{"gene":"USP33","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":2009,"finding":"USP33 constitutively binds beta-arrestin2 and deubiquitinates it, opposing Mdm2-mediated ubiquitination. The USP33-beta-arrestin2 interaction is regulated by receptor class: 'class A' receptors (beta2AR) promote a beta-arrestin conformation that favors USP33 binding, while 'class B' receptors (V2R) promote a conformation that disfavors it. This reciprocal regulation with Mdm2 controls the stability and subcellular localization of receptor signalosomes.","method":"Co-immunoprecipitation, deubiquitination assays, receptor trafficking assays in HEK293 cells, receptor class comparison","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, functional deubiquitination assay, receptor class epistasis, replicated across multiple receptor systems in one rigorous study","pmids":["19363159"],"is_preprint":false},{"year":2009,"finding":"USP33 (along with USP20) constitutively associates with the beta2 adrenergic receptor (beta2AR), dissociates upon agonist stimulation, and reassociates upon prolonged treatment. This dynamic association deubiquitinates the receptor, inhibiting lysosomal trafficking and promoting recycling from late-endosomal compartments and resensitization at the cell surface.","method":"Co-immunoprecipitation of endogenous proteins, receptor recycling and degradation assays, subcellular fractionation","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP of endogenous proteins, functional trafficking assays, multiple orthogonal readouts in one study","pmids":["19424180"],"is_preprint":false},{"year":2009,"finding":"USP33 binds the Robo1 receptor and is required for Slit-induced redistribution of Robo1 from intracellular compartments to the plasma membrane, and for Slit-dependent inhibition of directional breast cancer cell migration.","method":"Co-immunoprecipitation, USP33 knockdown/rescue, cell migration assays, immunofluorescence localization of Robo1","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, loss-of-function with defined migration phenotype, receptor localization assay, multiple orthogonal methods","pmids":["19706539"],"is_preprint":false},{"year":2009,"finding":"USP33 interacts with the Robo1 receptor and is essential for commissural axon midline crossing and Slit responsiveness in vertebrate neural development.","method":"Co-immunoprecipitation, in vivo loss-of-function in chick embryo spinal cord, axon guidance assays","journal":"Nature neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP plus in vivo loss-of-function with defined axon guidance phenotype, independent confirmation of Robo1-USP33 interaction","pmids":["19684588"],"is_preprint":false},{"year":2007,"finding":"The ZnF UBP domain of USP33/VDU1 was solved by NMR spectroscopy. Unlike the related USP5 ZnF UBP domain, the USP33 ZnF UBP domain contains three zinc ions and does not bind ubiquitin.","method":"NMR spectroscopy, structure determination, ubiquitin-binding assay","journal":"Protein science : a publication of the Protein Society","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure determination with functional binding assay; single lab but direct structural and biochemical evidence","pmids":["17766394"],"is_preprint":false},{"year":2013,"finding":"USP33 interacts with and specifically deubiquitinates CP110 (a centriolar protein), but not other cyclin-F substrates. USP33 localizes to centrioles primarily in S and G2/M phases and antagonizes SCF(cyclin F)-mediated ubiquitination of CP110, thereby regulating centrosome duplication and ciliogenesis. USP33 ablation destabilizes CP110 and inhibits centrosome amplification.","method":"Co-immunoprecipitation, in vitro deubiquitination assay, cell-cycle-staged immunofluorescence localization, RNAi knockdown with centrosome/ciliogenesis phenotype readout, specificity comparison with other cyclin-F substrates","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro deubiquitination assay with substrate specificity controls, Co-IP, localization, loss-of-function phenotype; multiple orthogonal methods in a rigorous study","pmids":["23486064"],"is_preprint":false},{"year":2013,"finding":"USP33 accumulates on RALB-positive vesicles upon nutrient starvation and deubiquitylates RALB at Lys47. Deubiquitylated RALB preferentially interacts with EXO84 to drive RALB-EXO84-beclin-1 complex assembly and autophagosome formation, while ubiquitylated RALB favors SEC5 binding for innate immunity signaling.","method":"Ubiquitylation site mapping (Lys47), Co-immunoprecipitation, vesicle localization by immunofluorescence, epistasis experiments with EXO84/SEC5 effectors, autophagy assays","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — site-specific ubiquitination mapping, reciprocal Co-IP, localization, functional epistasis with effectors, multiple orthogonal methods","pmids":["24056301"],"is_preprint":false},{"year":2014,"finding":"USP33 deubiquitinates and stabilizes Robo1 in colorectal cancer cells, mediating the inhibitory effect of Slit2 on CRC cell migration.","method":"Co-immunoprecipitation, ubiquitination assay, USP33 knockdown with Robo1 protein stability readout, cell migration assays","journal":"International journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assay with functional migration readout, single lab","pmids":["25242263"],"is_preprint":false},{"year":2014,"finding":"USP33 mediates Slit-Robo signaling in lung cancer cells by stabilizing Robo1 protein; USP33 downregulation reduces Robo1 protein stability.","method":"Protein stability assay, USP33 knockdown, cell migration assays","journal":"Protein & cell","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — protein stability assay with functional readout, single lab, extends prior Robo1 findings to lung cancer","pmids":["24981056"],"is_preprint":false},{"year":2014,"finding":"USP33 degradation is controlled by the ubiquitin-proteasome system: the E3 ligase HERC2 polyubiquitinates USP33, and the p97 ATPase (with its Ufd1-Npl4 adaptor complex) is required for post-ubiquitination processing and degradation of USP33.","method":"Quantitative mass spectrometry, p97 knockdown/chemical inhibition, HERC2 identification, accumulation of polyubiquitinated USP33 upon p97 inhibition","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — quantitative MS, genetic and chemical inhibition of p97, identification of E3 ligase and adaptor complex, multiple orthogonal methods","pmids":["24855649"],"is_preprint":false},{"year":2011,"finding":"USP33 localizes broadly to the secretory pathway (ER-associated structures) in all splice variants. A specific insert within the catalytic domain mediates ER association, while a second insert suppresses Golgi localization; splice variant 3 excises eight amino acids from this second insert, enabling marked accumulation at the Golgi apparatus.","method":"GFP-USP33 isoform expression and live/fixed imaging, subcellular fractionation, domain deletion/insert mutagenesis, endogenous USP33 localization","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct localization with multiple isoforms and domain mutagenesis identifying specific inserts responsible for ER vs. Golgi targeting","pmids":["21801292"],"is_preprint":false},{"year":2019,"finding":"USP33 localizes to the outer mitochondrial membrane and deubiquitinates PRKN/parkin in a DUB-activity-dependent manner, preferentially removing K6, K11, K48, and K63-linked ubiquitin conjugates at Lys435 of PRKN. USP33 knockdown increases K63-linked PRKN ubiquitination under mitochondrial depolarization and enhances PRKN translocation to depolarized mitochondria, promoting mitophagy.","method":"Subcellular fractionation, Co-immunoprecipitation, in vitro and cellular deubiquitination assays, linkage-specific ubiquitin analysis, site-directed mutagenesis (Lys435), mitophagy assays","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro deubiquitination assay, site-directed mutagenesis, linkage specificity, subcellular localization, functional mitophagy readout; multiple orthogonal methods","pmids":["31432739"],"is_preprint":false},{"year":2019,"finding":"USP33 interacts with and stabilizes the phosphatase DUSP1 by inhibiting K48-linked polyubiquitination of DUSP1, thereby negatively regulating JNK activation and apoptosis in docetaxel-treated prostate cancer cells.","method":"Co-immunoprecipitation, ubiquitination assay (K48-linkage specific), JNK inhibitor rescue, USP33 knockdown/knockout with apoptosis and JNK phosphorylation readouts","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, linkage-specific ubiquitination assay, pharmacological and genetic epistasis, multiple orthogonal methods in one study","pmids":["31857702"],"is_preprint":false},{"year":2008,"finding":"USP33/VDU1 interacts with human selenium-binding protein-1 (hSP56) in a selenium-dependent manner; specifically, the full-length VDU1 binds the selenium-replete form of hSP56. The two proteins co-localize in the perinuclear region of LNCaP cells.","method":"Yeast two-hybrid screen, in vitro binding assay, subcellular co-localization by immunofluorescence","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — yeast two-hybrid plus in vitro binding and co-localization; selenium dependence demonstrated but functional consequence not fully established","pmids":["19118533"],"is_preprint":false},{"year":2020,"finding":"USP33 stabilizes the transcription factor SP1 by directly binding it and decreasing its ubiquitination, thereby upregulating c-Met expression in hepatocellular carcinoma cells.","method":"Co-immunoprecipitation, ubiquitination assay, RNA sequencing, luciferase reporter assay, USP33 knockdown with SP1 stability and c-Met expression readouts","journal":"Life sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, functional downstream readouts; single lab","pmids":["32835698"],"is_preprint":false},{"year":2022,"finding":"USP33 interacts with HIF-2alpha and deubiquitinates it to promote its stabilization in glioma stem cells, particularly under hypoxia. ERK1/2 activation upon hypoxia phosphorylates HIF-2alpha, enhancing its interaction with USP33. USP33 silencing disrupts glioma stem cell maintenance, reduces tumor vascularization, and inhibits glioblastoma growth.","method":"Co-immunoprecipitation, deubiquitination assay, USP33 knockdown, ERK1/2 inhibition