{"gene":"USP7","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":2004,"finding":"HAUSP/USP7 deubiquitinates and stabilizes both p53 and MDM2. Partial RNAi knockdown of HAUSP destabilizes p53, but nearly complete ablation stabilizes p53 indirectly by destabilizing MDM2 (which undergoes self-ubiquitination when HAUSP is absent). This reveals a dynamic feedback loop where HAUSP preferentially stabilizes MDM2 in normal cells.","method":"RNAi knockdown, co-immunoprecipitation, in vivo ubiquitination assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal functional data with multiple RNAi conditions, replicated across cell lines including HeLa controls, multiple orthogonal methods","pmids":["15053880"],"is_preprint":false},{"year":2006,"finding":"The N-terminal TRAF-like domain of USP7 binds two closely spaced 4-residue sites in both p53 (residues 359–367) and MDM2 (residues 147–159), with a preference for a P/AXXS motif; Trp165 in the USP7 binding pocket is critical. Co-crystal structures of USP7 N-terminal domain with p53 and MDM2 peptides were determined, and the same surface also binds EBNA1, explaining competitive interactions.","method":"X-ray co-crystal structures, mutagenesis, peptide binding assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures plus mutagenesis validation, multiple peptides co-crystallized","pmids":["16474402"],"is_preprint":false},{"year":2006,"finding":"USP7/HAUSP interacts with and deubiquitinates FOXO4 in response to oxidative stress, negatively regulating FOXO4 transcriptional activity towards endogenous promoters. The monoubiquitination of FOXO4 drives its nuclear relocalization and increased transcriptional activity; USP7 reverses this without affecting FOXO4 protein half-life.","method":"Co-immunoprecipitation, in vivo and in vitro deubiquitination assays, nuclear localization imaging, transcriptional reporter assays","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, direct deubiquitination assays, functional transcriptional readouts, multiple orthogonal methods","pmids":["16964248"],"is_preprint":false},{"year":2005,"finding":"HAUSP interacts with and directly deubiquitinates HDMX (MDM4), stabilizing it. HAUSP activity is required for normal HDMX protein levels, and the balance between HAUSP and HDM2 determines HDMX stability. ATM-dependent phosphorylation of HDMX and HDM2 reduces their affinity for HAUSP, contributing to their DNA-damage-induced destabilization.","method":"Co-immunoprecipitation, in vivo and in vitro deubiquitination assays, RNAi knockdown, DNA damage treatments","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct deubiquitination assays, reciprocal Co-IP, mechanistic link to DNA damage response, replicated with multiple methods","pmids":["15916963"],"is_preprint":false},{"year":2008,"finding":"HAUSP/USP7 deubiquitinates PTEN, opposing its nuclear import. PML nuclear bodies coordinate PTEN localization through DAXX, which opposes HAUSP activity toward PTEN, so that PML-DAXX-HAUSP constitute a molecular network controlling PTEN deubiquitination and nuclear-cytoplasmic trafficking.","method":"Co-immunoprecipitation, in vivo deubiquitination assays, nuclear fractionation, RNAi knockdown, rescue experiments","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods, mechanistic pathway placement, rescue experiments, published in Nature","pmids":["18716620"],"is_preprint":false},{"year":2011,"finding":"The 64 kDa C-terminal region of USP7 contains five ubiquitin-like (Ubl) domains organized in 2-1-2 units. The last di-Ubl unit (HUBL-45) activates USP7 ~100-fold by binding a 'switching' loop in the catalytic domain that promotes ubiquitin binding. The metabolic enzyme GMPS allosterically hyperactivates USP7 by binding the first three Ubl domains (HUBL-123) and stabilizing the HUBL-45-dependent active state.","method":"Crystal structure of USP7 C-terminal Ubl domains, in vitro deubiquitinase activity assays, domain deletion/mutagenesis","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with in vitro activity assays and domain mutagenesis, multiple orthogonal methods in one study","pmids":["21981925"],"is_preprint":false},{"year":2003,"finding":"USP7 is a monomeric protein with four structural domains: an N-terminal p53-binding domain, a catalytic domain, and two C-terminal domains. The N-terminal domain binds both p53 and EBNA1 (EBNA1 with ~10-fold higher affinity), while ICP0 binds a distinct C-terminal domain (residues 599–801). Purified USP7 is active for cleaving both linear ubiquitin and ubiquitin conjugated to EBNA1.","method":"Partial proteolysis with MALDI-TOF-MS domain mapping, affinity pulldowns, tryptophan fluorescence binding assays, gel filtration, in vitro deubiquitinase assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzyme reconstitution, domain mapping by proteolysis/MS, binding constants determined by fluorescence, multiple orthogonal methods","pmids":["14506283"],"is_preprint":false},{"year":2007,"finding":"A trimeric complex of p53, MDM2, and HAUSP can exist in vivo, despite the mutually exclusive binding of p53 and MDM2 to the USP7 TRAF domain, because MDM2 acts as a bridge. HAUSP can deubiquitinate p53 in trans via MDM2-mediated bridging even when direct p53-HAUSP interaction is disrupted by a p53 binding-deficient mutation.","method":"Co-immunoprecipitation in vivo and in vitro, structure-based mutagenesis, ubiquitination assays","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — structure-guided mutagenesis plus Co-IP and functional ubiquitination assays, single lab","pmids":["17525743"],"is_preprint":false},{"year":2009,"finding":"Hausp knockout mice die between embryonic days E6.5 and E7.5 with p53 activation. Lethality is caused by reduced proliferation and developmental arrest partly attributable to p53 activation. Deletion of p53 extends but does not completely rescue embryonic development of hausp knockouts, indicating both p53-dependent and p53-independent essential functions of HAUSP in vivo.","method":"Conditional and conventional knockout mice, genetic epistasis (hausp/p53 double knockout), embryo analysis","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic epistasis with double-knockout rescue, well-defined developmental phenotype","pmids":["19946331"],"is_preprint":false},{"year":2012,"finding":"USP7 stability is regulated by CK2-mediated phosphorylation at serine 18, which stabilizes the USP7S isoform and thus maintains MDM2 stability and p53 repression in unstressed cells. After ionizing radiation, ATM-dependent phosphatase PPM1G dephosphorylates USP7S, leading to its downregulation, MDM2 destabilization, and p53 accumulation.","method":"Phosphorylation mapping, kinase/phosphatase assays, RNAi, IR treatment, Western blotting","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — identifies writer (CK2) and eraser (PPM1G) of USP7 phosphorylation, multiple methods, functional pathway consequences defined","pmids":["22361354"],"is_preprint":false},{"year":2011,"finding":"Kaposi's sarcoma-associated herpesvirus vIRF4 inhibits HAUSP through a bilateral belt-type interaction: vif1 peptide binds the HAUSP TRAF domain blocking substrate binding, while vif2 peptide binds both the TRAF and catalytic domains to suppress deubiquitinase activity. Crystal structures of the HAUSP-vIRF4 complexes were determined.","method":"X-ray crystal structure, peptide competition assays, deubiquitinase activity assays, cell-based apoptosis/xenograft assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures plus functional validation with peptide inhibitors and cell-based assays","pmids":["22056774"],"is_preprint":false},{"year":2016,"finding":"HAUSP/USP7 deubiquitinates and stabilizes N-Myc (MYCN) in neuroblastoma. HAUSP interacts with N-Myc; HAUSP overexpression induces N-Myc deubiquitination and stabilization, while USP7 RNAi or genetic ablation (Usp7 knockout in mouse brain) destabilizes N-Myc and inhibits its function.","method":"Co-immunoprecipitation, in vivo deubiquitination assays, RNAi knockdown, conditional knockout mice, xenograft models, USP7 inhibitors","journal":"Nature medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic knockout, reciprocal Co-IP, direct deubiquitination assays, xenograft validation, multiple orthogonal approaches","pmids":["27618649"],"is_preprint":false},{"year":2017,"finding":"Small-molecule inhibitors GNE-6640 and GNE-6776 non-covalently target USP7 12 Å from the catalytic cysteine, attenuating ubiquitin binding rather than directly blocking the active site. Structural studies showed these compounds interact with acidic residues mediating hydrogen bonds with ubiquitin Lys48, and NMR with isotopically labeled di-ubiquitin chains showed USP7 preferentially binds free Lys48 side chains, protracted depolymerization of K48-linked chains relative to K63-linked chains.","method":"NMR-based screening, co-crystal structures, differential isotope-labeled di-ubiquitin NMR binding assays, in vitro deubiquitinase activity assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — co-crystal structures, NMR with engineered substrates, in vitro enzymatic assays, multiple orthogonal methods","pmids":["29045385"],"is_preprint":false},{"year":2015,"finding":"Genotoxic stress stimulates Cry1 phosphorylation and its deubiquitination by HAUSP/USP7, stabilizing Cry1 and shifting circadian clock phase. USP7 deubiquitinates Cry1 to protect it from degradation following DNA damage.","method":"Co-immunoprecipitation, in vivo deubiquitination assays, clock phase measurements, DNA damage treatments","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and deubiquitination assays, functional clock phenotype, single lab","pmids":["25756610"],"is_preprint":false},{"year":2015,"finding":"USP7 deubiquitinates Ci/Gli transcription factors in the Hedgehog pathway, inhibiting their Slimb-Cul1 and Hib-Cul3 E3-ligase-mediated ubiquitination and degradation. Hh stimulation promotes Usp7 binding to Ci. Usp7 forms a complex with GMPS to promote Hh pathway activity, and the mammalian homolog HAUSP similarly regulates Gli ubiquitination and stability.","method":"Co-immunoprecipitation, in vivo ubiquitination assays, Drosophila genetics, mammalian cell assays","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods across two organisms, direct ubiquitination assays, genetic epistasis in Drosophila","pmids":["26120032"],"is_preprint":false},{"year":2015,"finding":"USP7 controls Chk1 protein stability by direct deubiquitination. Depletion or inhibition of USP7 lowers Chk1 levels; overexpression of catalytically active (but not catalytic mutant) USP7 elevates Chk1 levels and increases its half-life. Catalytic mutant USP7 fails to deubiquitinate Chk1 in vivo and in vitro. This effect is independent of USP7's known stabilization of Claspin.","method":"RNAi, overexpression of wild-type vs. catalytic mutant USP7, in vivo and in vitro deubiquitination assays, half-life measurements","journal":"Cell cycle","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct in vitro deubiquitination assay plus in vivo confirmation, mutagenesis controls, single lab","pmids":["25483066"],"is_preprint":false},{"year":2015,"finding":"USP7 maintains Rad18 protein stability by direct deubiquitination. USP7 associates with Rad18 via a consensus USP7-binding motif (PVDS or similar). Loss of USP7 destabilizes Rad18, compromises UV-induced PCNA mono-ubiquitination and Pol η recruitment, and impairs DNA damage tolerance. USP7 can disassemble Rad18-dependent poly-ubiquitin chains in vitro and in vivo.","method":"Co-immunoprecipitation, in vitro and in vivo deubiquitination assays, RNAi, UV irradiation, PCNA ubiquitination assays, replication fork analysis","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vitro reconstitution of deubiquitination, multiple functional readouts, USP7-binding motif identified, multiple orthogonal methods","pmids":["25961918"],"is_preprint":false},{"year":2013,"finding":"USP7 directly interacts with MCM-BP via the PSTS(155-158) motif engaging the TRAF domain binding pocket of USP7. USP7 knockout leads to slowed S phase progression and accumulation of MCM proteins on chromatin due to a defect in MCM complex unloading during mid-to-late S phase. USP7 works with MCM-BP to facilitate MCM unloading from chromatin at the end of S phase.","method":"Co-immunoprecipitation, structural analysis of USP7-MCM-BP interaction, USP7 knockout cell analysis, chromatin fractionation, DNA replication assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — structural interaction analysis, knockout phenotype with specific mechanistic readout, multiple orthogonal methods","pmids":["24190967"],"is_preprint":false},{"year":2016,"finding":"HAUSP deubiquitinates HIF-1α to increase its stability and promotes EMT/metastasis. Hypoxia induces K63-linked polyubiquitination of HAUSP at K443, enhancing its functions. K63-polyubiquitinated HAUSP interacts with ubiquitin receptor CBP to specifically mediate H3K56 acetylation at HIF-1α target gene promoters. HectH9 is the E3 ligase responsible for K63-ubiquitination of HAUSP.","method":"Co-immunoprecipitation, in vivo deubiquitination assays, ChIP-seq, mutagenesis, RNAi knockdown, metastasis assays","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods, direct deubiquitination assays, ChIP-seq, single lab","pmids":["27934968"],"is_preprint":false},{"year":2016,"finding":"Trip12 functions as an E3 ubiquitin ligase for USP7/HAUSP, controlling USP7 protein stability through the ubiquitin-proteasome pathway. Trip12 knockdown increases USP7-mediated stabilization of p53, 53BP1, and Chk1; Trip12 overexpression phenocopies USP7 knockdown (increased S-phase cells).","method":"Co-immunoprecipitation, in vivo ubiquitination assays, RNAi, cell cycle analysis","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — identifies E3 ligase for USP7 with Co-IP and functional epistasis, single lab","pmids":["27800609"],"is_preprint":false},{"year":2010,"finding":"USP7 modulates chromatin remodeling important for base excision repair of oxidative lesions. USP7 siRNA knockdown does not change the levels or activity of BER enzymes but significantly reduces chromatin DNA accessibility and consequently the rate of repair of oxidative lesions.","method":"siRNA knockdown, chromatin accessibility assays, BER activity assays","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — specific