{"gene":"USP50","run_date":"2026-04-28T23:00:23","timeline":{"discoveries":[{"year":2010,"finding":"USP50 was identified as a deubiquitinating enzyme involved in the G2/M DNA damage checkpoint; it interacts with HSP90 (identified by TAP-tag/mass spectrometry and yeast two-hybrid), and its depletion causes loss of the HSP90 client Wee1, thereby preventing Wee1 stabilization and allowing CDC25B-driven mitotic entry. USP50 also accumulates in the nucleus in response to DNA damaging agents.","method":"siRNA screen, TAP-tag/mass spectrometry, yeast two-hybrid, siRNA knockdown with G2/M checkpoint bypass assay","journal":"Cell cycle (Georgetown, Tex.)","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (siRNA screen, TAP-MS, Y2H, functional checkpoint assay) in a single study","pmids":["20930503"],"is_preprint":false},{"year":2017,"finding":"USP50 binds to the inflammasome adaptor protein ASC and deubiquitinates K63-linked polyubiquitin chains on ASC, thereby promoting NLRP3 inflammasome activation; USP50 knockdown in THP-1 cells and bone marrow-derived macrophages reduced procaspase-1 cleavage, IL-1β and IL-18 secretion, and ASC speck formation.","method":"Co-immunoprecipitation, deubiquitination assay, siRNA knockdown, inflammasome activation assays (ELISA, caspase-1 cleavage, ASC speck formation)","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, deubiquitination assay, clean KD with defined cellular phenotype across two cell types","pmids":["28094437"],"is_preprint":false},{"year":2017,"finding":"USP50 associates with Ku70 (a DNA-binding protein) as revealed by co-immunoprecipitation and proteomics; overexpression of USP50 reduces Ku70 protein levels by promoting Ku70 degradation without affecting its mRNA, suggesting indirect regulation of Ku70 stability. USP50 protein itself is unstable in a manner independent of proteasomal and lysosomal degradation pathways.","method":"Co-immunoprecipitation, proteomics, overexpression, mRNA/protein level analysis, proteasome/lysosome inhibitor assays","journal":"Journal of investigative medicine","confidence":"Medium","confidence_rationale":"Tier 3 — single Co-IP/proteomics with partial mechanistic follow-up; indirect regulation of Ku70 not fully characterized","pmids":["29101126"],"is_preprint":false},{"year":2022,"finding":"USP50 interacts with CPT1a (carnitine palmitoyl transferase 1a) in LPS-treated macrophages and stabilizes CPT1a by preventing its ubiquitination and degradation; NF-κB was shown by ChIP assay to bind the USP50 promoter, placing USP50 downstream of mitochondrial STAT3/NF-κB signaling. USP50 knockdown decreased CPT1a expression and fatty acid oxidation mediated by mitochondrial STAT3.","method":"Co-immunoprecipitation, siRNA knockdown, metabolic assays (FAO), ChIP assay, cytokine array, mass spectrometry","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP, ChIP, and functional metabolic readout, but single study","pmids":["34976224"],"is_preprint":false},{"year":2023,"finding":"USP50 is a crucial regulator of ACE2 protein levels; it directly interacts with ACE2 and its deubiquitinase activity counteracts K48-linked polyubiquitination of ACE2 at Lys788. Vitamin C blocks the USP50-ACE2 interaction, thereby promoting ACE2 K48-linked polyubiquitination and proteasomal degradation without affecting ACE2 transcription, reducing SARS-CoV-2 infection in vitro and in mice.","method":"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (Lys788), protein stability assay, in vivo mouse model, viral infection assay","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods including mutagenesis, ubiquitination assay, Co-IP, and in vivo validation","pmids":["36876523"],"is_preprint":false},{"year":2023,"finding":"Pristimerin covalently binds the Cys53 site of USP50 (via Michael-acceptor mechanism) and blocks USP50 binding to PYCARD/ASC, thereby reducing USP50-mediated cleavage of K63-linked ubiquitin chains on ASC, which promotes SQSTM1/p62-mediated autophagic degradation of the AIM2-ASC complex.","method":"Drug affinity responsive target stability (DARTS), co-immunoprecipitation, deubiquitination assay, site-directed identification of Cys53, autophagic flux assays","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 — active-site covalent binding identified and validated with multiple assays in a single study","pmids":["37647255"],"is_preprint":false},{"year":2024,"finding":"USP50 is a chromatin-associated protein (identified as catalytically inactive but ubiquitin-binding) required for ongoing DNA replication and fork restart. It supports proper WRN-FEN1 localization at or near stalled replication forks; depletion of USP50 leads to increased association of DNA2 nuclease and RECQL4/RECQL5 helicases with nascent DNA, causing DNA breaks near GC-rich sequences and telomere instability. Suppression of DNA2 