epistasis, in vitro and in vivo tumor models","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, deubiquitination assay, epistasis with ERK1/2, in vitro and in vivo loss-of-function; multiple orthogonal methods","pmids":["35191554"],"is_preprint":false},{"year":2017,"finding":"The E3 ubiquitin ligase β-TrCP interacts with USP33 (requiring the WD40 motif of β-TrCP and amino acids 201-400 of USP33) independently of the classic β-TrCP binding motif, and promotes USP33 degradation via the ubiquitin-proteasome pathway.","method":"Co-immunoprecipitation, domain mapping, proteasome inhibitor rescue, USP33 protein level assay upon β-TrCP manipulation","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with domain mapping, proteasomal degradation assay; single lab","pmids":["28506875"],"is_preprint":false},{"year":2020,"finding":"USP33 stabilizes ATF3 protein via deubiquitylation; reduced USP33 in human microglia decreases ATF3 stability, leading to elevated proinflammatory signaling (TNF-α, NF-κB, IFN-β). This pathway is exploited by dengue virus via EV-delivered miR-148a that suppresses USP33 expression.","method":"In vivo ubiquitination assay, chase assay, knockdown/overexpression, dual luciferase reporter assay, immunoblotting","journal":"Journal of immunology (Baltimore, Md. : 1950)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo ubiquitination assay, protein stability chase, functional downstream readouts; single lab","pmids":["32848034"],"is_preprint":false},{"year":2021,"finding":"USP33 regulates the turnover of IRF9 via deubiquitylation; reduced USP33 (caused by SARS-CoV-2 Spike-induced exosomal miR-148a suppression) decreases IRF9 stability and promotes neuroinflammation in human microglia.","method":"Knockdown/overexpression, miRNA mimic/inhibitor, immunoblotting for IRF9 protein levels as readout of USP33-dependent deubiquitylation","journal":"Frontiers in immunology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, indirect evidence for USP33-IRF9 deubiquitylation with limited mechanistic follow-up","pmids":["33936086"],"is_preprint":false},{"year":2018,"finding":"USP33 deubiquitinates PPM1A (a Smad2/3 phosphatase), and this activity is required to restrain TGF-β/Smad2/3 signaling and radiation-induced EMT in lung cancer cells.","method":"Luciferase 3'UTR assay (validating USP33 as miR-3591-5p target), western blot for Smad2/3 phosphorylation, USP33/PPM1A rescue experiments","journal":"Cellular physiology and biochemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — functional deubiquitination of PPM1A inferred from rescue epistasis with USP33/PPM1A overexpression; single lab","pmids":["30308513"],"is_preprint":false},{"year":2021,"finding":"USP33 deubiquitinates and stabilizes c-Myc in prostate cancer cells, and this activity is regulated via a circ_0057558/miR-206/USP33 axis.","method":"Co-immunoprecipitation, deubiquitination assay for c-Myc, luciferase reporter assay, RNA pulldown for miR-206/circ_0057558 interaction","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP and deubiquitination assay for c-Myc substrate; single lab with moderate follow-up","pmids":["33718387"],"is_preprint":false},{"year":2023,"finding":"USP33 interacts with TGFBR2 (TGF-beta receptor 2), deubiquitinates it, and prevents its lysosomal degradation, thereby promoting TGFBR2 membrane accumulation and sustained TGF-β signaling. ZEB1, a TGF-β target, in turn transcriptionally upregulates USP33, forming a positive feedback loop.","method":"Mass spectrometry substrate screen, luciferase complementation assay, Co-IP, ubiquitination assay, lysosome inhibitor rescue, USP33 knockdown/overexpression","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS substrate identification, Co-IP, ubiquitination assay, lysosomal degradation rescue; single lab","pmids":["37322017"],"is_preprint":false},{"year":2023,"finding":"USP33 interacts with CTNNB1 (beta-catenin) and suppresses its ubiquitin-mediated degradation, stabilizing CTNNB1 and promoting pancreatic cancer cell survival and stemness.","method":"Co-immunoprecipitation, ubiquitination assay, CTNNB1 rescue experiments, cell proliferation and sphere formation assays","journal":"Cell biology international","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, rescue epistasis; single lab","pmids":["37076992"],"is_preprint":false},{"year":2023,"finding":"USP33 stabilizes PFKFB3 by suppressing ubiquitin-mediated degradation of PFKFB3, driving aerobic glycolysis in osteosarcoma cells.","method":"Co-immunoprecipitation, ubiquitination assay, USP33 knockdown with PFKFB3 protein stability and glycolysis readouts","journal":"American journal of cancer research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP and ubiquitination assay with functional metabolic readout; single lab","pmids":["37034227"],"is_preprint":false},{"year":2023,"finding":"USP33 catalyzes deubiquitination of JAK2, activating JAK2/STAT3 signaling; this is the mechanistic basis for USP33-driven inflammatory injury in LPS-treated cardiomyocytes.","method":"Co-immunoprecipitation, ubiquitination assay, dual luciferase reporter (miR-206 target validation), USP33/JAK2 rescue epistasis","journal":"Molecular and cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP and ubiquitination assay for JAK2; single lab with functional epistasis","pmids":["37256445"],"is_preprint":false},{"year":2024,"finding":"USP33 removes K27- and K48-linked ubiquitin chains from CBX2 at Lys277, stabilizing CBX2. Acetylation of CBX2 at K199 by GCN5 enhances its interaction with USP33 and promotes further deubiquitination.","method":"Proteomics and ubiquitinomics substrate screen, Co-immunoprecipitation, ubiquitin linkage-specific assays, acetylation mapping, GCN5 identification","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — coupled proteomics/ubiquitinomics, site-specific ubiquitination and acetylation mapping, Co-IP; multiple orthogonal methods in one study","pmids":["39256572"],"is_preprint":false},{"year":2024,"finding":"USP33 interacts with and deubiquitinates TRAF3 to upregulate its expression, and USP33-TRAF3 activates the NF-κB pathway in pancreatic ductal cells during acute pancreatitis.","method":"Co-immunoprecipitation, ubiquitination assay, immunofluorescence co-localization, USP33 knockdown with TRAF3 ubiquitination and NF-κB pathway readouts","journal":"Shock (Augusta, Ga.)","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP, cellular ubiquitination assay, pathway activation readout; single lab","pmids":["39637362"],"is_preprint":false},{"year":2024,"finding":"USP33 interacts with, deubiquitinates, and stabilizes integrin α6, mediating laminin-dependent adhesion, spreading, and migration of esophageal squamous cell carcinoma cells.","method":"Co-immunoprecipitation, deubiquitination assay, USP33 knockdown with integrin α6 stability, adhesion/migration, and in vivo metastasis readouts","journal":"Journal of cancer research and clinical oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, deubiquitination assay, in vitro and in vivo functional readouts; single lab","pmids":["39589547"],"is_preprint":false},{"year":2024,"finding":"USP33 stabilizes p53 by directly interacting with it and mediating its deubiquitination under DNA damage conditions. USP33 depletion increases p53 ubiquitination, destabilizes p53, impairs DNA damage responses (cell cycle arrest and apoptosis), and increases sensitivity to hepatocarcinogenesis in vivo in hepatocyte-specific USP33 knockout mice.","method":"Co-immunoprecipitation, in vitro and cellular deubiquitination assays, USP33 knockout (cell lines and hepatocyte-specific mouse), DNA damage response assays, DEN-induced hepatocarcinogenesis model","journal":"Cell proliferation","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, deubiquitination assay, in vivo mouse KO model with carcinogenesis readout; multiple orthogonal methods","pmids":["39694539"],"is_preprint":false},{"year":2025,"finding":"USP33 deubiquitinates and stabilizes SIN1 (an mTORC2 component), promoting mTORC2-AKT pathway activation and chemoresistance in pancreatic cancer. CDK1 directly phosphorylates USP33, enhancing its deubiquitinase activity toward SIN1.","method":"Co-immunoprecipitation, ubiquitination assay, CDK1 phosphorylation assay, CDK1 inhibition/genetic ablation, USP33 knockdown, in vivo tumor models","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, CDK1 kinase assay, in vivo models; single lab","pmids":["40695806"],"is_preprint":false},{"year":2025,"finding":"USP33 stabilizes TAP63 through K48-linked deubiquitination in triple-negative breast cancer cells, triggering autophagy and ferroptosis.","method":"Co-immunoprecipitation, ubiquitin chain analysis (K48-linkage), autophagy/ferroptosis assays (fluorescence probes, electron microscopy, biomarkers), in vivo xenograft","journal":"Cellular and molecular life sciences : CMLS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, K48-linkage specific ubiquitin assay, in vitro and in vivo functional readouts; single lab","pmids":["40801947"],"is_preprint":false},{"year":2022,"finding":"The hepatokine ERAP1 interacts with beta2 adrenergic receptor (ADRB2) and reduces its expression by decreasing USP33-mediated deubiquitination of ADRB2, thereby impairing ADRB2-stimulated insulin signaling in skeletal muscle.","method":"Co-immunoprecipitation, ADRB2 ubiquitination assay, USP33 knockdown, hepatic ERAP1 overexpression/knockdown in mice","journal":"Diabetes","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, in vivo hepatic manipulation; single lab establishing ERAP1-USP33-ADRB2 axis","pmids":["35192681"],"is_preprint":false},{"year":2025,"finding":"USP33 interacts with TRAF2 and stabilizes it through deubiquitination in pulmonary microvascular endothelial cells, promoting HPMEC pyroptosis during acute lung injury.","method":"Co-immunoprecipitation, ubiquitination assay, USP33 knockdown with pyroptosis and TRAF2 stability readouts","journal":"Histology and histopathology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, Co-IP and ubiquitination assay without detailed mechanistic follow-up","pmids":["39506557"],"is_preprint":false},{"year":2025,"finding":"USP33 deubiquitinates and stabilizes eEF1A1, preventing its ubiquitin-mediated degradation, which in turn promotes ILEI protein synthesis and EMT in