{"gene":"USP32","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":2010,"finding":"USP32 is an active deubiquitinating enzyme with conserved catalytic residues (asparagine, cysteine, histidine, and aspartic acid domains); GFP-fused USP32 localizes to the Golgi apparatus as determined by fluorescent protection assay and BODIPY-TR staining; stable silencing of USP32 causes significant decrease in cell proliferation and migration.","method":"Fluorescent protection assay, BODIPY-TR staining, stable siRNA silencing with proliferation/migration readouts","journal":"Mammalian genome","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — direct localization experiment with functional consequence (Golgi), confirmed catalytic domain conservation; single lab, two orthogonal localization methods","pmids":["20549504"],"is_preprint":false},{"year":2019,"finding":"USP32 deubiquitylates the small GTPase Rab7, a master regulator of late endosome (LE) biology. Loss of USP32 inhibits LE transport and recycling of LE cargos, causing dispersion and swelling of late endosomes. Mechanistically, the LE transport effector RILP prefers ubiquitylation-deficient Rab7, while retromer-mediated LE recycling benefits from an intact cycle of Rab7 ubiquitylation, suggesting reversible ubiquitylation switches Rab7 between its various functions.","method":"SILAC-based ubiquitome profiling, USP32 knockdown/knockout with late endosome morphology and cargo recycling readouts, mechanistic studies with RILP and retromer","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — SILAC ubiquitome substrate identification combined with functional loss-of-function assays and mechanistic dissection of effector preferences; single lab but multiple orthogonal methods","pmids":["30926795"],"is_preprint":false},{"year":2020,"finding":"USP32 silencing decreases SMAD2 expression in gastric cancer cells, and restoration of SMAD2 rescues the proliferation, motility, and chemoresistance phenotypes caused by USP32 knockdown, placing SMAD2 downstream of USP32 in this pathway.","method":"siRNA knockdown of USP32, western blot for SMAD2, in vitro and in vivo proliferation/migration/chemoresistance assays with rescue experiments","journal":"International journal of biological sciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — functional epistasis via knockdown and rescue but no direct deubiquitination of SMAD2 demonstrated; single lab, single mechanism approach","pmids":["32226309"],"is_preprint":false},{"year":2021,"finding":"USP32 promotes ER-associated degradation (ERAD) of the solute carrier protein SLC35F2, thereby destabilizing SLC35F2 protein levels. Loss of USP32 stabilizes SLC35F2, increasing uptake of the anticancer compound YM155. This identifies USP32 as a determinant of SLC35F2 protein stability.","method":"CRISPR-Cas9 genome-scale USP knockout screen for YM155 resistance, targeted validation with in vitro and in vivo SLC35F2 protein stability assays","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR genome-scale screen plus targeted validation in vitro and in vivo; single lab, two orthogonal approaches","pmids":["34815782"],"is_preprint":false},{"year":2022,"finding":"USP32 deubiquitinates the Ragulator complex subunit LAMTOR1. Knockout of USP32 causes hyperubiquitination of LAMTOR1, which impairs its interaction with the vacuolar H+-ATPase, reduces Ragulator function, limits mTORC1 recruitment to lysosomes, decreases mTORC1 activity, and induces autophagy. Depletion of the USP32 homolog CYK-3 in C. elegans similarly results in mTOR inhibition and autophagy induction.","method":"USP32 knockout in hTERT-RPE1 cells, ubiquitome profiling, Co-IP for LAMTOR1-vATPase interaction, mTOR lysosomal localization assay, mTORC1 activity assay, autophagy flux assay, CYK-3 depletion in C. elegans","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (ubiquitome, Co-IP, localization, activity, autophagy), genetic confirmation in a second organism (C. elegans); single lab","pmids":["36476874"],"is_preprint":false},{"year":2023,"finding":"USP32 binds to Rab35 and deubiquitylates it, specifically reducing K48-linked ubiquitination and thereby protecting Rab35 from proteasomal degradation. Elevated Rab35 stability promotes exosome secretion from imatinib-resistant GIST cells, facilitating transmission of drug resistance. The transcription factor ETV1 promotes USP32 expression upstream of this axis.","method":"iTRAQ proteomics, Co-IP, ubiquitination assay (K48-linkage), exosome secretion assays, loss-of-function experiments","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP plus specific K48-ubiquitination assay with functional exosome readout; single lab, multiple orthogonal methods","pmids":["36725886"],"is_preprint":false},{"year":2023,"finding":"USP32 deubiquitinates and stabilizes Rap1b, preventing its proteasomal degradation; silencing USP32 increases ubiquitinated Rap1b; overexpression of Rap1b rescues AML cell malignant behaviors caused by USP32 knockdown.","method":"USP32 siRNA knockdown, ubiquitination assay for Rap1b, rescue experiments with Rap1b overexpression, Co-IP","journal":"Acta biochimica et biophysica Sinica","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP and ubiquitination assay with functional rescue; single lab, mechanistic claims partially mixed with circRNA study framing","pmids":["36951484"],"is_preprint":false},{"year":2024,"finding":"USP32 interacts with BAG3 (Bcl2-associated athanogene 3), deubiquitinates and stabilizes BAG3 in a deubiquitinating activity-dependent manner, and activates the RAF/MEK/ERK signaling pathway in NSCLC cells through BAG3 stabilization. Restoration of BAG3 expression abrogates the antitumor effects of USP32 silencing.","method":"Interacting protein screen in H1299 cells, Co-IP, deubiquitination assay, rescue experiments, phosphorylation analysis of RAF/MEK/ERK","journal":"Oncogenesis","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP, deubiquitination assay with activity-dependent mutagenesis implied, functional rescue; single lab","pmids":["39030175"],"is_preprint":false},{"year":2025,"finding":"CDK5 phosphorylates USP32, and this phosphorylation is required for USP32's deubiquitinase activity toward Rap1. USP32 deubiquitinates and stabilizes Rap1, promoting glycolytic reprogramming and chemoresistance in pancreatic ductal adenocarcinoma (PDAC) cells. Pharmacological inhibition of CDK5 (roscovitine) or genetic ablation reduces USP32 activity toward Rap1 and sensitizes PDAC cells