or RECQL4/5 rescues USP50-depletion phenotypes.","method":"Chromatin fractionation, iPOND/nascent DNA association, proximity ligation assay, genetic epistasis (siRNA double knockdown), replication fork assays (DNA fiber), comet assay, immunofluorescence","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including genetic epistasis, nascent DNA proteomics, and replication assays, replicated in preprint and published paper","pmids":["39284827"],"is_preprint":false},{"year":2024,"finding":"USP50 interacts with and deubiquitinates ASC to activate the NLRP3 inflammasome in the context of bile acid-induced gastric carcinogenesis, and USP50 deficiency reduces NLRP3 inflammasome activation and HMGB1 release.","method":"Co-immunoprecipitation, deubiquitination assay, USP50 overexpression/knockdown, NLRP3 inflammasome activation assays, in vivo DGR mouse model","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP and deubiquitination assay with in vivo validation, single study","pmids":["38469295"],"is_preprint":false},{"year":2025,"finding":"USP50 directly interacts with NLRP3 and removes K48-linked polyubiquitin chains from NLRP3, preventing its proteasomal degradation and stabilizing NLRP3 protein; USP50 overexpression activates NLRP3 inflammasome and NF-κB signaling, and negatively regulates β-catenin via a NLRP3-GSK3β axis to inhibit EMT and HCC metastasis.","method":"Co-immunoprecipitation, deubiquitination assay (K48-linkage specific), protein stability assay, in vivo overexpression experiments, mechanistic pathway analysis","journal":"Journal of pharmaceutical analysis","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP, linkage-specific deubiquitination assay, in vivo validation; single study","pmids":["41404189"],"is_preprint":false},{"year":2025,"finding":"USP50 promotes NLRP3 stability by removing K48-linked ubiquitin chains from NLRP3, thereby preventing proteasomal degradation; USP50 knockdown enhances NLRP3 K48-linked ubiquitination and degradation, suppresses macrophage pyroptosis, and reduces lung injury in a CLP sepsis mouse model.","method":"Immunoprecipitation, immunoblotting, protein stability assays, flow cytometry (pyroptosis), in vivo CLP mouse model, NLRP3 overexpression rescue","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP, ubiquitination linkage assay, rescue experiment, and in vivo validation; single study","pmids":["40702108"],"is_preprint":false},{"year":2024,"finding":"USP50 is catalytically inactive as a protease but functions as a ubiquitin-binding protein on chromatin; genetic epistasis shows that replication defects in USP50-depleted cells are driven by DNA2, RECQL4, and RECQL5, placing USP50 upstream of these nuclease/helicase activities at stalled forks.","method":"Genetic epistasis (siRNA double knockdown), ubiquitin-binding assays, chromatin fractionation; published as preprint","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — epistasis and biochemical characterization, but preprint version superseded by published paper","pmids":["38260523"],"is_preprint":true}],"current_model":"USP50 is a ubiquitin-specific protease (catalytically inactive as a protease but functional as a ubiquitin-binding deubiquitinase in some contexts) that acts at multiple cellular nodes: it stabilizes Wee1 via HSP90 to enforce the G2/M DNA damage checkpoint; it deubiquitinates ASC (removing K63-linked chains) and NLRP3 (removing K48-linked chains) to regulate inflammasome activation; it stabilizes CPT1a to promote fatty acid oxidation downstream of mitochondrial STAT3/NF-κB; it deubiquitinates ACE2 to prevent its K48-linked ubiquitination and degradation; and at replication forks it suppresses aberrant DNA2 nuclease and RECQL4/5 helicase activity by supporting WRN-FEN1 localization, thereby maintaining replication fidelity."},"narrative":{"teleology":[{"year":2010,"claim":"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","pmids":["20930503"],"confidence":"High","gaps":["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"]},{"year":2017,"claim":"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","pmids":["28094437"],"confidence":"High","gaps":["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"]},{"year":2017,"claim":"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","pmids":["29101126"],"confidence":"Medium","gaps":["Mechanism of USP50-induced Ku70 degradation is undefined","No reciprocal validation of the Ku70 interaction","USP50 protein instability mechanism remains uncharacterized"]},{"year":2022,"claim":"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","pmids":["34976224"],"confidence":"Medium","gaps":["Direct deubiquitination of CPT1a by USP50 not demonstrated with purified components","Ubiquitin chain linkage type on CPT1a not defined","Single study without independent