USP50

USP50 is a ubiquitin-specific protease family member that operates as both a deubiquitinase and a ubiquitin-binding scaffold to regulate innate immune signaling, DNA replication integrity, and the DNA damage checkpoint. USP50 activates the NLRP3 inflammasome by removing K63-linked polyubiquitin chains from the adaptor ASC and K48-linked chains from NLRP3 itself, thereby stabilizing both components and promoting caspase-1 cleavage, IL-1β secretion, and pyroptosis (PMID:28094437, PMID:41404189, PMID:40702108). At replication forks, USP50 is catalytically inactive as a protease but binds ubiquitin on chromatin and supports WRN–FEN1 localization, preventing aberrant DNA2 nuclease and RECQL4/5 helicase activity that otherwise causes DNA breaks at GC-rich regions and telomere instability (PMID:39284827). USP50 additionally stabilizes the HSP90 client Wee1 to enforce the G2/M DNA damage checkpoint (PMID:20930503), deubiquitinates ACE2 at K788 to prevent its K48-linked ubiquitination and proteasomal degradation (PMID:36876523), and stabilizes CPT1a downstream of NF-κB signaling to promote fatty acid oxidation in macrophages (PMID:34976224).

1. 2010 High

   Establishing USP50's first known cellular role: the question of how the G2/M DNA damage checkpoint is maintained was addressed by showing that USP50 interacts with HSP90 and stabilizes the checkpoint kinase Wee1, preventing premature mitotic entry after DNA damage.

   Evidence siRNA screen, TAP-tag/mass spectrometry, yeast two-hybrid, and G2/M checkpoint bypass assay in human cells

   PMID:20930503

   Open questions at the time

   • Whether USP50 directly deubiquitinates Wee1 or acts indirectly via HSP90 was not resolved
   • Catalytic activity versus scaffold function was not distinguished
   • No structural characterization of the USP50–HSP90 interaction
2. 2017 High

   Expanding USP50 into innate immunity: the question of which deubiquitinase activates the NLRP3 inflammasome via ASC was answered by showing USP50 binds ASC and removes K63-linked polyubiquitin chains, promoting ASC speck formation, caspase-1 cleavage, and IL-1β/IL-18 secretion.

   Evidence Reciprocal co-immunoprecipitation, deubiquitination assay, siRNA knockdown in THP-1 cells and bone marrow-derived macrophages

   PMID:28094437

   Open questions at the time

   • The precise ubiquitin-chain linkage sites on ASC targeted by USP50 were not mapped
   • Whether USP50 also acts on other inflammasome components was unknown at this point
   • Catalytic mechanism of USP50 not biochemically defined
3. 2017 Medium

   The question of whether USP50 has additional nuclear interaction partners was addressed by identifying an association with the DNA repair protein Ku70, though USP50 overexpression paradoxically promoted Ku70 degradation, suggesting indirect regulation.

   Evidence Co-immunoprecipitation and proteomics with overexpression in human cells

   PMID:29101126

   Open questions at the time

   • Mechanism of USP50-induced Ku70 degradation is undefined
   • No reciprocal validation of the Ku70 interaction
   • USP50 protein instability mechanism remains uncharacterized
4. 2022 Medium

   Linking USP50 to metabolic reprogramming: the question of how mitochondrial STAT3/NF-κB signaling sustains fatty acid oxidation in macrophages was answered by showing NF-κB transcriptionally induces USP50, which then stabilizes CPT1a by preventing its ubiquitination and degradation.

   Evidence Co-immunoprecipitation, ChIP assay, siRNA knockdown with fatty acid oxidation measurements in LPS-treated macrophages

   PMID:34976224

   Open questions at the time

   • Direct deubiquitination of CPT1a by USP50 not demonstrated with purified components
   • Ubiquitin chain linkage type on CPT1a not defined
   • Single study without independent replication
5. 2023 High

   Defining a pharmacologically targetable substrate: USP50 was shown to directly deubiquitinate ACE2 at K788 (removing K48-linked chains), stabilizing ACE2 and facilitating SARS-CoV-2 entry; vitamin C disrupts the USP50–ACE2 interaction and promotes ACE2 degradation, reducing viral infection in vivo.