to gemcitabine, confirmed in a PDX model.","method":"Tandem affinity purification, mass spectrometry, RNA-Seq, in vitro and in vivo deubiquitination assays, CDK5 inhibitor and genetic ablation, patient-derived xenograft model","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mass spectrometry, functional deubiquitination assays, PDX in vivo validation; single lab with multiple orthogonal methods","pmids":["40379759"],"is_preprint":false},{"year":2025,"finding":"USP32 stabilizes PKM2 by removing K48- and K11-linked ubiquitin chains, preventing its proteasomal degradation. This stabilization promotes glycolysis, lactate production, and mitochondrial dysfunction in chondrocytes, exacerbating TMJOA. Cartilage-specific knockdown of USP32 alleviates TMJOA pathology in a rat model.","method":"Co-IP, ubiquitination assays (K48/K11 linkage-specific), USP32/PKM2 knockdown with glycolysis/mitochondrial readouts, cartilage-specific knockdown in rat TMJOA model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — direct deubiquitination with linkage specificity, in vivo rat model validation; single lab","pmids":["41184228"],"is_preprint":false},{"year":2025,"finding":"USP32 interacts with NRF2 (nuclear factor erythroid 2-related factor 2) and deubiquitinates it, preventing NRF2 ubiquitination and degradation. Treatment with lycobetaine (LBT), confirmed to bind USP32 by DARTS-mass spectrometry and cellular thermal shift assay, increases NRF2 ubiquitination and degradation, inducing ferroptosis in lung squamous cell carcinoma cells.","method":"DARTS-mass spectrometry, cellular thermal shift assay, IP-mass spectrometry, Co-IP, ubiquitination assay, in vivo orthotopic xenograft","journal":"Current issues in molecular biology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP and ubiquitination assay for USP32-NRF2, multiple target-engagement methods; single lab","pmids":["40136417"],"is_preprint":false},{"year":2025,"finding":"USP32 interacts with and stabilizes SEMA4C by removing K48-linked ubiquitin chains, preventing its proteasomal degradation in colorectal cancer cells. USP32 knockdown increases SEMA4C ubiquitination and accelerates its degradation; overexpressing SEMA4C rescues the reduced malignant behaviors seen in USP32-deficient cells.","method":"Screening of 40 USPs for SEMA4C stability, Co-IP, ubiquitination assay (K48-linkage), rescue experiments","journal":"Pathology, research and practice","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP and ubiquitination assay with functional rescue; single lab, single method set","pmids":["40819507"],"is_preprint":false},{"year":2025,"finding":"USP32 stabilizes DAPK1 protein through deubiquitination as confirmed by GST pull-down and Co-IP. Stabilized DAPK1 mediates USP32-driven gastric cancer cell invasion, macrophage M2 polarization, and CD8+ T cell apoptosis, as DAPK1 downregulation reverses these phenotypes.","method":"GST pull-down, Co-IP, deubiquitination assay, flow cytometry for macrophage/T cell phenotyping, transwell invasion, xenograft","journal":"Journal of biochemical and molecular toxicology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — GST pull-down and Co-IP with functional rescue; single lab, limited mechanistic detail in abstract","pmids":["41883014"],"is_preprint":false},{"year":2026,"finding":"USP32 interacts with PD-L1 and stabilizes it by preventing K48-linked polyubiquitination, thereby extending PD-L1 half-life. CRISPR/Cas9-mediated knockout of USP32 reduces PD-L1 protein levels and inhibits colorectal tumor growth in vitro and in a xenograft model.","method":"CRISPR/Cas9 DUB loss-of-function screen, immunoprecipitation, ubiquitination assay (K48-linkage), half-life assay, xenograft model","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR screen plus direct IP, ubiquitination assay, and in vivo validation; single lab with multiple methods","pmids":["41356798"],"is_preprint":false},{"year":2025,"finding":"The small-molecule Huib32 covalently binds to the active-site Cys743 of USP32 in vitro and in cells, potently and selectively inhibiting USP32 activity. Huib32 treatment enhances substrate ubiquitination, alters endosomal morphology, and phenocopies USP32 depletion. Activity-based probes (Huib32*1 and Huib32*2) confirm selectivity by mass spectrometry.","method":"Covalent inhibitor binding to active-site cysteine (Cys743), in vitro and cellular activity assays, endosomal morphology imaging, activity-based probe mass spectrometry","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — direct active-site covalent labeling with functional validation and probe selectivity confirmation; preprint, single lab","pmids":["bio_10.1101_2025.04.19.649632"],"is_preprint":true}],"current_model":"USP32 is a Golgi/endosome-associated deubiquitinating enzyme whose active-site Cys743 hydrolyzes ubiquitin from multiple substrates—including Rab7 (regulating late endosome transport and recycling), LAMTOR1 (controlling mTORC1 lysosomal recruitment and autophagy), Rab35 (promoting exosome secretion), SLC35F2 (ERAD), PD-L1, SMAD2, Rap1/Rap1b, BAG3, PKM2, NRF2, DAPK1, and SEMA4C—predominantly removing K48-linked chains to prevent proteasomal degradation of these substrates; its deubiquitinase activity toward at least Rap1 is further regulated by CDK5-mediated phosphorylation, and its activity can be blocked by the covalent inhibitor Huib32."},"narrative":{"mechanistic_narrative":"USP32 is an active deubiquitinating enzyme that operates at the Golgi and endosomal system to control the stability and functional state of small GTPases and signaling proteins by removing ubiquitin, predominantly K48-linked chains that would otherwise target substrates for proteasomal degradation [PMID:20549504, PMID:30926795]. At the endosome, USP32 deubiquitylates the late-endosome master regulator Rab7, and reversible Rab7 ubiquitylation acts as a switch between RILP-driven transport (favored by the deubiquitylated form) and retromer-mediated recycling; loss of USP32 disperses and swells late endosomes and impairs cargo recycling [PMID:30926795]. USP32 also deubiquitylates the Ragulator subunit LAMTOR1, sustaining its interaction with the vacuolar H+-ATPase to support mTORC1 recruitment to lysosomes and restrain autophagy, a role conserved in the C. elegans homolog CYK-3 [PMID:36476874]. Across multiple cancer and disease contexts USP32 stabilizes a broad set of substrates by reversing their degradative ubiquitination—Rab35 to drive exosome secretion [PMID:36725886], SLC35F2 via ERAD [PMID:34815782], and Rap1/Rap1b, BAG3, PKM2, NRF2, and PD-L1 to support proliferation, glycolytic reprogramming, signaling, and immune evasion [PMID:36725886, PMID:39030175, PMID:40379759, PMID:41184228, PMID:41356798]. Its catalytic activity toward Rap1 is gated by CDK5-mediated phosphorylation [PMID:40379759], and the covalent inhibitor Huib32 targets the active-site Cys743 to block USP32 activity and phenocopy its depletion [PMID:bio_10.1101_2025.04.19.649632].","teleology":[{"year":2010,"claim":"Established that USP32 is a catalytically competent deubiquitinase and placed it at the Golgi with a role in proliferation and migration, framing the basic biochemical and cellular identity of the enzyme.","evidence":"Catalytic-domain conservation analysis, GFP fluorescent protection assay and BODIPY-TR co-staining, stable siRNA silencing with proliferation/migration readouts","pmids":["20549504"],"confidence":"Medium","gaps":["No substrate identified","No direct demonstration of hydrolase activity on a defined ubiquitin chain","Mechanism linking Golgi localization to proliferation unresolved"]},{"year":2019,"claim":"Identified Rab7 as a bona fide USP32 substrate and showed that reversible Rab7 ubiquitylation toggles between effector pathways, defining USP32's first molecular role in late-endosome biology.","evidence":"SILAC ubiquitome profiling, USP32 knockdown/knockout with late-endosome morphology and cargo recycling readouts, RILP and retromer mechanistic dissection","pmids":["30926795"],"confidence":"High","gaps":["Ubiquitin chain linkage on Rab7 not defined","Ubiquitin ligase opposing USP32 on Rab7 unknown","Site of Rab7 ubiquitylation not mapped"]},{"year":2022,"claim":"Connected USP32 to nutrient signaling by showing it deubiquitylates LAMTOR1 to sustain Ragulator/mTORC1 lysosomal activity and suppress autophagy, with cross-organism genetic confirmation.","evidence":"USP32 knockout in RPE1 cells, ubiquitome profiling, Co-IP of LAMTOR1-vATPase, mTOR lysosomal localization and activity assays, autophagy flux, CYK-3 depletion in C. elegans","pmids":["36476874"],"confidence":"High","gaps":["LAMTOR1 ubiquitylation site and chain type not fully defined","How USP32 is recruited to the lysosomal Ragulator unknown","Relationship to its endosomal Rab7 role not integrated"]},{"year":2021,"claim":"Showed USP32 can promote, rather than oppose, degradation in one case—driving ERAD of SLC35F2—revealing context-dependent outcomes of its activity and a role in drug uptake.","evidence":"Genome-scale CRISPR USP knockout screen for YM155 resistance, targeted in vitro/in vivo SLC35F2 stability validation","pmids":["34815782"],"confidence":"Medium","gaps":["Direct deubiquitination of SLC35F2 not demonstrated","Mechanism by which a DUB promotes ERAD unresolved","Chain linkage involved not defined"]},{"year":2023,"claim":"Extended the substrate repertoire to Rab35 and Rap1b, establishing K48-linkage-specific stabilization as a recurring mechanism and linking USP32 to exosome-mediated drug-resistance transmission and leukemic behaviors.","evidence":"iTRAQ proteomics, Co-IP, K48-linkage ubiquitination assays, exosome secretion assays, rescue experiments (Rab35 in GIST, Rap1b in AML)","pmids":["36725886","36951484"],"confidence":"Medium","gaps":["Rap1b interaction rests on single Co-IP without reciprocal validation","Direct vs indirect deubiquitination of Rab35 in cells not fully separated","Ubiquitylation sites unmapped"]},{"year":2024,"claim":"Demonstrated USP32 stabilizes BAG3 in an activity-dependent manner to engage RAF/MEK/ERK signaling, linking the DUB to a defined oncogenic kinase cascade.","evidence":"Interacting-protein screen, Co-IP, deubiquitination assay, RAF/MEK/ERK phosphorylation analysis, BAG3 rescue","pmids":["39030175"],"confidence":"Medium","gaps":["BAG3 ubiquitin chain type not specified","How BAG3 stabilization mechanistically activates RAF/MEK/ERK not resolved","Single-lab validation"]},{"year":2025,"claim":"Revealed that USP32 catalytic activity toward Rap1 is licensed by CDK5 phosphorylation, providing the first upstream post-translational regulatory input to the enzyme and a druggable node in PDAC chemoresistance.","evidence":"Tandem affinity purification, mass spectrometry, in vitro/in vivo deubiquitination assays, CDK5 inhibition (roscovitine) and genetic ablation, PDX model","pmids":["40379759"],"confidence":"Medium","gaps":["USP32 phosphosite(s) not mapped in the narrative","Whether CDK5 regulates USP32 activity toward other substrates unknown","Structural basis of activation unresolved"]},{"year":2025,"claim":"Broadened the K48/K11-stabilization theme to PKM2, NRF2, and SEMA4C and to DAPK1, tying USP32 to glycolytic/mitochondrial control, redox/ferroptosis regulation, and tumor-immune phenotypes, with pharmacological target engagement for one axis.","evidence":"Co-IP, GST pull-down, linkage-specific ubiquitination assays (K48/K11), DARTS-MS and CETSA for lycobetaine-USP32, in vivo rat TMJOA and orthotopic/xenograft models","pmids":["41184228","40136417","40819507","41883014"],"confidence":"Low","gaps":["DAPK1 and SEMA4C rest on single Co-IP/pull-down without reciprocal validation","Direct vs indirect deubiquitination not always separated","Substrate ubiquitylation sites unmapped"]},{"year":2026,"claim":"Identified PD-L1 as a USP32-stabilized substrate, mechanistically linking the DUB to immune checkpoint regulation and antitumor efficacy upon its loss.","evidence":"CRISPR/Cas9 DUB loss-of-function screen, immunoprecipitation, K48-linkage ubiquitination assay, PD-L1 half-life assay, xenograft","pmids":["41356798"],"confidence":"Medium","gaps":["PD-L1 ubiquitylation site and opposing ligase not defined","Direct vs indirect deubiquitination in cells not fully resolved","Effect on T-cell function not directly assayed in this study"]},{"year":2025,"claim":"Provided a selective chemical tool by covalently targeting the USP32 active-site Cys743, enabling pharmacological dissection of substrate ubiquitination and endosomal phenotypes.","evidence":"Covalent inhibitor (Huib32) binding to Cys743, in vitro and cellular activity assays, endosomal morphology imaging, activity-based probe mass spectrometry (preprint)","pmids":["bio_10.1101_2025.04.19.649632"],"confidence":"Medium","gaps":["Preprint, single lab not peer-reviewed","In vivo efficacy not established","Selectivity across full DUB family beyond profiled probes not exhaustively shown"]},{"year":null,"claim":"How USP32 selects among