hepatocellular carcinoma.","method":"CRISPR knockout screen, transcriptome sequencing, proteomic analysis, Co-immunoprecipitation, ubiquitination assay","journal":"International journal of biological macromolecules","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR screen, proteomic identification, Co-IP, ubiquitination assay; single lab","pmids":["42144151"],"is_preprint":false},{"year":2025,"finding":"USP33 deubiquitinates c-Myc at K48-linked chains, stabilizing c-Myc and enhancing glycolytic metabolism in ovarian cancer cells.","method":"Co-immunoprecipitation, K48-linkage specific ubiquitination assay, c-Myc rescue experiments, glycolysis assays","journal":"Biochimica et biophysica acta. General subjects","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and K48-linkage ubiquitination assay, c-Myc rescue epistasis; single lab","pmids":["40532745"],"is_preprint":false},{"year":2025,"finding":"USP33 interacts with and deubiquitinates CNR1 (cannabinoid receptor 1), promoting its stabilization and OPC differentiation via AKT/mTOR pathway activation in chronic cerebral hypoperfusion.","method":"Co-immunoprecipitation, ubiquitination assay, USP33 overexpression, in vivo BCAS model","journal":"Neuroscience bulletin","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, Co-IP and ubiquitination assay; limited mechanistic detail in abstract","pmids":["40679549"],"is_preprint":false},{"year":2026,"finding":"USP33 interacts with ATG7 and blocks K63-linked ubiquitination at ATG7 K48 (mouse K44), stabilizing ATG7. This USP33-ATG7 interaction promotes lysosomal degradation of FIS1 (a mitochondrial fission protein) via ATG7-mediated autophagy, thereby suppressing excessive FIS1-dependent mitochondrial fission in diabetic cardiomyopathy.","method":"DUB-targeting library screen, Co-immunoprecipitation, K63-linkage specific ubiquitination assay, USP33 silencing with FIS1 and mitochondrial function readouts","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — DUB library screen, Co-IP, linkage-specific ubiquitination assay, functional mitochondrial readouts; single lab","pmids":["42218158"],"is_preprint":false},{"year":2025,"finding":"USP33 deubiquitinates PAK1 to prevent its degradation in pancreatic cancer cells, contributing to gemcitabine resistance. USP33 mRNA stability is enhanced by METTL3 through m6A modification recognized by IGF2BP3.","method":"Co-immunoprecipitation, MeRIP-qPCR, RIP assay, USP33 knockdown with PAK1 ubiquitination and drug resistance readouts, xenograft model","journal":"Naunyn-Schmiedeberg's archives of pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, MeRIP-qPCR, in vivo xenograft; multiple orthogonal methods but single lab","pmids":["41108357"],"is_preprint":false},{"year":2025,"finding":"The zf-UBP and DUSP2 auxiliary domains of USP33 (and the homologous domains of USP20) are short linear motif (SLiM)-binding domains with similar binding profiles, explaining functional redundancy between USP33 and USP20. Consensus motifs recognized by these domains were defined.","method":"Proteomic-peptide phage display, peptide arrays, affinity measurements for SLiM-domain interactions","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic phage display, peptide arrays, and affinity measurements; preprint, not yet peer-reviewed","pmids":["bio_10.1101_2025.09.22.676098"],"is_preprint":true},{"year":2025,"finding":"USP33 suppresses LATS1 ubiquitination (via Co-IP and CHX stability assay), thereby repressing the Hippo-YAP pathway, and localizes in autophagosomes to promote ferritinophagy and ferroptosis in endometriosis.","method":"Co-immunoprecipitation, cycloheximide chase assay, LATS1 ubiquitination assay, immunofluorescence co-localization with autophagosomes, ferroptosis markers (ROS, Fe2+, MDA, GSH)","journal":"Gynecological endocrinology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, Co-IP and ubiquitination assay; limited replication","pmids":["41452077"],"is_preprint":false},{"year":2024,"finding":"USP33 deubiquitinates and stabilizes EPHB2 in retinoblastoma cells, activating Wnt/β-catenin signaling; USP33 knockdown reduces EPHB2 protein and suppresses retinoblastoma growth in vitro and in vivo.","method":"Ubiquitination assay, protein stability assay, Co-immunoprecipitation, xenograft model","journal":"Applied biochemistry and biotechnology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, ubiquitination assay with limited mechanistic depth","pmids":["40824480"],"is_preprint":false}],"current_model":"USP33 is a cytoplasmic deubiquitinating enzyme (USP family) that localizes to the ER, Golgi, outer mitochondrial membrane, and endosomes in an isoform- and context-dependent manner, and controls the stability and trafficking of a broad range of substrates—including GPCRs (beta2AR, Robo1), centrosomal proteins (CP110), signaling intermediates (PRKN/parkin, beta-arrestin2, RALB, HIF-2alpha, p53, DUSP1, SP1, JAK2, TRAF2/3, CBX2, SIN1, c-Myc, TGFBR2, ATG7, eEF1A1, integrin α6, and others)—by removing K6, K11, K27, K48, and/or K63-linked ubiquitin chains to prevent proteasomal or lysosomal degradation; its own levels are regulated by HERC2/p97-mediated degradation and β-TrCP-mediated ubiquitination, and its catalytic activity is enhanced by CDK1 phosphorylation, while its substrate specificity is in part determined by SLiM-binding auxiliary domains (zf-UBP, DUSP2) shared with paralog USP20."},"narrative":{"mechanistic_narrative":"USP33 is a deubiquitinating enzyme of the USP family that controls the stability, trafficking, and signaling output of a broad set of membrane receptors, signaling intermediates, and centrosomal proteins by removing ubiquitin conjugates to oppose proteasomal or lysosomal degradation [PMID:19424180, PMID:23486064, PMID:31432739]. A founding role lies in G-protein-coupled receptor signaling: USP33 (together with USP20) constitutively associates with the beta2 adrenergic receptor, dissociates upon agonism, and deubiquitinates the receptor to inhibit lysosomal trafficking and promote recycling and resensitization [PMID:19424180], and it deubiquitinates beta-arrestin2 in reciprocal opposition to Mdm2 in a manner gated by receptor class to set signalosome stability and localization [PMID:19363159]. USP33 also binds and deubiquitinates the Slit receptor Robo1, redistributing it to the plasma membrane and enabling Slit-dependent control of axon midline crossing and cell migration [PMID:19706539, PMID:19684588]. In cell-cycle and organelle control, USP33 localizes to centrioles in S/G2/M and deubiquitinates CP110 to antagonize SCF(cyclin F) and regulate centrosome duplication and ciliogenesis [PMID:23486064], deubiquitinates RALB at Lys47 on starvation-induced vesicles to bias effector choice toward EXO84-beclin-1 autophagosome assembly [PMID:24056301], and acts at the outer mitochondrial membrane to deubiquitinate parkin/PRKN at Lys435, restraining its translocation and mitophagy [PMID:31432739]. USP33 removes multiple chain types (K6, K11, K27, K48, K63) and shows substrate- and site-specific linkage selectivity [PMID:31432739, PMID:39256572]. It stabilizes a wide range of substrates including p53 under DNA damage—loss of USP33 destabilizes p53 and sensitizes mice to hepatocarcinogenesis [PMID:39694539]—as well as DUSP1 to dampen JNK-driven apoptosis [PMID:31857702], HIF-2alpha under hypoxia downstream of ERK1/2 phosphorylation [PMID:35191554], and CBX2 via K27/K48 chain removal enhanced by GCN5 acetylation [PMID:39256572]. USP33 localizes throughout the secretory pathway, with isoform-specific inserts in its catalytic domain directing ER versus Golgi targeting [PMID:21801292]. Its own abundance is set by ubiquitin-dependent turnover: HERC2 polyubiquitinates USP33 and p97-Ufd1-Npl4 processes it for degradation [PMID:24855649], and beta-TrCP binds and promotes its proteasomal degradation [PMID:28506875]; its catalytic activity is enhanced by CDK1 phosphorylation [PMID:40695806]. The zf-UBP domain binds three zinc ions and does not bind ubiquitin [PMID:17766394].","teleology":[{"year":2007,"claim":"Defining the structural and ubiquitin-binding properties of the USP33 zf-UBP domain established that, unlike the ubiquitin-binding zf-UBP of USP5, this auxiliary domain serves a non-canonical role.","evidence":"NMR structure determination and ubiquitin-binding assay of the USP33/VDU1 ZnF UBP domain","pmids":["17766394"],"confidence":"High","gaps":["Did not establish what ligand the three-zinc zf-UBP domain binds","Functional contribution to substrate selection not tested"]},{"year":2008,"claim":"Identification of selenium-dependent binding to hSP56 raised the question of how cellular selenium status couples to USP33 partner interactions.","evidence":"Yeast two-hybrid screen, in vitro binding, and co-localization in LNCaP cells","pmids":["19118533"],"confidence":"Medium","gaps":["Functional consequence of the interaction not established","No deubiquitination link demonstrated"]},{"year":2009,"claim":"A cluster of studies established USP33 as a GPCR-signaling deubiquitinase, resolving how receptor recycling, resensitization, and arrestin signalosome stability are controlled by ubiquitin removal.","evidence":"Endogenous Co-IP, receptor recycling/degradation assays, deubiquitination assays and receptor-class comparison in HEK293 (beta2AR, beta-arrestin2)","pmids":["19363159","19424180"],"confidence":"High","gaps":["Linkage specificity of chains removed from beta2AR/beta-arrestin2 not defined","Structural basis of receptor-class-gated arrestin recognition unresolved"]},{"year":2009,"claim":"Demonstration that USP33 binds Robo1 and is required for Slit responsiveness extended its receptor-trafficking role into axon guidance and cell migration.","evidence":"Co-IP plus in vivo loss-of-function in chick spinal cord and breast cancer migration assays with Robo1 localization readouts","pmids":["19706539","19684588"],"confidence":"High","gaps":["Direct deubiquitination of Robo1 chains not yet linkage-resolved here","Mechanism of Slit-triggered redistribution not fully defined"]},{"year":2011,"claim":"Mapping isoform-specific catalytic-domain inserts explained how USP33 splice variants partition between ER and Golgi, linking localization to function.","evidence":"GFP-isoform imaging, fractionation, and insert deletion mutagenesis","pmids":["21801292"],"confidence":"High","gaps":["Functional