replication"]},{"year":2023,"claim":"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","pmids":["36876523"],"confidence":"High","gaps":["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"]},{"year":2023,"claim":"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","pmids":["37647255"],"confidence":"Medium","gaps":["Whether Cys53 is the catalytic cysteine or an allosteric site not definitively resolved","Selectivity of pristimerin across USP family members not characterized","Single study"]},{"year":2024,"claim":"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","pmids":["39284827"],"confidence":"High","gaps":["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"]},{"year":2024,"claim":"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","pmids":["38469295"],"confidence":"Medium","gaps":["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"]},{"year":2025,"claim":"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","pmids":["41404189","40702108"],"confidence":"Medium","gaps":["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"]},{"year":null,"claim":"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.","evidence":"","pmids":[],"confidence":"Medium","gaps":["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"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[1,4,5,7,8,9]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,4,8,9]}],"localization":[{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[6,10]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,6]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1,5,7,8,9]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[0,6]},{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[6,10]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[3]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[8,9]}],"complexes":[],"partners":["HSP90","ASC","NLRP3","ACE2","CPT1A","WRN","FEN1","XRCC6"],"other_free_text":[]},"mechanistic_narrative":"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]."},"prefetch_data":{"uniprot":{"accession":"Q70EL3","full_name":"Ubiquitin carboxyl-terminal hydrolase 50","aliases":["Ubiquitin-specific peptidase 50"],"length_aa":339,"mass_kda":39.0,"function":"Deubiquitinating enzyme that removes conjugated ubiquitin from specific proteins to regulate different cellular processes. Regulates the inflammasome signaling pathway by deubiquitinating 'Lys-63'-linked polyubiquitination of the PYCARD/ASC adapter protein (PubMed:28094437). Regulates the ubiquitination and stability of the ACE2 protein (PubMed:36876523). Acts as a negative regulator of the G2/M checkpoint pathway, by preventing serine/threonine kinase WEE1 degradation, thereby repressing entry into mitosis following activation of the G2/M DNA damage checkpoint (PubMed:20930503)","subcellular_location":"Cytoplasm; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q70EL3/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/USP50","classification":"Not Classified","n_dependent_lines":11,"n_total_lines":1208,"dependency_fraction":0.009105960264900662},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/USP50","total_profiled":1310},"omim":[{"mim_id":"620563","title":"UBIQUITIN-SPECIFIC PEPTIDASE 50; USP50","url":"https://www.omim.org/entry/620563"},{"mim_id":"606216","title":"METHIONINE SULFOXIDE REDUCTASE B1; MSRB1","url":"https://www.omim.org/entry/606216"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in single","driving_tissues":[{"tissue":"testis","ntpm":1.3}],"url":"https://www.proteinatlas.org/search/USP50"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"Q70EL3","domains":[{"cath_id":"3.90.70.10","chopping":"14-24_42-159_174-333","consensus_level":"medium","plddt":88.1412,"start":14,"end":333}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q70EL3","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q70EL3-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q70EL3-F1-predicted_aligned_error_v6.png","plddt_mean":80.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=USP50","jax_strain_url":"https://www.jax.org/strain/search?query=USP50"},"sequence":{"accession":"Q70EL3","fasta_url":"https://rest.uniprot.org/uniprotkb/Q70EL3.