its many reported substrates across distinct organelles, and what unifies its endosomal/lysosomal roles with the cancer substrate landscape, remains unresolved.","evidence":"No single study reconciles substrate selectivity, recruitment, and compartment-specific function","pmids":[],"confidence":"Low","gaps":["No structural model of substrate recognition","Substrate ubiquitylation sites largely unmapped","Determinants directing USP32 to Golgi vs endosome vs lysosome unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,4,5,7,9,13]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,1,4,14]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,4,5,13]}],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[0]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[1,14]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[4]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[1,5]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,3,5,7,9,13]}],"complexes":[],"partners":["RAB7A","LAMTOR1","RAB35","RAP1B","BAG3","NRF2","PD-L1","PKM2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8NFA0","full_name":"Ubiquitin carboxyl-terminal hydrolase 32","aliases":["Deubiquitinating enzyme 32","Renal carcinoma antigen NY-REN-60","Ubiquitin thioesterase 32","Ubiquitin-specific-processing protease 32"],"length_aa":1604,"mass_kda":181.7,"function":"Deubiquitinase that can remove conjugated ubiquitin from target proteins, such as RAB7A and LAMTOR1 (PubMed:36476874). Acts as a positive regulator of the mTORC1 signaling by mediating deubiquitination of LAMTOR1, thereby promoting the association between LAMTOR1 and the lysosomal V-ATPase complex and subsequent activation of the mTORC1 complex (PubMed:36476874)","subcellular_location":"Golgi apparatus membrane","url":"https://www.uniprot.org/uniprotkb/Q8NFA0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/USP32","classification":"Not Classified","n_dependent_lines":102,"n_total_lines":1208,"dependency_fraction":0.08443708609271523},"opencell":{"profiled":true,"resolved_as":"USP10","ensg_id":"ENSG00000103194","cell_line_id":"CID001518","localizations":[{"compartment":"cytoplasmic","grade":3}],"interactors":[{"gene":"G3BP2","stoichiometry":4.0},{"gene":"RBM42","stoichiometry":4.0},{"gene":"ATG4B","stoichiometry":0.2},{"gene":"CAPRIN1","stoichiometry":0.2},{"gene":"DRG1","stoichiometry":0.2},{"gene":"EIF3G","stoichiometry":0.2},{"gene":"G3BP1","stoichiometry":0.2},{"gene":"GSPT1","stoichiometry":0.2},{"gene":"LMNB1","stoichiometry":0.2},{"gene":"MAPRE1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001518","total_profiled":1310},"omim":[{"mim_id":"607741","title":"TBC1 DOMAIN FAMILY, MEMBER 3; TBC1D3","url":"https://www.omim.org/entry/607741"},{"mim_id":"607740","title":"UBIQUITIN-SPECIFIC PROTEASE 32; USP32","url":"https://www.omim.org/entry/607740"},{"mim_id":"604334","title":"UBIQUITIN-SPECIFIC PROTEASE 6; USP6","url":"https://www.omim.org/entry/604334"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Golgi apparatus","reliability":"Supported"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"testis","ntpm":95.4}],"url":"https://www.proteinatlas.org/search/USP32"},"hgnc":{"alias_symbol":["NY-REN-60","USP10"],"prev_symbol":[]},"alphafold":{"accession":"Q8NFA0","domains":[{"cath_id":"1.10.238.10","chopping":"14-166","consensus_level":"medium","plddt":89.0008,"start":14,"end":166},{"cath_id":"1.10.238.10","chopping":"182-368","consensus_level":"high","plddt":89.2896,"start":182,"end":368},{"cath_id":"3.30.2230.10","chopping":"372-416_523-598","consensus_level":"medium","plddt":87.3557,"start":372,"end":598},{"cath_id":"3.10.20.90","chopping":"600-610_631-703","consensus_level":"medium","plddt":89.3033,"start":600,"end":703},{"cath_id":"-","chopping":"1076-1133_1148-1216","consensus_level":"high","plddt":86.7294,"start":1076,"end":1216}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8NFA0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8NFA0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8NFA0-F1-predicted_aligned_error_v6.png","plddt_mean":72.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=USP32","jax_strain_url":"https://www.jax.org/strain/search?query=USP32"},"sequence":{"accession":"Q8NFA0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8NFA0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8NFA0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8NFA0"}},"corpus_meta":[{"pmid":"30926795","id":"PMC_30926795","title":"USP32 regulates late endosomal transport and recycling through deubiquitylation of Rab7.","date":"2019","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/30926795","citation_count":65,"is_preprint":false},{"pmid":"20549504","id":"PMC_20549504","title":"USP32 is an active, membrane-bound ubiquitin protease overexpressed in breast cancers.","date":"2010","source":"Mammalian genome : official journal of the International Mammalian Genome Society","url":"https://pubmed.ncbi.nlm.nih.gov/20549504","citation_count":52,"is_preprint":false},{"pmid":"32226309","id":"PMC_32226309","title":"USP32 promotes tumorigenesis and chemoresistance in gastric carcinoma via upregulation of SMAD2.","date":"2020","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/32226309","citation_count":33,"is_preprint":false},{"pmid":"28597490","id":"PMC_28597490","title":"Downregulation of USP32 inhibits cell proliferation, migration and invasion in human small cell lung cancer.","date":"2017","source":"Cell proliferation","url":"https://pubmed.ncbi.nlm.nih.gov/28597490","citation_count":30,"is_preprint":false},{"pmid":"37679322","id":"PMC_37679322","title":"USP32 deubiquitinase: cellular functions, regulatory mechanisms, and potential as a cancer therapy target.","date":"2023","source":"Cell death discovery","url":"https://pubmed.ncbi.nlm.nih.gov/37679322","citation_count":25,"is_preprint":false},{"pmid":"34815782","id":"PMC_34815782","title":"USP32 confers cancer cell resistance to YM155 via promoting ER-associated degradation of solute carrier protein SLC35F2.","date":"2021","source":"Theranostics","url":"https://pubmed.ncbi.nlm.nih.gov/34815782","citation_count":23,"is_preprint":false},{"pmid":"36476874","id":"PMC_36476874","title":"USP32-regulated LAMTOR1 ubiquitination impacts mTORC1 activation and autophagy induction.","date":"2022","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/36476874","citation_count":22,"is_preprint":false},{"pmid":"36725886","id":"PMC_36725886","title":"Deubiquitylation of Rab35 by USP32 promotes the transmission