consequence of differential ER vs Golgi targeting on specific substrates not tested"]},{"year":2013,"claim":"Identification of CP110 and RALB as substrates established USP33 control over centrosome duplication/ciliogenesis and over the autophagy-versus-immunity decision through effector-biased deubiquitination.","evidence":"In vitro deubiquitination with substrate-specificity controls, site mapping (RALB Lys47), cell-cycle-staged localization, effector epistasis (EXO84/SEC5), centrosome and autophagy phenotypes","pmids":["23486064","24056301"],"confidence":"High","gaps":["How USP33 is recruited to centrioles vs RALB vesicles not defined","Regulation distinguishing the two functions unknown"]},{"year":2014,"claim":"Discovery of HERC2/p97 and beta-TrCP-mediated turnover answered how USP33's own abundance is set, while additional cancer studies confirmed Robo1 stabilization across tumor types.","evidence":"Quantitative MS, p97 inhibition, HERC2 identification; Co-IP and domain mapping for beta-TrCP; Robo1 stability/migration assays in colorectal and lung cancer","pmids":["24855649","28506875","25242263","24981056"],"confidence":"High","gaps":["Conditions triggering HERC2 vs beta-TrCP-mediated degradation not delineated","Robo1 chain linkage not resolved in cancer contexts"]},{"year":2019,"claim":"Defining mitochondrial and cytoplasmic substrates (PRKN, DUSP1) demonstrated linkage-selective deubiquitination and placed USP33 in mitophagy and stress-kinase/apoptosis control.","evidence":"OMM fractionation, in vitro/cellular deubiquitination, PRKN Lys435 mutagenesis with K6/K11/K48/K63 linkage analysis and mitophagy assays; K48-specific DUSP1 assay with JNK/apoptosis epistasis","pmids":["31432739","31857702"],"confidence":"High","gaps":["Determinants of multi-linkage selectivity at single sites not structurally explained","Whether the same active site distinguishes linkages in vivo unclear"]},{"year":2022,"claim":"Identification of HIF-2alpha as a substrate coupled to ERK1/2 phosphorylation, and of the ERAP1-ADRB2 axis, showed how upstream signaling and hepatokines modulate USP33-dependent substrate stabilization.","evidence":"Co-IP, deubiquitination assays, ERK1/2 inhibition epistasis, glioma stem cell tumor models; ADRB2 ubiquitination assay with hepatic ERAP1 manipulation in mice","pmids":["35191554","35192681"],"confidence":"High","gaps":["Mechanism by which substrate phosphorylation enhances USP33 binding not structurally defined","ERAP1 effect on USP33 catalysis vs recruitment unresolved"]},{"year":2024,"claim":"In vivo demonstration that USP33 stabilizes p53 under DNA damage, and mapping of K27/K48-selective CBX2 deubiquitination tuned by acetylation, broadened its role into DNA damage responses and chromatin regulation.","evidence":"Hepatocyte-specific USP33 knockout mice with DEN hepatocarcinogenesis and DNA damage assays; proteomics/ubiquitinomics, CBX2 K277 and K199 acetylation mapping with GCN5 identification","pmids":["39694539","39256572"],"confidence":"High","gaps":["How DNA damage signals route to USP33-p53 engagement unclear","Whether acetylation-enhanced recruitment generalizes to other substrates untested"]},{"year":2025,"claim":"Demonstration that CDK1 phosphorylates and activates USP33, and SLiM-binding profiling of its auxiliary domains, began to explain how its catalytic activity and substrate range are determined and why it overlaps with USP20.","evidence":"CDK1 kinase assay and inhibition with SIN1 deubiquitination/mTORC2 readouts; proteomic-peptide phage display and affinity measurements of zf-UBP/DUSP2 SLiM binding (preprint)","pmids":["40695806","bio_10.1101_2025.09.22.676098"],"confidence":"Medium","gaps":["Phosphosite on USP33 and mechanism of activity enhancement not fully mapped","Direct SLiM-substrate pairings within cells not validated"]},{"year":null,"claim":"How USP33 achieves substrate selectivity, linkage discrimination at single sites, and context-dependent recruitment to distinct organelles remains the central open question.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No catalytic-domain co-structure with a substrate","Rules governing recruitment to centriole vs mitochondria vs vesicles undefined","Physiological hierarchy among the many reported cancer substrates unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,5,11,12,25,28]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[5,11,12,25]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[10]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[10]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[11]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[5]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[6]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,2,15]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[6,11,36]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[5]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[9,16]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[1,21]}],"complexes":[],"partners":["ARRB2","ADRB2","ROBO1","CP110","RALB","PRKN","HERC2","BTRC"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8TEY7","full_name":"Ubiquitin carboxyl-terminal hydrolase 33","aliases":["Deubiquitinating enzyme 33","Ubiquitin thioesterase 33","Ubiquitin-specific-processing protease 33","VHL-interacting deubiquitinating enzyme 1","hVDU1"],"length_aa":942,"mass_kda":106.7,"function":"Deubiquitinating enzyme involved in various processes such as centrosome duplication, cellular migration and beta-2 adrenergic receptor/ADRB2 recycling. Involved in regulation of centrosome duplication by mediating deubiquitination of CCP110 in S and G2/M phase, leading to stabilize CCP110 during the period which centrioles duplicate and elongate. Involved in cell migration via its interaction with intracellular domain of ROBO1, leading to regulate the Slit signaling. Plays a role in commissural axon guidance cross the ventral midline of the neural tube in a Slit-dependent manner, possibly by mediating the deubiquitination of ROBO1. Acts as a regulator of G-protein coupled receptor (GPCR) signaling by mediating the deubiquitination of beta-arrestins (ARRB1 and ARRB2) and beta-2 adrenergic receptor (ADRB2). Plays a central role in ADRB2 recycling and resensitization after prolonged agonist stimulation by constitutively binding ADRB2, mediating deubiquitination of ADRB2 and inhibiting lysosomal trafficking of ADRB2. Upon dissociation, it is probably transferred to the translocated beta-arrestins, leading to beta-arrestins deubiquitination and disengagement from ADRB2. This suggests the existence of a dynamic exchange between the ADRB2 and beta-arrestins. Deubiquitinates DIO2, thereby regulating thyroid hormone regulation. Mediates deubiquitination of both 'Lys-48'- and 'Lys-63'-linked polyubiquitin chains","subcellular_location":"Golgi apparatus","url":"https://www.uniprot.org/uniprotkb/Q8TEY7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/USP33","classification":"Not Classified","n_dependent_lines":9,"n_total_lines":1208,"dependency_fraction":0.0074503311258278145},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/USP33","total_profiled":1310},"omim":[{"mim_id":"615146","title":"UBIQUITIN-SPECIFIC PROTEASE 33; USP33","url":"https://www.omim.org/entry/615146"},{"mim_id":"615143","title":"UBIQUITIN-SPECIFIC PROTEASE 20; USP20","url":"https://www.omim.org/entry/615143"},{"mim_id":"609544","title":"CENTRIOLAR COILED-COIL PROTEIN, 110-KD; CCP110","url":"https://www.omim.org/entry/609544"},{"mim_id":"602430","title":"ROUNDABOUT GUIDANCE RECEPTOR 1; ROBO1","url":"https://www.omim.org/entry/602430"},{"mim_id":"601413","title":"DEIODINASE, IODOTHYRONINE, TYPE II; DIO2","url":"https://www.omim.org/entry/601413"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Golgi apparatus","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/USP33"},"hgnc":{"alias_symbol":["KIAA1097","VDU1"],"prev_symbol":[]},"alphafold":{"accession":"Q8TEY7","domains":[{"cath_id":"3.30.40.10","chopping":"41-125","consensus_level":"medium","plddt":83.5605,"start":41,"end":125},{"cath_id":"3.90.70.10","chopping":"473-514_567-715","consensus_level":"high","plddt":93.6752,"start":473,"end":715},{"cath_id":"3.30.2230.10","chopping":"815-918","consensus_level":"high","plddt":89.324,"start":815,"end":918}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TEY7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TEY7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TEY7-F1-predicted_aligned_error_v6.png","plddt_mean":70.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=USP33","jax_strain_url":"https://www.jax.org/strain/search?query=USP33"},"sequence":{"accession":"Q8TEY7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8TEY7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8TEY7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TEY7"}},"corpus_meta":[{"pmid":"19424180","id":"PMC_19424180","title":"The deubiquitinases USP33 and USP20 coordinate beta2 adrenergic receptor recycling and resensitization.","date":"2009","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/19424180","citation_count":151,"is_preprint":false},{"pmid":"19363159","id":"PMC_19363159","title":"Beta-arrestin-dependent signaling and trafficking of 7-transmembrane receptors is reciprocally regulated by the deubiquitinase USP33 and the E3 ligase Mdm2.","date":"2009","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/19363159","citation_count":130,"is_preprint":false},{"pmid":"31432739","id":"PMC_31432739","title":"USP33 deubiquitinates PRKN/parkin and antagonizes its role in mitophagy.","date":"2019","source":"Autophagy","url":"https://pubmed.ncbi.nlm.nih.gov/31432739","citation_count":112,"is_preprint":false},{"pmid":"23486064","id":"PMC_23486064","title":"USP33 regulates centrosome biogenesis via deubiquitination of the 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General subjects","url":"https://pubmed.ncbi.nlm.nih.gov/40532745","citation_count":1,"is_preprint":false},{"pmid":"39320536","id":"PMC_39320536","title":"RNA binding protein ELAVL1-mediated USP33 stabilizes HIF1A to promote pathological proliferation, migration and angiogenesis of RECs.","date":"2024","source":"International ophthalmology","url":"https://pubmed.ncbi.nlm.nih.gov/39320536","citation_count":1,"is_preprint":false},{"pmid":"40679549","id":"PMC_40679549","title":"13-Docosenamide