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q70EL3/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q70EL3"}},"corpus_meta":[{"pmid":"34976224","id":"PMC_34976224","title":"Mitochondrial STAT3 exacerbates LPS-induced sepsis by driving CPT1a-mediated fatty acid oxidation.","date":"2022","source":"Theranostics","url":"https://pubmed.ncbi.nlm.nih.gov/34976224","citation_count":123,"is_preprint":false},{"pmid":"20930503","id":"PMC_20930503","title":"A screen for deubiquitinating enzymes involved in the G₂/M checkpoint identifies USP50 as a regulator of HSP90-dependent Wee1 stability.","date":"2010","source":"Cell cycle (Georgetown, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/20930503","citation_count":42,"is_preprint":false},{"pmid":"28094437","id":"PMC_28094437","title":"The deubiquitinating enzyme, ubiquitin-specific peptidase 50, regulates inflammasome activation by targeting the ASC adaptor protein.","date":"2017","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/28094437","citation_count":38,"is_preprint":false},{"pmid":"37647255","id":"PMC_37647255","title":"Pristimerin suppresses AIM2 inflammasome by modulating AIM2-PYCARD/ASC stability via selective autophagy to alleviate tendinopathy.","date":"2023","source":"Autophagy","url":"https://pubmed.ncbi.nlm.nih.gov/37647255","citation_count":30,"is_preprint":false},{"pmid":"37608279","id":"PMC_37608279","title":"ACE2 in chronic disease and COVID-19: gene regulation and post-translational modification.","date":"2023","source":"Journal of biomedical science","url":"https://pubmed.ncbi.nlm.nih.gov/37608279","citation_count":25,"is_preprint":false},{"pmid":"27988371","id":"PMC_27988371","title":"Fine mapping of QT interval regions in global populations refines previously identified QT interval loci and identifies signals unique to African and Hispanic descent populations.","date":"2016","source":"Heart rhythm","url":"https://pubmed.ncbi.nlm.nih.gov/27988371","citation_count":20,"is_preprint":false},{"pmid":"36876523","id":"PMC_36876523","title":"Vitamin C promotes ACE2 degradation and protects against SARS-CoV-2 infection.","date":"2023","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/36876523","citation_count":17,"is_preprint":false},{"pmid":"29101126","id":"PMC_29101126","title":"Induction of deubiquitinating enzyme USP50 during erythropoiesis and its potential role in the regulation of Ku70 stability.","date":"2017","source":"Journal of investigative medicine : the official publication of the American Federation for Clinical Research","url":"https://pubmed.ncbi.nlm.nih.gov/29101126","citation_count":15,"is_preprint":false},{"pmid":"38469295","id":"PMC_38469295","title":"USP50 regulates NLRP3 inflammasome activation in duodenogastric reflux-induced gastric tumorigenesis.","date":"2024","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/38469295","citation_count":14,"is_preprint":false},{"pmid":"26596346","id":"PMC_26596346","title":"ESCRT-Dependent Cell Death in a Caenorhabditis elegans Model of the Lysosomal Storage Disorder Mucolipidosis Type IV.","date":"2015","source":"Genetics","url":"https://pubmed.ncbi.nlm.nih.gov/26596346","citation_count":10,"is_preprint":false},{"pmid":"38203295","id":"PMC_38203295","title":"Genome-Wide Association Analysis of Heat Tolerance in F2 Progeny from the Hybridization between Two Congeneric Oyster Species.","date":"2023","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/38203295","citation_count":6,"is_preprint":false},{"pmid":"39284827","id":"PMC_39284827","title":"USP50 suppresses alternative RecQ helicase use and deleterious DNA2 activity during replication.","date":"2024","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/39284827","citation_count":3,"is_preprint":false},{"pmid":"40702108","id":"PMC_40702108","title":"Interfering with USP50 expression inhibits macrophage pyroptosis in sepsis-induced acute lung injury by degrading NLRP3 protein.","date":"2025","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/40702108","citation_count":1,"is_preprint":false},{"pmid":"38260523","id":"PMC_38260523","title":"USP50 suppresses alternative RecQ helicase use and deleterious DNA2 activity during replication.","date":"2024","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/38260523","citation_count":0,"is_preprint":false},{"pmid":"41404189","id":"PMC_41404189","title":"USP50-mediated NLRP3 deubiquitination enhances NLRP3 inflammasome activation to suppress HCC metastasis.","date":"2025","source":"Journal of pharmaceutical analysis","url":"https://pubmed.ncbi.nlm.nih.gov/41404189","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9152,"output_tokens":3012,"usd":0.036318},"stage2":{"model":"claude-opus-4-6","input_tokens":6362,"output_tokens":3139,"usd":0.165428},"total_usd":0.201746,"stage1_batch_id":"msgbatch_01BparvgRZ9S2LMidvaoo86K","stage2_batch_id":"msgbatch_01NkBfv9SVZWthhS7j6mU6G3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2010,\n      \"finding\": \"USP50 was identified as a deubiquitinating enzyme involved in the G2/M DNA damage checkpoint; it interacts with HSP90 (identified by TAP-tag/mass spectrometry and yeast two-hybrid), and its depletion causes loss of the HSP90 client Wee1, thereby preventing Wee1 stabilization and allowing CDC25B-driven mitotic entry. USP50 also accumulates in the nucleus in response to DNA damaging agents.