The Molecular Mechanisms Of The Ubiquitin Ligase HERC4 In Modulating The Maf Transcription Factors

Multiple myeloma(MM)is an incurable malignant blood disease.Both the morbidity and mortality of MM is listed as the second highest among the hematological malignancies.The pathophysiology of MM is featured with the overexpression/overactivation of several key genes,of which are the Maf transcription factors include c-Maf,MafA,and MafB.These Maf genes are found in chromosomal translocations in MM cells,in addition,their expression and function are modulated by post-translational modifications,including ubiquitination.Ubiquitination is a biological process mediated by a series of enzymes,including the ubiquitin-activating enzymes(El),ubiquitin-conjugating enzymes(E2),ubiquitin ligases(E3),and deubiquitinases(DUBs).But the specific enzymes midiating Maf ubiquitination are not yet reported.Our previous studies have found the E3 ligase HERC4,a HERC family protein,was present in the immunoprecipitates of c-Maf analyzed by the LC/MS/MS strategy.But how HERC4 modulates the Maf proteins is yet to know.In the present thesis,the detailed mechanisms involved in HERC4 modulating Maf proteins were investigated.Part 1 The molecular mechanisms of c-Maf ubiquitination mediated by the ubiquitin ligase HERC4Aims:The purpose of this section was aimed(1)to identify whether HERC4 is the E3 ligase of c-Maf,(2)to dissect the detailed molecular mechanisms of c-Maf ubquitination mediated by HERC4,and(3)to explore the roles of HERC4 in MM pathogenesis.Methods:1.Co-immunoprecipitation(Co-IP),immunofluorescence(IF),and Western blotting(WB)methods were applied to verify the interaction of HERC4 and c-Maf.2.The Co-IP,ubiquitination assay in vitro,and WB methods were used to measure the ubiquitination level of c-Maf in the presence of HERC4.3.The stability and half-lifes of c-Maf,MafA,and MafB mediated by HERC4 were evaluated by WB.4.The WB and IP methods were applied to identify the key lysine residues for c-Maf ubiquitination mediated by HERC4.5.The effects of the deubiquitinase USP5 on c-Maf ubiquitination and degradation regulated by HERC4 were analyzed by IP and WB.6.The expression profile of HERC4 was analyzed through the Gene Expression Omnibus(GEO)datasets(http://www.ncbi.nlm.nih.gov/gds).And the expression of HERC4 was evaluated in MM cell lines,solid tumors,and primary MM samples by RT-PCR.7.MM cell lines RPMI-8226 and MM1.S were infected with HERC4 lentivirus and the cell viability was measured by MTT.RPMI-8226 cells expressing HERC4 were inoculated into SCID mice subcutaneously and the tumor volume and c-Maf protein were analyzed.Results:1.IP and WB showed that HERC4 interacted with c-Maf in HEK293T cells.To confirm this interaction,lentivirus-mediated HERC4 was infected into MM1.S cells,subsequent IP and WB analyses revealed that HERC4 was present in the c-Maf immunoprecipitated complex.The IF analysis revealed that HERC4 was mainly expressed in the cytoplasm while c-Maf was localized to nuclei,however,HERC4 was relocated to the nucleus when cells were co-expressed with c-Maf.2.HERC4 induced the K48-linked ubiquitination which resulting in the degradation of c-Maf,but not MafA and MafB.Knockdown of the expression of HERC4 blocked the ubiquitination of c-Maf mediated by HERC4.5.HERC4 induced the degradation of c-Maf except the mutant c-Maf-K85R,c-Maf-K297R,and c-Maf-K345R.HERC4 failed to mediate the ubiquitination of c-Maf mutants,including c-Maf-K85R and c-Maf-K297R.5.USP5 was identified by LC/MS/MS method,and the IP results showed USP5 abolished the ubiquitination and degradation of c-Maf induced by HERC4 in vivo and in vitro.6.HERC4 was steadily decreased during the development of MM as demonstrated from a series of DNA microarray datasets generated from healthy donors and MM patients with monoclonal gammopathy of undetermined significance(MGUS),smoldering MM(SMM),or MM.7.When representative MM cell lines RPMI-8226 and MM1.S were infected with lentiviral HERC4,proliferation of both cell lines were significantly inhibited in a dose-dependent manner.Notably,restoration of HERC4 markedly delayed MM tumor growth in nude mice,and this changes were associated with increased HERC4 expression and concurrent downregulation of c-Maf as well as Cyclin D2,the downstream gene of c-Maf.Summary:HERC4 is the ubiquitin ligase of c-Maf,and it induces K48-linked polyubiquitination at the lysine 85 and lysine 297 of c-Maf.HERC4 induces c-Maf instability via the proteasomal degradation.USP5 as the deubiquitinase abolishes the role of HERC4 