OTUB1 Inhibits The Polyubiquitination Of C-Maf And Increases Multiple Myeloma Cell Viability

Objectivesc-Maf,originally identified as a homolog of the avian viral oncogene v-maf,belongs to the family of AP-1 basic leucine zipper transcription factors.It forms homo-or hetero-dimers to modulate the transcription of its target genes through binding to the consensus Maf responsive element(MARE).It has reported that c-Maf overexpression is not only associated with chromosome translocation but also associated with the protein ubiquitination system.The aim of the present study is to identify the specific deubiquitinating enzyme that decreases c-Maf polyubiquitination and stabilizes c-Maf.We will also explore the underlying mechanisms and the effects of OTUB1 on c-Maf activity and MM cell proliferation.Methods(1)An affinity purification coupled with tandem mass spectrometry(AP-MS)was used to identify c-Maf-related proteins,followed by co-IP to verify the MS findings.(2)The distribution of c-Maf and OTUB1 protein in HEK293 T cells was analyzed by a confocal microscope to verify the co-localization.(3)HEK293T cells with overexpressed c-Maf and OTUB1 were treated with CHX to detect the half-life and stability of c-Maf.(4)Site-directed mutagenesis was used to generate OTUB1 mutants with replaced amino acid residues to explore the key functional amino acid in OTUB1 deubiquitinating activity.(5)To find out the ubiquitination site(s)on c-Maf modulated by OTUB1,14 c-Maf mutants with lysine to arginine(K > R)were co-transfected with OTUB1,followed by WB to determine c-Maf protein stability.(6)The reporter of Maf-responsive element-driving luciferase was constructed.The effect of OTUB1 on c-Maf transcriptional activity was analyzed by luciferase activity assay.(7)OTUB1 was packed into lentivirus that were used to infect MM cells to examine the effects of OTUB1 on endogenous c-Maf as well as the viability of MM cells.Results(1)The AP-MS identified individual proteins interacting with c-Maf from the c-Maf co-immunoprecipitated complex,of which OTUB1 was singled out.OTUB1 is a deubiquitinase and 6 unique OTUB1 peptides were identified and the coverage was up to 26%,which suggested that OTUB1 might interact with c-Maf.(2)HEK293T cells were co-transfected with OTUB1 and c-Maf plasmids and Western blot found that OTUB1 interacted with c-Maf.More importantly,OTUB1 lentivirus infection into MM cells showed that OTUB1 interacted with endogenous c-Maf.Moreover,HEK293 T cells were co-transfected with c-Maf and OTUB1 for 24 hrs followed by confocal microscope analysis and it showed that OTUB1 and c-Maf was co-localized in the nuclei.(3)OTUB1 decreased the polyubiquitination level of c-Maf in both HEK293 T cells and MM cells.When OTUB1 was knocked down by shRNA,c-Maf ubiquitination level could be partly recovered.Notably,although MafA and MafB belong to the Maf family with c-Maf,OTUB1 decreased the ubiquitination level of MafA but had no effect on MafB.(4)HEK293T cells were co-transfected with OTUB1 and c-Maf plasmids for 24 hrs,followed by WB and it showed that OTUB1 upregulated c-Maf protein.Moreover,when MM cell line RPMI-8226 was infected with lentiviral OTUB1,c-Maf in RPMI-8226 cells were also increased.When cells were treated with CHX,an inhibitor of protein synthesis de novo,OTUB1 markedly delayed c-Maf degradation.These results showed that OTUB1 stabilized c-Maf via the deubiquitination manner.(5)The K to R mutant c-Maf plasmids were co-transfected with OTUB1,respectively,for 24 hrs,the Western blot assays showed that OTUB1 increased the K85 R,K283R and K347 R c-Maf proteins.When several potent amino acid residues in OTUB1 were mutated followed in co-transfected with c-Maf and Western blot assays.The results showed that K71 was essential for OTUB1 to prevent c-Maf ubiquitination.(6)The luciferase assay showed that OTUB1 upregulated transcriptional activity of c-Maf and MafA in association with the stability of c-Maf and MafA.(7)When MM cell lines were infected with shOTUB1,c-Maf was decreased.In contrast,when cells were infected with OTUB1 lentivirus,c-Maf was stabilized.More importantly,OTUB1 promoted MM cell proliferation.ConclusionsThe AP/MS and subsequent ubiquitination and protein stability analyses revealed that OTUB1 binds to c-Maf and inhibits the polyubiquitination of c-Maf.OTUB1 stabilizes c-Maf and upregulates its transcriptional activity.Specifically OTUB1 precludes the ubiquitination at K85,K283 and K347 of c-Maf.Moreover,K71 is essential for OTUB1 deubiquitinating activity.OTUB1 increases the endogenous c-Maf level and increases MM cell proliferation.The study collectively suggests that OTUB1 modulates c-Maf stability and activity.Targeting at OTUB1 could be a novel strategy for MM treatment.