OTUD6B

OTUD6B is a catalytically active OTU-family deubiquitinase that controls the stability of diverse substrates to regulate cell proliferation, proteasome function, and stress responses (PMID:21267069, PMID:28343629). Its catalytic cysteine is required for ubiquitin chain hydrolysis, and enforced expression arrests cells in G1 by downregulating cyclin D2 (PMID:21267069). Biallelic loss-of-function variants in humans impair incorporation of 19S subunits into the 26S proteasome and cause accumulation of ubiquitin-protein conjugates, defining a Mendelian disorder with congenital heart defects recapitulated in Otud6b knockout mice (PMID:28343629). A recurring mechanistic theme is catalysis-dependent removal of K48-linked polyubiquitin to stabilize pro-proliferative substrates: OTUD6B deubiquitinates LIN28B in a G1/S-restricted manner to drive MYC expression and S-phase entry (PMID:36059274, PMID:37634410), and stabilizes RIPK1, PTK2/FAK, and FXR1 to promote tumor proliferation and metastasis through downstream STAT3 and ERK signaling (PMID:38880876, PMID:39192576, PMID:42056075). Beyond catalysis, OTUD6B can act as a scaffold: it stabilizes pVHL independently of its enzymatic activity by coupling pVHL with elongin B/C into a more stable CBC-VHL E3 ligase complex, thereby restraining HIF accumulation (PMID:32328410, PMID:35110537). OTUD6B also localizes to centrosomes and the mitotic spindle, where it protects the kinesin KIFC1/HSET from premature mitotic degradation to enable bipolar spindle formation in centrosome-amplified cancer cells (PMID:39789388), and to stress granules, where it associates with the ATPase VCP/p97 to modulate granule assembly and clearance (PMID:41651815). Two splice isoforms exert opposing control over the 48S preinitiation complex and protein synthesis downstream of mTORC1 (PMID:27864334).

1. 2011 Medium

   Established that OTUD6B is an enzymatically active deubiquitinase and linked its activity to cell-cycle control, defining the core biochemical identity of the protein.

   Evidence In vitro deubiquitinating assay with catalytic Cys mutant plus flow-cytometry cell-cycle analysis in Ba/F3 cells

   PMID:21267069

   Open questions at the time

   • No physiological substrate identified at this stage
   • Mechanism linking DUB activity to cyclin D2 downregulation unresolved
2. 2016 Medium

   Connected OTUD6B to translational control, showing its two splice isoforms oppositely regulate the 48S preinitiation complex and protein synthesis downstream of mTORC1.

   Evidence Co-IP with initiation complex components, [35S]-methionine and BrdU incorporation, isoform overexpression/knockdown in NSCLC cells

   PMID:27864334

   Open questions at the time

   • Direct deubiquitination target within the initiation machinery not defined
   • Structural basis of isoform-specific opposing activity unknown
3. 2017 High

   Defined OTUD6B as a human disease gene and tied it to proteasome biogenesis, showing loss-of-function impairs 26S assembly.

   Evidence Proteasome activity assays and native PAGE in patient PBMCs, ubiquitin-conjugate immunoblot, and Otud6b knockout mouse model

   PMID:28343629

   Open questions at the time

   • Molecular mechanism by which OTUD6B promotes 19S incorporation not established
   • Substrate responsible for proteasome assembly phenotype unidentified
4. 2020 High

   Revealed a catalysis-independent scaffolding function, showing OTUD6B stabilizes pVHL by assembling the CBC-VHL E3 complex, expanding its mechanistic repertoire beyond deubiquitination.

   Evidence Reciprocal Co-IP, ubiquitylation assays, catalytic mutant analysis, HIF-1α reporter and ChIP in HCC cells

   PMID:32328410

   Open questions at the time

   • Structural detail of how OTUD6B couples pVHL to elongin B/C not resolved
   • Relationship between scaffolding and catalytic substrate pools unclear
5. 2021 Medium

   Identified a direct OTUD6B–OTUB1 DUB-DUB interaction and, in zebrafish, an antiviral signaling role, broadening the partner and pathway landscape.

