Mechanistic Study Of The Regulation Of Immunomodulatory Drugs And Cereblon On The Ubiquitination And Degradation Of The Downstream Proteins

Background and AimsIntellectual disability occurs in 0.6%of the population in China.The causes of ID are highly heterogeneous,but most of them are induced by genetic mutations.Two cereblon(CRBN)mutants,R419X and C391R,have been reported to cause intellectual disability.CRBN mainly plays a role by forming cullin-4 RING E3 ubiquitin ligase(CRL4)complexes with DNA damage-binding protein 1(DDB1),cullin 4A(Cul4A)and regulator of cullins-1(ROC1).CRBN mediates the ubiquitination and subsequent proteasomal degradation of target proteins.Preliminary experiments found that thalidomide and its structural analogues lenalidomide and pomalidomide can interact with CRBN and regulate CRL4cRBN E3 ubiquitin ligase activity.CRBN can regulate the stability of many proteins,such as Nogo A,chlorine ion channel CLC-1,transcription factor IKZF1,and oncoprotein c-Jun,in the presence or in the abscence of IMiDs.But how CRBN and its mutants regulate these downstream proteins,how IMiDs influence this regulation,and whether the interacting domains in CRBN play roles in this regulation are still unclear.This thesis will try to answer these questions.MethodsHEK293T cells were transfected with CRBN and its mutants and CRBN-interacting proteins to explore the effect of CRBN mutants on CRBN-interacting proteins.HEK293T cells were treated with cycloheximide(CHX)and the degradation rates of CRBN and its mutants were detected by immunoblotting,and the effects of CRBN on the stability of Nogo A were also studied.The stability of CRBN and its mutants and the role of MG 132 in the regulation of proteins by IMiDs were measured by immunoblotting using HEK293T cells.The interacting region between CRBN and its substrate was detected by immunoprecipitation and Western blotting.The effects of IMiDs on the interaction between CRBN and its substrates and the ubiquitination of CRBN interacting proteins were investigated.Overexpression or knockdown of CRBN in Drosophila melanogaster and expression of green fluorescent protein(GFP)in different neurons were observed by confocal microscopy to explore whether CRBN could affect neuronal growth.ResultsThe degradation rate of C391R CRBN in HEK293T cells was slower than that of WT and R419X CRBN.When treated with proteasome inhibitor MG132,compared with WT and R419X,the protein levels of the four cysteine mutants in zinc finger domain increased slightly,indicating that they were relatively stable,of which C391R CRBN was the most stable mutant.WT and R419X CRBN could significantly reduce the protein level of Nogo A,but C391R mutant has little effect on Nogo A protein level.Nogo A protein degraded faster in HEK293T cells co-transfected with C391R CRBN than co-transfected with WT and R419X CRBN.The effect of C391R mutants on Nogo A reduction was weaker than that of WT,and the effect of zinc finger mutants on Nogo A was also reduced.Because of the improper control of the experimental parameters in Drosophila brain dissection experiment,the preliminary experiment cannot result in reliable and scientific conclusion and subsequent experiments are required for further study.Immunoprecipitation and immunoblotting experiments found that the 82-186 region in CRBN binds to CLC-1 and IKZF1.IMiDs can significantly reduce the protein level of IKZF1,but can increase the protein level of MEIS2.The protein levels of both IKZF1 and MEIS2 were decreased upon CRBN expression.IMiDs could decrease CLC-1 protein level,but had no significant influence on c-Jun protein level.CRBN expression reduces CLC-1 and c-Jun protein level.IMiDs do not affect c-Jun ubiquitination,but decrease MEIS2 ubiquitination and increase CLC-1 ubiquitination.IMiDs do not alter the interaction between c-Jun and CRBN,but enhance the interaction between IKZF1 and CRBN and reduce the interaction between CLC-1(MEIS2)and CRBN.Proteasome inhibitor MG132 can increase the protein level of these CRBN-interacting proteins.ConclusionsThe zinc finger structure in CRBN can promote the ubiquitination and proteasome degradation of Nogo A.C391R mutant has no zinc finger structure and thus inhibits Nogo A ubiquitination and leads to the accumulation of Nogo A protein.In the absence of IMiDs,CRBN binds to c-Jun,CLC-1,and MEIS2 to promote their ubiquitination and proteasome degradation,thereby reducing their protein levels.In the presence of IMiDs,IMiDs enhance the binding of CRBN to CLC-1/IKZF1 and reduce the binding of CRBN and MEIS2,thus affecting their ubiquitination and protein level in opposite direction.However,the interaction between CRBN and c-Jun is not affected.This study confirmed that zinc finger structure has an important role on the stability and function of CRBN and its substrates.The interacting region in CRBN is a key factor that affects the ubiquitination and stability of CRBN-interacting proteins mediaited by IMiDs.