A Study Of The Mechanisms By Which Neddylation Facilitates Andovirus E4orf6-mediated P53 Degradation

Objective:To investigate the mechanism of adenovirus E4orf6-CRL5 E3 ligase-mediated p53 degradation,and to provide a theoretical basis for the development of specific drugs targeting human adenovirus.Method:Cellular immunofluorescence staining and Western blots were used to examine the effects of adenovirus protein E4orf6 on the cell localization of Cullin5 and p53,and the effects of neddylation on E4orf6 for both exogenous and endogenous Cullin5 proteins.Real-time PCR and Western blot were used to examine the effects of E4orf6 on the expression of genes related to neddylation,and detected the effects of E4orf6 on the expression levels and transcription levels of SENP8 and p53 proteins.We generated E2 binding enzymes UBE2 M and UBE2 F stable knockdown HEK293 T cell lines using shRNA targeting UBE2 M and UBE2 F.The effects of UBE2 M and UBE2 F and the mutants on E4orf6-induced p53 degradation were detected using Western blots.We used Western blots to examine the effects of MLN4924 on the neddylation of E4orf6-dependent Cullin5 and the effects on E4orf6-induced p53 degradation.Results:E4orf6 induces nuclear localization of Cullin5 and enhances neddylation of Cullin5;SENP8 inhibits E4orf6-induced p53 degradation,and is down-regulated by E4orf6;Cullin5-mediated E4orf6 induced p53 degradation requires UBE2M;MLN4924 inhibits the neddylation of E4orf6-dependent Cullin5 and inhibits the degradation of p53.Conclusion:E4orf6 enhances the neddylation of Cullin5 and induces the degradation of p53 with the participation of UBE2 M.SENP8 inhibits Cullin5-mediated E4orf6 degraded p53 and is down-regulated by E4orf6,which is a natural limited factor for E4orf6-induced p53 degradation.MLN4924 can inhibit the neddylation of Cullin5,thereby inhibiting the degradation of E4orf6-induced p53.