Identification Of USP5 As A Deubiquitinase Of The Transcription Factor C-Maf

Objectivec-Maf is highly expressed in multiple myeloma(MM) and is one of the main pathogenic proteins of MM. It can be degraded through ubiquitin-proteasome system,therefore looking for enzymes mediating c-Maf ubiquitination and deubiquitination has great significance for the treatment of MM. But these enzymes have not been studied clearly. In the present study, affinity purification coupled with tandem mass spectrometry(AP-MS) strategy is performed to find and identify the deubiquitinase(DUB) USP5 and to further study its effect on c-Maf ubiquitination and protein stability. This study will provide a theoretical basis for in depth understanding of c-Maf stability and MM therapy.Methods1. HEK293 T cells were transfected with c-Maf plasmids, then AP-MS was applied to find the proteins interacted with c-Maf and these results were verified with Scaffold software to discover DUBs among them.2. To verify the interaction of USP5 and c-Maf, individual plasmids were co-transfected into HEK293 T cells followed by immunoprecipitation(IP) with specific antibodies; Ubiquitin(Ub) was overexpressed to detect the function of USP5 in mediating c-Maf deubiquitination.3. USP5 and c-Maf plasmids were co-transfected into HEK293 T cells and Western blot(WB) was applied to detect USP5 dose- and time-effect on c-Maf protein stability;After c-Maf and USP5 were overexpressed for 36 hr, HEK293 T cells were treated with Cycloheximide(CHX) for different time and then cell lysates were detected by WB to analyze c-Maf half time.4. c-Maf belongs to the large Maf family including MafA and MafB, all of which are related to MM. To understand whether USP5 is specific in mediating c-Maf stability, WB and CHX were performed to examine MafA and MafB stability.5. c-Maf modulates the transcription of several genes including cyclin D2(CCND2).To study the effect of USP5 on c-Maf transcriptional activity, pGL4-CCND2-Luci plasmids were cloned and transfected with c-Maf and USP5 plasmids for 48 hr. Luciferase activity were measured using Bright-Glo substrate system.6. There are 14 lysine residues in the c-Maf protein. To find out which ubiquitination site(s) on c-Maf could be modulated by USP5, we constructed 14 c-Maf mutants encoding specific lysine residues and co-transfected with USP5 plasmids, respectively. WB was applied to analyze the probable lysine residue(s).7. USP5 has 5 domains. To find out which domain(s) is important for USP5, we cloned individual domains and examined their effects on c-Maf stability.8. Overexpression of c-Maf is frequently found in MM and can induce poor prognosis. Previous studies demonstrate that c-Maf protein is highly expressed in most MM cell lines. Therefore, we evaluated USP5 expression profile in MM and other cancer cell lines using WB.Results1. The AP-MS assay identified 104 specific proteins in the c-Maf IP which suggested that these proteins might interact with c-Maf, including USP5. There were 21 specific USP5 peptides identified by MS, covered 32% of the total amino acids in USP5.2. When USP5 and c-Maf plasmids were co-transfected, IP assay confirmed that there was a strong interaction between USP5 and c-Maf. Moreover, USP5 decreased the c-Maf ubiquitination level.3. c-Maf expression was increased by USP5 in a concentration- and time-dependent manner. USP5 also prolonged the half time of c-Maf. Notably, USP5 also increased MafB protein stability but did not show effects on MafA protein. This suggested that USP5 may have some specific effect on c-Maf.4. USP5 increased the protein level of c-Maf and its transcriptional activity in terms of CCND2 promoter.5. USP5 increased the stability of c-Maf mutant K347 and K308 in a concentration-dependent manner.6. c-Maf stability was increased by the UBA1-UBA2 domains of USP5, but was not affected by other specific domains.7. USP5 was expressed higher in MM than other tumor cell lines.ConclusionIn this study, we identified that the deubiquitinase USP5 interacts with c-Maf and decreases its polyubiquitination level, thus stabilizing its protein stability, prolonging its half time and increasing its transcriptional activity. Further studies show that USP5 mainly removed ubiquitin chains at the K347 and K308 of c-Maf and the UBA1-UBA2 domains are crucial in USP5. Meanwhile, compared with leukemia and other cancer cell lines,USP5 was expressed higher in MM, suggesting its potential role in regulating MM pathophysiology.