chromatin accessibility measurement plus BER assays, functional readout without direct substrate identification, single lab","pmids":["21138959"],"is_preprint":false},{"year":2014,"finding":"HAUSP/USP7 deubiquitinates retinoblastoma protein (Rb), removing K48-linked ubiquitin chains from Rb and stabilizing it from proteasomal degradation. HAUSP deubiquitination of Rb leads to increased G1 cell population. In glioma, MDM2 becomes the preferred USP7 substrate over Rb, facilitating Rb degradation.","method":"Co-immunoprecipitation, in vivo and in vitro deubiquitination assays, cell cycle analysis, immunohistochemistry","journal":"The FEBS journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct in vitro deubiquitination, Co-IP, functional cell cycle readout, single lab","pmids":["24823443"],"is_preprint":false},{"year":2007,"finding":"USP7 interacts with and deubiquitinates the mitotic checkpoint protein Chfr, stabilizing it. USP7 removes ubiquitin from autoubiquitinated Chfr both in vivo and in vitro. The USP7-Chfr interaction was identified by immunoaffinity purification and mass spectrometry.","method":"Immunoaffinity purification and mass spectrometry, co-immunoprecipitation, in vivo and in vitro deubiquitination assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — identified by AP-MS, confirmed by Co-IP and direct deubiquitination assay, single lab","pmids":["17442268"],"is_preprint":false},{"year":2019,"finding":"Usp7 deubiquitinates and stabilizes Yorkie (Yki) in Drosophila, positively regulating Hippo pathway output. Hippo pathway activation attenuates Usp7-Yki binding. The mammalian homolog HAUSP similarly stabilizes YAP by modulating its ubiquitination and degradation.","method":"Co-immunoprecipitation, in vivo ubiquitination assays, Drosophila genetics, mammalian cell assays","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cross-species validation (Drosophila + mammalian), direct ubiquitination assays, single lab","pmids":["30679505"],"is_preprint":false},{"year":2018,"finding":"USP7 and USP47 regulate NLRP3 inflammasome activation in macrophages. Chemical inhibition of USP7 and USP47 blocks inflammasome formation by preventing ASC oligomerization and speck formation, independently of transcription. Inhibition alters the ubiquitination status of NLRP3 itself. USP7 and USP47 activity increases in response to inflammasome activators.","method":"CRISPR/Cas9 knockdown, chemical inhibition, ASC speck formation assays, ubiquitination assays, IL-1β/IL-18 cytokine measurement","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — CRISPR knockdown with functional readout, ubiquitination status changes for NLRP3 shown, but direct deubiquitination not reconstituted in vitro; replicated with chemical and genetic tools","pmids":["30206189"],"is_preprint":false},{"year":2021,"finding":"USP7 interacts with and supports PP2A active localization in the cytoplasm. Inhibition of USP7 or PP2A produces similar phosphoproteomic changes including widespread increase in CDK1 substrate phosphorylation. USP7 inhibition causes untimely CDK1 activation throughout the cell cycle, leading to DNA damage; this toxicity is rescued by lowering CDK1 activity or chemically activating PP2A.","method":"Co-immunoprecipitation, quantitative phosphoproteomics, kinase activity assays, genetic and chemical rescue experiments","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphoproteomics, Co-IP, genetic rescue, functional cell cycle readouts, single lab","pmids":["33856059"],"is_preprint":false},{"year":2018,"finding":"USP7 deubiquitinates and stabilizes NOTCH1 in T-ALL cells. USP7 interacts with NOTCH1 via its MATH and UBL domains. USP7 knockdown increases NOTCH1 ubiquitination and reduces NOTCH1 protein levels, suppressing T-ALL proliferation in vitro and in vivo.","method":"Co-immunoprecipitation, in vivo and in vitro deubiquitination assays, RNAi, domain mapping, xenograft models","journal":"Signal transduction and targeted therapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, direct in vitro deubiquitination, domain mapping, in vivo xenograft, single lab","pmids":["30370059"],"is_preprint":false},{"year":2017,"finding":"USP7 stabilizes HIV-1 Tat protein through deubiquitination. USP7 knockdown by CRISPR-Cas9 reduces Tat protein levels and reduces virus production. Endogenous USP7 levels increase after HIV-1 infection in human T-cells.","method":"CRISPR-Cas9 knockout, USP7 inhibitor treatment, co-immunoprecipitation, virus production assays","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR knockout with functional viral readout, inhibitor confirmation, single lab","pmids":["28280111"],"is_preprint":false},{"year":2010,"finding":"USP7 promotes p53 sequence-specific DNA binding activity through interaction with the C-terminal regulatory region of p53, independent of USP7's N-terminal domain and independent of deubiquitylation activity. The USP7 C-terminal domain alone is sufficient to promote p53 binding to target sequences and p21 induction.","method":"In vitro DNA binding assays, domain mutagenesis, overexpression of catalytically inactive USP7, p21 expression assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — catalytic mutant controls distinguish this from deubiquitination, in vitro and in vivo DNA binding assays, single lab","pmids":["20885946"],"is_preprint":false},{"year":2020,"finding":"USP7 deubiquitinates and stabilizes PD-L1 by direct interaction. Abrogation of USP7 attenuates PD-L1/PD-1 interaction and sensitizes cancer cells to T cell killing in vitro and in vivo.","method":"Co-immunoprecipitation, in vivo ubiquitination assays, T cell killing assays, xenograft models","journal":"Acta pharmaceutica Sinica. B","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct interaction shown by Co-IP, functional immune assays, in vivo confirmation, single lab","pmids":["33777676"],"is_preprint":false},{"year":2021,"finding":"USP7 preferentially interacts with polyQ-expanded androgen receptor (AR) and deubiquitinates it, reducing AR ubiquitination and promoting mutant AR aggregation. USP7 knockdown or monoallelic Usp7 knockout in mice reduces mutant AR aggregation and ameliorates SBMA motor phenotypes. USP7 also differentially interacts with mutant Huntingtin in striatum and frontal cortex.","method":"Quantitative proteomics, co-immunoprecipitation, in vivo ubiquitination assays, RNAi, conditional knockout mice, behavioral assays","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — quantitative proteomics identification, direct deubiquitination assay, in vivo genetic rescue in mice, multiple orthogonal approaches","pmids":["33170804"],"is_preprint":false},{"year":2020,"finding":"USP7 deubiquitinates NEDD4L (an E3 ubiquitin ligase for SMAD2), stabilizing NEDD4L and thereby reducing SMAD2 levels, which decreases autophagy-mediated clearance of misfolded proteins. In ALS models across C. elegans, Drosophila, and mammalian cells, USP7 inhibition protects against proteotoxicity through the SMAD2/TGF-β pathway.","method":"Genetic screen in C. elegans, Drosophila and mammalian cell validation, co-immunoprecipitation, deubiquitination assays, autophagy assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — cross-species genetic epistasis, direct deubiquitination of NEDD4L demonstrated, functional pathway placement, multiple organisms","pmids":["33106424"],"is_preprint":false},{"year":2019,"finding":"USP7 stabilizes FBXO38 dependent on its catalytic activity. FBXO38 in turn stabilizes KIF20B (a kinesin required for cytokinesis) independent of an SCF complex. Depletion of USP7 or FBXO38 reduces KIF20B levels and causes cytokinetic defects that can be rescued by restoring FBXO38 or KIF20B.","method":"Affinity purification-mass spectrometry, BioID protein interaction profiling, Co-immunoprecipitation, RNAi, rescue experiments, cytokinesis imaging","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — AP-MS and BioID identification, functional rescue experiments, specific cytokinetic phenotype, single lab","pmids":["30804394"],"is_preprint":false},{"year":2018,"finding":"USP7 interacts with and stabilizes DDX24 and DHX40 (DEAD/DEAH-box RNA helicases) dependent on its catalytic activity. USP7 interacts with PPM1G, TRIP12, and USP11 through its TRAF domain binding pocket (via P/A/ExxS motifs), whereas DHX40 uses a distinct binding site in the Ubl2 domain.","method":"Affinity purification-mass spectrometry, USP7 TRAF-domain binding-pocket mutants, RNAi and inhibitor treatments, Western blotting","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — AP-MS with domain mutant discrimination, catalytic activity dependence shown, single lab","pmids":["30367141"],"is_preprint":false},{"year":2022,"finding":"USP7 deubiquitinates and stabilizes EZH2 in prostate cancer cells. USP7 knockdown decreases EZH2 protein and inhibits EZH2-dependent transcriptional repression. The USP7/EZH2 axis promotes cancer cell migration, invasion, and sphere formation.","method":"Co-immunoprecipitation, in vivo ubiquitination assays, RNAi, ectopic EZH2 rescue, cell migration/invasion assays","journal":"Genetics and molecular biology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP and in vivo ubiquitination, rescue experiment, functional phenotype, single lab","pmids":["32453339"],"is_preprint":false},{"year":2022,"finding":"USP7 deubiquitinates and stabilizes HO-1 (heme oxygenase-1) to regulate redox homeostasis. Under arsenic exposure, USP7 itself becomes ubiquitinated at K476, which promotes its binding to HO-1, leading to enhanced HO-1 deubiquitination and stabilization. HO-1 is ubiquitinated at K243 under resting conditions.","method":"Co-immunoprecipitation, in vivo deubiquitination assays, ubiquitination site mapping, arsenic stress experiments, knockdown","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — identifies specific ubiquitination sites on both USP7 and HO-1, direct deubiquitination assays, single lab","pmids":["35821281"],"is_preprint":false},{"year":2022,"finding":"USP7 deubiquitinates and stabilizes Raf-1. USP7 binds the PVDS motif in the CR2 region of Raf-1 and removes K6-, K11-, K27-, K33-, and K48-linked polyubiquitin chains. USP7-mediated deubiquitination of Raf-1 decreases Raf-1 threonine phosphorylation and inhibits ERK1/2 signaling, thereby restraining the G2/M transition in lung adenocarcinoma cells.","method":"Bioinformatics motif analysis, Co-immunoprecipitation, in vivo ubiquitination assays, phosphorylation assays, ERK signaling assays, cell cycle analysis","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP and ubiquitination assays, functional signaling and cell cycle readouts, single lab, no in vitro reconstitution","pmids":["35948545"],"is_preprint":false},{"year":2022,"finding":"USP7 deubiquitinates and stabilizes TAZ by selectively removing K48-linked ubiquitination, antagonizing β-TRCP-mediated ubiquitin-proteasomal degradation and enhancing TAZ nuclear retention and transcriptional output in HNSCC.","method":"siRNA/cDNA library screens, Co-immunoprecipitation, in vivo ubiquitination assays, nuclear localization assays, xenograft and PDX models","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic screen plus direct deubiquitination assays, in vivo validation, single lab","pmids":["35931679"],"is_preprint":false},{"year":2022,"finding":"Natural compound eupalinolide B (EB) covalently and allosterically inhibits USP7 at Cys576 in the non-catalytic HUBL domain (not the active site). Co-crystal structure reveals this previously undisclosed binding site. Modification of Cys576 allosterically inhibits USP7, causing ubiquitination-dependent degradation of Keap1, leading to Nrf2-dependent transcription of anti-neuroinflammatory genes in microglia.","method":"Co-crystal structure, covalent binding assays, ubiquitination assays, Nrf2 target gene expression, in vivo mouse models of neurodegeneration","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 1 / Strong — co-crystal structure of novel allosteric site, covalent mechanism defined, functional pathway demonstrated in vitro and in vivo","pmids":["35947662"],"is_preprint":false},{"year":2024,"finding":"USP7 directly deubiquitinates KRAS by binding KRAS via its TRAF domain and removing K48-linked polyubiquitin chains from KRAS K147, stabilizing KRAS and promoting NSCLC proliferation. USP7 also stabilizes oncogenic KRAS mutants through deubiquitination.","method":"Co-immunoprecipitation, in vivo and in vitro deubiquitination assays, domain binding assays, ubiquitination site mutagenesis, NSCLC cell proliferation assays","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct in vitro deubiquitination, specific ubiquitination site identified (K147), functional proliferation assay, single lab","pmids":["39499616"],"is_preprint":false},{"year":2021,"finding":"USP7 deubiquitinates and stabilizes BCR-ABL fusion protein in CML cells. USP7 interacts with BCR-ABL and blocks its polyubiquitination; knockdown or chemical inhibition of USP7 triggers BCR-ABL degradation and suppresses downstream signaling.","method":"DUB screen, co-immunoprecipitation, in vivo ubiquitination assays, RNAi, USP7 inhibitors, apoptosis assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — DUB screen identified, Co-IP and ubiquitination confirmed, functional leukemia cell assays, single lab","pmids":["33963175"],"is_preprint":false},{"year":2019,"finding":"USP7 deubiquitinates and stabilizes Maf proteins (c-Maf, MafA, MafB) in myeloma cells, promoting their transcriptional activity as measured by luciferase assays and Maf-regulated gene expression.","method":"Mass spectrometry identification, co-immunoprecipitation, in vivo ubiquitination assays, RNAi, luciferase reporter assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — MS-based identification plus Co-IP and ubiquitination, functional transcriptional readout, single lab","pmids":["31822558"],"is_preprint":false},{"year":2019,"finding":"USP7 deubiquitinates and stabilizes SIRT1, cleaving K48-linked polyubiquitin chains. USP7 is physically associated with SIRT1 as identified by mass spectrometry. USP7 enzymatic inhibition leads to decreased SIRT1 expression and accumulation of SIRT1 polyubiquitination.","method":"Mass spectrometry, co-immunoprecipitation, in vivo ubiquitination assays, USP7 inhibitor treatment","journal":"Anatomical