Cereblon Suppresses LPS-induced Inflammatory Response And Apoptosis Through Promoting The Ubiquitination And Degradation Of C-Jun

Background and aims:Cereblon(CRBN)is a substrate receptor for the cullin-4 RING E3 ubiquitin ligase(CRL4)complex and couples the ubiquitin to specific targets for their ubiquitination and subsequent degradation.Oncoprotein c-Jun is a component of the activator protein-1(AP-1)transcription factor complex,which regulates the inflammatory response.One of the mechanisms controlling the activation of AP-1 is to regulate the AP-1 complex components,particularly c-Jun.In our work,using mass spectrometric and biochemical approaches,we found that CRBN overexpression attenuated c-Jun protein level through the ubiquitin-proteasome pathway.c-Jun/AP-1 was the downstream factor in the inflammatory response pathway.Therefore,we ask whether CRBN regulates inflammatory response through regulating c-Jun/AP-1 activation.Although it has been reported that CRBN regulates inflammation through its non-enzymatic function,the relationship between its enzymatic activity and inflammation is not explored.Is the enzymatic activity of CRBN and its associated E3 ligase also involved in mediating inflammation? In this thesis,we perform a series of biochemical experiments to explore the effect of CRBN and its associated E3 ligase on inflammation and apoptosis.Methods:Stable cell lines expressing GFP or CRBN were constructed in HEK293 T cells.Using stable isotope labeling by amino acids in cell culture(SILAC)and quantitative proteomics,we found that c-Jun was significantly down-regulated in the CRBN-expressing HEK293 T cells.Then,we used biological approaches to further confirm the regulation of CRBN on c-Jun protein level.We further used the immunoprecipitation,immunofluorescence,and immunoblotting to examine whether c-Jun and CRBN interact with each other.Meanwhile,biochemical approaches were used to explore the molecular mechanism of c-Jun ubiquitination and stability regulated by CRBN.The effect of CRBN on the activity of c-Jun/AP-1 and the level of m RNA and protein of inflammatory cytokines was examined in inflammatory cells.We further used flow cytometry to study the effect of CRBN on LPS-induced THP-1 cell apoptosis and to explore the role of c-Jun/AP-1 in this regulation.Results:Mass spectrometric analysis discovered that CRBN down-regulated c-Jun protein level.The regulation of CRBN on c-Jun protein level was further verified by biochemical analyses.In HEK293 T cells,CRBN overexpression significantly reduced c-Jun protein level while this effect was rescued by the addition of proteasome inhibitor(MG132).The c-Jun protein half-life was remarkably shortened by CRBN expression in HEK293 T cells treated with protein synthesis inhibitor,cycloheximide(CHX).Meanwhile,using q-PCR experiment,we found that CRBN had no effect on c-Jun m RNA level.Ubiquitination assay showed that CRBN promoted the formation of K48-linked polyubiquitin chains on c-Jun,and thus accelerated its degradation.These results suggested that CRBN regulated c-Jun protein level through the ubiquitin-proteasome pathway but not through the transcription.Dual-luciferase reporter assay showed that CRBN clearly down-regulated AP-1 activity in the model inflammatory cells.We further revealed that CRBN attenuated the production of inflammatory cytokines and this effect was blocked by c-Jun knockdown in the presence of NF-?B inhibitors.Moreover,flow cytometry and Western blotting analyses revealed that CRBN diminished the LPS-induced cell apoptosis and the effect of CRBN expression or knockdown on cell apoptosis could be completely suppressed by c-Jun knockdown by the addition of NF-?B inhibitors.These results demonstrated that the enzymatic function of CRBN and its associated E3 ligase also played important roles in the regulation of inflammatory response and apoptosis by promoting the ubiquitination and degradation of c-Jun.Conclusion:In this work,using mass spectrometric,we discovered the regulation of CRBN on c-Jun protein level.Biochemical experiments verified that CRBN interacted with c-Jun,promoted the c-Jun degradation,and reduced its stability through enhancing its ubiquitination and degradation but did not affect its m RNA level.These effects in turn inhibited AP-1 activity,leading to the suppression of inflammatory response and cell apoptosis induced by LPS.Together,we discovered a new molecular mechanism by which CBRN regulated inflammatory response and cell apoptosis.