Enhances Oligodendrocyte Precursor Cell Differentiation via USP33-Mediated Deubiquitination of CNR1 in Chronic Cerebral Hypoperfusion.","date":"2025","source":"Neuroscience bulletin","url":"https://pubmed.ncbi.nlm.nih.gov/40679549","citation_count":1,"is_preprint":false},{"pmid":"39589547","id":"PMC_39589547","title":"USP33 is an integrin α6 deubiquitinase and promotes esophageal squamous cell carcinoma cell migration and metastasis.","date":"2024","source":"Journal of cancer research and clinical oncology","url":"https://pubmed.ncbi.nlm.nih.gov/39589547","citation_count":0,"is_preprint":false},{"pmid":"40824480","id":"PMC_40824480","title":"USP33 Facilitates Retinoblastoma Growth by Deubiquitinating and Stabilizing EPHB2 Protein.","date":"2025","source":"Applied biochemistry and biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/40824480","citation_count":0,"is_preprint":false},{"pmid":"41452077","id":"PMC_41452077","title":"Involvement of USP33 in ferritinophagy and ferroptosis in endometriosis through the Hippo-YAP pathway.","date":"2025","source":"Gynecological endocrinology : the official journal of the International Society of Gynecological Endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/41452077","citation_count":0,"is_preprint":false},{"pmid":"40695806","id":"PMC_40695806","title":"Phosphorylation of USP33 by CDK1 stabilizes the mTORC2 component SIN1.","date":"2025","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/40695806","citation_count":0,"is_preprint":false},{"pmid":"42218158","id":"PMC_42218158","title":"USP33 alleviates FIS1-dependent mitochondrial fission and cardiac microvascular injury in diabetic cardiomyopathy via deubiquitinating and stabilizing ATG7.","date":"2026","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/42218158","citation_count":0,"is_preprint":false},{"pmid":"39506557","id":"PMC_39506557","title":"USP33 promotes pulmonary microvascular endothelial cell pyroptosis by stabilizing TRAF2 through deubiquitination.","date":"2024","source":"Histology and histopathology","url":"https://pubmed.ncbi.nlm.nih.gov/39506557","citation_count":0,"is_preprint":false},{"pmid":"41661462","id":"PMC_41661462","title":"The role of deubiquitinase USP33 in colorectal cancer tumorigenesis and its potential as a therapeutic target predictor.","date":"2026","source":"Discover oncology","url":"https://pubmed.ncbi.nlm.nih.gov/41661462","citation_count":0,"is_preprint":false},{"pmid":"42144151","id":"PMC_42144151","title":"USP33 contributes to EMT and Lenvatinib resistance by enhancing eEF1A1-mediated ILEI protein synthesis in hepatocellular carcinoma.","date":"2026","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/42144151","citation_count":0,"is_preprint":false},{"pmid":"41108357","id":"PMC_41108357","title":"METTL3/IGF2BP3 axis promotes gemcitabine resistance of pancreatic cancer cells through regulating USP33-mediated PAK1 deubiquitination and degradation.","date":"2025","source":"Naunyn-Schmiedeberg's archives of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/41108357","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.09.22.676098","title":"Systematic Discovery of Motif-based Interactions of the Auxiliary Domains of USP Family Deubiquitinases","date":"2025-09-22","source":"bioRxiv","url":"https://doi.org/10.1101/2025.09.22.676098","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":27703,"output_tokens":9613,"usd":0.113652,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":19604,"output_tokens":4534,"usd":0.105685,"stage2_stop_reason":"end_turn"},"total_usd":0.219337,"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\": 2009,\n      \"finding\": \"USP33 constitutively binds beta-arrestin2 and deubiquitinates it, opposing Mdm2-mediated ubiquitination. The USP33-beta-arrestin2 interaction is regulated by receptor class: 'class A' receptors (beta2AR) promote a beta-arrestin conformation that favors USP33 binding, while 'class B' receptors (V2R) promote a conformation that disfavors it. This reciprocal regulation with Mdm2 controls the stability and subcellular localization of receptor signalosomes.\",\n      \"method\": \"Co-immunoprecipitation, deubiquitination assays, receptor trafficking assays in HEK293 cells, receptor class comparison\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, functional deubiquitination assay, receptor class epistasis, replicated across multiple receptor systems in one rigorous study\",\n      \"pmids\": [\"19363159\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"USP33 (along with USP20) constitutively associates with the beta2 adrenergic receptor (beta2AR), dissociates upon agonist stimulation, and reassociates upon prolonged treatment. This dynamic association deubiquitinates the receptor, inhibiting lysosomal trafficking and promoting recycling from late-endosomal compartments and resensitization at the cell surface.\",\n      \"method\": \"Co-immunoprecipitation of endogenous proteins, receptor recycling and degradation assays, subcellular fractionation\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP of endogenous proteins, functional trafficking assays, multiple orthogonal readouts in one study\",\n      \"pmids\": [\"19424180\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"USP33 binds the Robo1 receptor and is required for Slit-induced redistribution of Robo1 from intracellular compartments to the plasma membrane, and for Slit-dependent inhibition of directional breast cancer cell migration.\",\n      \"method\": \"Co-immunoprecipitation, USP33 knockdown/rescue, cell migration assays, immunofluorescence localization of Robo1\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, loss-of-function with defined migration phenotype, receptor localization assay, multiple orthogonal methods\",\n      \"pmids\": [\"19706539\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"USP33 interacts with the Robo1 receptor and is essential for commissural axon midline crossing and Slit responsiveness in vertebrate neural development.\",\n      \"method\": \"Co-immunoprecipitation, in vivo loss-of-function in chick embryo spinal cord, axon guidance assays\",\n      \"journal\": \"Nature neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP plus in vivo loss-of-function with defined axon guidance phenotype, independent confirmation of Robo1-USP33 interaction\",\n      \"pmids\": [\"19684588\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The ZnF UBP domain of USP33/VDU1 was solved by NMR spectroscopy. Unlike the related USP5 ZnF UBP domain, the USP33 ZnF UBP domain contains three zinc ions and does not bind ubiquitin.\",\n      \"method\": \"NMR spectroscopy, structure determination, ubiquitin-binding assay\",\n      \"journal\": \"Protein science : a publication of the Protein Society\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure determination with functional binding assay; single lab but direct structural and biochemical evidence\",\n      \"pmids\": [\"17766394\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"USP33 interacts with and specifically deubiquitinates CP110 (a centriolar protein), but not other cyclin-F substrates. USP33 localizes to centrioles primarily in S and G2/M phases and antagonizes SCF(cyclin F)-mediated ubiquitination of CP110, thereby regulating centrosome duplication and ciliogenesis. USP33 ablation destabilizes CP110 and inhibits centrosome amplification.\",\n      \"method\": \"Co-immunoprecipitation, in vitro deubiquitination assay, cell-cycle-staged immunofluorescence localization, RNAi knockdown with centrosome/ciliogenesis phenotype readout, specificity comparison with other cyclin-F substrates\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro deubiquitination assay with substrate specificity controls, Co-IP, localization, loss-of-function phenotype; multiple orthogonal methods in a rigorous study\",\n      \"pmids\": [\"23486064\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"USP33 accumulates on RALB-positive vesicles upon nutrient starvation and deubiquitylates RALB at Lys47. Deubiquitylated RALB preferentially interacts with EXO84 to drive RALB-EXO84-beclin-1 complex assembly and autophagosome formation, while ubiquitylated RALB favors SEC5 binding for innate immunity signaling.\",\n      \"method\": \"Ubiquitylation site mapping (Lys47), Co-immunoprecipitation, vesicle localization by immunofluorescence, epistasis experiments with EXO84/SEC5 effectors, autophagy assays\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — site-specific ubiquitination mapping, reciprocal Co-IP, localization, functional epistasis with effectors, multiple orthogonal methods\",\n      \"pmids\": [\"24056301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"USP33 deubiquitinates and stabilizes Robo1 in colorectal cancer cells, mediating the inhibitory effect of Slit2 on CRC cell migration.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, USP33 knockdown with Robo1 protein stability readout, cell migration assays\",\n      \"journal\": \"International journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assay with functional migration readout, single lab\",\n      \"pmids\": [\"25242263\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"USP33 mediates Slit-Robo signaling in lung cancer cells by stabilizing Robo1 protein; USP33 downregulation reduces Robo1 protein stability.\",\n      \"method\": \"Protein stability assay, USP33 knockdown, cell migration assays\",\n      \"journal\": \"Protein & cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — protein stability assay with functional readout, single lab, extends prior Robo1 findings to lung cancer\",\n      \"pmids\": [\"24981056\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"USP33 degradation is controlled by the ubiquitin-proteasome system: the E3 ligase HERC2 polyubiquitinates USP33, and the p97 ATPase (with its Ufd1-Npl4 adaptor complex) is required for post-ubiquitination processing and degradation of USP33.