Affinage

USP32

Ubiquitin carboxyl-terminal hydrolase 32 · UniProt Q8NFA0

Length
1604 aa
Mass
181.7 kDa
Annotated
2026-06-11
20 papers in source corpus 15 papers cited in narrative 15 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 5/5 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

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].

Mechanistic history

Synthesis pass · year-by-year structured walk · 10 steps
  1. 2010 Medium

    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

    PMID:20549504

    Open questions at the time
    • No substrate identified
    • No direct demonstration of hydrolase activity on a defined ubiquitin chain
    • Mechanism linking Golgi localization to proliferation unresolved
  2. 2019 High

    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

    PMID:30926795

    Open questions at the time
    • Ubiquitin chain linkage on Rab7 not defined
    • Ubiquitin ligase opposing USP32 on Rab7 unknown
    • Site of Rab7 ubiquitylation not mapped
  3. 2022 High

    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

    PMID:36476874

    Open questions at the time
    • 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
  4. 2021 Medium

    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

    PMID:34815782

    Open questions at the time
    • Direct deubiquitination of SLC35F2 not demonstrated
    • Mechanism by which a DUB promotes ERAD unresolved
    • Chain linkage involved not defined
  5. 2023 Medium

    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)

    PMID:36725886 PMID:36951484

    Open questions at the time
    • Rap1b interaction rests on single Co-IP without reciprocal validation
    • Direct vs indirect deubiquitination of Rab35 in cells not fully separated
    • Ubiquitylation sites unmapped
  6. 2024 Medium

    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

    PMID:39030175

    Open questions at the time
    • BAG3 ubiquitin chain type not specified
    • How BAG3 stabilization mechanistically activates RAF/MEK/ERK not resolved
    • Single-lab validation
  7. 2025 Medium

    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

    PMID:40379759

    Open questions at the time
    • USP32 phosphosite(s) not mapped in the narrative
    • Whether CDK5 regulates USP32 activity toward other substrates unknown
    • Structural basis of activation unresolved
  8. 2025 Low

    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

    PMID:40136417 PMID:40819507 PMID:41184228 PMID:41883014

    Open questions at the time
    • DAPK1 and SEMA4C rest on single Co-IP/pull-down without reciprocal validation
    • Direct vs indirect deubiquitination not always separated
    • Substrate ubiquitylation sites unmapped
  9. 2026 Medium

    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

    PMID:41356798

    Open questions at the time
    • 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
  10. 2025 Medium

    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)

    PMID:bio_10.1101_2025.04.19.649632

    Open questions at the time
    • Preprint, single lab not peer-reviewed
    • In vivo efficacy not established
    • Selectivity across full DUB family beyond profiled probes not exhaustively shown

Open questions

Synthesis pass · forward-looking unresolved questions
  • 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.
  • No structural model of substrate recognition
  • Substrate ubiquitylation sites largely unmapped
  • Determinants directing USP32 to Golgi vs endosome vs lysosome unknown

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140096 catalytic activity, acting on a protein 7 GO:0016787 hydrolase activity 4 GO:0098772 molecular function regulator activity 4
Localization
GO:0005768 endosome 2 GO:0005764 lysosome 1 GO:0005794 Golgi apparatus 1
Pathway
R-HSA-392499 Metabolism of proteins 6 R-HSA-5653656 Vesicle-mediated transport 2 R-HSA-9612973 Autophagy 1
Partners
BAG3LAMTOR1NRF2PD-L1PKM2RAB35RAB7ARAP1B