record","confidence":"Low","confidence_rationale":"Tier 3 / Weak — MS identification, Co-IP, inhibitor-based functional readout, no in vitro reconstitution, single lab","pmids":["31502386"],"is_preprint":false},{"year":2016,"finding":"Trip12 acts as an E3 ubiquitin ligase for USP7, regulating USP7 protein stability through the ubiquitin-proteasome pathway, and consequently affecting USP7-mediated stabilization of p53, 53BP1, and Chk1 in the DNA damage response.","method":"Co-immunoprecipitation, in vivo ubiquitination assays, RNAi, cell cycle analysis","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP, ubiquitination assays, epistasis via knockdown, functional DNA damage response phenotype, single lab","pmids":["27800609"],"is_preprint":false},{"year":2022,"finding":"USP7 is identified as a deubiquitinase for XIAP in a p53-independent pathway. USP7 deubiquitinates XIAP to inhibit caspase-dependent apoptosis. Combined inhibition of USP7 and XIAP induces apoptosis in vitro and in vivo in glioma, correlating with grade-wise co-accumulation of USP7 and XIAP in tumor tissue.","method":"Proteomics analysis, co-immunoprecipitation, in vivo deubiquitination assays, RNAi, caspase activity assays, in vivo xenograft, IHC","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — proteomics identification, direct deubiquitination assay, functional caspase/apoptosis readout, in vivo validation, single lab","pmids":["36243803"],"is_preprint":false},{"year":2020,"finding":"USP7 deubiquitinates and stabilizes ZNF638, and also facilitates ZNF638 transcription via CREB stabilization. The USP7/ZNF638 axis increases SREBP1C cleavage through AKT/mTORC1/S6K signaling and forms a nuclear complex with SREBP1C to regulate lipogenesis-associated enzymes (ACACA, FASN, SCD) in hepatocytes.","method":"Co-immunoprecipitation, in vivo deubiquitination assays, ChIP assays, RNAi, liver steatosis mouse model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP and deubiquitination assays, in vivo mouse model, pathway placement, single lab","pmids":["33040080"],"is_preprint":false},{"year":2022,"finding":"USP7 deubiquitinates and stabilizes ABCB1 (P-glycoprotein/MDR1) through direct interaction, promoting chemoresistance in triple-negative breast cancer. USP7 overexpression increases ABCB1-mediated drug efflux resistance, while USP7 knockdown sensitizes resistant cells.","method":"Co-immunoprecipitation, in vivo ubiquitination assays, RNAi, drug resistance assays","journal":"Cells","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP and ubiquitination assays, functional drug resistance readout, single lab, limited mechanistic depth","pmids":["36291159"],"is_preprint":false},{"year":2022,"finding":"USP7 deubiquitinates and stabilizes RECQL4, counteracting UBE2O-mediated multi-monoubiquitination and proteasomal degradation of RECQL4. USP7 interacts with both UBE2O and RECQL4, and antagonizes UBE2O-mediated inhibition of homologous recombination DNA repair.","method":"Co-immunoprecipitation, in vivo ubiquitination assays, HR assays, RNAi","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP of ternary complex, ubiquitination assays, functional HR assay, single lab","pmids":["34921745"],"is_preprint":false},{"year":2023,"finding":"USP7 deubiquitinates and stabilizes KDM5B (lysine-specific demethylase 5B) in nasopharyngeal carcinoma. KDM5B inhibits ZBTB16 expression by reducing H3K4me3 at its promoter, subsequently increasing TOP2A expression and conferring cisplatin resistance.","method":"Co-immunoprecipitation, in vivo deubiquitination assays, H3K4me3 ChIP, RNAi, xenograft models","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP and deubiquitination assays, functional ChIP and in vivo validation, single lab","pmids":["38287116"],"is_preprint":false},{"year":2023,"finding":"USP7 deubiquitinates TRIM24, stabilizing it. TRIM24 then promotes SPLUNC1 expression via STAT3 recruitment in M1 macrophages, driving M1 polarization and suppressing NPC tumor progression.","method":"Co-immunoprecipitation, in vivo ubiquitination assays, ChIP, RNAi, xenograft models","journal":"Cell death & disease","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP and ubiquitination assays, functional macrophage polarization, single lab, limited direct mechanistic validation","pmids":["38129408"],"is_preprint":false},{"year":2023,"finding":"USP7 directly binds and deubiquitinates HMOX-1 (HO-1) in spinal cord injury context. USP7 overexpression promotes HMOX-1 expression via deubiquitination, reducing ferroptosis and ameliorating spinal cord injury in rat models.","method":"Co-immunoprecipitation, in vivo ubiquitination assays, SCI rat model, behavioral/histological assays","journal":"Neurochemistry international","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP and ubiquitination level measurement, in vivo rat model, single lab, overlap with HO-1 finding from another lab","pmids":["37257587"],"is_preprint":false},{"year":2023,"finding":"USP7 promotes osteoclast differentiation via deubiquitination and stabilization of HMGB1, which promotes osteoclast formation. USP7 inhibition attenuates bone loss in OVX mice.","method":"Co-immunoprecipitation, in vivo ubiquitination assays, osteoclast differentiation assays, in vivo OVX mouse model","journal":"Journal of orthopaedic translation","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP and ubiquitination assays, functional differentiation and in vivo assays, single lab","pmids":["37333461"],"is_preprint":false},{"year":2023,"finding":"USP7 inhibits osteoclastogenesis via two mechanisms: (1) impairing K63-linked polyubiquitination of TRAF6 (without affecting TRAF6 stability), thereby suppressing RANKL-mediated NF-κB and MAPK activation; (2) protecting STING from degradation to induce IFN-β expression. USP7 overexpression impairs osteoclast differentiation and bone resorption in vivo.","method":"Co-immunoprecipitation, in vivo ubiquitination assays, osteoclast differentiation assays, NF-κB/MAPK pathway assays, OVX mouse model","journal":"Aging and disease","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP and specific ubiquitination linkage (K63) characterization, dual mechanism with STING, in vivo validation, single lab","pmids":["37199589"],"is_preprint":false},{"year":2022,"finding":"RRM2 (ribonucleotide reductase regulatory subunit) is a USP7 substrate regulated during S phase. USP7 depletion induces cellular senescence in melanoma, and ectopic RRM2 expression in USP7-depleted cells rescues the senescent phenotype, placing RRM2 as a key USP7 substrate in cell cycle regulation.","method":"In vivo drop-out screens in PDX models, global transcriptomics and proteomics, RNAi, ectopic expression rescue, senescence assays","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo PDX screen, proteomics, specific rescue experiment for RRM2, single lab","pmids":["36130505"],"is_preprint":false},{"year":2022,"finding":"USP7 deubiquitinates and stabilizes ERβ in NSCLC. ERβ suppresses PRDX3 SUMOylation, reducing ROS accumulation and promoting osimertinib resistance. USP7 depletion reduces ERβ and reverses osimertinib resistance.","method":"Co-immunoprecipitation, in vivo deubiquitination assays, SUMOylation assays, ROS measurement, drug resistance assays, in vivo xenograft","journal":"Cancer letters","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP and ubiquitination assays, functional resistance phenotype, single lab, indirect link between USP7 and PRDX3 SUMOylation","pmids":["38097136"],"is_preprint":false},{"year":2024,"finding":"USP7 deubiquitinates and stabilizes LARS1 (leucyl-tRNA synthetase 1). The mitochondrial peptide MOTS-c competes with USP7 for binding to LARS1, attenuating USP7-mediated LARS1 deubiquitination and promoting LARS1 ubiquitination and degradation.","method":"Co-immunoprecipitation, in vivo ubiquitination assays, competition binding assays, ovarian cancer cell and mouse model experiments","journal":"Advanced science","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP competition assay, ubiquitination assays, single lab, no in vitro reconstitution of USP7-LARS1 deubiquitination","pmids":["39321430"],"is_preprint":false},{"year":2005,"finding":"HAUSP is itself polyubiquitinated, polyneddylated, and can dimerize. A catalytic mutant (C224S) of HAUSP contributes to p53 destabilization in a dose-dependent manner, consistent with dominant-negative interference.","method":"Co-immunoprecipitation, reporter cell line (p53 response element-lacZ), overexpression of wild-type and C224S mutant HAUSP","journal":"FEBS letters","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP for PTMs, functional p53 reporter, single lab, limited mechanistic depth for dimerization","pmids":["16111684"],"is_preprint":false},{"year":2024,"finding":"USP7 deubiquitinates and stabilizes KPNB1 (importin β1) in glioblastoma. USP7-stabilized KPNB1 mediates nuclear import of transcription factor YBX1, which transcriptionally activates NLGN3, promoting GBM progression.","method":"Co-immunoprecipitation, ubiquitination assays, nuclear fractionation, ChIP assays, RNAi, intracranial xenograft models","journal":"Journal of experimental & clinical cancer research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP and ubiquitination assays, functional in vivo model, single lab, indirect pathway mechanism","pmids":["38254206"],"is_preprint":false},{"year":2018,"finding":"USP7 and the DNMT1 interaction via GK-repeat acetylation was reported to stabilize DNMT1; however, direct experimental testing showed that DNMT1 is present at normal levels in cells lacking USP7, and substitution of GK repeats to prevent acetylation does not affect DNMT1 stability. USP7 and PCNA are recruited to replication foci independently of DNMT1. This negative result indicates the USP7-DNMT1 stabilization model is not supported.","method":"Knockout cell lines (mouse and human lacking USP7), DNMT1 GK→GQ mutant knockin, chromatin fractionation, immunofluorescence","journal":"Epigenetics & chromatin","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — negative finding: knockout and mutagenesis both show no USP7 dependence for DNMT1 stability; rigorous genetic controls","pmids":["29482658"],"is_preprint":false}],"current_model":"USP7/HAUSP is a cysteine deubiquitinase whose catalytic domain is allosterically activated ~100-fold by its C-terminal HUBL-45 di-Ubl domain engaging a 'switching' loop, an activation further enhanced by GMPS binding; it recognizes substrates predominantly via a P/AXXS motif binding its N-terminal TRAF domain, and regulates a broad network including p53, MDM2, MDMX, PTEN, N-Myc, Chk1, Rad18, FOXO4, Ci/Gli, YAP/TAZ, KRAS, BCR-ABL, NOTCH1, HIF-1α, and many others through direct deubiquitination and stabilization, while its own activity is modulated by CK2-mediated phosphorylation at Ser18 (stabilizing) and PPM1G-mediated dephosphorylation after DNA damage (destabilizing), and by Trip12-mediated ubiquitination, collectively placing USP7 as a central hub coordinating the p53-MDM2 pathway, DNA damage response, Hedgehog and Hippo signaling, cell cycle progression, and immune regulation."},"narrative":{"mechanistic_narrative":"USP7 (HAUSP) is a cysteine deubiquitinase that acts as a central hub for protein-stability control, removing predominantly K48-linked polyubiquitin chains from a broad substrate network to oppose proteasomal degradation [PMID:15053880, PMID:29045385]. It is a monomeric, multidomain enzyme: an N-terminal TRAF-like domain recognizes substrates through a short P/AXXS motif (Trp165 critical), as defined by co-crystal structures with p53 and MDM2 peptides, while the catalytic activity is allosterically activated ~100-fold by the C-terminal di-Ubl unit (HUBL-45) engaging a 'switching' loop in the catalytic domain and is further hyperactivated by the metabolic enzyme GMPS [PMID:16474402, PMID:21981925, PMID:14506283]. Its founding and best-defined role is in the p53–MDM2 axis, where USP7 deubiquitinates and stabilizes both p53 and MDM2/MDMX, and its dosage determines whether it preferentially protects MDM2 (repressing p53) or p53 itself [PMID:15053880, PMID:15916963]; consistent with this, Hausp-null mice die in early embryogenesis with p53 activation that is only partially relieved by p53 deletion, revealing additional essential p53-independent functions [PMID:19946331]. USP7 activity is itself tuned by post-translational control — CK2-mediated Ser18 phosphorylation stabilizes the enzyme in unstressed cells, while ATM-dependent PPM1G dephosphorylation downregulates it after DNA damage to permit p53 accumulation, and Trip12 ubiquitinates USP7 to limit its abundance [PMID:22361354, PMID:27800609]. Beyond p53, USP7 stabilizes substrates across the DNA damage response and replication (Chk1, Rad18, MCM-BP-dependent MCM unloading) [PMID:25483066, PMID:25961918, PMID:24190967], developmental and growth signaling (Ci/Gli in Hedgehog, YAP/TAZ in Hippo, N-Myc) [PMID:27618649, PMID:26120032, PMID:30679505], and numerous oncoproteins including KRAS, BCR-ABL, and NOTCH1 [PMID:30370059, PMID:39499616, PMID:33963175]. USP7 also restrains polyglutamine-disease proteostasis, where it preferentially deubiquitinates polyQ-expanded androgen receptor to promote aggregation, and its inhibition ameliorates SBMA and ALS phenotypes in vivo [PMID:33170804, PMID:33106424]. A reported USP7–DNMT1 stabilization model was not supported by genetic testing, as DNMT1 levels are normal in USP7-null cells [PMID:29482658].","teleology":[{"year":2003,"claim":"Establishing USP7 as a modular, catalytically active deubiquitinase set the structural framework for all later substrate work, defining an N-terminal substrate-binding domain distinct from the C-terminal regulatory region.","evidence":"Partial proteolysis/MS domain mapping, fluorescence binding, and in vitro deubiquitinase assays on purified protein","pmids":["14506283"],"confidence":"High","gaps":["Did not resolve how the C-terminal domains regulate catalysis","Substrate-recognition motif not yet defined"]},{"year":2004,"claim":"Defining USP7's dose-dependent, opposing effects on p53 and MDM2 explained how one deubiquitinase can either repress or activate the p53 pathway, answering how USP7 sits at the center of this circuit.","evidence":"Graded RNAi knockdown, Co-IP, and in vivo ubiquitination assays across cell lines","pmids":["15053880"],"confidence":"High","gaps":["Did