Ubiquitin-conjugating Enzyme UBE2O Regulates Biological Rhythm By Promoting BMAL1 Ubiquitination And Degradation

Background and aimsCircadian clock is an important internal mechanism for bodies to synchronize with the environment.Its disorders seriously affect the human health and survival.The central circadian clock is regulated by a Transcriptional/Translational Feedback Loop(TTFL)wich consists of a positive limb containing two core clock proteins,circadian locomoter output cycles protein kaput(CLOCK)and aryl hydrocarbon receptor nuclear translocator-like protein 1(ARNTL or BMAL1),and a negative limb containing Period(PER)and Cryptochrome(CRY).Many post-translational modifications are involved in the regulation of TTFL.Among them,ubiquitination plays important roles in the regulation of protein stability and cell signal transduction.The role of ubiquitination in the regulation of circadian clock has been widely reported.However,this kind of study was mianly focused on the regulation of the negative components of the TTFL.Ubiquitin protein ligase E3A(UBE3A)is the only enzyme in the ubiquitin conjugation system that has been reported to enhance BMAL1 ubiquitination and degradation.This limits our understanding of the biological significance of the ubiquitination on the regulation of biological rhythm.Therefore,in this work,we use a quantitative proteomic approach to systematically identify proteins associated with BMAL1 and to elucidate the underlying mechanism by which a BMAL1-interacting protein,UBE2 O,regulates biological rhythm.MethodsWe transfected FLAG-BMAL1 and pcDNA3.1,respectively,to HEK293 T cells and obtained the whole cell lysates.FLAG M2 beads were used to immunoprecipitate BMAL1 and its interacting proteins.The purified samples were validated by immunoblotting,separated by SDS-PAGE,and digested with trypsin to obtain peptides for mass spectrometry identification of BMAL1-interacting proteins.We further focused on the proteins that were associated with the ubiquitin conjugation system.Pulldown assay,immunostaining,and immunofluorescence were used to further validate their interaction.A series of biochemical experiments were utilized to explore the regulation of a ubiquitin conjugating enzyme,UBE2 O,on BMAL1 function.Dual-luciferase report assay and qPCR were applied to detect the effect of UBE2 O on BMAL1 transcriptional activity.At last,we examined the circadian rhythm after knocking down UBE2 O in the Per2::luciferase U2 OS cell line.LumiCycle experiment was used to study the regulation of this ubiquitin-conjugating enzyme on the circadian rhythm.ResultsUBE2O was the only E2 conjugating enzyme identified from LC-MS/MS analysis of BMAL1-interacting proteins.We found that UBE2 O indeed interacted with BMAL1.BMAL1 protein level was significantly reduced by UBE2 O overexpression and increased by UBE2 O knockdown in HEK293 T and N2 a cells.We further foud that the reduction of BMAL1 protein could be rescued by the addition of a proteasome inhibitor MG132.Expectedly,we found that UBE2 O reduced the transcription of Per1,Cry1,Nr1d1,and Rora but had little effect on the transcription of Bmal1 itself.The results suggested that UBE2 O affected the BMAL1 protein level through its ubiquitination and degradation but not through its transcription.Circadian rhythm measurement in U2 OS cells showed that the amputide of the circadian rhythm was significantly increased but the period had little changed.These results demonstrated that UBE2 O played an important role in the regulation of circadian behavior in cells.ConclusionIn this work,using mass spectrometric and bioinformatic analyses,we identified an E2 ubiquitin conjugating enzyme,UBE2 O,which potentially interacts with the core circadian protein BMAL1.Biochemical experiments discovered that UBE2 O regulated the BMAL1 protein level in two cell lines.Further studies demonstrated that UBE2 O enhanced BMAL1 ubiquitination and reduced its stability,leading to the reduction of its transcriptional activity.Lumi Cycle studies discovered that the amplitude of circadian clock was regulated upon UBE2 O knockdown.Together,we disovered a new regulator that modulates the circadian behavior through enhancing the ubiquitination and proteasomal degradation of the core clock protein BMAL1.