   Evidence GFP-Trap Co-IP and AlphaScreen for OTUB1; Co-IP, K63-specific ubiquitination assays and zebrafish knockout for IRF3/IRF7 regulation

   PMID:33421002 PMID:34183367

   Open questions at the time

   • Functional consequence of OTUB1 interaction not established
   • Zebrafish ortholog function (negative IFN regulation) conflicts with later human data
6. 2022 High

   Defined a cell-cycle-restricted substrate, LIN28B, establishing how OTUD6B drives MYC-dependent S-phase entry, and confirmed pVHL stabilization across tumor-derived mutants.

   Evidence DUB siRNA screen, reciprocal Co-IP, ubiquitination assays, cell-cycle synchronization and xenografts for LIN28B; Co-IP and migration assays across pVHL mutants in ccRCC

   PMID:35110537 PMID:36059274

   Open questions at the time

   • Mechanism restricting LIN28B deubiquitination to G1/S not defined
   • Determinants of the pVHL I151 dependence for binding unresolved
7. 2023 Medium

   Resolved a species-distinct antiviral role and expanded the catalytic substrate set, showing human OTUD6B cleaves K33-linked chains on IRF3 and K48 chains on β-TrCP and LIN28B (with MCTS1 as a coactivator).

   Evidence Linkage- and site-specific ubiquitination assays with IRF3 Lys315 mutagenesis and in vivo mouse virus challenge; Co-IP and ATRA studies for β-TrCP; MCTS1 Co-IP and K48 assays for LIN28B

   PMID:37038094 PMID:37634410 PMID:37650650

   Open questions at the time

   • Reason for opposing human vs zebrafish IFN regulation mechanistically unexplained
   • How OTUD6B selects among K33 and K48 linkages on different substrates unknown
8. 2024 Medium

   Extended the substrate catalog to RIPK1 and PTK2/FAK in cancer and implicated OTUD6B in a Calpain-1/HIF-1α loop, linking it to proliferation, metastasis, and vascular pathology.

   Evidence IP-MS, Co-IP and ubiquitination assays with xenografts for RIPK1 and PTK2; proteomics and siRNA in a mouse PAH model with Calpain-1 KO

   PMID:38878112 PMID:38880876 PMID:39192576

   Open questions at the time

   • Direct deubiquitination of Calpain-1 not demonstrated
   • Whether RIPK1 and PTK2 stabilization share a common recognition mechanism unknown
9. 2025 High

   Localized OTUD6B to centrosomes and the mitotic spindle and to stress granules, establishing roles in spindle integrity via KIFC1 and in condensate dynamics via VCP.

   Evidence siRNA screen, Co-IP, ubiquitination assays, CRISPR KO, catalytic-mutant and KIFC1 overexpression rescue with imaging; proximity proteomics and Co-IP with VCP plus SG dynamics assays; Co-IP and G-quadruplex assays for DDX5

   PMID:39789388 PMID:40651961 PMID:41651815

   Open questions at the time

   • How OTUD6B is recruited to centrosomes vs stress granules not defined
   • Whether the proteasome-assembly defect connects to centrosome/spindle phenotypes unexplored
10. 2026 Medium

    Defined FXR1 as a domain-specific K48 substrate, linking OTUD6B to an FXR1–MEK2–ERK feed-forward oncogenic loop in colorectal liver metastasis.

    Evidence Domain-mapping Co-IP, K48-specific ubiquitination assays, MEK2 mRNA stability assays, catalytic mutant and in vivo liver metastasis models

    PMID:42056075

    Open questions at the time

    • Generalizability of the N-terminal/KH-domain recognition mode to other substrates untested
    • Direct structural basis of OTUD6B–FXR1 binding unresolved

• How OTUD6B achieves substrate and linkage selectivity (K48 vs K33), and how its catalytic, scaffolding, translational, and condensate functions are coordinated within a single protein, remains unresolved.
• No structural model of substrate or linkage recognition
• Mechanism integrating proteasome-assembly, spindle, and stress-granule roles unknown
• Determinants switching between catalytic and scaffold-only modes undefined