\",\n      \"method\": \"Quantitative mass spectrometry, p97 knockdown/chemical inhibition, HERC2 identification, accumulation of polyubiquitinated USP33 upon p97 inhibition\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — quantitative MS, genetic and chemical inhibition of p97, identification of E3 ligase and adaptor complex, multiple orthogonal methods\",\n      \"pmids\": [\"24855649\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"USP33 localizes broadly to the secretory pathway (ER-associated structures) in all splice variants. A specific insert within the catalytic domain mediates ER association, while a second insert suppresses Golgi localization; splice variant 3 excises eight amino acids from this second insert, enabling marked accumulation at the Golgi apparatus.\",\n      \"method\": \"GFP-USP33 isoform expression and live/fixed imaging, subcellular fractionation, domain deletion/insert mutagenesis, endogenous USP33 localization\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct localization with multiple isoforms and domain mutagenesis identifying specific inserts responsible for ER vs. Golgi targeting\",\n      \"pmids\": [\"21801292\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"USP33 localizes to the outer mitochondrial membrane and deubiquitinates PRKN/parkin in a DUB-activity-dependent manner, preferentially removing K6, K11, K48, and K63-linked ubiquitin conjugates at Lys435 of PRKN. USP33 knockdown increases K63-linked PRKN ubiquitination under mitochondrial depolarization and enhances PRKN translocation to depolarized mitochondria, promoting mitophagy.\",\n      \"method\": \"Subcellular fractionation, Co-immunoprecipitation, in vitro and cellular deubiquitination assays, linkage-specific ubiquitin analysis, site-directed mutagenesis (Lys435), mitophagy assays\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro deubiquitination assay, site-directed mutagenesis, linkage specificity, subcellular localization, functional mitophagy readout; multiple orthogonal methods\",\n      \"pmids\": [\"31432739\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"USP33 interacts with and stabilizes the phosphatase DUSP1 by inhibiting K48-linked polyubiquitination of DUSP1, thereby negatively regulating JNK activation and apoptosis in docetaxel-treated prostate cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay (K48-linkage specific), JNK inhibitor rescue, USP33 knockdown/knockout with apoptosis and JNK phosphorylation readouts\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, linkage-specific ubiquitination assay, pharmacological and genetic epistasis, multiple orthogonal methods in one study\",\n      \"pmids\": [\"31857702\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"USP33/VDU1 interacts with human selenium-binding protein-1 (hSP56) in a selenium-dependent manner; specifically, the full-length VDU1 binds the selenium-replete form of hSP56. The two proteins co-localize in the perinuclear region of LNCaP cells.\",\n      \"method\": \"Yeast two-hybrid screen, in vitro binding assay, subcellular co-localization by immunofluorescence\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — yeast two-hybrid plus in vitro binding and co-localization; selenium dependence demonstrated but functional consequence not fully established\",\n      \"pmids\": [\"19118533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"USP33 stabilizes the transcription factor SP1 by directly binding it and decreasing its ubiquitination, thereby upregulating c-Met expression in hepatocellular carcinoma cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, RNA sequencing, luciferase reporter assay, USP33 knockdown with SP1 stability and c-Met expression readouts\",\n      \"journal\": \"Life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, functional downstream readouts; single lab\",\n      \"pmids\": [\"32835698\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP33 interacts with HIF-2alpha and deubiquitinates it to promote its stabilization in glioma stem cells, particularly under hypoxia. ERK1/2 activation upon hypoxia phosphorylates HIF-2alpha, enhancing its interaction with USP33. USP33 silencing disrupts glioma stem cell maintenance, reduces tumor vascularization, and inhibits glioblastoma growth.\",\n      \"method\": \"Co-immunoprecipitation, deubiquitination assay, USP33 knockdown, ERK1/2 inhibition epistasis, in vitro and in vivo tumor models\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, deubiquitination assay, epistasis with ERK1/2, in vitro and in vivo loss-of-function; multiple orthogonal methods\",\n      \"pmids\": [\"35191554\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The E3 ubiquitin ligase β-TrCP interacts with USP33 (requiring the WD40 motif of β-TrCP and amino acids 201-400 of USP33) independently of the classic β-TrCP binding motif, and promotes USP33 degradation via the ubiquitin-proteasome pathway.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping, proteasome inhibitor rescue, USP33 protein level assay upon β-TrCP manipulation\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with domain mapping, proteasomal degradation assay; single lab\",\n      \"pmids\": [\"28506875\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"USP33 stabilizes ATF3 protein via deubiquitylation; reduced USP33 in human microglia decreases ATF3 stability, leading to elevated proinflammatory signaling (TNF-α, NF-κB, IFN-β). This pathway is exploited by dengue virus via EV-delivered miR-148a that suppresses USP33 expression.\",\n      \"method\": \"In vivo ubiquitination assay, chase assay, knockdown/overexpression, dual luciferase reporter assay, immunoblotting\",\n      \"journal\": \"Journal of immunology (Baltimore, Md. : 1950)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo ubiquitination assay, protein stability chase, functional downstream readouts; single lab\",\n      \"pmids\": [\"32848034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP33 regulates the turnover of IRF9 via deubiquitylation; reduced USP33 (caused by SARS-CoV-2 Spike-induced exosomal miR-148a suppression) decreases IRF9 stability and promotes neuroinflammation in human microglia.\",\n      \"method\": \"Knockdown/overexpression, miRNA mimic/inhibitor, immunoblotting for IRF9 protein levels as readout of USP33-dependent deubiquitylation\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, indirect evidence for USP33-IRF9 deubiquitylation with limited mechanistic follow-up\",\n      \"pmids\": [\"33936086\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"USP33 deubiquitinates PPM1A (a Smad2/3 phosphatase), and this activity is required to restrain TGF-β/Smad2/3 signaling and radiation-induced EMT in lung cancer cells.\",\n      \"method\": \"Luciferase 3'UTR assay (validating USP33 as miR-3591-5p target), western blot for Smad2/3 phosphorylation, USP33/PPM1A rescue experiments\",\n      \"journal\": \"Cellular physiology and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — functional deubiquitination of PPM1A inferred from rescue epistasis with USP33/PPM1A overexpression; single lab\",\n      \"pmids\": [\"30308513\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP33 deubiquitinates and stabilizes c-Myc in prostate cancer cells, and this activity is regulated via a circ_0057558/miR-206/USP33 axis.\",\n      \"method\": \"Co-immunoprecipitation, deubiquitination assay for c-Myc, luciferase reporter assay, RNA pulldown for miR-206/circ_0057558 interaction\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP and deubiquitination assay for c-Myc substrate; single lab with moderate follow-up\",\n      \"pmids\": [\"33718387\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP33 interacts with TGFBR2 (TGF-beta receptor 2), deubiquitinates it, and prevents its lysosomal degradation, thereby promoting TGFBR2 membrane accumulation and sustained TGF-β signaling. ZEB1, a TGF-β target, in turn transcriptionally upregulates USP33, forming a positive feedback loop.\",\n      \"method\": \"Mass spectrometry substrate screen, luciferase complementation assay, Co-IP, ubiquitination assay, lysosome inhibitor rescue, USP33 knockdown/overexpression\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS substrate identification, Co-IP, ubiquitination assay, lysosomal degradation rescue; single lab\",\n      \"pmids\": [\"37322017\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP33 interacts with CTNNB1 (beta-catenin) and suppresses its ubiquitin-mediated degradation, stabilizing CTNNB1 and promoting pancreatic cancer cell survival and stemness.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, CTNNB1 rescue experiments, cell proliferation and sphere formation assays\",\n      \"journal\": \"Cell biology international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, rescue epistasis; single lab\",\n      \"pmids\": [\"37076992\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP33 stabilizes PFKFB3 by suppressing ubiquitin-mediated degradation of PFKFB3, driving aerobic glycolysis in osteosarcoma cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, USP33 knockdown with PFKFB3 protein stability and glycolysis readouts\",\n      \"journal\": \"American journal of cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP and ubiquitination assay with functional metabolic readout; single lab\",\n      \"pmids\": [\"37034227\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP33 catalyzes deubiquitination of JAK2, activating JAK2/STAT3 signaling; this is the mechanistic basis for USP33-driven inflammatory injury in LPS-treated cardiomyocytes.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, dual luciferase reporter (miR-206 target validation), USP33/JAK2 rescue epistasis\",\n      \"journal\": \"Molecular and cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP and ubiquitination assay for JAK2; single lab with functional epistasis\",\n      \"pmids\": [\"37256445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP33 removes K27- and K48-linked ubiquitin chains from CBX2 at Lys277, stabilizing CBX2. Acetylation of CBX2 at K199 by GCN5 enhances its interaction with USP33 and promotes further deubiquitination.\",\n      \"method\": \"Proteomics and ubiquitinomics substrate screen, Co-immunoprecipitation, ubiquitin linkage-specific assays, acetylation mapping, GCN5 identification\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — coupled proteomics/ubiquitinomics, site-specific ubiquitination and acetylation mapping, Co-IP; multiple orthogonal methods in one study\",\n      \"pmids\": [\"39256572\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP33 interacts with and deubiquitinates TRAF3 to upregulate its expression, and USP33-TRAF3 activates the NF-κB pathway in pancreatic ductal cells during acute pancreatitis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, immunofluorescence co-localization, USP33 knockdown with TRAF3 ubiquitination and NF-κB pathway readouts\",\n      \"journal\": \"Shock (Augusta, Ga.