Human Ubiquitin Conjugating Enzyme UBE2T UBE2W And UBE2Z Functional Studies

Ubiquitination means the covalent attachment of the protein ubiquitin (Ub) to other cellular proteins.This kind of posttranslational modification has been implicated in a number of important physiological processes,besides targeting proteins for degradation through the 26S proteasome.Ubiquitin conjugating enzyme(E2) plays an important role in the ubiquitin system,as an essential component.Here we report the research about three human putative ubiquitin conjugating enzyme genes in human liver cDNA library, UBE2T,UBE2W and UBE2Z.Bioinformatics analysis showed that UBE2T,UBE2W and UBE2Z all contain highly conserved intact ubiquitin conjugating enzyme domains(UBCc domains). UBE2T and UBE2Z belong to the classâ…¡ubiquitin conjugating enzyme E2, according to their C-terminal extension,while UBE2W consisting of only one core UBCc domain is a member of the classâ… ubiquitin conjugating enzyme E2. In this study,His-UBE2T/UBE2W/UBE2Z fusion proteins were expressed successfully in E.coli cells and purified with high homogenicity.The conjunction ability of UBE2T and UBE2W with the ubiquitin in the system with UBE1 was testified in vitro,which indicated that they are two novel members of the uhiquitin conjugating enzyme family.Regretfully we did not detect the conjunction of UBE2Z and ubiquitin protein in the system with UBE1.The results of subcellular localization showed that the C-terminal extension of UBE2T was implicated in nuclear import,as well as the catalytic cysteine residue of UBE2W.The Yeast two-hybrid analysis screened out several potential interacting proteins with UBE2W.Two of those proteins RNF8 and RNF167 which contain RING domains,the special mark of RING family ubiquitin ligase E3,were chosen as the E3 ligase candidates of UBE2W.The interactions between UBE2W and RNF8 or RNF167 were detected in both Pulldown and co-Immuneprecipitation assays. UBE2W was supposed to regulate RNF167 protein cellular stability through promotion of its autoubiquitination.Furthermore,overexpression of UBE2W in AD293 cells was found to inhibit the transcriptional activity of p53,the well-kown tumor suppressor protein.The results suggested that UBE2W might be involved in the tumorigenesis.Although the final substrate proteins of UBE2W have not been identified yet and the molecular mechanism of its inhibition to p53 signal pathway is still a mystery,it is sure that UBE2W plays an important role in physiological processes.We will continue our research to further unveil the critical cellular function of UBE2W.