\",\n      \"method\": \"siRNA screen, TAP-tag/mass spectrometry, yeast two-hybrid, siRNA knockdown with G2/M checkpoint bypass assay\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (siRNA screen, TAP-MS, Y2H, functional checkpoint assay) in a single study\",\n      \"pmids\": [\"20930503\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"USP50 binds to the inflammasome adaptor protein ASC and deubiquitinates K63-linked polyubiquitin chains on ASC, thereby promoting NLRP3 inflammasome activation; USP50 knockdown in THP-1 cells and bone marrow-derived macrophages reduced procaspase-1 cleavage, IL-1β and IL-18 secretion, and ASC speck formation.\",\n      \"method\": \"Co-immunoprecipitation, deubiquitination assay, siRNA knockdown, inflammasome activation assays (ELISA, caspase-1 cleavage, ASC speck formation)\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, deubiquitination assay, clean KD with defined cellular phenotype across two cell types\",\n      \"pmids\": [\"28094437\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"USP50 associates with Ku70 (a DNA-binding protein) as revealed by co-immunoprecipitation and proteomics; overexpression of USP50 reduces Ku70 protein levels by promoting Ku70 degradation without affecting its mRNA, suggesting indirect regulation of Ku70 stability. USP50 protein itself is unstable in a manner independent of proteasomal and lysosomal degradation pathways.\",\n      \"method\": \"Co-immunoprecipitation, proteomics, overexpression, mRNA/protein level analysis, proteasome/lysosome inhibitor assays\",\n      \"journal\": \"Journal of investigative medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP/proteomics with partial mechanistic follow-up; indirect regulation of Ku70 not fully characterized\",\n      \"pmids\": [\"29101126\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP50 interacts with CPT1a (carnitine palmitoyl transferase 1a) in LPS-treated macrophages and stabilizes CPT1a by preventing its ubiquitination and degradation; NF-κB was shown by ChIP assay to bind the USP50 promoter, placing USP50 downstream of mitochondrial STAT3/NF-κB signaling. USP50 knockdown decreased CPT1a expression and fatty acid oxidation mediated by mitochondrial STAT3.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, metabolic assays (FAO), ChIP assay, cytokine array, mass spectrometry\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, ChIP, and functional metabolic readout, but single study\",\n      \"pmids\": [\"34976224\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP50 is a crucial regulator of ACE2 protein levels; it directly interacts with ACE2 and its deubiquitinase activity counteracts K48-linked polyubiquitination of ACE2 at Lys788. Vitamin C blocks the USP50-ACE2 interaction, thereby promoting ACE2 K48-linked polyubiquitination and proteasomal degradation without affecting ACE2 transcription, reducing SARS-CoV-2 infection in vitro and in mice.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (Lys788), protein stability assay, in vivo mouse model, viral infection assay\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods including mutagenesis, ubiquitination assay, Co-IP, and in vivo validation\",\n      \"pmids\": [\"36876523\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Pristimerin covalently binds the Cys53 site of USP50 (via Michael-acceptor mechanism) and blocks USP50 binding to PYCARD/ASC, thereby reducing USP50-mediated cleavage of K63-linked ubiquitin chains on ASC, which promotes SQSTM1/p62-mediated autophagic degradation of the AIM2-ASC complex.\",\n      \"method\": \"Drug affinity responsive target stability (DARTS), co-immunoprecipitation, deubiquitination assay, site-directed identification of Cys53, autophagic flux assays\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — active-site covalent binding identified and validated with multiple assays in a single study\",\n      \"pmids\": [\"37647255\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP50 is a chromatin-associated protein (identified as catalytically inactive but ubiquitin-binding) required for ongoing DNA replication and fork restart. It supports proper WRN-FEN1 localization at or near stalled replication forks; depletion of USP50 leads to increased association of DNA2 nuclease and RECQL4/RECQL5 helicases with nascent DNA, causing DNA breaks near GC-rich sequences and telomere instability. Suppression of DNA2 or RECQL4/5 rescues USP50-depletion phenotypes.\",\n      \"method\": \"Chromatin fractionation, iPOND/nascent DNA association, proximity ligation assay, genetic epistasis (siRNA double knockdown), replication fork assays (DNA fiber), comet assay, immunofluorescence\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including genetic epistasis, nascent DNA proteomics, and replication assays, replicated in preprint and published paper\",\n      \"pmids\": [\"39284827\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP50 interacts with and deubiquitinates ASC to activate the NLRP3 inflammasome in the context of bile acid-induced gastric carcinogenesis, and USP50 deficiency reduces NLRP3 inflammasome activation and HMGB1 release.