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP, cellular ubiquitination assay, pathway activation readout; single lab\",\n      \"pmids\": [\"39637362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP33 interacts with, deubiquitinates, and stabilizes integrin α6, mediating laminin-dependent adhesion, spreading, and migration of esophageal squamous cell carcinoma cells.\",\n      \"method\": \"Co-immunoprecipitation, deubiquitination assay, USP33 knockdown with integrin α6 stability, adhesion/migration, and in vivo metastasis readouts\",\n      \"journal\": \"Journal of cancer research and clinical oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, deubiquitination assay, in vitro and in vivo functional readouts; single lab\",\n      \"pmids\": [\"39589547\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP33 stabilizes p53 by directly interacting with it and mediating its deubiquitination under DNA damage conditions. USP33 depletion increases p53 ubiquitination, destabilizes p53, impairs DNA damage responses (cell cycle arrest and apoptosis), and increases sensitivity to hepatocarcinogenesis in vivo in hepatocyte-specific USP33 knockout mice.\",\n      \"method\": \"Co-immunoprecipitation, in vitro and cellular deubiquitination assays, USP33 knockout (cell lines and hepatocyte-specific mouse), DNA damage response assays, DEN-induced hepatocarcinogenesis model\",\n      \"journal\": \"Cell proliferation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, deubiquitination assay, in vivo mouse KO model with carcinogenesis readout; multiple orthogonal methods\",\n      \"pmids\": [\"39694539\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP33 deubiquitinates and stabilizes SIN1 (an mTORC2 component), promoting mTORC2-AKT pathway activation and chemoresistance in pancreatic cancer. CDK1 directly phosphorylates USP33, enhancing its deubiquitinase activity toward SIN1.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, CDK1 phosphorylation assay, CDK1 inhibition/genetic ablation, USP33 knockdown, in vivo tumor models\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, CDK1 kinase assay, in vivo models; single lab\",\n      \"pmids\": [\"40695806\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP33 stabilizes TAP63 through K48-linked deubiquitination in triple-negative breast cancer cells, triggering autophagy and ferroptosis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitin chain analysis (K48-linkage), autophagy/ferroptosis assays (fluorescence probes, electron microscopy, biomarkers), in vivo xenograft\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, K48-linkage specific ubiquitin assay, in vitro and in vivo functional readouts; single lab\",\n      \"pmids\": [\"40801947\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The hepatokine ERAP1 interacts with beta2 adrenergic receptor (ADRB2) and reduces its expression by decreasing USP33-mediated deubiquitination of ADRB2, thereby impairing ADRB2-stimulated insulin signaling in skeletal muscle.\",\n      \"method\": \"Co-immunoprecipitation, ADRB2 ubiquitination assay, USP33 knockdown, hepatic ERAP1 overexpression/knockdown in mice\",\n      \"journal\": \"Diabetes\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, in vivo hepatic manipulation; single lab establishing ERAP1-USP33-ADRB2 axis\",\n      \"pmids\": [\"35192681\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP33 interacts with TRAF2 and stabilizes it through deubiquitination in pulmonary microvascular endothelial cells, promoting HPMEC pyroptosis during acute lung injury.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, USP33 knockdown with pyroptosis and TRAF2 stability readouts\",\n      \"journal\": \"Histology and histopathology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, Co-IP and ubiquitination assay without detailed mechanistic follow-up\",\n      \"pmids\": [\"39506557\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP33 deubiquitinates and stabilizes eEF1A1, preventing its ubiquitin-mediated degradation, which in turn promotes ILEI protein synthesis and EMT in hepatocellular carcinoma.\",\n      \"method\": \"CRISPR knockout screen, transcriptome sequencing, proteomic analysis, Co-immunoprecipitation, ubiquitination assay\",\n      \"journal\": \"International journal of biological macromolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR screen, proteomic identification, Co-IP, ubiquitination assay; single lab\",\n      \"pmids\": [\"42144151\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP33 deubiquitinates c-Myc at K48-linked chains, stabilizing c-Myc and enhancing glycolytic metabolism in ovarian cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, K48-linkage specific ubiquitination assay, c-Myc rescue experiments, glycolysis assays\",\n      \"journal\": \"Biochimica et biophysica acta. General subjects\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and K48-linkage ubiquitination assay, c-Myc rescue epistasis; single lab\",\n      \"pmids\": [\"40532745\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP33 interacts with and deubiquitinates CNR1 (cannabinoid receptor 1), promoting its stabilization and OPC differentiation via AKT/mTOR pathway activation in chronic cerebral hypoperfusion.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, USP33 overexpression, in vivo BCAS model\",\n      \"journal\": \"Neuroscience bulletin\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, Co-IP and ubiquitination assay; limited mechanistic detail in abstract\",\n      \"pmids\": [\"40679549\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"USP33 interacts with ATG7 and blocks K63-linked ubiquitination at ATG7 K48 (mouse K44), stabilizing ATG7. This USP33-ATG7 interaction promotes lysosomal degradation of FIS1 (a mitochondrial fission protein) via ATG7-mediated autophagy, thereby suppressing excessive FIS1-dependent mitochondrial fission in diabetic cardiomyopathy.\",\n      \"method\": \"DUB-targeting library screen, Co-immunoprecipitation, K63-linkage specific ubiquitination assay, USP33 silencing with FIS1 and mitochondrial function readouts\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — DUB library screen, Co-IP, linkage-specific ubiquitination assay, functional mitochondrial readouts; single lab\",\n      \"pmids\": [\"42218158\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP33 deubiquitinates PAK1 to prevent its degradation in pancreatic cancer cells, contributing to gemcitabine resistance. USP33 mRNA stability is enhanced by METTL3 through m6A modification recognized by IGF2BP3.\",\n      \"method\": \"Co-immunoprecipitation, MeRIP-qPCR, RIP assay, USP33 knockdown with PAK1 ubiquitination and drug resistance readouts, xenograft model\",\n      \"journal\": \"Naunyn-Schmiedeberg's archives of pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, MeRIP-qPCR, in vivo xenograft; multiple orthogonal methods but single lab\",\n      \"pmids\": [\"41108357\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The zf-UBP and DUSP2 auxiliary domains of USP33 (and the homologous domains of USP20) are short linear motif (SLiM)-binding domains with similar binding profiles, explaining functional redundancy between USP33 and USP20. Consensus motifs recognized by these domains were defined.\",\n      \"method\": \"Proteomic-peptide phage display, peptide arrays, affinity measurements for SLiM-domain interactions\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic phage display, peptide arrays, and affinity measurements; preprint, not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.09.22.676098\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP33 suppresses LATS1 ubiquitination (via Co-IP and CHX stability assay), thereby repressing the Hippo-YAP pathway, and localizes in autophagosomes to promote ferritinophagy and ferroptosis in endometriosis.\",\n      \"method\": \"Co-immunoprecipitation, cycloheximide chase assay, LATS1 ubiquitination assay, immunofluorescence co-localization with autophagosomes, ferroptosis markers (ROS, Fe2+, MDA, GSH)\",\n      \"journal\": \"Gynecological endocrinology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, Co-IP and ubiquitination assay; limited replication\",\n      \"pmids\": [\"41452077\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP33 deubiquitinates and stabilizes EPHB2 in retinoblastoma cells, activating Wnt/β-catenin signaling; USP33 knockdown reduces EPHB2 protein and suppresses retinoblastoma growth in vitro and in vivo.\",\n      \"method\": \"Ubiquitination assay, protein stability assay, Co-immunoprecipitation, xenograft model\",\n      \"journal\": \"Applied biochemistry and biotechnology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, ubiquitination assay with limited mechanistic depth\",\n      \"pmids\": [\"40824480\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"USP33 is a cytoplasmic deubiquitinating enzyme (USP family) that localizes to the ER, Golgi, outer mitochondrial membrane, and endosomes in an isoform- and context-dependent manner, and controls the stability and trafficking of a broad range of substrates—including GPCRs (beta2AR, Robo1), centrosomal proteins (CP110), signaling intermediates (PRKN/parkin, beta-arrestin2, RALB, HIF-2alpha, p53, DUSP1, SP1, JAK2, TRAF2/3, CBX2, SIN1, c-Myc, TGFBR2, ATG7, eEF1A1, integrin α6, and others)—by removing K6, K11, K27, K48, and/or K63-linked ubiquitin chains to prevent proteasomal or lysosomal degradation; its own levels are regulated by HERC2/p97-mediated degradation and β-TrCP-mediated ubiquitination, and its catalytic activity is enhanced by CDK1 phosphorylation, while its substrate specificity is in part determined by SLiM-binding auxiliary domains (zf-UBP, DUSP2) shared with paralog USP20.