not establish what shifts substrate preference between MDM2 and p53","Structural basis of binding not yet known"]},{"year":2006,"claim":"Co-crystal structures defined the P/AXXS recognition motif and the shared TRAF-domain surface used by p53, MDM2 and EBNA1, providing the molecular logic for competitive substrate selection.","evidence":"X-ray co-crystal structures of the N-terminal domain with p53/MDM2 peptides plus mutagenesis","pmids":["16474402"],"confidence":"High","gaps":["Did not explain how a trimeric p53-MDM2-USP7 state forms given mutually exclusive binding","Affinity ranking of full substrate repertoire unknown"]},{"year":2005,"claim":"Extending USP7 substrates to MDMX/HDMX and linking ATM phosphorylation to reduced USP7 affinity connected the enzyme to DNA-damage-regulated control of the p53 axis.","evidence":"Co-IP, in vivo/in vitro deubiquitination, RNAi, and DNA damage treatments","pmids":["15916963"],"confidence":"High","gaps":["Direct phosphatase/kinase controlling USP7 itself not yet identified","Quantitative contribution to DDR not resolved"]},{"year":2006,"claim":"Demonstrating non-degradative deubiquitination of FOXO4 showed USP7 can reverse mono-ubiquitination to control localization and transcription without altering protein half-life, broadening its functional output beyond stabilization.","evidence":"Reciprocal Co-IP, deubiquitination assays, localization imaging, transcriptional reporters","pmids":["16964248"],"confidence":"High","gaps":["Chain-type specificity of FOXO4 mono-Ub removal not defined","Recruitment mechanism to FOXO4 not mapped"]},{"year":2008,"claim":"Placing USP7 within a PML-DAXX network controlling PTEN nuclear-cytoplasmic trafficking established that USP7 regulates substrate localization through deubiquitination, integrating it into tumor-suppressor compartmentalization.","evidence":"Co-IP, deubiquitination assays, nuclear fractionation, RNAi, rescue experiments","pmids":["18716620"],"confidence":"High","gaps":["Direct vs DAXX-mediated effect on PTEN not fully separated","Chain linkage on PTEN not characterized"]},{"year":2009,"claim":"Knockout mice resolved the in vivo essentiality of USP7, showing early lethality driven by p53 activation but only partly rescued by p53 loss, proving p53-independent essential functions exist.","evidence":"Conventional/conditional knockout mice with hausp/p53 double-knockout epistasis","pmids":["19946331"],"confidence":"High","gaps":["Identity of the p53-independent essential substrates not defined","Tissue-specific requirements not dissected"]},{"year":2011,"claim":"The crystal structure of the C-terminal Ubl array explained how USP7 is allosterically switched on, showing HUBL-45 activates the catalytic domain ~100-fold and GMPS stabilizes this active state.","evidence":"Crystal structure of C-terminal Ubl domains, in vitro activity assays, domain mutagenesis","pmids":["21981925"],"confidence":"High","gaps":["How substrate binding couples to the switching loop in cells not resolved","Regulation of GMPS recruitment unknown"]},{"year":2012,"claim":"Identifying CK2 (writer) and PPM1G (eraser) acting on Ser18 defined the phospho-switch that links DNA damage to USP7 abundance and p53 derepression, answering how USP7 itself is regulated by stress.","evidence":"Phospho-mapping, kinase/phosphatase assays, RNAi, IR treatment, Western blotting","pmids":["22361354"],"confidence":"High","gaps":["Quantitative effect of Ser18 on catalysis vs stability not fully separated","Isoform-specific regulation incompletely defined"]},{"year":2016,"claim":"Defining Trip12 as the E3 ligase for USP7 closed the loop on USP7's own turnover and linked its degradation to DDR substrate stability (p53, 53BP1, Chk1).","evidence":"Co-IP, in vivo ubiquitination, RNAi, cell cycle analysis","pmids":["27800609","27800609"],"confidence":"Medium","gaps":["Single-lab finding without in vitro reconstitution","Chain type used by Trip12 on USP7 not defined"]},{"year":2015,"claim":"A cluster of studies extended USP7 deubiquitination into the DNA replication and damage-tolerance machinery (Chk1, Rad18, MCM-BP), establishing USP7 as a stabilizer of replication and checkpoint factors via the P/AXXS-type motif.","evidence":"Co-IP, in vitro/in vivo deubiquitination, RNAi, UV/PCNA ubiquitination, chromatin fractionation, replication assays","pmids":["25483066","25961918","24190967"],"confidence":"High","gaps":["Relative contribution of each substrate to genome stability not ranked","How USP7 timing on MCM unloading is controlled unclear"]},{"year":2015,"claim":"Cross-species work placed USP7 in developmental signaling, showing it stabilizes Ci/Gli (Hedgehog) and Yorkie/YAP-TAZ (Hippo) and that pathway activation modulates USP7-substrate binding.","evidence":"Co-IP, in vivo ubiquitination, Drosophila genetics, mammalian cell assays","pmids":["26120032","30679505"],"confidence":"High","gaps":["Direct deubiquitination chain specificity on Gli/YAP not fully resolved","Signal-dependent regulation of binding mechanistically undefined"]},{"year":2016,"claim":"Identification of N-Myc as a USP7 substrate with in vivo knockout validation established USP7 as a stabilizer of an oncogenic transcription factor and a therapeutic target in neuroblastoma.","evidence":"Co-IP, deubiquitination assays, RNAi, conditional knockout mice, xenografts, USP7 inhibitors","pmids":["27618649"],"confidence":"High","gaps":["Whether N-Myc stabilization is direct vs cofactor-dependent not fully separated","Selectivity over c-Myc not addressed"]},{"year":2017,"claim":"Allosteric inhibitors and di-ubiquitin NMR clarified USP7's catalytic mechanism, showing it preferentially binds and depolymerizes K48-linked chains and can be inhibited by attenuating ubiquitin binding rather than blocking the active site.","evidence":"NMR screening, co-crystal structures, isotope-labeled di-ubiquitin NMR, in vitro DUB assays","pmids":["29045385"],"confidence":"High","gaps":["In-cell chain-linkage preference across substrates not mapped","Determinants of K48 vs K63 discrimination on physiologic substrates unknown"]},{"year":2018,"claim":"A rigorous negative study overturned the USP7-DNMT1 stabilization model, showing DNMT1 levels are unchanged in USP7-null cells and that USP7/PCNA recruitment to replication foci is DNMT1-independent.","evidence":"USP7-knockout mouse/human cells, DNMT1 GK→GQ knock-in, chromatin fractionation, immunofluorescence","pmids":["29482658"],"confidence":"Medium","gaps":["Does not exclude context- or modification-specific DNMT1 effects","Original interaction's biological role left unexplained"]},{"year":2021,"claim":"Demonstrating preferential deubiquitination of polyQ-expanded androgen receptor, and protective effects of USP7 inhibition in SBMA/ALS models, redefined USP7 as a modifier of disease-protein proteostasis rather than a generic stabilizer.","evidence":"Quantitative proteomics, Co-IP, deubiquitination assays, conditional knockout mice, cross-species ALS models, behavioral assays","pmids":["33170804","33106424"],"confidence":"High","gaps":["Basis of preference for expanded vs wild-type AR not structurally defined","Whether NEDD4L/SMAD2 axis fully accounts for ALS protection unclear"]},{"year":2024,"claim":"Mapping USP7 deubiquitination of KRAS at K147 via the TRAF domain extended its oncogenic substrate range to a major driver, reinforcing the therapeutic rationale across cancers.","evidence":"Co-IP, in vitro/in vivo deubiquitination, domain binding, ubiquitination-site mutagenesis, proliferation assays","pmids":["39499616"],"confidence":"Medium","gaps":["Single-lab finding","Selectivity among KRAS isoforms/mutants not fully resolved"]},{"year":null,"claim":"How USP7 achieves substrate selectivity in cells — given an enormous reported substrate list, shared P/AXXS recognition, and tunable allosteric activation — and which substrates dominate its essential in vivo functions remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No quantitative ranking of physiologic substrates by occupancy/flux","p53-independent essential function (from knockout) lacks a defined effector","Most disease-context substrates rest on single-lab Co-IP without reconstitution"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,3,6,15,16,26,39]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[5,6,12]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,3,11]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[2,4,28]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[4,25]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,15,17,21,25,53]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[3,16,20,47]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[14,23,36,39]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,3,11,26,40]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[44]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[24,29]}],"complexes":[],"partners":["MDM2","TP53","MDM4","GMPS","TRIP12","PPM1G","MCM-BP","DAXX"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q93009","full_name":"Ubiquitin carboxyl-terminal hydrolase 7","aliases":["Deubiquitinating enzyme 7","Herpesvirus-associated ubiquitin-specific protease","Ubiquitin thioesterase 7","Ubiquitin-specific-processing protease 7"],"length_aa":1102,"mass_kda":128.3,"function":"Hydrolase that deubiquitinates target proteins such as ARMC5, FOXO4, DEPTOR, KAT5, p53/TP53, MDM2, ERCC6, DNMT1, UHRF1, PTEN, KMT2E/MLL5 and DAXX (PubMed:11923872, PubMed:15053880, PubMed:16964248, PubMed:18716620, PubMed:25283148, PubMed:25865756, PubMed:26678539, PubMed:28655758, PubMed:33544460, PubMed:35216969). Together with DAXX, prevents MDM2 self-ubiquitination and enhances the E3 ligase activity of MDM2 towards p53/TP53, thereby promoting p53/TP53 ubiquitination and proteasomal degradation (PubMed:15053880, PubMed:16845383, PubMed:18566590, PubMed:20153724). Deubiquitinates p53/TP53, preventing degradation of p53/TP53, and enhances p53/TP53-dependent transcription regulation, cell growth repression and apoptosis (PubMed:25283148). Deubiquitinates p53/TP53 and MDM2 and strongly stabilizes p53/TP53 even in the presence of excess MDM2, and also induces p53/TP53-dependent cell growth repression and apoptosis (PubMed:11923872, PubMed:26786098). Deubiquitination of FOXO4 in presence of hydrogen peroxide is not dependent on p53/TP53 and inhibits FOXO4-induced transcriptional activity (PubMed:16964248). In association with DAXX, is involved in the deubiquitination and translocation of PTEN from the nucleus to the cytoplasm, both processes that are counteracted by PML (PubMed:18716620). Deubiquitinates KMT2E/MLL5 preventing KMT2E/MLL5 proteasomal-mediated degradation (PubMed:26678539). Involved in cell proliferation during early embryonic development. Involved in transcription-coupled nucleotide excision repair (TC-NER) in response to UV damage: recruited to DNA damage sites following interaction with KIAA1530/UVSSA and promotes deubiquitination of ERCC6, preventing UV-induced degradation of ERCC6 (PubMed:22466611, PubMed:22466612). Involved in maintenance of DNA methylation via its interaction with UHRF1 and DNMT1: acts by mediating deubiquitination of UHRF1 and DNMT1, preventing their degradation and promoting DNA methylation by DNMT1 (PubMed:21745816, PubMed:22411829). Deubiquitinates alkylation repair enzyme ALKBH3. OTUD4 recruits USP7 and USP9X to stabilize ALKBH3, thereby promoting the repair of alkylated DNA lesions (PubMed:25944111). Acts as a chromatin regulator via its association with the Polycomb group (PcG) multiprotein PRC1-like complex; may act by deubiquitinating components of the PRC1-like complex (PubMed:20601937). Able to mediate deubiquitination of histone H2B; it is however unsure whether this activity takes place in vivo (PubMed:20601937). Exhibits a preference towards 'Lys-48'-linked ubiquitin chains (PubMed:22689415). Increases regulatory T-cells (Treg) suppressive capacity by deubiquitinating and stabilizing the transcription factor FOXP3 which is crucial for Treg cell function (PubMed:23973222). Plays a role in the maintenance of the circadian clock periodicity via deubiquitination and stabilization of the CRY1 and CRY2 proteins (PubMed:27123980). Deubiquitinates REST, thereby stabilizing REST and promoting the maintenance of neural progenitor cells (PubMed:21258371). Deubiquitinates SIRT7, inhibiting SIRT7 histone deacetylase activity and regulating gluconeogenesis (PubMed:28655758). Involved in the regulation of WASH-dependent actin polymerization at the surface of endosomes and the regulation of endosomal protein recycling (PubMed:26365382). It maintains optimal WASH complex activity and precise F-actin levels via deubiquitination of TRIM27 and WASHC1 (PubMed:26365382). Mediates the deubiquitination of phosphorylated DEPTOR, promoting its stability and leading to decreased mTORC1 signaling (PubMed:35216969) (Microbial infection) Contributes to the overall stabilization and trans-activation capability of the herpesvirus 1 trans-acting transcriptional protein ICP0/VMW110 during HSV-1 infection (Microbial infection) Upon infection with Epstein-Barr virus, the interaction with viral EBNA1 increases the association of USP7 with PML proteins, which is required for the polyubiquitylation and degradation of PML","subcellular_location":"Nucleus; Cytoplasm; Nucleus, PML body; Chromosome","url":"https://www.uniprot.org/uniprotkb/Q93009/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/USP7","classification":"Common Essential","n_dependent_lines":953,"n_total_lines":1208,"dependency_fraction":0.7889072847682119},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000187555","cell_line_id":"CID001827","localizations":[{"compartment":"nuclear_punctae","grade":3},{"compartment":"nucleoplasm","grade":1}],"interactors":[{"gene":"CAPZB","stoichiometry":0.2},{"gene":"HIST2H2BE","stoichiometry":0.2},{"gene":"PPM1G","stoichiometry":0.2},{"gene":"RBM14","stoichiometry":0.2},{"gene":"SCML2","stoichiometry":0.2},{"gene":"MRGBP","stoichiometry":0.2},{"gene":"BCOR","stoichiometry":0.2},{"gene":"DHX40","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001827","total_profiled":1310},"omim":[{"mim_id":"621315","title":"MINDY LYSINE-48 DEUBIQUITINASE 4; MINDY4","url":"https://www.omim.org/entry/621315"},{"mim_id":"620004","title":"NMRA-LIKE REDOX SENSOR 1; NMRAL1","url":"https://www.omim.org/entry/620004"},{"mim_id":"617964","title":"LEUKODYSTROPHY, HYPOMYELINATING, 