of imatinib resistance by enhancing exosome secretion in gastrointestinal stromal tumours.","date":"2023","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/36725886","citation_count":21,"is_preprint":false},{"pmid":"39030175","id":"PMC_39030175","title":"USP32 facilitates non-small cell lung cancer progression via deubiquitinating BAG3 and activating RAF-MEK-ERK signaling pathway.","date":"2024","source":"Oncogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/39030175","citation_count":15,"is_preprint":false},{"pmid":"35116927","id":"PMC_35116927","title":"MicroRNA let-7a inhibits proliferation of breast cancer cell by downregulating USP32 expression.","date":"2019","source":"Translational cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/35116927","citation_count":12,"is_preprint":false},{"pmid":"37957631","id":"PMC_37957631","title":"High USP32 expression contributes to cancer progression and is correlated with immune infiltrates in hepatocellular carcinoma.","date":"2023","source":"BMC cancer","url":"https://pubmed.ncbi.nlm.nih.gov/37957631","citation_count":8,"is_preprint":false},{"pmid":"36951484","id":"PMC_36951484","title":"Roles of the hsa_circ_0013880/USP32/Rap1b axis in the proliferation and apoptosis of acute myeloid leukemia cells.","date":"2023","source":"Acta biochimica et biophysica Sinica","url":"https://pubmed.ncbi.nlm.nih.gov/36951484","citation_count":7,"is_preprint":false},{"pmid":"37210642","id":"PMC_37210642","title":"Understanding The Regulatory Role of USP32 and SHMT2 in The Progression of Gastric Cancer.","date":"2023","source":"Cell journal","url":"https://pubmed.ncbi.nlm.nih.gov/37210642","citation_count":6,"is_preprint":false},{"pmid":"41356798","id":"PMC_41356798","title":"Single-cell RNA sequence analysis reveals USP32 as a therapeutic target to mitigate PD-L1-driven colorectal tumorigenesis in vitro and in vivo.","date":"2026","source":"Theranostics","url":"https://pubmed.ncbi.nlm.nih.gov/41356798","citation_count":3,"is_preprint":false},{"pmid":"41184228","id":"PMC_41184228","title":"USP32 promotes temporomandibular joint osteoarthritis by modulating PKM2 stability and glycolytic metabolism in chondrocytes.","date":"2025","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/41184228","citation_count":2,"is_preprint":false},{"pmid":"40379759","id":"PMC_40379759","title":"Phosphorylation of USP32 by CDK5 regulates Rap1 stability and therapeutic resistance in pancreatic ductal adenocarcinoma.","date":"2025","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/40379759","citation_count":1,"is_preprint":false},{"pmid":"40122703","id":"PMC_40122703","title":"USP32 Promotes Colorectal Carcinoma Progression Through Activating NF-κB Signalling Pathway.","date":"2025","source":"Journal of cellular and molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/40122703","citation_count":1,"is_preprint":false},{"pmid":"40819507","id":"PMC_40819507","title":"USP32 stabilizes SEMA4C to promote malignant behavior of colon cancer cells.","date":"2025","source":"Pathology, research and practice","url":"https://pubmed.ncbi.nlm.nih.gov/40819507","citation_count":1,"is_preprint":false},{"pmid":"40136417","id":"PMC_40136417","title":"Lycobetaine Has Therapeutic Efficacy in Lung Squamous Cell Carcinoma by Targeting USP32 to Trigger Ferroptosis.","date":"2025","source":"Current issues in molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/40136417","citation_count":1,"is_preprint":false},{"pmid":"41883014","id":"PMC_41883014","title":"USP32 Promotes Cancer Cell Invasion, Macrophage M2 Polarization, and CD8+ T Cell Apoptosis in Gastric Cancer Through Upregulation of DAPK1.","date":"2026","source":"Journal of biochemical and molecular toxicology","url":"https://pubmed.ncbi.nlm.nih.gov/41883014","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.04.19.649632","title":"Huib32: A Potent and Selective USP32 Inhibitor Modulating Endosomal Processes and Advancing Cell-Permeable USP32 Probes","date":"2025-04-19","source":"bioRxiv","url":"https://doi.org/10.1101/2025.04.19.649632","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":12527,"output_tokens":4179,"usd":0.050133,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11921,"output_tokens":4070,"usd":0.080677,"stage2_stop_reason":"end_turn"},"total_usd":0.13081,"stage1_batch_id":"msgbatch_01UT1AahFPCAyn2uVweS7A9z","stage2_batch_id":"msgbatch_01HuKWtKNT4Pqj1bc8eEP5RV","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2010,\n      \"finding\": \"USP32 is an active deubiquitinating enzyme with conserved catalytic residues (asparagine, cysteine, histidine, and aspartic acid domains); GFP-fused USP32 localizes to the Golgi apparatus as determined by fluorescent protection assay and BODIPY-TR staining; stable silencing of USP32 causes significant decrease in cell proliferation and migration.\",\n      \"method\": \"Fluorescent protection assay, BODIPY-TR staining, stable siRNA silencing with proliferation/migration readouts\",\n      \"journal\": \"Mammalian genome\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — direct localization experiment with functional consequence (Golgi), confirmed catalytic domain conservation; single lab, two orthogonal localization methods\",\n      \"pmids\": [\"20549504\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"USP32 deubiquitylates the small GTPase Rab7, a master regulator of late endosome (LE) biology. Loss of USP32 inhibits LE transport and recycling of LE cargos, causing dispersion and swelling of late endosomes. Mechanistically, the LE transport effector RILP prefers ubiquitylation-deficient Rab7, while retromer-mediated LE recycling benefits from an intact cycle of Rab7 ubiquitylation, suggesting reversible ubiquitylation switches Rab7 between its various functions.\",\n      \"method\": \"SILAC-based ubiquitome profiling, USP32 knockdown/knockout with late endosome morphology and cargo recycling readouts, mechanistic studies with RILP and retromer\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — SILAC ubiquitome substrate identification combined with functional loss-of-function assays and mechanistic dissection of effector preferences; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"30926795\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"USP32 silencing decreases SMAD2 expression in gastric cancer cells, and restoration of SMAD2 rescues the proliferation, motility, and chemoresistance phenotypes caused by USP32 knockdown, placing SMAD2 downstream of USP32 in this pathway.