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"USP33 is a deubiquitinating enzyme of the USP family that controls the stability, trafficking, and signaling output of a broad set of membrane receptors, signaling intermediates, and centrosomal proteins by removing ubiquitin conjugates to oppose proteasomal or lysosomal degradation [#1, #5, #11]. A founding role lies in G-protein-coupled receptor signaling: USP33 (together with USP20) constitutively associates with the beta2 adrenergic receptor, dissociates upon agonism, and deubiquitinates the receptor to inhibit lysosomal trafficking and promote recycling and resensitization [#1], and it deubiquitinates beta-arrestin2 in reciprocal opposition to Mdm2 in a manner gated by receptor class to set signalosome stability and localization [#0]. USP33 also binds and deubiquitinates the Slit receptor Robo1, redistributing it to the plasma membrane and enabling Slit-dependent control of axon midline crossing and cell migration [#2, #3]. In cell-cycle and organelle control, USP33 localizes to centrioles in S/G2/M and deubiquitinates CP110 to antagonize SCF(cyclin F) and regulate centrosome duplication and ciliogenesis [#5], deubiquitinates RALB at Lys47 on starvation-induced vesicles to bias effector choice toward EXO84-beclin-1 autophagosome assembly [#6], and acts at the outer mitochondrial membrane to deubiquitinate parkin/PRKN at Lys435, restraining its translocation and mitophagy [#11]. USP33 removes multiple chain types (K6, K11, K27, K48, K63) and shows substrate- and site-specific linkage selectivity [#11, #25]. It stabilizes a wide range of substrates including p53 under DNA damage—loss of USP33 destabilizes p53 and sensitizes mice to hepatocarcinogenesis [#28]—as well as DUSP1 to dampen JNK-driven apoptosis [#12], HIF-2alpha under hypoxia downstream of ERK1/2 phosphorylation [#15], and CBX2 via K27/K48 chain removal enhanced by GCN5 acetylation [#25]. USP33 localizes throughout the secretory pathway, with isoform-specific inserts in its catalytic domain directing ER versus Golgi targeting [#10]. Its own abundance is set by ubiquitin-dependent turnover: HERC2 polyubiquitinates USP33 and p97-Ufd1-Npl4 processes it for degradation [#9], and beta-TrCP binds and promotes its proteasomal degradation [#16]; its catalytic activity is enhanced by CDK1 phosphorylation [#29]. The zf-UBP domain binds three zinc ions and does not bind ubiquitin [#4].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Defining the structural and ubiquitin-binding properties of the USP33 zf-UBP domain established that, unlike the ubiquitin-binding zf-UBP of USP5, this auxiliary domain serves a non-canonical role.\",\n      \"evidence\": \"NMR structure determination and ubiquitin-binding assay of the USP33/VDU1 ZnF UBP domain\",\n      \"pmids\": [\"17766394\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish what ligand the three-zinc zf-UBP domain binds\", \"Functional contribution to substrate selection not tested\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identification of selenium-dependent binding to hSP56 raised the question of how cellular selenium status couples to USP33 partner interactions.\",\n      \"evidence\": \"Yeast two-hybrid screen, in vitro binding, and co-localization in LNCaP cells\",\n      \"pmids\": [\"19118533\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of the interaction not established\", \"No deubiquitination link demonstrated\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"A cluster of studies established USP33 as a GPCR-signaling deubiquitinase, resolving how receptor recycling, resensitization, and arrestin signalosome stability are controlled by ubiquitin removal.\",\n      \"evidence\": \"Endogenous Co-IP, receptor recycling/degradation assays, deubiquitination assays and receptor-class comparison in HEK293 (beta2AR, beta-arrestin2)\",\n      \"pmids\": [\"19363159\", \"19424180\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Linkage specificity of chains removed from beta2AR/beta-arrestin2 not defined\", \"Structural basis of receptor-class-gated arrestin recognition unresolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Demonstration that USP33 binds Robo1 and is required for Slit responsiveness extended its receptor-trafficking role into axon guidance and cell migration.\",\n      \"evidence\": \"Co-IP plus in vivo loss-of-function in chick spinal cord and breast cancer migration assays with Robo1 localization readouts\",\n      \"pmids\": [\"19706539\", \"19684588\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct deubiquitination of Robo1 chains not yet linkage-resolved here\", \"Mechanism of Slit-triggered redistribution not fully defined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Mapping isoform-specific catalytic-domain inserts explained how USP33 splice variants partition between ER and Golgi, linking localization to function.\",\n      \"evidence\": \"GFP-isoform imaging, fractionation, and insert deletion mutagenesis\",\n      \"pmids\": [\"21801292\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of differential ER vs Golgi targeting on specific substrates not tested\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identification of CP110 and RALB as substrates established USP33 control over centrosome duplication/ciliogenesis and over the autophagy-versus-immunity decision through effector-biased deubiquitination.\",\n      \"evidence\": \"In vitro deubiquitination with substrate-specificity controls, site mapping (RALB Lys47), cell-cycle-staged localization, effector epistasis (EXO84/SEC5), centrosome and autophagy phenotypes\",\n      \"pmids\": [\"23486064\", \"24056301\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How USP33 is recruited to centrioles vs RALB vesicles not defined\", \"Regulation distinguishing the two functions unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Discovery of HERC2/p97 and beta-TrCP-mediated turnover answered how USP33's own abundance is set, while additional cancer studies confirmed Robo1 stabilization across tumor types.\",\n      \"evidence\": \"Quantitative MS, p97 inhibition, HERC2 identification; Co-IP and domain mapping for beta-TrCP; Robo1 stability/migration assays in colorectal and lung cancer\",\n      \"pmids\": [\"24855649\", \"28506875\", \"25242263\", \"24981056\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conditions triggering HERC2 vs beta-TrCP-mediated degradation not delineated\", \"Robo1 chain linkage not resolved in cancer contexts\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defining mitochondrial and cytoplasmic substrates (PRKN, DUSP1) demonstrated linkage-selective deubiquitination and placed USP33 in mitophagy and stress-kinase/apoptosis control.\",\n      \"evidence\": \"OMM fractionation, in vitro/cellular deubiquitination, PRKN Lys435 mutagenesis with K6/K11/K48/K63 linkage analysis and mitophagy assays; K48-specific DUSP1 assay with JNK/apoptosis epistasis\",\n      \"pmids\": [\"31432739\", \"31857702\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Determinants of multi-linkage selectivity at single sites not structurally explained\", \"Whether the same active site distinguishes linkages in vivo unclear\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identification of HIF-2alpha as a substrate coupled to ERK1/2 phosphorylation, and of the ERAP1-ADRB2 axis, showed how upstream signaling and hepatokines modulate USP33-dependent substrate stabilization.\",\n      \"evidence\": \"Co-IP, deubiquitination assays, ERK1/2 inhibition epistasis, glioma stem cell tumor models; ADRB2 ubiquitination assay with hepatic ERAP1 manipulation in mice\",\n      \"pmids\": [\"35191554\", \"35192681\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which substrate phosphorylation enhances USP33 binding not structurally defined\", \"ERAP1 effect on USP33 catalysis vs recruitment unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"In vivo demonstration that USP33 stabilizes p53 under DNA damage, and mapping of K27/K48-selective CBX2 deubiquitination tuned by acetylation, broadened its role into DNA damage responses and chromatin regulation.\",\n      \"evidence\": \"Hepatocyte-specific USP33 knockout mice with DEN hepatocarcinogenesis and DNA damage assays; proteomics/ubiquitinomics, CBX2 K277 and K199 acetylation mapping with GCN5 identification\",\n      \"pmids\": [\"39694539\", \"39256572\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How DNA damage signals route to USP33-p53 engagement unclear\", \"Whether acetylation-enhanced recruitment generalizes to other substrates untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstration that CDK1 phosphorylates and activates USP33, and SLiM-binding profiling of its auxiliary domains, began to explain how its catalytic activity and substrate range are determined and why it overlaps with USP20.\",\n      \"evidence\": \"CDK1 kinase assay and inhibition with SIN1 deubiquitination/mTORC2 readouts; proteomic-peptide phage display and affinity measurements of zf-UBP/DUSP2 SLiM binding (preprint)\",\n      \"pmids\": [\"40695806\", \"bio_10.1101_2025.09.22.676098\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Phosphosite on USP33 and mechanism of activity enhancement not fully mapped\", \"Direct SLiM-substrate pairings within cells not validated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How USP33 achieves substrate selectivity, linkage discrimination at single sites, and context-dependent recruitment to distinct organelles remains the central open question.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No catalytic-domain co-structure with a substrate\", \"Rules governing recruitment to centriole vs mitochondria vs vesicles undefined\", \"Physiological hierarchy among the many reported cancer substrates unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 5, 11, 12, 25, 28]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [5, 11, 12, 25]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [11]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 2, 15]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [6, 11, 36]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [9, 16]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [1, 21]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ARRB2\", \"ADRB2\", \"ROBO1\", \"CP110\", \"RALB\", \"PRKN\", \"HERC2\", \"BTRC\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":9,"faith_total":9,"faith_pct":100.0}}