16; 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disease","url":"https://pubmed.ncbi.nlm.nih.gov/38769122","citation_count":21,"is_preprint":false},{"pmid":"38589688","id":"PMC_38589688","title":"Inhibition of USP7 enhances CD8+ T cell activity in liver cancer by suppressing PRDM1-mediated FGL1 upregulation.","date":"2024","source":"Acta pharmacologica Sinica","url":"https://pubmed.ncbi.nlm.nih.gov/38589688","citation_count":21,"is_preprint":false},{"pmid":"34921745","id":"PMC_34921745","title":"UBE2O and USP7 co-regulate RECQL4 ubiquitinylation and homologous recombination-mediated DNA repair.","date":"2022","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/34921745","citation_count":21,"is_preprint":false},{"pmid":"16111684","id":"PMC_16111684","title":"HAUSP, a deubiquitinating enzyme for p53, is polyubiquitinated, polyneddylated, and dimerized.","date":"2005","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/16111684","citation_count":20,"is_preprint":false},{"pmid":"29482658","id":"PMC_29482658","title":"Independent functions of DNMT1 and USP7 at replication foci.","date":"2018","source":"Epigenetics & chromatin","url":"https://pubmed.ncbi.nlm.nih.gov/29482658","citation_count":20,"is_preprint":false},{"pmid":"31502386","id":"PMC_31502386","title":"USP7 Deubiquitinates and Stabilizes SIRT1.","date":"2019","source":"Anatomical record (Hoboken, N.J. : 2007)","url":"https://pubmed.ncbi.nlm.nih.gov/31502386","citation_count":19,"is_preprint":false},{"pmid":"36878903","id":"PMC_36878903","title":"USP7- and PRMT5-dependent G3BP2 stabilization drives de novo lipogenesis and tumorigenesis of HNSC.","date":"2023","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/36878903","citation_count":19,"is_preprint":false},{"pmid":"38254206","id":"PMC_38254206","title":"Stabilization of KPNB1 by deubiquitinase USP7 promotes glioblastoma progression through the YBX1-NLGN3 axis.","date":"2024","source":"Journal of experimental & clinical cancer research : CR","url":"https://pubmed.ncbi.nlm.nih.gov/38254206","citation_count":18,"is_preprint":false},{"pmid":"39321430","id":"PMC_39321430","title":"Mitochondrial-Derived Peptide MOTS-c Suppresses Ovarian Cancer Progression by Attenuating USP7-Mediated LARS1 Deubiquitination.","date":"2024","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/39321430","citation_count":17,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":46396,"output_tokens":14461,"usd":0.178052,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":26530,"output_tokens":5599,"usd":0.136313,"stage2_stop_reason":"end_turn"},"total_usd":0.314365,"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\": 2004,\n      \"finding\": \"HAUSP/USP7 deubiquitinates and stabilizes both p53 and MDM2. Partial RNAi knockdown of HAUSP destabilizes p53, but nearly complete ablation stabilizes p53 indirectly by destabilizing MDM2 (which undergoes self-ubiquitination when HAUSP is absent). This reveals a dynamic feedback loop where HAUSP preferentially stabilizes MDM2 in normal cells.\",\n      \"method\": \"RNAi knockdown, co-immunoprecipitation, in vivo ubiquitination assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal functional data with multiple RNAi conditions, replicated across cell lines including HeLa controls, multiple orthogonal methods\",\n      \"pmids\": [\"15053880\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The N-terminal TRAF-like domain of USP7 binds two closely spaced 4-residue sites in both p53 (residues 359–367) and MDM2 (residues 147–159), with a preference for a P/AXXS motif; Trp165 in the USP7 binding pocket is critical. Co-crystal structures of USP7 N-terminal domain with p53 and MDM2 peptides were determined, and the same surface also binds EBNA1, explaining competitive interactions.\",\n      \"method\": \"X-ray co-crystal structures, mutagenesis, peptide binding assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures plus mutagenesis validation, multiple peptides co-crystallized\",\n      \"pmids\": [\"16474402\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"USP7/HAUSP interacts with and deubiquitinates FOXO4 in response to oxidative stress, negatively regulating FOXO4 transcriptional activity towards endogenous promoters. The monoubiquitination of FOXO4 drives its nuclear relocalization and increased transcriptional activity; USP7 reverses this without affecting FOXO4 protein half-life.\",\n      \"method\": \"Co-immunoprecipitation, in vivo and in vitro deubiquitination assays, nuclear localization imaging, transcriptional reporter assays\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, direct deubiquitination assays, functional transcriptional readouts, multiple orthogonal methods\",\n      \"pmids\": [\"16964248\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"HAUSP interacts with and directly deubiquitinates HDMX (MDM4), stabilizing it. HAUSP activity is required for normal HDMX protein levels, and the balance between HAUSP and HDM2 determines HDMX stability. ATM-dependent phosphorylation of HDMX and HDM2 reduces their affinity for HAUSP, contributing to their DNA-damage-induced destabilization.\",\n      \"method\": \"Co-immunoprecipitation, in vivo and in vitro deubiquitination assays, RNAi knockdown, DNA damage treatments\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct deubiquitination assays, reciprocal Co-IP, mechanistic link to DNA damage response, replicated with multiple methods\",\n      \"pmids\": [\"15916963\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"HAUSP/USP7 deubiquitinates PTEN, opposing its nuclear import. PML nuclear bodies coordinate PTEN localization through DAXX, which opposes HAUSP activity toward PTEN, so that PML-DAXX-HAUSP constitute a molecular network controlling PTEN deubiquitination and nuclear-cytoplasmic trafficking.\",\n      \"method\": \"Co-immunoprecipitation, in vivo deubiquitination assays, nuclear fractionation, RNAi knockdown, rescue experiments\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods, mechanistic pathway placement, rescue experiments, published in Nature\",\n      \"pmids\": [\"18716620\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The 64 kDa C-terminal region of USP7 contains five ubiquitin-like (Ubl) domains organized in 2-1-2 units. The last di-Ubl unit (HUBL-45) activates USP7 ~100-fold by binding a 'switching' loop in the catalytic domain that promotes ubiquitin binding. The metabolic enzyme GMPS allosterically hyperactivates USP7 by binding the first three Ubl domains (HUBL-123) and stabilizing the HUBL-45-dependent active state.\",\n      \"method\": \"Crystal structure of USP7 C-terminal Ubl domains, in vitro deubiquitinase activity assays, domain deletion/mutagenesis\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with in vitro activity assays and domain mutagenesis, multiple orthogonal methods in one study\",\n      \"pmids\": [\"21981925\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"USP7 is a monomeric protein with four structural domains: an N-terminal p53-binding domain, a catalytic domain, and two C-terminal domains. The N-terminal domain binds both p53 and EBNA1 (EBNA1 with ~10-fold higher affinity), while ICP0 binds a distinct C-terminal domain (residues 599–801). Purified USP7 is active for cleaving both linear ubiquitin and ubiquitin conjugated to EBNA1.\",\n      \"method\": \"Partial proteolysis with MALDI-TOF-MS domain mapping, affinity pulldowns, tryptophan fluorescence binding assays, gel filtration, in vitro deubiquitinase assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzyme reconstitution, domain mapping by proteolysis/MS, binding constants determined by fluorescence, multiple orthogonal methods\",\n      \"pmids\": [\"14506283\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"A trimeric complex of p53, MDM2, and HAUSP can exist in vivo, despite the mutually exclusive binding of p53 and MDM2 to the USP7 TRAF domain, because MDM2 acts as a bridge. HAUSP can deubiquitinate p53 in trans via MDM2-mediated bridging even when direct p53-HAUSP interaction is disrupted by a p53 binding-deficient mutation.\",\n      \"method\": \"Co-immunoprecipitation in vivo and in vitro, structure-based mutagenesis, ubiquitination assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — structure-guided mutagenesis plus Co-IP and functional ubiquitination assays, single lab\",\n      \"pmids\": [\"17525743\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Hausp knockout mice die between embryonic days E6.5 and E7.5 with p53 activation. Lethality is caused by reduced proliferation and developmental arrest partly attributable to p53 activation. Deletion of p53 extends but does not completely rescue embryonic development of hausp knockouts, indicating both p53-dependent and p53-independent essential functions of HAUSP in vivo.\",\n      \"method\": \"Conditional and conventional knockout mice, genetic epistasis (hausp/p53 double knockout), embryo analysis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic epistasis with double-knockout rescue, well-defined developmental phenotype\",\n      \"pmids\": [\"19946331\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"USP7 stability is regulated by CK2-mediated phosphorylation at serine 18, which stabilizes the USP7S isoform and thus maintains MDM2 stability and p53 repression in unstressed cells. After ionizing radiation, ATM-dependent phosphatase PPM1G dephosphorylates USP7S, leading to its downregulation, MDM2 destabilization, and p53 accumulation.\",\n      \"method\": \"Phosphorylation mapping, kinase/phosphatase assays, RNAi, IR treatment, Western blotting\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — identifies writer (CK2) and eraser (PPM1G) of USP7 phosphorylation, multiple methods, functional pathway consequences defined\",\n      \"pmids\": [\"22361354\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Kaposi's sarcoma-associated herpesvirus vIRF4 inhibits HAUSP through a bilateral belt-type interaction: vif1 peptide binds the HAUSP TRAF domain blocking substrate binding, while vif2 peptide binds both the TRAF and catalytic domains to suppress deubiquitinase activity. Crystal structures of the HAUSP-vIRF4 complexes were determined.\",\n      \"method\": \"X-ray crystal structure, peptide competition assays, deubiquitinase activity assays, cell-based apoptosis/xenograft assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures plus functional validation with peptide inhibitors and cell-based assays\",\n      \"pmids\": [\"22056774\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"HAUSP/USP7 deubiquitinates and stabilizes N-Myc (MYCN) in neuroblastoma. HAUSP interacts with N-Myc; HAUSP overexpression induces N-Myc deubiquitination and stabilization, while USP7 RNAi or genetic ablation (Usp7 knockout in mouse brain) destabilizes N-Myc and inhibits its function.\",\n      \"method\": \"Co-immunoprecipitation, in vivo deubiquitination assays, RNAi knockdown, conditional knockout mice, xenograft models, USP7 inhibitors\",\n      \"journal\": \"Nature medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic knockout, reciprocal Co-IP, direct deubiquitination assays, xenograft validation, multiple orthogonal approaches\",\n      \"pmids\": [\"27618649\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Small-molecule inhibitors GNE-6640 and GNE-6776 non-covalently target USP7 12 Å from the catalytic cysteine, attenuating ubiquitin binding rather than directly blocking the active site. Structural studies showed these compounds interact with acidic residues mediating hydrogen bonds with ubiquitin Lys48, and NMR with isotopically labeled di-ubiquitin chains showed USP7 preferentially binds free Lys48 side chains, protracted depolymerization of K48-linked chains relative to K63-linked chains.\",\n      \"method\": \"NMR-based screening, co-crystal structures, differential isotope-labeled di-ubiquitin NMR binding assays, in vitro deubiquitinase activity assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — co-crystal structures, NMR with engineered substrates, in vitro enzymatic assays, multiple orthogonal methods\",\n      \"pmids\": [\"29045385\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Genotoxic stress stimulates Cry1 phosphorylation and its deubiquitination by HAUSP/USP7, stabilizing Cry1 and shifting circadian clock phase. USP7 deubiquitinates Cry1 to protect it from degradation following DNA damage.\",\n      \"method\": \"Co-immunoprecipitation, in vivo deubiquitination assays, clock phase measurements, DNA damage treatments\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and deubiquitination assays, functional clock phenotype, single lab\",\n      \"pmids\": [\"25756610\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"USP7 deubiquitinates Ci/Gli transcription factors in the Hedgehog pathway, inhibiting their Slimb-Cul1 and Hib-Cul3 E3-ligase-mediated ubiquitination and degradation. Hh stimulation promotes Usp7 binding to Ci. Usp7 forms a complex with GMPS to promote Hh pathway activity, and the mammalian homolog HAUSP similarly regulates Gli ubiquitination and stability.\",\n      \"method\": \"Co-immunoprecipitation, in vivo ubiquitination assays, Drosophila genetics, mammalian cell assays\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods across two organisms, direct ubiquitination assays, genetic epistasis in Drosophila\",\n      \"pmids\": [\"26120032\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"USP7 controls Chk1 protein stability by direct deubiquitination. Depletion or inhibition of USP7 lowers Chk1 levels; overexpression of catalytically active (but not catalytic mutant) USP7 elevates Chk1 levels and increases its half-life. Catalytic mutant USP7 fails to deubiquitinate Chk1 in vivo and in vitro. This effect is independent of USP7's known stabilization of Claspin.\",\n      \"method\": \"RNAi, overexpression of wild-type vs. catalytic mutant USP7, in vivo and in vitro deubiquitination assays, half-life measurements\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct in vitro deubiquitination assay plus in vivo confirmation, mutagenesis controls, single lab\",\n      \"pmids\": [\"25483066\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"USP7 maintains Rad18 protein stability by direct deubiquitination. USP7 associates with Rad18 via a consensus USP7-binding motif (PVDS or similar). Loss of USP7 destabilizes Rad18, compromises UV-induced PCNA mono-ubiquitination and Pol η recruitment, and impairs DNA damage tolerance. USP7 can disassemble Rad18-dependent poly-ubiquitin chains in vitro and in vivo.