The Mechanism Of The Deubiquitinase USP7 Enhancing The Stability Of The Transcription Factor MafB

ObjectivesThe transcription factor MafB is frequently overexpressed in multiple tumors including multiple myeloma(MM),and it is associated with malignancy and poor prognosis of MM.The stability of MafB protein is modulated by the ubiquitin proteasome pathway(UPP),a system involved in multiple enzymes,including ubiquitin-activating enzymes,ubiquitin-conjugating enzymes,and ubiquitin ligases.In addition,there are a class of deubiquitinases(DUBs)that reduce the ubiquitination of target proteins.However,the enzymes mediate MafB ubiquitination are not yet known.The aim of the present study is to identify specific DUB(s)preventing MafB ubiquitination and to dissect the underlying molecular mechanism and its significance in MM.Methods1.The affinity purification coupled mass spectrometry(AP-MS)was applied to isolate and purify MafB associated proteins.A Mass database was analyzed.2.The interaction between USP7 and MafB and MafB polyubiquitination was verified by Co-Immunoprecipitation(Co-IP)and Western blot(WB).3.The effects of USP7 on the stability of MafB and its family members MafA and c-Maf were detected by WB method.The half-life of Maf proteins in the presence of USP7 was evaluated after treatment of cycloheximide(CHX),an inhibitor of protein synthesis de novo.4.The truncated USP7 plasmids were constructed using molecular cloning method.The interactions between specific truncation of USP7 and MafB were determined by IP and WB methods.5.Specific recognition element of MafB was constructed by molecular cloning method.6.The effects of USP7 on MafB transcriptional activities were detected by luciferase assay.7.The expression profiles in a panel of cancer cell lines and primary MM samples were detected by reverse transcription-polymerase chain reaction(RT-PCR).8.P5091,a small molecule inhibitor of USP7,was used to treat cells for determine the effects of inhibited USP7 on MafB stability and ubiquitination.Results1.The AP-MS analyses on the MafB interacting proteins identified several key ubiquitination enzymes including the ligase TRIM26 and the deubiquitinase USP7.The coverage rate of MafB-and USP7-specific peptides was 50% and 12%,respectively.The interaction between USP7 and MafB was confirmed in HEK293 T cells co-transfected with the USP7 and MafB plasmids,as well as in HeLa cells which expressed endogenous MafB.2.When a USP7 plasmid was introduced into HEK293 T cells transfected with MafB or HeLa cells expressing endogenous MafB,the ubiquitination levels of both cells were markedly decreased.3.USP7 significantly stabilized MafB as well as c-Maf and MafA in a time and concentration-dependent manner.USP7 markedly prolonged the half-lives of MafB and c-Maf.4.To evaluate the effects of USP7 on MafB function,a construct with the luciferase as a reporter under the Maf responsive element(MARE)was made and was co-transfected into HEK293 T cells along with MafB and USP7.The results showed that USP7 significantly increased the luciferase expression,indicating that USP7 promoted the transcription activity of MafB.5.A series of USP7 truncated constructs were made to be co-transfected with MafB,followed by IP and WB.The results showed that the TRAF and HUBL domains of USP7 were sufficient to bind to MafB protein.Moreover,the TRAF domain significantly reduced the ubiquitination level of MafB protein.6.The protein levels of USP7 and MafB were highly consistent in a panel of cancer cell lines.The higher of USP7,the higher of the MafB proteins.The mRNA expressions of USP7 and MafB in MM patients were also higher than healthy controls.The results suggest that there was a correlation between the expression of USP7 and MafB.7.MM cell line RPMI-8226 was treated with P5091,a reported USP7 inhibitor.WB analysis showed that P5091 led to a higher level of MafB ubiquitination.P5091 decreased MafB at the protein level and suppressed the transcriptional activity of MafB.ConclusionsIn the present study,we applied AP-MS,WB and IP methods to identify USP7 as a deubiquitinase of MafB.USP7 interacts with MafB through the TRAF and HUBL domains,thus preventing MafB polyubiquitination and degradation via the proteasomes.Inhibition of USP7 leads to MafB instability and downregulates transcriptional activity.Targeting the USP7/MafB might be a novel strategy for the treatment of MM.