The deubiquitinase USP7 stabilizes Maf proteins to promote myeloma cell survival

Abstract

<jats:p>The Maf proteins, including c-Maf, MafA, and MafB, are critical transcription factors in myelomagenesis. Previous studies demonstrated that Maf proteins are processed by the ubiquitin–proteasome pathway, but the mechanisms remain elusive. This study applied MS to identify MafB ubiquitination-associated proteins and found that the ubiquitin-specific protease USP7 was present in the MafB interactome. Moreover, USP7 also interacted with c-Maf and MafA and blocked their polyubiquitination and degradation. Consistently, knockdown of USP7 resulted in Maf protein degradation along with increased polyubiquitination levels. The action of USP7 thus promoted Maf transcriptional activity as evidenced by luciferase assays and by the up-regulation of the expression of Maf-modulated genes. Furthermore, USP7 was up-regulated in myeloma cells, and it was negatively associated with the survival of myeloma patients. USP7 promoted myeloma cell survival, and when it was inhibited by its specific inhibitor P5091, myeloma cell lines underwent apoptosis. These results therefore demonstrated that USP7 is a deubiquitinase of Maf proteins and promotes MM cell survival in association with Maf stability. Given the significance of USP7 and Maf proteins in myeloma genesis, targeting the USP7/Maf axle is a potential strategy to the precision therapy of MM.</jats:p>

Anti-bacterial and anti-viral nanchangmycin displays anti-myeloma activity by targeting Otub1 and c-Maf

“…After being treated with Nam for 24 h, cells were harvested to prepare whole cell lysates, and the IP assay was conducted as described previously 17 . After IP, proteins were re-suspended in RIPA buffer and used for an IB assay.…”

“…MM cells were treated with Nam for 24–72 h before being subjected to the MTT assay as described previously 17 . Apoptosis was measured by flow cytometry to determine Annexin V and propidium iodide (MultiSciences Biotech Co., Ltd, Hangzhou, China) staining as described previously 17 .…”

“…The Otub1 cDNA generated by using the following primers: 5′-CCGCTCGAGATGGCGGCGGAGGAACCTCAG-3′ (forward) and 5′-CGCGGATCCTCTTTGTAGAGGATATCG-3′ (reverse) was inserted into pLVX-AcGFP lentiviral vector (Clontech Laboratories) within the Xho I and BamH I sites. To generate lentiviral particles, HEK293T cells at 80% confluence were transfected with pLVX-AcGFP-Otub1 (10 µg) as well as plasmids for VSV-G envelope glycoprotein (3.5 µg), Rev (2.5 µg), and ΔR8.74 packaging proteins (6.5 µg) using Lipofectamine2000 (Invitrogen) as described as previously 17 . Forty hours later, the lentiviral particle-enriched supernatants were harvested, filtered and stored frozen at −80 °C for further use.…”

“…c-Maf undergoes degradation in proteasomes after the K48-linked polyubiquitination mediated by the ubiquitin ligase HERC4 13 , the ubiquitin-conjugating enzyme UBE2O 14 or the TMEPAI/NEDD4 ligase 15 . Moreover, c-Maf ubiquitination is a dynamic process, deubiquitinases USP5 and USP7 could prevent c-Maf from ubiquitination therefore stabilizing c-Maf and promoting MM cell survival and proliferation 16 , 17 . The ubiquitin thioesterase Otub1 is recently found to stabilize c-Maf by preventing its K48-linked polyubiquitination 18 .…”

Ubiquitin–proteasome system (UPS) as a target for anticancer treatment

“…USP7 promotes MM cell survival by deubiquitinating Maf proteins, including c-Maf, MafA, and MafB, which are critical transcription factors in myelomagenesis. Indeed, USP7 is up-regulated in MM cell line and MM patients with high USP7 expression exhibit lower survival rate (He et al 2020 ).…”