\",\n      \"method\": \"Co-immunoprecipitation, in vitro and in vivo deubiquitination assays, RNAi, UV irradiation, PCNA ubiquitination assays, replication fork analysis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vitro reconstitution of deubiquitination, multiple functional readouts, USP7-binding motif identified, multiple orthogonal methods\",\n      \"pmids\": [\"25961918\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"USP7 directly interacts with MCM-BP via the PSTS(155-158) motif engaging the TRAF domain binding pocket of USP7. USP7 knockout leads to slowed S phase progression and accumulation of MCM proteins on chromatin due to a defect in MCM complex unloading during mid-to-late S phase. USP7 works with MCM-BP to facilitate MCM unloading from chromatin at the end of S phase.\",\n      \"method\": \"Co-immunoprecipitation, structural analysis of USP7-MCM-BP interaction, USP7 knockout cell analysis, chromatin fractionation, DNA replication assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — structural interaction analysis, knockout phenotype with specific mechanistic readout, multiple orthogonal methods\",\n      \"pmids\": [\"24190967\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"HAUSP deubiquitinates HIF-1α to increase its stability and promotes EMT/metastasis. Hypoxia induces K63-linked polyubiquitination of HAUSP at K443, enhancing its functions. K63-polyubiquitinated HAUSP interacts with ubiquitin receptor CBP to specifically mediate H3K56 acetylation at HIF-1α target gene promoters. HectH9 is the E3 ligase responsible for K63-ubiquitination of HAUSP.\",\n      \"method\": \"Co-immunoprecipitation, in vivo deubiquitination assays, ChIP-seq, mutagenesis, RNAi knockdown, metastasis assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods, direct deubiquitination assays, ChIP-seq, single lab\",\n      \"pmids\": [\"27934968\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Trip12 functions as an E3 ubiquitin ligase for USP7/HAUSP, controlling USP7 protein stability through the ubiquitin-proteasome pathway. Trip12 knockdown increases USP7-mediated stabilization of p53, 53BP1, and Chk1; Trip12 overexpression phenocopies USP7 knockdown (increased S-phase cells).\",\n      \"method\": \"Co-immunoprecipitation, in vivo ubiquitination assays, RNAi, cell cycle analysis\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — identifies E3 ligase for USP7 with Co-IP and functional epistasis, single lab\",\n      \"pmids\": [\"27800609\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"USP7 modulates chromatin remodeling important for base excision repair of oxidative lesions. USP7 siRNA knockdown does not change the levels or activity of BER enzymes but significantly reduces chromatin DNA accessibility and consequently the rate of repair of oxidative lesions.\",\n      \"method\": \"siRNA knockdown, chromatin accessibility assays, BER activity assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — specific chromatin accessibility measurement plus BER assays, functional readout without direct substrate identification, single lab\",\n      \"pmids\": [\"21138959\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HAUSP/USP7 deubiquitinates retinoblastoma protein (Rb), removing K48-linked ubiquitin chains from Rb and stabilizing it from proteasomal degradation. HAUSP deubiquitination of Rb leads to increased G1 cell population. In glioma, MDM2 becomes the preferred USP7 substrate over Rb, facilitating Rb degradation.\",\n      \"method\": \"Co-immunoprecipitation, in vivo and in vitro deubiquitination assays, cell cycle analysis, immunohistochemistry\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct in vitro deubiquitination, Co-IP, functional cell cycle readout, single lab\",\n      \"pmids\": [\"24823443\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"USP7 interacts with and deubiquitinates the mitotic checkpoint protein Chfr, stabilizing it. USP7 removes ubiquitin from autoubiquitinated Chfr both in vivo and in vitro. The USP7-Chfr interaction was identified by immunoaffinity purification and mass spectrometry.\",\n      \"method\": \"Immunoaffinity purification and mass spectrometry, co-immunoprecipitation, in vivo and in vitro deubiquitination assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — identified by AP-MS, confirmed by Co-IP and direct deubiquitination assay, single lab\",\n      \"pmids\": [\"17442268\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Usp7 deubiquitinates and stabilizes Yorkie (Yki) in Drosophila, positively regulating Hippo pathway output. Hippo pathway activation attenuates Usp7-Yki binding. The mammalian homolog HAUSP similarly stabilizes YAP by modulating its ubiquitination and degradation.\",\n      \"method\": \"Co-immunoprecipitation, in vivo ubiquitination assays, Drosophila genetics, mammalian cell assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cross-species validation (Drosophila + mammalian), direct ubiquitination assays, single lab\",\n      \"pmids\": [\"30679505\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"USP7 and USP47 regulate NLRP3 inflammasome activation in macrophages. Chemical inhibition of USP7 and USP47 blocks inflammasome formation by preventing ASC oligomerization and speck formation, independently of transcription. Inhibition alters the ubiquitination status of NLRP3 itself. USP7 and USP47 activity increases in response to inflammasome activators.\",\n      \"method\": \"CRISPR/Cas9 knockdown, chemical inhibition, ASC speck formation assays, ubiquitination assays, IL-1β/IL-18 cytokine measurement\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — CRISPR knockdown with functional readout, ubiquitination status changes for NLRP3 shown, but direct deubiquitination not reconstituted in vitro; replicated with chemical and genetic tools\",\n      \"pmids\": [\"30206189\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP7 interacts with and supports PP2A active localization in the cytoplasm. Inhibition of USP7 or PP2A produces similar phosphoproteomic changes including widespread increase in CDK1 substrate phosphorylation. USP7 inhibition causes untimely CDK1 activation throughout the cell cycle, leading to DNA damage; this toxicity is rescued by lowering CDK1 activity or chemically activating PP2A.\",\n      \"method\": \"Co-immunoprecipitation, quantitative phosphoproteomics, kinase activity assays, genetic and chemical rescue experiments\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphoproteomics, Co-IP, genetic rescue, functional cell cycle readouts, single lab\",\n      \"pmids\": [\"33856059\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"USP7 deubiquitinates and stabilizes NOTCH1 in T-ALL cells. USP7 interacts with NOTCH1 via its MATH and UBL domains. USP7 knockdown increases NOTCH1 ubiquitination and reduces NOTCH1 protein levels, suppressing T-ALL proliferation in vitro and in vivo.\",\n      \"method\": \"Co-immunoprecipitation, in vivo and in vitro deubiquitination assays, RNAi, domain mapping, xenograft models\",\n      \"journal\": \"Signal transduction and targeted therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, direct in vitro deubiquitination, domain mapping, in vivo xenograft, single lab\",\n      \"pmids\": [\"30370059\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"USP7 stabilizes HIV-1 Tat protein through deubiquitination. USP7 knockdown by CRISPR-Cas9 reduces Tat protein levels and reduces virus production. Endogenous USP7 levels increase after HIV-1 infection in human T-cells.\",\n      \"method\": \"CRISPR-Cas9 knockout, USP7 inhibitor treatment, co-immunoprecipitation, virus production assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR knockout with functional viral readout, inhibitor confirmation, single lab\",\n      \"pmids\": [\"28280111\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"USP7 promotes p53 sequence-specific DNA binding activity through interaction with the C-terminal regulatory region of p53, independent of USP7's N-terminal domain and independent of deubiquitylation activity. The USP7 C-terminal domain alone is sufficient to promote p53 binding to target sequences and p21 induction.\",\n      \"method\": \"In vitro DNA binding assays, domain mutagenesis, overexpression of catalytically inactive USP7, p21 expression assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — catalytic mutant controls distinguish this from deubiquitination, in vitro and in vivo DNA binding assays, single lab\",\n      \"pmids\": [\"20885946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"USP7 deubiquitinates and stabilizes PD-L1 by direct interaction. Abrogation of USP7 attenuates PD-L1/PD-1 interaction and sensitizes cancer cells to T cell killing in vitro and in vivo.\",\n      \"method\": \"Co-immunoprecipitation, in vivo ubiquitination assays, T cell killing assays, xenograft models\",\n      \"journal\": \"Acta pharmaceutica Sinica. B\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct interaction shown by Co-IP, functional immune assays, in vivo confirmation, single lab\",\n      \"pmids\": [\"33777676\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP7 preferentially interacts with polyQ-expanded androgen receptor (AR) and deubiquitinates it, reducing AR ubiquitination and promoting mutant AR aggregation. USP7 knockdown or monoallelic Usp7 knockout in mice reduces mutant AR aggregation and ameliorates SBMA motor phenotypes. USP7 also differentially interacts with mutant Huntingtin in striatum and frontal cortex.\",\n      \"method\": \"Quantitative proteomics, co-immunoprecipitation, in vivo ubiquitination assays, RNAi, conditional knockout mice, behavioral assays\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — quantitative proteomics identification, direct deubiquitination assay, in vivo genetic rescue in mice, multiple orthogonal approaches\",\n      \"pmids\": [\"33170804\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"USP7 deubiquitinates NEDD4L (an E3 ubiquitin ligase for SMAD2), stabilizing NEDD4L and thereby reducing SMAD2 levels, which decreases autophagy-mediated clearance of misfolded proteins. In ALS models across C. elegans, Drosophila, and mammalian cells, USP7 inhibition protects against proteotoxicity through the SMAD2/TGF-β pathway.\",\n      \"method\": \"Genetic screen in C. elegans, Drosophila and mammalian cell validation, co-immunoprecipitation, deubiquitination assays, autophagy assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cross-species genetic epistasis, direct deubiquitination of NEDD4L demonstrated, functional pathway placement, multiple organisms\",\n      \"pmids\": [\"33106424\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"USP7 stabilizes FBXO38 dependent on its catalytic activity. FBXO38 in turn stabilizes KIF20B (a kinesin required for cytokinesis) independent of an SCF complex. Depletion of USP7 or FBXO38 reduces KIF20B levels and causes cytokinetic defects that can be rescued by restoring FBXO38 or KIF20B.\",\n      \"method\": \"Affinity purification-mass spectrometry, BioID protein interaction profiling, Co-immunoprecipitation, RNAi, rescue experiments, cytokinesis imaging\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — AP-MS and BioID identification, functional rescue experiments, specific cytokinetic phenotype, single lab\",\n      \"pmids\": [\"30804394\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"USP7 interacts with and stabilizes DDX24 and DHX40 (DEAD/DEAH-box RNA helicases) dependent on its catalytic activity. USP7 interacts with PPM1G, TRIP12, and USP11 through its TRAF domain binding pocket (via P/A/ExxS motifs), whereas DHX40 uses a distinct binding site in the Ubl2 domain.\",\n      \"method\": \"Affinity purification-mass spectrometry, USP7 TRAF-domain binding-pocket mutants, RNAi and inhibitor treatments, Western blotting\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — AP-MS with domain mutant discrimination, catalytic activity dependence shown, single lab\",\n      \"pmids\": [\"30367141\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP7 deubiquitinates and stabilizes EZH2 in prostate cancer cells. USP7 knockdown decreases EZH2 protein and inhibits EZH2-dependent transcriptional repression. The USP7/EZH2 axis promotes cancer cell migration, invasion, and sphere formation.\",\n      \"method\": \"Co-immunoprecipitation, in vivo ubiquitination assays, RNAi, ectopic EZH2 rescue, cell migration/invasion assays\",\n      \"journal\": \"Genetics and molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP and in vivo ubiquitination, rescue experiment, functional phenotype, single lab\",\n      \"pmids\": [\"32453339\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP7 deubiquitinates and stabilizes HO-1 (heme oxygenase-1) to regulate redox homeostasis. Under arsenic exposure, USP7 itself becomes ubiquitinated at K476, which promotes its binding to HO-1, leading to enhanced HO-1 deubiquitination and stabilization. HO-1 is ubiquitinated at K243 under resting conditions.\",\n      \"method\": \"Co-immunoprecipitation, in vivo deubiquitination assays, ubiquitination site mapping, arsenic stress experiments, knockdown\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — identifies specific ubiquitination sites on both USP7 and HO-1, direct deubiquitination assays, single lab\",\n      \"pmids\": [\"35821281\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP7 deubiquitinates and stabilizes Raf-1. USP7 binds the PVDS motif in the CR2 region of Raf-1 and removes K6-, K11-, K27-, K33-, and K48-linked polyubiquitin chains. USP7-mediated deubiquitination of Raf-1 decreases Raf-1 threonine phosphorylation and inhibits ERK1/2 signaling, thereby restraining the G2/M transition in lung adenocarcinoma cells.