both antiapoptotic and proapoptotic proteins. Bax is a mutidomain proapoptotic protein which served as the gateway to apoptosis. In our study, we discovered a new isoform of Bax, namely Bax-ω, which had the same carboxy-terminal but lacked amino acids1to78compared to Bax-β. Bax-ω could trigger strong apoptosis when it was overexpressed in HEK293cells though it lacked BH3domain. The unique carboxy-terminal of Bax-ω and Bax-β may play a role in its proapoptotic activity. mRNA expression analysis revealed that Bax-ω was expressed in a variety of human normal tissues. Our results suggested that Bax-ω, a novel variant of Bax, encodes a protein with a proapoptotic effect and BH3domain may not be the only function unit for inducing apoptosis.