\",\n      \"method\": \"Co-immunoprecipitation, deubiquitination assay, USP50 overexpression/knockdown, NLRP3 inflammasome activation assays, in vivo DGR mouse model\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP and deubiquitination assay with in vivo validation, single study\",\n      \"pmids\": [\"38469295\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP50 directly interacts with NLRP3 and removes K48-linked polyubiquitin chains from NLRP3, preventing its proteasomal degradation and stabilizing NLRP3 protein; USP50 overexpression activates NLRP3 inflammasome and NF-κB signaling, and negatively regulates β-catenin via a NLRP3-GSK3β axis to inhibit EMT and HCC metastasis.\",\n      \"method\": \"Co-immunoprecipitation, deubiquitination assay (K48-linkage specific), protein stability assay, in vivo overexpression experiments, mechanistic pathway analysis\",\n      \"journal\": \"Journal of pharmaceutical analysis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, linkage-specific deubiquitination assay, in vivo validation; single study\",\n      \"pmids\": [\"41404189\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP50 promotes NLRP3 stability by removing K48-linked ubiquitin chains from NLRP3, thereby preventing proteasomal degradation; USP50 knockdown enhances NLRP3 K48-linked ubiquitination and degradation, suppresses macrophage pyroptosis, and reduces lung injury in a CLP sepsis mouse model.\",\n      \"method\": \"Immunoprecipitation, immunoblotting, protein stability assays, flow cytometry (pyroptosis), in vivo CLP mouse model, NLRP3 overexpression rescue\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, ubiquitination linkage assay, rescue experiment, and in vivo validation; single study\",\n      \"pmids\": [\"40702108\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP50 is catalytically inactive as a protease but functions as a ubiquitin-binding protein on chromatin; genetic epistasis shows that replication defects in USP50-depleted cells are driven by DNA2, RECQL4, and RECQL5, placing USP50 upstream of these nuclease/helicase activities at stalled forks.\",\n      \"method\": \"Genetic epistasis (siRNA double knockdown), ubiquitin-binding assays, chromatin fractionation; published as preprint\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — epistasis and biochemical characterization, but preprint version superseded by published paper\",\n      \"pmids\": [\"38260523\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"USP50 is a ubiquitin-specific protease (catalytically inactive as a protease but functional as a ubiquitin-binding deubiquitinase in some contexts) that acts at multiple cellular nodes: it stabilizes Wee1 via HSP90 to enforce the G2/M DNA damage checkpoint; it deubiquitinates ASC (removing K63-linked chains) and NLRP3 (removing K48-linked chains) to regulate inflammasome activation; it stabilizes CPT1a to promote fatty acid oxidation downstream of mitochondrial STAT3/NF-κB; it deubiquitinates ACE2 to prevent its K48-linked ubiquitination and degradation; and at replication forks it suppresses aberrant DNA2 nuclease and RECQL4/5 helicase activity by supporting WRN-FEN1 localization, thereby maintaining replication fidelity.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"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].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"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.\",\n      \"evidence\": \"siRNA screen, TAP-tag/mass spectrometry, yeast two-hybrid, and G2/M checkpoint bypass assay in human cells\",\n      \"pmids\": [\"20930503\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether USP50 directly deubiquitinates Wee1 or acts indirectly via HSP90 was not resolved\",\n        \"Catalytic activity versus scaffold function was not distinguished\",\n        \"No structural characterization of the USP50–HSP90 interaction\"\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"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.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation, deubiquitination assay, siRNA knockdown in THP-1 cells and bone marrow-derived macrophages\",\n      \"pmids\": [\"28094437\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The precise ubiquitin-chain linkage sites on ASC targeted by USP50 were not mapped\",\n        \"Whether USP50 also acts on other inflammasome components was unknown at this point\",\n        \"Catalytic mechanism of USP50 not biochemically defined\"\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"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.