   Evidence Co-immunoprecipitation, site-directed mutagenesis (K788), K48-linkage-specific ubiquitination assay, mouse infection model

   PMID:36876523

   Open questions at the time

   • Crystal structure of USP50–ACE2 complex not available
   • Whether vitamin C effect is direct binding to USP50 or indirect
   • Physiological relevance beyond SARS-CoV-2 context not tested
6. 2023 Medium

   Identifying the USP50 active-site residue engaged by a covalent inhibitor: pristimerin binds Cys53 of USP50, blocking its interaction with ASC and rerouting the AIM2–ASC complex toward SQSTM1/p62-mediated autophagic degradation, establishing Cys53 as functionally critical.

   Evidence DARTS assay, co-immunoprecipitation, site-directed mutagenesis of Cys53, autophagic flux assays

   PMID:37647255

   Open questions at the time

   • Whether Cys53 is the catalytic cysteine or an allosteric site not definitively resolved
   • Selectivity of pristimerin across USP family members not characterized
   • Single study
7. 2024 High

   Resolving USP50's catalytic status and replication function: USP50 was shown to be catalytically inactive as a protease but to bind ubiquitin on chromatin, where it supports WRN–FEN1 localization at replication forks and suppresses aberrant DNA2 nuclease and RECQL4/5 helicase activity, preventing DNA breaks at GC-rich regions and telomere instability.

   Evidence iPOND/nascent DNA proteomics, chromatin fractionation, proximity ligation assay, genetic epistasis with DNA2/RECQL4/RECQL5 siRNA, DNA fiber assays in human cells

   PMID:39284827

   Open questions at the time

   • Mechanism by which ubiquitin binding by USP50 controls WRN–FEN1 fork localization is unknown
   • How USP50 catalytic inactivity is reconciled with its apparent DUB activity on ASC, NLRP3, and ACE2 in other studies
   • No structural model of USP50 ubiquitin-binding domain
8. 2024 Medium

   Extending the ASC–USP50 axis to gastric carcinogenesis: USP50-mediated deubiquitination of ASC activates the NLRP3 inflammasome in bile acid-induced gastric cancer, with USP50 deficiency reducing HMGB1 release in vivo.

   Evidence Co-immunoprecipitation, deubiquitination assay, USP50 knockdown/overexpression, DGR mouse model

   PMID:38469295

   Open questions at the time

   • Causal role of USP50 in human gastric cancer not established
   • Whether bile acids directly regulate USP50 expression or activity not defined
   • Single study without independent cohort validation
9. 2025 Medium

   Establishing dual inflammasome substrate specificity: USP50 directly deubiquitinates NLRP3 (removing K48-linked chains) to prevent its proteasomal degradation, with functional consequences for NF-κB activation, β-catenin regulation in HCC, and macrophage pyroptosis in sepsis models.

   Evidence K48-linkage-specific deubiquitination assay, co-immunoprecipitation, in vivo HCC and CLP sepsis mouse models, rescue experiments

   PMID:40702108 PMID:41404189

   Open questions at the time

   • Specific NLRP3 lysine residues targeted by USP50 not mapped
   • Whether USP50 acts on NLRP3 and ASC simultaneously or sequentially is unknown
   • Reconciliation of USP50 catalytic inactivity (replication studies) with its apparent DUB activity on NLRP3 remains unresolved

• A central unresolved question is whether USP50 possesses intrinsic deubiquitinase catalytic activity—its protease-dead status at replication forks contrasts with multiple reports of direct deubiquitination of ASC, NLRP3, and ACE2—and whether context-dependent cofactors or distinct biochemical mechanisms explain this discrepancy.
• No reconstituted in vitro DUB assay with purified USP50 alone has been reported
• No crystal or cryo-EM structure of USP50 exists
• Tissue-specific and context-dependent regulation of USP50 expression and activity is poorly characterized