The Function Of UBE2O In Mediating Ubiquitination Of C-Maf Protein

Part 1 Identification of important components in c-Maf ubiquitination and the discovery of E2Objectives: To screen the important components which participated in c-Maf ubiquitination, and to find ubiquitin conjugating enzyme(E2) involved in c-Maf ubiquitination.Methods:(1) The proteins interacted with c-Maf were pulled down by HA beads through immunoprecipitation method.(2) Protein samples were separated by SDS-PAGE gel electrophoresis and the gel was stained with a high sensitive coomassie stain.(3) Those proteins were identified by LC-MS/MS, and the proteins which related with the ubiquitination of c-Maf were analyzed using the KEGG protocol.(4) Finally, the ubiquitin conjugating enzyme(E2) was comfirmed by IP/WB.Results:(1) Five important components related with ubiquitination of c-Maf were found by immunoprecipitation(IP) and LC-MS/MS technology. They were Uba1(E1), UBE2O(E2), HUWE1(E3), UBR5(E3), HERC4(E3).(2) Five unique peptides of UBE2 O were identified by LC-MS/MS.(3) IP/WB comfirmed that UBE2 O interacted with c-Maf and mediated c-Maf ubiquitination.Conclusion: c-Maf could be degraded through the ubiquitin-proteasome pathway, and UBE2 O may be a potential ubiquitin conjugating enzyme of c-Maf.Part 2 The function of UBE2 O in mediating c-Maf polyubiquitinationObjectives: To study the effects of UBE2 O in mediating polyubiquitination of c-Maf protein.Methods:(1) The effect of UBE2 O on Maf family protein stability was detected by Western Blot and CHX chase assay.(2) The effect of UBE2 O on c-Maf transcriptional activity was analyzed by luciferase activity assay.(3) The role of over-expressed UBE2 O on endogenous c-Maf expression and apoptosis of multiple myeloma cells was detected through the mediation of lentivirus and Western blot.Results:(1) UBE2 O reduced expression and attenuated stability of c-Maf and Maf B, but had no significant effects on Maf A expression.(2) UBE2 O reduced c-Maf transcriptional activity on its downstream target gene CCND2 promoter.(3) Over-expressed UBE2 O promoted endogenous c-Maf degradation and induced apoptosis of multiple myeloma cells.Conclusion: UBE2 O is a potential ubiquitin conjugating enzyme of c-Maf.