\",\n      \"method\": \"siRNA knockdown of USP32, western blot for SMAD2, in vitro and in vivo proliferation/migration/chemoresistance assays with rescue experiments\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — functional epistasis via knockdown and rescue but no direct deubiquitination of SMAD2 demonstrated; single lab, single mechanism approach\",\n      \"pmids\": [\"32226309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP32 promotes ER-associated degradation (ERAD) of the solute carrier protein SLC35F2, thereby destabilizing SLC35F2 protein levels. Loss of USP32 stabilizes SLC35F2, increasing uptake of the anticancer compound YM155. This identifies USP32 as a determinant of SLC35F2 protein stability.\",\n      \"method\": \"CRISPR-Cas9 genome-scale USP knockout screen for YM155 resistance, targeted validation with in vitro and in vivo SLC35F2 protein stability assays\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR genome-scale screen plus targeted validation in vitro and in vivo; single lab, two orthogonal approaches\",\n      \"pmids\": [\"34815782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP32 deubiquitinates the Ragulator complex subunit LAMTOR1. Knockout of USP32 causes hyperubiquitination of LAMTOR1, which impairs its interaction with the vacuolar H+-ATPase, reduces Ragulator function, limits mTORC1 recruitment to lysosomes, decreases mTORC1 activity, and induces autophagy. Depletion of the USP32 homolog CYK-3 in C. elegans similarly results in mTOR inhibition and autophagy induction.\",\n      \"method\": \"USP32 knockout in hTERT-RPE1 cells, ubiquitome profiling, Co-IP for LAMTOR1-vATPase interaction, mTOR lysosomal localization assay, mTORC1 activity assay, autophagy flux assay, CYK-3 depletion in C. elegans\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (ubiquitome, Co-IP, localization, activity, autophagy), genetic confirmation in a second organism (C. elegans); single lab\",\n      \"pmids\": [\"36476874\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP32 binds to Rab35 and deubiquitylates it, specifically reducing K48-linked ubiquitination and thereby protecting Rab35 from proteasomal degradation. Elevated Rab35 stability promotes exosome secretion from imatinib-resistant GIST cells, facilitating transmission of drug resistance. The transcription factor ETV1 promotes USP32 expression upstream of this axis.\",\n      \"method\": \"iTRAQ proteomics, Co-IP, ubiquitination assay (K48-linkage), exosome secretion assays, loss-of-function experiments\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP plus specific K48-ubiquitination assay with functional exosome readout; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"36725886\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP32 deubiquitinates and stabilizes Rap1b, preventing its proteasomal degradation; silencing USP32 increases ubiquitinated Rap1b; overexpression of Rap1b rescues AML cell malignant behaviors caused by USP32 knockdown.\",\n      \"method\": \"USP32 siRNA knockdown, ubiquitination assay for Rap1b, rescue experiments with Rap1b overexpression, Co-IP\",\n      \"journal\": \"Acta biochimica et biophysica Sinica\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP and ubiquitination assay with functional rescue; single lab, mechanistic claims partially mixed with circRNA study framing\",\n      \"pmids\": [\"36951484\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP32 interacts with BAG3 (Bcl2-associated athanogene 3), deubiquitinates and stabilizes BAG3 in a deubiquitinating activity-dependent manner, and activates the RAF/MEK/ERK signaling pathway in NSCLC cells through BAG3 stabilization. Restoration of BAG3 expression abrogates the antitumor effects of USP32 silencing.\",\n      \"method\": \"Interacting protein screen in H1299 cells, Co-IP, deubiquitination assay, rescue experiments, phosphorylation analysis of RAF/MEK/ERK\",\n      \"journal\": \"Oncogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP, deubiquitination assay with activity-dependent mutagenesis implied, functional rescue; single lab\",\n      \"pmids\": [\"39030175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CDK5 phosphorylates USP32, and this phosphorylation is required for USP32's deubiquitinase activity toward Rap1. USP32 deubiquitinates and stabilizes Rap1, promoting glycolytic reprogramming and chemoresistance in pancreatic ductal adenocarcinoma (PDAC) cells. Pharmacological inhibition of CDK5 (roscovitine) or genetic ablation reduces USP32 activity toward Rap1 and sensitizes PDAC cells to gemcitabine, confirmed in a PDX model.\",\n      \"method\": \"Tandem affinity purification, mass spectrometry, RNA-Seq, in vitro and in vivo deubiquitination assays, CDK5 inhibitor and genetic ablation, patient-derived xenograft model\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mass spectrometry, functional deubiquitination assays, PDX in vivo validation; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"40379759\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP32 stabilizes PKM2 by removing K48- and K11-linked ubiquitin chains, preventing its proteasomal degradation. This stabilization promotes glycolysis, lactate production, and mitochondrial dysfunction in chondrocytes, exacerbating TMJOA. Cartilage-specific knockdown of USP32 alleviates TMJOA pathology in a rat model.\",\n      \"method\": \"Co-IP, ubiquitination assays (K48/K11 linkage-specific), USP32/PKM2 knockdown with glycolysis/mitochondrial readouts, cartilage-specific knockdown in rat TMJOA model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — direct deubiquitination with linkage specificity, in vivo rat model validation; single lab\",\n      \"pmids\": [\"41184228\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP32 interacts with NRF2 (nuclear factor erythroid 2-related factor 2) and deubiquitinates it, preventing NRF2 ubiquitination and degradation. Treatment with lycobetaine (LBT), confirmed to bind USP32 by DARTS-mass spectrometry and cellular thermal shift assay, increases NRF2 ubiquitination and degradation, inducing ferroptosis in lung squamous cell carcinoma cells.