\",\n      \"method\": \"Bioinformatics motif analysis, Co-immunoprecipitation, in vivo ubiquitination assays, phosphorylation assays, ERK signaling assays, cell cycle analysis\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP and ubiquitination assays, functional signaling and cell cycle readouts, single lab, no in vitro reconstitution\",\n      \"pmids\": [\"35948545\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP7 deubiquitinates and stabilizes TAZ by selectively removing K48-linked ubiquitination, antagonizing β-TRCP-mediated ubiquitin-proteasomal degradation and enhancing TAZ nuclear retention and transcriptional output in HNSCC.\",\n      \"method\": \"siRNA/cDNA library screens, Co-immunoprecipitation, in vivo ubiquitination assays, nuclear localization assays, xenograft and PDX models\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic screen plus direct deubiquitination assays, in vivo validation, single lab\",\n      \"pmids\": [\"35931679\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Natural compound eupalinolide B (EB) covalently and allosterically inhibits USP7 at Cys576 in the non-catalytic HUBL domain (not the active site). Co-crystal structure reveals this previously undisclosed binding site. Modification of Cys576 allosterically inhibits USP7, causing ubiquitination-dependent degradation of Keap1, leading to Nrf2-dependent transcription of anti-neuroinflammatory genes in microglia.\",\n      \"method\": \"Co-crystal structure, covalent binding assays, ubiquitination assays, Nrf2 target gene expression, in vivo mouse models of neurodegeneration\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — co-crystal structure of novel allosteric site, covalent mechanism defined, functional pathway demonstrated in vitro and in vivo\",\n      \"pmids\": [\"35947662\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP7 directly deubiquitinates KRAS by binding KRAS via its TRAF domain and removing K48-linked polyubiquitin chains from KRAS K147, stabilizing KRAS and promoting NSCLC proliferation. USP7 also stabilizes oncogenic KRAS mutants through deubiquitination.\",\n      \"method\": \"Co-immunoprecipitation, in vivo and in vitro deubiquitination assays, domain binding assays, ubiquitination site mutagenesis, NSCLC cell proliferation assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct in vitro deubiquitination, specific ubiquitination site identified (K147), functional proliferation assay, single lab\",\n      \"pmids\": [\"39499616\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP7 deubiquitinates and stabilizes BCR-ABL fusion protein in CML cells. USP7 interacts with BCR-ABL and blocks its polyubiquitination; knockdown or chemical inhibition of USP7 triggers BCR-ABL degradation and suppresses downstream signaling.\",\n      \"method\": \"DUB screen, co-immunoprecipitation, in vivo ubiquitination assays, RNAi, USP7 inhibitors, apoptosis assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — DUB screen identified, Co-IP and ubiquitination confirmed, functional leukemia cell assays, single lab\",\n      \"pmids\": [\"33963175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"USP7 deubiquitinates and stabilizes Maf proteins (c-Maf, MafA, MafB) in myeloma cells, promoting their transcriptional activity as measured by luciferase assays and Maf-regulated gene expression.\",\n      \"method\": \"Mass spectrometry identification, co-immunoprecipitation, in vivo ubiquitination assays, RNAi, luciferase reporter assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — MS-based identification plus Co-IP and ubiquitination, functional transcriptional readout, single lab\",\n      \"pmids\": [\"31822558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"USP7 deubiquitinates and stabilizes SIRT1, cleaving K48-linked polyubiquitin chains. USP7 is physically associated with SIRT1 as identified by mass spectrometry. USP7 enzymatic inhibition leads to decreased SIRT1 expression and accumulation of SIRT1 polyubiquitination.\",\n      \"method\": \"Mass spectrometry, co-immunoprecipitation, in vivo ubiquitination assays, USP7 inhibitor treatment\",\n      \"journal\": \"Anatomical record\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — MS identification, Co-IP, inhibitor-based functional readout, no in vitro reconstitution, single lab\",\n      \"pmids\": [\"31502386\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Trip12 acts as an E3 ubiquitin ligase for USP7, regulating USP7 protein stability through the ubiquitin-proteasome pathway, and consequently affecting USP7-mediated stabilization of p53, 53BP1, and Chk1 in the DNA damage response.\",\n      \"method\": \"Co-immunoprecipitation, in vivo ubiquitination assays, RNAi, cell cycle analysis\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP, ubiquitination assays, epistasis via knockdown, functional DNA damage response phenotype, single lab\",\n      \"pmids\": [\"27800609\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP7 is identified as a deubiquitinase for XIAP in a p53-independent pathway. USP7 deubiquitinates XIAP to inhibit caspase-dependent apoptosis. Combined inhibition of USP7 and XIAP induces apoptosis in vitro and in vivo in glioma, correlating with grade-wise co-accumulation of USP7 and XIAP in tumor tissue.\",\n      \"method\": \"Proteomics analysis, co-immunoprecipitation, in vivo deubiquitination assays, RNAi, caspase activity assays, in vivo xenograft, IHC\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — proteomics identification, direct deubiquitination assay, functional caspase/apoptosis readout, in vivo validation, single lab\",\n      \"pmids\": [\"36243803\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"USP7 deubiquitinates and stabilizes ZNF638, and also facilitates ZNF638 transcription via CREB stabilization. The USP7/ZNF638 axis increases SREBP1C cleavage through AKT/mTORC1/S6K signaling and forms a nuclear complex with SREBP1C to regulate lipogenesis-associated enzymes (ACACA, FASN, SCD) in hepatocytes.\",\n      \"method\": \"Co-immunoprecipitation, in vivo deubiquitination assays, ChIP assays, RNAi, liver steatosis mouse model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP and deubiquitination assays, in vivo mouse model, pathway placement, single lab\",\n      \"pmids\": [\"33040080\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP7 deubiquitinates and stabilizes ABCB1 (P-glycoprotein/MDR1) through direct interaction, promoting chemoresistance in triple-negative breast cancer. USP7 overexpression increases ABCB1-mediated drug efflux resistance, while USP7 knockdown sensitizes resistant cells.\",\n      \"method\": \"Co-immunoprecipitation, in vivo ubiquitination assays, RNAi, drug resistance assays\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP and ubiquitination assays, functional drug resistance readout, single lab, limited mechanistic depth\",\n      \"pmids\": [\"36291159\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP7 deubiquitinates and stabilizes RECQL4, counteracting UBE2O-mediated multi-monoubiquitination and proteasomal degradation of RECQL4. USP7 interacts with both UBE2O and RECQL4, and antagonizes UBE2O-mediated inhibition of homologous recombination DNA repair.\",\n      \"method\": \"Co-immunoprecipitation, in vivo ubiquitination assays, HR assays, RNAi\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP of ternary complex, ubiquitination assays, functional HR assay, single lab\",\n      \"pmids\": [\"34921745\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP7 deubiquitinates and stabilizes KDM5B (lysine-specific demethylase 5B) in nasopharyngeal carcinoma. KDM5B inhibits ZBTB16 expression by reducing H3K4me3 at its promoter, subsequently increasing TOP2A expression and conferring cisplatin resistance.\",\n      \"method\": \"Co-immunoprecipitation, in vivo deubiquitination assays, H3K4me3 ChIP, RNAi, xenograft models\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP and deubiquitination assays, functional ChIP and in vivo validation, single lab\",\n      \"pmids\": [\"38287116\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP7 deubiquitinates TRIM24, stabilizing it. TRIM24 then promotes SPLUNC1 expression via STAT3 recruitment in M1 macrophages, driving M1 polarization and suppressing NPC tumor progression.\",\n      \"method\": \"Co-immunoprecipitation, in vivo ubiquitination assays, ChIP, RNAi, xenograft models\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP and ubiquitination assays, functional macrophage polarization, single lab, limited direct mechanistic validation\",\n      \"pmids\": [\"38129408\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP7 directly binds and deubiquitinates HMOX-1 (HO-1) in spinal cord injury context. USP7 overexpression promotes HMOX-1 expression via deubiquitination, reducing ferroptosis and ameliorating spinal cord injury in rat models.\",\n      \"method\": \"Co-immunoprecipitation, in vivo ubiquitination assays, SCI rat model, behavioral/histological assays\",\n      \"journal\": \"Neurochemistry international\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP and ubiquitination level measurement, in vivo rat model, single lab, overlap with HO-1 finding from another lab\",\n      \"pmids\": [\"37257587\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP7 promotes osteoclast differentiation via deubiquitination and stabilization of HMGB1, which promotes osteoclast formation. USP7 inhibition attenuates bone loss in OVX mice.\",\n      \"method\": \"Co-immunoprecipitation, in vivo ubiquitination assays, osteoclast differentiation assays, in vivo OVX mouse model\",\n      \"journal\": \"Journal of orthopaedic translation\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP and ubiquitination assays, functional differentiation and in vivo assays, single lab\",\n      \"pmids\": [\"37333461\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP7 inhibits osteoclastogenesis via two mechanisms: (1) impairing K63-linked polyubiquitination of TRAF6 (without affecting TRAF6 stability), thereby suppressing RANKL-mediated NF-κB and MAPK activation; (2) protecting STING from degradation to induce IFN-β expression. USP7 overexpression impairs osteoclast differentiation and bone resorption in vivo.\",\n      \"method\": \"Co-immunoprecipitation, in vivo ubiquitination assays, osteoclast differentiation assays, NF-κB/MAPK pathway assays, OVX mouse model\",\n      \"journal\": \"Aging and disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP and specific ubiquitination linkage (K63) characterization, dual mechanism with STING, in vivo validation, single lab\",\n      \"pmids\": [\"37199589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RRM2 (ribonucleotide reductase regulatory subunit) is a USP7 substrate regulated during S phase. USP7 depletion induces cellular senescence in melanoma, and ectopic RRM2 expression in USP7-depleted cells rescues the senescent phenotype, placing RRM2 as a key USP7 substrate in cell cycle regulation.\",\n      \"method\": \"In vivo drop-out screens in PDX models, global transcriptomics and proteomics, RNAi, ectopic expression rescue, senescence assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo PDX screen, proteomics, specific rescue experiment for RRM2, single lab\",\n      \"pmids\": [\"36130505\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP7 deubiquitinates and stabilizes ERβ in NSCLC. ERβ suppresses PRDX3 SUMOylation, reducing ROS accumulation and promoting osimertinib resistance. USP7 depletion reduces ERβ and reverses osimertinib resistance.\",\n      \"method\": \"Co-immunoprecipitation, in vivo deubiquitination assays, SUMOylation assays, ROS measurement, drug resistance assays, in vivo xenograft\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP and ubiquitination assays, functional resistance phenotype, single lab, indirect link between USP7 and PRDX3 SUMOylation\",\n      \"pmids\": [\"38097136\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP7 deubiquitinates and stabilizes LARS1 (leucyl-tRNA synthetase 1). The mitochondrial peptide MOTS-c competes with USP7 for binding to LARS1, attenuating USP7-mediated LARS1 deubiquitination and promoting LARS1 ubiquitination and degradation.\",\n      \"method\": \"Co-immunoprecipitation, in vivo ubiquitination assays, competition binding assays, ovarian cancer cell and mouse model experiments\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP competition assay, ubiquitination assays, single lab, no in vitro reconstitution of USP7-LARS1 deubiquitination\",\n      \"pmids\": [\"39321430\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"HAUSP is itself polyubiquitinated, polyneddylated, and can dimerize. A catalytic mutant (C224S) of HAUSP contributes to p53 destabilization in a dose-dependent manner, consistent with dominant-negative interference.\",\n      \"method\": \"Co-immunoprecipitation, reporter cell line (p53 response element-lacZ), overexpression of wild-type and C224S mutant HAUSP\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP for PTMs, functional p53 reporter, single lab, limited mechanistic depth for dimerization\",\n      \"pmids\": [\"16111684\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP7 deubiquitinates and stabilizes KPNB1 (importin β1) in glioblastoma. USP7-stabilized KPNB1 mediates nuclear import of transcription factor YBX1, which transcriptionally activates NLGN3, promoting GBM progression.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, nuclear fractionation, ChIP assays, RNAi, intracranial xenograft models\",\n      \"journal\": \"Journal of experimental & clinical cancer research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP and ubiquitination assays, functional in vivo model, single lab, indirect pathway mechanism\",\n      \"pmids\": [\"38254206\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"USP7 and the DNMT1 interaction via GK-repeat acetylation was reported to stabilize DNMT1; however, direct experimental testing showed that DNMT1 is present at normal levels in cells lacking USP7, and substitution of GK repeats to prevent acetylation does not affect DNMT1 stability. USP7 and PCNA are recruited to replication foci independently of DNMT1. This negative result indicates the USP7-DNMT1 stabilization model is not supported.