\",\n      \"evidence\": \"Co-immunoprecipitation and proteomics with overexpression in human cells\",\n      \"pmids\": [\"29101126\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanism of USP50-induced Ku70 degradation is undefined\",\n        \"No reciprocal validation of the Ku70 interaction\",\n        \"USP50 protein instability mechanism remains uncharacterized\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"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.\",\n      \"evidence\": \"Co-immunoprecipitation, ChIP assay, siRNA knockdown with fatty acid oxidation measurements in LPS-treated macrophages\",\n      \"pmids\": [\"34976224\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct deubiquitination of CPT1a by USP50 not demonstrated with purified components\",\n        \"Ubiquitin chain linkage type on CPT1a not defined\",\n        \"Single study without independent replication\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"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.\",\n      \"evidence\": \"Co-immunoprecipitation, site-directed mutagenesis (K788), K48-linkage-specific ubiquitination assay, mouse infection model\",\n      \"pmids\": [\"36876523\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Crystal structure of USP50–ACE2 complex not available\",\n        \"Whether vitamin C effect is direct binding to USP50 or indirect\",\n        \"Physiological relevance beyond SARS-CoV-2 context not tested\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"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.\",\n      \"evidence\": \"DARTS assay, co-immunoprecipitation, site-directed mutagenesis of Cys53, autophagic flux assays\",\n      \"pmids\": [\"37647255\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether Cys53 is the catalytic cysteine or an allosteric site not definitively resolved\",\n        \"Selectivity of pristimerin across USP family members not characterized\",\n        \"Single study\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"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.\",\n      \"evidence\": \"iPOND/nascent DNA proteomics, chromatin fractionation, proximity ligation assay, genetic epistasis with DNA2/RECQL4/RECQL5 siRNA, DNA fiber assays in human cells\",\n      \"pmids\": [\"39284827\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Mechanism by which ubiquitin binding by USP50 controls WRN–FEN1 fork localization is unknown\",\n        \"How USP50 catalytic inactivity is reconciled with its apparent DUB activity on ASC, NLRP3, and ACE2 in other studies\",\n        \"No structural model of USP50 ubiquitin-binding domain\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"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.\",\n      \"evidence\": \"Co-immunoprecipitation, deubiquitination assay, USP50 knockdown/overexpression, DGR mouse model\",\n      \"pmids\": [\"38469295\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Causal role of USP50 in human gastric cancer not established\",\n        \"Whether bile acids directly regulate USP50 expression or activity not defined\",\n        \"Single study without independent cohort validation\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"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.\",\n      \"evidence\": \"K48-linkage-specific deubiquitination assay, co-immunoprecipitation, in vivo HCC and CLP sepsis mouse models, rescue experiments\",\n      \"pmids\": [\"41404189\", \"40702108\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Specific NLRP3 lysine residues targeted by USP50 not mapped\",\n        \"Whether USP50 acts on NLRP3 and ASC simultaneously or sequentially is unknown\",\n        \"Reconciliation of USP50 catalytic inactivity (replication studies) with its apparent DUB activity on NLRP3 remains unresolved\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"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.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No reconstituted in vitro DUB assay with purified USP50 alone has been reported\",\n        \"No crystal or cryo-EM structure of USP50 exists\",\n        \"Tissue-specific and context-dependent regulation of USP50 expression and activity is poorly characterized\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [1, 4, 5, 7, 8, 9]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 4, 8, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [6, 10]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 5, 7, 8, 9]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [6, 10]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [8, 9]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"HSP90\",\n      \"ASC\",\n      \"NLRP3\",\n      \"ACE2\",\n      \"CPT1A\",\n      \"WRN\",\n      \"FEN1\",\n      \"XRCC6\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}