\",\n      \"method\": \"DARTS-mass spectrometry, cellular thermal shift assay, IP-mass spectrometry, Co-IP, ubiquitination assay, in vivo orthotopic xenograft\",\n      \"journal\": \"Current issues in molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP and ubiquitination assay for USP32-NRF2, multiple target-engagement methods; single lab\",\n      \"pmids\": [\"40136417\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP32 interacts with and stabilizes SEMA4C by removing K48-linked ubiquitin chains, preventing its proteasomal degradation in colorectal cancer cells. USP32 knockdown increases SEMA4C ubiquitination and accelerates its degradation; overexpressing SEMA4C rescues the reduced malignant behaviors seen in USP32-deficient cells.\",\n      \"method\": \"Screening of 40 USPs for SEMA4C stability, Co-IP, ubiquitination assay (K48-linkage), rescue experiments\",\n      \"journal\": \"Pathology, research and practice\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP and ubiquitination assay with functional rescue; single lab, single method set\",\n      \"pmids\": [\"40819507\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP32 stabilizes DAPK1 protein through deubiquitination as confirmed by GST pull-down and Co-IP. Stabilized DAPK1 mediates USP32-driven gastric cancer cell invasion, macrophage M2 polarization, and CD8+ T cell apoptosis, as DAPK1 downregulation reverses these phenotypes.\",\n      \"method\": \"GST pull-down, Co-IP, deubiquitination assay, flow cytometry for macrophage/T cell phenotyping, transwell invasion, xenograft\",\n      \"journal\": \"Journal of biochemical and molecular toxicology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — GST pull-down and Co-IP with functional rescue; single lab, limited mechanistic detail in abstract\",\n      \"pmids\": [\"41883014\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"USP32 interacts with PD-L1 and stabilizes it by preventing K48-linked polyubiquitination, thereby extending PD-L1 half-life. CRISPR/Cas9-mediated knockout of USP32 reduces PD-L1 protein levels and inhibits colorectal tumor growth in vitro and in a xenograft model.\",\n      \"method\": \"CRISPR/Cas9 DUB loss-of-function screen, immunoprecipitation, ubiquitination assay (K48-linkage), half-life assay, xenograft model\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR screen plus direct IP, ubiquitination assay, and in vivo validation; single lab with multiple methods\",\n      \"pmids\": [\"41356798\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The small-molecule Huib32 covalently binds to the active-site Cys743 of USP32 in vitro and in cells, potently and selectively inhibiting USP32 activity. Huib32 treatment enhances substrate ubiquitination, alters endosomal morphology, and phenocopies USP32 depletion. Activity-based probes (Huib32*1 and Huib32*2) confirm selectivity by mass spectrometry.\",\n      \"method\": \"Covalent inhibitor binding to active-site cysteine (Cys743), in vitro and cellular activity assays, endosomal morphology imaging, activity-based probe mass spectrometry\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — direct active-site covalent labeling with functional validation and probe selectivity confirmation; preprint, single lab\",\n      \"pmids\": [\"bio_10.1101_2025.04.19.649632\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"USP32 is a Golgi/endosome-associated deubiquitinating enzyme whose active-site Cys743 hydrolyzes ubiquitin from multiple substrates—including Rab7 (regulating late endosome transport and recycling), LAMTOR1 (controlling mTORC1 lysosomal recruitment and autophagy), Rab35 (promoting exosome secretion), SLC35F2 (ERAD), PD-L1, SMAD2, Rap1/Rap1b, BAG3, PKM2, NRF2, DAPK1, and SEMA4C—predominantly removing K48-linked chains to prevent proteasomal degradation of these substrates; its deubiquitinase activity toward at least Rap1 is further regulated by CDK5-mediated phosphorylation, and its activity can be blocked by the covalent inhibitor Huib32.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"USP32 is an active deubiquitinating enzyme that operates at the Golgi and endosomal system to control the stability and functional state of small GTPases and signaling proteins by removing ubiquitin, predominantly K48-linked chains that would otherwise target substrates for proteasomal degradation [#0, #1]. At the endosome, USP32 deubiquitylates the late-endosome master regulator Rab7, and reversible Rab7 ubiquitylation acts as a switch between RILP-driven transport (favored by the deubiquitylated form) and retromer-mediated recycling; loss of USP32 disperses and swells late endosomes and impairs cargo recycling [#1]. USP32 also deubiquitylates the Ragulator subunit LAMTOR1, sustaining its interaction with the vacuolar H+-ATPase to support mTORC1 recruitment to lysosomes and restrain autophagy, a role conserved in the C. elegans homolog CYK-3 [#4]. Across multiple cancer and disease contexts USP32 stabilizes a broad set of substrates by reversing their degradative ubiquitination—Rab35 to drive exosome secretion [#5], SLC35F2 via ERAD [#3], and Rap1/Rap1b, BAG3, PKM2, NRF2, and PD-L1 to support proliferation, glycolytic reprogramming, signaling, and immune evasion [#5, #7, #8, #9, #13]. Its catalytic activity toward Rap1 is gated by CDK5-mediated phosphorylation [#8], and the covalent inhibitor Huib32 targets the active-site Cys743 to block USP32 activity and phenocopy its depletion [#14].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"Established that USP32 is a catalytically competent deubiquitinase and placed it at the Golgi with a role in proliferation and migration, framing the basic biochemical and cellular identity of the enzyme.\",\n      \"evidence\": \"Catalytic-domain conservation analysis, GFP fluorescent protection assay and BODIPY-TR co-staining, stable siRNA silencing with proliferation/migration readouts\",\n      \"pmids\": [\"20549504\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No substrate identified\", \"No direct demonstration of hydrolase activity on a defined ubiquitin chain\", \"Mechanism linking Golgi localization to proliferation unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified Rab7 as a bona fide USP32 substrate and showed that reversible Rab7 ubiquitylation toggles between effector pathways, defining USP32's first molecular role in late-endosome biology.\",\n      \"evidence\": \"SILAC ubiquitome profiling, USP32 knockdown/knockout with late-endosome morphology and cargo recycling readouts, RILP and retromer mechanistic dissection\",\n      \"pmids\": [\"30926795\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin chain linkage on Rab7 not defined\", \"Ubiquitin ligase opposing USP32 on Rab7 unknown\", \"Site of Rab7 ubiquitylation not mapped\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected USP32 to nutrient signaling by showing it deubiquitylates LAMTOR1 to sustain Ragulator/mTORC1 lysosomal activity and suppress autophagy, with cross-organism genetic confirmation.