The E3 Ligase Hrd1 Regulates Circadian Clock By The Ubiquitination And Degradation Of BMAL1

Aim:To observe the degradation of core clock protein BMAL1 induced by E3 ligase Hrdl and on this basis to explore the change of the inherent rhythm in cells.Methods:We overexpressed or knocked down the expression of Hrdl in Human Embryonic Kidney 293 cells(HEK 293)or Mouse Neuroblastoma cells(N2a),and then detected the changes of BMAL1 protein levels by western blot;In order to analyze protein stability,we treated the transfected cells with CHX(100 ug/ml)and then collected them at different time points and analyzed the changes of BMAL1 protein by western blot;To explore the way that Hrdl influenced BMAL1 protein,cells were co-transfected with FLAG-Hrdl and GFP-N3-BMAL1,after 48 h we treated the transfected cells with proteasome inhibitor MG 132 for 12 h,then they were harvested for immunoprecipitation experiments to detect whether Hrdl and BMAL1 can be co-immunoprecipitated with each other,and the changes of the ubiquitin levels of BMAL1;Further,in HEK 293 cell line we respectively co-transfected FLAG-Hrdl,GFP-N3-BMAL1 and HA-Ub(WT)or its mutant types,cells were treated with MG 132 for 12 h after transfected,then harvested for immunoprecipitation experiments;we overexpressed Hrdl in N2a cells,24 h later we collected them and used quantitative real-time PCR(qRT-PCR)to estimate the mRNA levels of Perl and Dbp;in N2a cell line,cells were transfected with FLAG-Hrdl for 24 h and exposed to 50%horse serum diluted in DMEM for synchronization study,16 h later cells were harvested at different time points for mRNA analysis.Result:We found that both of overexpression and deletion of Hrd1 would significantly influence the protein level of BMAL1.Using CHX(a kind of protein synthesis inhibitor)to treat Hrd1-transfected cells,we found that the half-life of BMAL1 protein was shorten,which suggested us Hrdl promotes the degradation of BMAL1.Using in vitro or in vivo immunoprecipitation assays and immunoblot,we found Hrdl could co-purify with BMAL1,further,the results of immunoprecipitation also showed that E3 ligase Hrdl could increase the ubiquitination of BMAL1.Using K48 mutant ubiquitin,Hrdl was unable to increase the ubiquitination of BMAL1.Using q-PCR,we found that overexpression of Hrdl significantly suppressed the expression of Dbp and Per1.Ulteriorly,in a whole oscillation period,overexpression of Hrdl caused a greatly reduction in the amplitude of Per1 mRNA level.Conclusion:Our results demonstrate the E3 ligase,Hrdl,is an endogenous regulator of circadian clock by the modulation of BMAL1.Here,BMAL1 is a substrate of Hrd1,which promotes ubiquitination of BMAL1 protein and mediates its degradation through UPS.