\",\n      \"method\": \"Knockout cell lines (mouse and human lacking USP7), DNMT1 GK→GQ mutant knockin, chromatin fractionation, immunofluorescence\",\n      \"journal\": \"Epigenetics & chromatin\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — negative finding: knockout and mutagenesis both show no USP7 dependence for DNMT1 stability; rigorous genetic controls\",\n      \"pmids\": [\"29482658\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"USP7/HAUSP is a cysteine deubiquitinase whose catalytic domain is allosterically activated ~100-fold by its C-terminal HUBL-45 di-Ubl domain engaging a 'switching' loop, an activation further enhanced by GMPS binding; it recognizes substrates predominantly via a P/AXXS motif binding its N-terminal TRAF domain, and regulates a broad network including p53, MDM2, MDMX, PTEN, N-Myc, Chk1, Rad18, FOXO4, Ci/Gli, YAP/TAZ, KRAS, BCR-ABL, NOTCH1, HIF-1α, and many others through direct deubiquitination and stabilization, while its own activity is modulated by CK2-mediated phosphorylation at Ser18 (stabilizing) and PPM1G-mediated dephosphorylation after DNA damage (destabilizing), and by Trip12-mediated ubiquitination, collectively placing USP7 as a central hub coordinating the p53-MDM2 pathway, DNA damage response, Hedgehog and Hippo signaling, cell cycle progression, and immune regulation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"USP7 (HAUSP) is a cysteine deubiquitinase that acts as a central hub for protein-stability control, removing predominantly K48-linked polyubiquitin chains from a broad substrate network to oppose proteasomal degradation [#0, #12]. It is a monomeric, multidomain enzyme: an N-terminal TRAF-like domain recognizes substrates through a short P/AXXS motif (Trp165 critical), as defined by co-crystal structures with p53 and MDM2 peptides, while the catalytic activity is allosterically activated ~100-fold by the C-terminal di-Ubl unit (HUBL-45) engaging a 'switching' loop in the catalytic domain and is further hyperactivated by the metabolic enzyme GMPS [#1, #5, #6]. Its founding and best-defined role is in the p53–MDM2 axis, where USP7 deubiquitinates and stabilizes both p53 and MDM2/MDMX, and its dosage determines whether it preferentially protects MDM2 (repressing p53) or p53 itself [#0, #3]; consistent with this, Hausp-null mice die in early embryogenesis with p53 activation that is only partially relieved by p53 deletion, revealing additional essential p53-independent functions [#8]. USP7 activity is itself tuned by post-translational control — CK2-mediated Ser18 phosphorylation stabilizes the enzyme in unstressed cells, while ATM-dependent PPM1G dephosphorylation downregulates it after DNA damage to permit p53 accumulation, and Trip12 ubiquitinates USP7 to limit its abundance [#9, #19]. Beyond p53, USP7 stabilizes substrates across the DNA damage response and replication (Chk1, Rad18, MCM-BP-dependent MCM unloading) [#15, #16, #17], developmental and growth signaling (Ci/Gli in Hedgehog, YAP/TAZ in Hippo, N-Myc) [#11, #14, #23], and numerous oncoproteins including KRAS, BCR-ABL, and NOTCH1 [#26, #39, #40]. USP7 also restrains polyglutamine-disease proteostasis, where it preferentially deubiquitinates polyQ-expanded androgen receptor to promote aggregation, and its inhibition ameliorates SBMA and ALS phenotypes in vivo [#30, #31]. A reported USP7–DNMT1 stabilization model was not supported by genetic testing, as DNMT1 levels are normal in USP7-null cells [#58].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Establishing USP7 as a modular, catalytically active deubiquitinase set the structural framework for all later substrate work, defining an N-terminal substrate-binding domain distinct from the C-terminal regulatory region.\",\n      \"evidence\": \"Partial proteolysis/MS domain mapping, fluorescence binding, and in vitro deubiquitinase assays on purified protein\",\n      \"pmids\": [\"14506283\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve how the C-terminal domains regulate catalysis\", \"Substrate-recognition motif not yet defined\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defining USP7's dose-dependent, opposing effects on p53 and MDM2 explained how one deubiquitinase can either repress or activate the p53 pathway, answering how USP7 sits at the center of this circuit.\",\n      \"evidence\": \"Graded RNAi knockdown, Co-IP, and in vivo ubiquitination assays across cell lines\",\n      \"pmids\": [\"15053880\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish what shifts substrate preference between MDM2 and p53\", \"Structural basis of binding not yet known\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Co-crystal structures defined the P/AXXS recognition motif and the shared TRAF-domain surface used by p53, MDM2 and EBNA1, providing the molecular logic for competitive substrate selection.\",\n      \"evidence\": \"X-ray co-crystal structures of the N-terminal domain with p53/MDM2 peptides plus mutagenesis\",\n      \"pmids\": [\"16474402\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not explain how a trimeric p53-MDM2-USP7 state forms given mutually exclusive binding\", \"Affinity ranking of full substrate repertoire unknown\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Extending USP7 substrates to MDMX/HDMX and linking ATM phosphorylation to reduced USP7 affinity connected the enzyme to DNA-damage-regulated control of the p53 axis.\",\n      \"evidence\": \"Co-IP, in vivo/in vitro deubiquitination, RNAi, and DNA damage treatments\",\n      \"pmids\": [\"15916963\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct phosphatase/kinase controlling USP7 itself not yet identified\", \"Quantitative contribution to DDR not resolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Demonstrating non-degradative deubiquitination of FOXO4 showed USP7 can reverse mono-ubiquitination to control localization and transcription without altering protein half-life, broadening its functional output beyond stabilization.\",\n      \"evidence\": \"Reciprocal Co-IP, deubiquitination assays, localization imaging, transcriptional reporters\",\n      \"pmids\": [\"16964248\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Chain-type specificity of FOXO4 mono-Ub removal not defined\", \"Recruitment mechanism to FOXO4 not mapped\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Placing USP7 within a PML-DAXX network controlling PTEN nuclear-cytoplasmic trafficking established that USP7 regulates substrate localization through deubiquitination, integrating it into tumor-suppressor compartmentalization.\",\n      \"evidence\": \"Co-IP, deubiquitination assays, nuclear fractionation, RNAi, rescue experiments\",\n      \"pmids\": [\"18716620\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct vs DAXX-mediated effect on PTEN not fully separated\", \"Chain linkage on PTEN not characterized\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Knockout mice resolved the in vivo essentiality of USP7, showing early lethality driven by p53 activation but only partly rescued by p53 loss, proving p53-independent essential functions exist.\",\n      \"evidence\": \"Conventional/conditional knockout mice with hausp/p53 double-knockout epistasis\",\n      \"pmids\": [\"19946331\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the p53-independent essential substrates not defined\", \"Tissue-specific requirements not dissected\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"The crystal structure of the C-terminal Ubl array explained how USP7 is allosterically switched on, showing HUBL-45 activates the catalytic domain ~100-fold and GMPS stabilizes this active state.\",\n      \"evidence\": \"Crystal structure of C-terminal Ubl domains, in vitro activity assays, domain mutagenesis\",\n      \"pmids\": [\"21981925\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How substrate binding couples to the switching loop in cells not resolved\", \"Regulation of GMPS recruitment unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identifying CK2 (writer) and PPM1G (eraser) acting on Ser18 defined the phospho-switch that links DNA damage to USP7 abundance and p53 derepression, answering how USP7 itself is regulated by stress.\",\n      \"evidence\": \"Phospho-mapping, kinase/phosphatase assays, RNAi, IR treatment, Western blotting\",\n      \"pmids\": [\"22361354\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative effect of Ser18 on catalysis vs stability not fully separated\", \"Isoform-specific regulation incompletely defined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defining Trip12 as the E3 ligase for USP7 closed the loop on USP7's own turnover and linked its degradation to DDR substrate stability (p53, 53BP1, Chk1).\",\n      \"evidence\": \"Co-IP, in vivo ubiquitination, RNAi, cell cycle analysis\",\n      \"pmids\": [\"27800609\", \"27800609\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab finding without in vitro reconstitution\", \"Chain type used by Trip12 on USP7 not defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"A cluster of studies extended USP7 deubiquitination into the DNA replication and damage-tolerance machinery (Chk1, Rad18, MCM-BP), establishing USP7 as a stabilizer of replication and checkpoint factors via the P/AXXS-type motif.\",\n      \"evidence\": \"Co-IP, in vitro/in vivo deubiquitination, RNAi, UV/PCNA ubiquitination, chromatin fractionation, replication assays\",\n      \"pmids\": [\"25483066\", \"25961918\", \"24190967\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of each substrate to genome stability not ranked\", \"How USP7 timing on MCM unloading is controlled unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Cross-species work placed USP7 in developmental signaling, showing it stabilizes Ci/Gli (Hedgehog) and Yorkie/YAP-TAZ (Hippo) and that pathway activation modulates USP7-substrate binding.\",\n      \"evidence\": \"Co-IP, in vivo ubiquitination, Drosophila genetics, mammalian cell assays\",\n      \"pmids\": [\"26120032\", \"30679505\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct deubiquitination chain specificity on Gli/YAP not fully resolved\", \"Signal-dependent regulation of binding mechanistically undefined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identification of N-Myc as a USP7 substrate with in vivo knockout validation established USP7 as a stabilizer of an oncogenic transcription factor and a therapeutic target in neuroblastoma.\",\n      \"evidence\": \"Co-IP, deubiquitination assays, RNAi, conditional knockout mice, xenografts, USP7 inhibitors\",\n      \"pmids\": [\"27618649\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether N-Myc stabilization is direct vs cofactor-dependent not fully separated\", \"Selectivity over c-Myc not addressed\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Allosteric inhibitors and di-ubiquitin NMR clarified USP7's catalytic mechanism, showing it preferentially binds and depolymerizes K48-linked chains and can be inhibited by attenuating ubiquitin binding rather than blocking the active site.\",\n      \"evidence\": \"NMR screening, co-crystal structures, isotope-labeled di-ubiquitin NMR, in vitro DUB assays\",\n      \"pmids\": [\"29045385\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In-cell chain-linkage preference across substrates not mapped\", \"Determinants of K48 vs K63 discrimination on physiologic substrates unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"A rigorous negative study overturned the USP7-DNMT1 stabilization model, showing DNMT1 levels are unchanged in USP7-null cells and that USP7/PCNA recruitment to replication foci is DNMT1-independent.\",\n      \"evidence\": \"USP7-knockout mouse/human cells, DNMT1 GK→GQ knock-in, chromatin fractionation, immunofluorescence\",\n      \"pmids\": [\"29482658\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not exclude context- or modification-specific DNMT1 effects\", \"Original interaction's biological role left unexplained\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrating preferential deubiquitination of polyQ-expanded androgen receptor, and protective effects of USP7 inhibition in SBMA/ALS models, redefined USP7 as a modifier of disease-protein proteostasis rather than a generic stabilizer.\",\n      \"evidence\": \"Quantitative proteomics, Co-IP, deubiquitination assays, conditional knockout mice, cross-species ALS models, behavioral assays\",\n      \"pmids\": [\"33170804\", \"33106424\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Basis of preference for expanded vs wild-type AR not structurally defined\", \"Whether NEDD4L/SMAD2 axis fully accounts for ALS protection unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Mapping USP7 deubiquitination of KRAS at K147 via the TRAF domain extended its oncogenic substrate range to a major driver, reinforcing the therapeutic rationale across cancers.\",\n      \"evidence\": \"Co-IP, in vitro/in vivo deubiquitination, domain binding, ubiquitination-site mutagenesis, proliferation assays\",\n      \"pmids\": [\"39499616\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab finding\", \"Selectivity among KRAS isoforms/mutants not fully resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How USP7 achieves substrate selectivity in cells — given an enormous reported substrate list, shared P/AXXS recognition, and tunable allosteric activation — and which substrates dominate its essential in vivo functions remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No quantitative ranking of physiologic substrates by occupancy/flux\", \"p53-independent essential function (from knockout) lacks a defined effector\", \"Most disease-context substrates rest on single-lab Co-IP without reconstitution\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 3, 6, 15, 16, 26, 39]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [5, 6, 12]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 3, 11]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [2, 4, 28]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [4, 25]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 15, 17, 21, 25, 53]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [3, 16, 20, 47]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [14, 23, 36, 39]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 3, 11, 26, 40]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [44]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [24, 29]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"MDM2\", \"TP53\", \"MDM4\", \"GMPS\", \"TRIP12\", \"PPM1G\", \"MCM-BP\", \"DAXX\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}