in mediating c-Maf ubiquitination.HERC4 is steadily decreased during the progress of MM development.HERC4 inhibits MM cell proliferation and delays MM tumor growth.Induction of HERC4 to degrade c-Maf is a potential strategy for MM treatment.Part 2 The molecular mechanisms of MafA ubiquitination and stability regulated by the ubiquitin ligase HERC4Aims:The above studies have clearly demonstrated that HERC4 induces the ubiquitination and degradation of c-Maf,but it upregulates the protein expression of MafA.The aim of this section was to dissect how HERC4 upregulates MafA.stability.Methods:1.WB was used to detect the stability and half-life of MafA.2.The HERC4 truncates were constructed and the interaction of MafA and HERC4 was explored through the Co-IP/B method.3.The IP and ubiquitination assay in vitro methods were used to examine the ubiquitination of MafA induced by HERC4.4.The IP method was performed to assess the role of the ubiquitin conjugating UBE20 in the ubiquitination of MafA induced by HERC4.Identification of the key domain(s)of UBE20 was performed by constructing UBE20 truncates followed by IP analysis.5.The IP method was used to detect the role of GSK3? in the ubiquitination of MafA mediated by HERC4.6.The IP method was carried out to analyze the phosphorylation of MafA mediated by HERC4.7.The pGL4-MARE-luciferase plasmid was constructed and the luciferase assay method was used to analyze the transcriptional activity of MafA mediated by HERC4.Results:1.HERC4 increased the protein of MafA in a dose-dependent manner.When the de novo synthesis of MafA was inhibited by cycloheximide(CHX),the half-life of MafA was not markedly changed by HERC4 although total MafA level was raised.2.HERC4 selectively interacted with MafA but not MafB,another member in the Maf family,which was consistent with the protein changes by HERC4.More specifically,the fragment between 376-728 amino acids was a key domain for HERC4 interaction with MafA because when this fragment was deleted,HERC4 failed to interact with MafA.3.HERC4 induced the K63-linked polyubiquitination of MafA in vivo and in vitro,when a K63R ubiquitin plasmid was cotransfected with MafA and HERC4,no polyUbiquitination on MafA.was detected.Notably,HERC4 knockdown markedly reduced the ubiquitination of MafA.4.The ubiquitin conjugating domain but not the coiled-coil domain of UBE20 was responsible for the interaction of UBE20 with HERC4,while the fragment between 376-728 amino acids and the HECT domain in HERC4 were critical for HERC4 interaction with UBE20.UBE20 inhibited the ubiquitination of MafA mediated by HERC4.5.GSK3? induced the phosphorylation of HERC4 and enhanced the interaction of HERC4 and MafA,which strengthening the ubiquitination of MafA mediated by HERC4.HERC4 inhibited the phosphorylation of MafA and GSK3p enhanced the role ofHERC4.6.The MARE-luciferase activity was marked increased when cotransfected with MafA or c-Maf.When cotransfected HERC4 with MafA or c-Maf,the MARE-luciferase activity was decreased markedly.And in the presence of GSK3p,HERC4 has a greater inhibitive effect on the transcriptional activity of MafA.Summary;HERC4 upregulates the MafPA protein level.HERC4 interacts with and induces the K63-linked polyubiquitination of MafA.HERC4 inhibits the transcriptional activity of MafA.UBE20 negatively regulates the ubiquitination of MafA induced by HERC4.GSK3? enhances HERC4 action in the process of ubiquitination of MafA.ConclusionsAlthough it has been proposed as a ubiquitin ligase since 2005,there are no ubiquitination substrates reported for HERC4.The present study demonstrated that HERC4 mediates the ubiquitination of both c-Maf and MafA,two critical transcription factors in MM pathophysiology,however,the detailed mechanisms are quite different.HERC4 mediated the K48-linked ubiquitination on c-Maf but K63-linked ubiquitination on MafA.HERC4 induces the degradation of c-Maf but has no effects on the half-life of MafA.HERC4 inhibits the transcriptional activity of c-Maf and MafA.The deubiquitinase USP5 partly abolishes HERC4-mediated c-Maf ubiquitination while the ubiquitin-conjugating enzyme UBE20 negatively regulates the ubiquitination of MafA induced by HERC4.HERC4 is steadily decreased during the progress of MM development.Restoration of HERC4 inhibits MM cell proliferation and delays MM tumor growth.These findings will help further understanding the molecular pathophysiology of MM and targeting the Maf ubiquitination is a potential strategy for the treatment of a subset of MM patients expressing high Maf proteins.