Evidence

Reading pass · 15 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2010 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. Fluorescent protection assay, BODIPY-TR staining, stable siRNA silencing with proliferation/migration readouts Mammalian genome Medium 20549504
2019 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. SILAC-based ubiquitome profiling, USP32 knockdown/knockout with late endosome morphology and cargo recycling readouts, mechanistic studies with RILP and retromer Nature communications High 30926795
2020 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. siRNA knockdown of USP32, western blot for SMAD2, in vitro and in vivo proliferation/migration/chemoresistance assays with rescue experiments International journal of biological sciences Low 32226309
2021 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. CRISPR-Cas9 genome-scale USP knockout screen for YM155 resistance, targeted validation with in vitro and in vivo SLC35F2 protein stability assays Theranostics Medium 34815782
2022 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. 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 Cell reports High 36476874
2023 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. iTRAQ proteomics, Co-IP, ubiquitination assay (K48-linkage), exosome secretion assays, loss-of-function experiments Oncogene Medium 36725886
2023 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. USP32 siRNA knockdown, ubiquitination assay for Rap1b, rescue experiments with Rap1b overexpression, Co-IP Acta biochimica et biophysica Sinica Low 36951484
2024 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. Interacting protein screen in H1299 cells, Co-IP, deubiquitination assay, rescue experiments, phosphorylation analysis of RAF/MEK/ERK Oncogenesis Medium 39030175
2025 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. Tandem affinity purification, mass spectrometry, RNA-Seq, in vitro and in vivo deubiquitination assays, CDK5 inhibitor and genetic ablation, patient-derived xenograft model Oncogene Medium 40379759
2025 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. Co-IP, ubiquitination assays (K48/K11 linkage-specific), USP32/PKM2 knockdown with glycolysis/mitochondrial readouts, cartilage-specific knockdown in rat TMJOA model Cell death & disease Medium 41184228
2025 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. DARTS-mass spectrometry, cellular thermal shift assay, IP-mass spectrometry, Co-IP, ubiquitination assay, in vivo orthotopic xenograft Current issues in molecular biology Medium 40136417
2025 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. Screening of 40 USPs for SEMA4C stability, Co-IP, ubiquitination assay (K48-linkage), rescue experiments Pathology, research and practice Low 40819507
2025 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. GST pull-down, Co-IP, deubiquitination assay, flow cytometry for macrophage/T cell phenotyping, transwell invasion, xenograft Journal of biochemical and molecular toxicology Low 41883014
2026 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. CRISPR/Cas9 DUB loss-of-function screen, immunoprecipitation, ubiquitination assay (K48-linkage), half-life assay, xenograft model Theranostics Medium 41356798
2025 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. Covalent inhibitor binding to active-site cysteine (Cys743), in vitro and cellular activity assays, endosomal morphology imaging, activity-based probe mass spectrometry bioRxivpreprint Medium bio_10.1101_2025.04.19.649632

Source papers

Stage 0 corpus · 20 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2019 USP32 regulates late endosomal transport and recycling through deubiquitylation of Rab7. Nature communications 65 30926795
2010 USP32 is an active, membrane-bound ubiquitin protease overexpressed in breast cancers. Mammalian genome : official journal of the International Mammalian Genome Society 52 20549504
2020 USP32 promotes tumorigenesis and chemoresistance in gastric carcinoma via upregulation of SMAD2. International journal of biological sciences 33 32226309
2017 Downregulation of USP32 inhibits cell proliferation, migration and invasion in human small cell lung cancer. Cell proliferation 30 28597490
2023 USP32 deubiquitinase: cellular functions, regulatory mechanisms, and potential as a cancer therapy target. Cell death discovery 25 37679322
2021 USP32 confers cancer cell resistance to YM155 via promoting ER-associated degradation of solute carrier protein SLC35F2. Theranostics 23 34815782
2022 USP32-regulated LAMTOR1 ubiquitination impacts mTORC1 activation and autophagy induction. Cell reports 22 36476874
2023 Deubiquitylation of Rab35 by USP32 promotes the transmission of imatinib resistance by enhancing exosome secretion in gastrointestinal stromal tumours. Oncogene 21 36725886
2024 USP32 facilitates non-small cell lung cancer progression via deubiquitinating BAG3 and activating RAF-MEK-ERK signaling pathway. Oncogenesis 15 39030175
2019 MicroRNA let-7a inhibits proliferation of breast cancer cell by downregulating USP32 expression. Translational cancer research 12 35116927
2023 High USP32 expression contributes to cancer progression and is correlated with immune infiltrates in hepatocellular carcinoma. BMC cancer 8 37957631
2023 Roles of the hsa_circ_0013880/USP32/Rap1b axis in the proliferation and apoptosis of acute myeloid leukemia cells. Acta biochimica et biophysica Sinica 7 36951484
2023 Understanding The Regulatory Role of USP32 and SHMT2 in The Progression of Gastric Cancer. Cell journal 6 37210642
2026 Single-cell RNA sequence analysis reveals USP32 as a therapeutic target to mitigate PD-L1-driven colorectal tumorigenesis in vitro and in vivo. Theranostics 3 41356798
2025 USP32 promotes temporomandibular joint osteoarthritis by modulating PKM2 stability and glycolytic metabolism in chondrocytes. Cell death & disease 2 41184228
2025 USP32 Promotes Colorectal Carcinoma Progression Through Activating NF-κB Signalling Pathway. Journal of cellular and molecular medicine 1 40122703
2025 Lycobetaine Has Therapeutic Efficacy in Lung Squamous Cell Carcinoma by Targeting USP32 to Trigger Ferroptosis. Current issues in molecular biology 1 40136417
2025 Phosphorylation of USP32 by CDK5 regulates Rap1 stability and therapeutic resistance in pancreatic ductal adenocarcinoma. Oncogene 1 40379759
2025 USP32 stabilizes SEMA4C to promote malignant behavior of colon cancer cells. Pathology, research and practice 1 40819507
2026 USP32 Promotes Cancer Cell Invasion, Macrophage M2 Polarization, and CD8+ T Cell Apoptosis in Gastric Cancer Through Upregulation of DAPK1. Journal of biochemical and molecular toxicology 0 41883014

Missed literature

Know a paper Affinage missed for USP32? Flag it for the maintainers and the community.

No submissions yet.