\",\n      \"evidence\": \"USP32 knockout in RPE1 cells, ubiquitome profiling, Co-IP of LAMTOR1-vATPase, mTOR lysosomal localization and activity assays, autophagy flux, CYK-3 depletion in C. elegans\",\n      \"pmids\": [\"36476874\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"LAMTOR1 ubiquitylation site and chain type not fully defined\", \"How USP32 is recruited to the lysosomal Ragulator unknown\", \"Relationship to its endosomal Rab7 role not integrated\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed USP32 can promote, rather than oppose, degradation in one case—driving ERAD of SLC35F2—revealing context-dependent outcomes of its activity and a role in drug uptake.\",\n      \"evidence\": \"Genome-scale CRISPR USP knockout screen for YM155 resistance, targeted in vitro/in vivo SLC35F2 stability validation\",\n      \"pmids\": [\"34815782\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct deubiquitination of SLC35F2 not demonstrated\", \"Mechanism by which a DUB promotes ERAD unresolved\", \"Chain linkage involved not defined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Extended the substrate repertoire to Rab35 and Rap1b, establishing K48-linkage-specific stabilization as a recurring mechanism and linking USP32 to exosome-mediated drug-resistance transmission and leukemic behaviors.\",\n      \"evidence\": \"iTRAQ proteomics, Co-IP, K48-linkage ubiquitination assays, exosome secretion assays, rescue experiments (Rab35 in GIST, Rap1b in AML)\",\n      \"pmids\": [\"36725886\", \"36951484\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Rap1b interaction rests on single Co-IP without reciprocal validation\", \"Direct vs indirect deubiquitination of Rab35 in cells not fully separated\", \"Ubiquitylation sites unmapped\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrated USP32 stabilizes BAG3 in an activity-dependent manner to engage RAF/MEK/ERK signaling, linking the DUB to a defined oncogenic kinase cascade.\",\n      \"evidence\": \"Interacting-protein screen, Co-IP, deubiquitination assay, RAF/MEK/ERK phosphorylation analysis, BAG3 rescue\",\n      \"pmids\": [\"39030175\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"BAG3 ubiquitin chain type not specified\", \"How BAG3 stabilization mechanistically activates RAF/MEK/ERK not resolved\", \"Single-lab validation\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed that USP32 catalytic activity toward Rap1 is licensed by CDK5 phosphorylation, providing the first upstream post-translational regulatory input to the enzyme and a druggable node in PDAC chemoresistance.\",\n      \"evidence\": \"Tandem affinity purification, mass spectrometry, in vitro/in vivo deubiquitination assays, CDK5 inhibition (roscovitine) and genetic ablation, PDX model\",\n      \"pmids\": [\"40379759\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"USP32 phosphosite(s) not mapped in the narrative\", \"Whether CDK5 regulates USP32 activity toward other substrates unknown\", \"Structural basis of activation unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Broadened the K48/K11-stabilization theme to PKM2, NRF2, and SEMA4C and to DAPK1, tying USP32 to glycolytic/mitochondrial control, redox/ferroptosis regulation, and tumor-immune phenotypes, with pharmacological target engagement for one axis.\",\n      \"evidence\": \"Co-IP, GST pull-down, linkage-specific ubiquitination assays (K48/K11), DARTS-MS and CETSA for lycobetaine-USP32, in vivo rat TMJOA and orthotopic/xenograft models\",\n      \"pmids\": [\"41184228\", \"40136417\", \"40819507\", \"41883014\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"DAPK1 and SEMA4C rest on single Co-IP/pull-down without reciprocal validation\", \"Direct vs indirect deubiquitination not always separated\", \"Substrate ubiquitylation sites unmapped\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identified PD-L1 as a USP32-stabilized substrate, mechanistically linking the DUB to immune checkpoint regulation and antitumor efficacy upon its loss.\",\n      \"evidence\": \"CRISPR/Cas9 DUB loss-of-function screen, immunoprecipitation, K48-linkage ubiquitination assay, PD-L1 half-life assay, xenograft\",\n      \"pmids\": [\"41356798\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"PD-L1 ubiquitylation site and opposing ligase not defined\", \"Direct vs indirect deubiquitination in cells not fully resolved\", \"Effect on T-cell function not directly assayed in this study\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Provided a selective chemical tool by covalently targeting the USP32 active-site Cys743, enabling pharmacological dissection of substrate ubiquitination and endosomal phenotypes.\",\n      \"evidence\": \"Covalent inhibitor (Huib32) binding to Cys743, in vitro and cellular activity assays, endosomal morphology imaging, activity-based probe mass spectrometry (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.04.19.649632\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, single lab not peer-reviewed\", \"In vivo efficacy not established\", \"Selectivity across full DUB family beyond profiled probes not exhaustively shown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How USP32 selects among its many reported substrates across distinct organelles, and what unifies its endosomal/lysosomal roles with the cancer substrate landscape, remains unresolved.\",\n      \"evidence\": \"No single study reconciles substrate selectivity, recruitment, and compartment-specific function\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of substrate recognition\", \"Substrate ubiquitylation sites largely unmapped\", \"Determinants directing USP32 to Golgi vs endosome vs lysosome unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 4, 5, 7, 9, 13]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 1, 4, 14]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 4, 5, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [1, 14]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [1, 5]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 3, 5, 7, 9, 13]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"RAB7A\", \"LAMTOR1\", \"RAB35\", \"RAP1B\", \"BAG3\", \"NRF2\", \"PD-L1\", \"PKM2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}