{"gene":"UBR5","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2002,"finding":"EDD/UBR5 contains a UBA domain, UBR1 zinc finger motif, and HECT domain; localizes to cell nuclei via importin alpha5 through consensus NLS; binds progesterone receptor (PR) and potentiates progestin-mediated gene transactivation; also binds CIB/DNAPK-interacting protein with altered association after DNA damage.","method":"Co-immunoprecipitation, nuclear localization assay, transactivation reporter assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — reciprocal Co-IP and functional reporter assays in single lab, multiple orthogonal methods","pmids":["12011095"],"is_preprint":false},{"year":2004,"finding":"Homozygous knockout of murine Edd is lethal by E10.5; Edd-deficient embryos display failed yolk sac and allantoic vascular development and defective chorioallantoic fusion, establishing an essential role in extraembryonic vascular development.","method":"Gene targeting/knockout mouse, embryo phenotyping","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with defined developmental phenotype, rigorous genetic model","pmids":["15282321"],"is_preprint":false},{"year":2006,"finding":"EDD/UBR5 interacts with CHK2 via a phospho-dependent interaction involving the CHK2 Forkhead-associated (FHA) domain and EDD's FHA-binding threonines; EDD is required upstream of CHK2 for efficient activating phosphorylation of CHK2 after ionizing radiation or radiomimetic treatment.","method":"Co-immunoprecipitation, RNA interference (RNAi) knockdown, kinase activity assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNAi epistasis and Co-IP in single lab with two orthogonal methods","pmids":["17074762"],"is_preprint":false},{"year":2006,"finding":"The PABC/MLLE domain of UBR5/HYD binds PAM2 peptide motifs with micromolar affinity, similar to PABP's PABC domain; UBR5 PABC domain interacts with anti-proliferative Tob2 protein, linking UBR5 to translation regulation and cell cycle control.","method":"Isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), NMR chemical shift perturbation, GST pulldown, co-immunoprecipitation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple biophysical methods (ITC, SPR, NMR) plus pulldown and Co-IP, single study with orthogonal validation","pmids":["16554297"],"is_preprint":false},{"year":2007,"finding":"The UBA domain of EDD/UBR5 binds ubiquitin; crystal structure at 1.85 Å reveals recognition via UBA helices α1 and α3 with hydrogen bonds including ordered water molecules; site-directed mutagenesis confirmed functional importance of interface residues; SPR showed no strong preference for polyubiquitin chains over monoubiquitin.","method":"ITC, NMR titrations, pulldown, 1.85 Å crystal structure, site-directed mutagenesis, SPR","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with mutagenesis validation and multiple biophysical methods in one study","pmids":["17897937"],"is_preprint":false},{"year":2007,"finding":"EDD/UBR5 interacts with APC tumor suppressor protein; EDD overexpression increases APC and Axin protein levels and inhibits β-catenin/LEF1 Wnt signaling; EDD knockdown reduces APC protein level without altering its mRNA, increasing β-catenin.","method":"Mass spectrometry of APC immunocomplexes, co-immunoprecipitation, siRNA knockdown, immunofluorescence co-localization, reporter assays","journal":"Genes to cells","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — MS identification plus Co-IP and siRNA knockdown with functional readout, single lab","pmids":["18076571"],"is_preprint":false},{"year":2007,"finding":"EDD/UBR5 is necessary for G1/S and intra-S phase DNA damage checkpoint activation and for maintenance of G2/M arrest after DSBs; EDD depletion leads to radioresistant DNA synthesis, premature mitotic entry, polyploidy, and mitotic catastrophe.","method":"RNAi knockdown, cell cycle analysis (flow cytometry), DNA damage assays","journal":"Cell cycle","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined cellular phenotype with KD, checkpoint assays, single lab","pmids":["18073532"],"is_preprint":false},{"year":2010,"finding":"UBR5 associates with CDK9 subunit of P-TEFb and mediates its polyubiquitination; TFIIS binds UBR5 and stimulates CDK9 polyubiquitination; UBR5, CDK9, and TFIIS co-localize along the γ-fibrinogen gene; TFIIS overexpression increases CDK9 association with gene regions and enhances RNAPII Ser2-CTD phosphorylation.","method":"Co-immunoprecipitation, chromatin immunoprecipitation (ChIP), ubiquitination assay, co-localization","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus Co-IP and ubiquitination assays, single lab, multiple orthogonal methods","pmids":["21127351"],"is_preprint":false},{"year":2010,"finding":"UBR5 localizes to smooth muscle cell nuclei and forms a complex with myocardin both in vivo and in vitro; UBR5 enhances transactivation of smooth muscle-specific promoters by myocardin family proteins and stabilizes myocardin protein (attenuates its degradation) independently of E3 ligase activity, requiring only HECT and UBR1 domains.","method":"Yeast two-hybrid, co-immunoprecipitation, siRNA knockdown, promoter-luciferase reporter, western blot protein stability assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus Co-IP, reporter assays, and siRNA with functional readout, single lab","pmids":["20167605"],"is_preprint":false},{"year":2010,"finding":"EDD/UBR5 interacts with GSK-3β and β-catenin; EDD expression promotes nuclear accumulation of both proteins and enhances β-catenin stability and activity by ubiquitinating β-catenin through Lys29- or Lys11-linked ubiquitin chains, leading to increased β-catenin protein levels.","method":"Co-immunoprecipitation, ubiquitination assay (K29/K11 chain linkage), nuclear fractionation, luciferase reporter assay","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and in-cell ubiquitination assay with chain-linkage analysis, reporter assay, single lab","pmids":["21118991"],"is_preprint":false},{"year":2011,"finding":"PEPCK1 is acetylated by P300 acetyltransferase; acetylated PEPCK1 interacts with UBR5 HECT E3 ligase, promoting PEPCK1 ubiquitination and degradation; SIRT2 deacetylates PEPCK1, stabilizing it; high glucose destabilizes PEPCK1 via this acetylation-UBR5 axis.","method":"Co-immunoprecipitation, in vitro ubiquitination assay, protein stability assay, acetylation mapping","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro ubiquitination, Co-IP, and acetyltransferase/deacetylase assays with mechanistic dissection, published in high-tier journal","pmids":["21726808"],"is_preprint":false},{"year":2011,"finding":"EDD/UBR5 is a key component of the miRNA silencing pathway; EDD interacts with GW182 proteins in Argonaute-miRNA complexes; EDD E3 ligase activity is dispensable for miRNA silencing; the PABC domain of EDD is essential for silencing function by recruiting DDX6 and Tob1/2.","method":"Genetic screen in mouse ES cells, co-immunoprecipitation, domain deletion analysis, RNAi silencing assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic screen plus Co-IP, domain mutants, and functional silencing assays, replicated with multiple miRNA targets","pmids":["21726813"],"is_preprint":false},{"year":2011,"finding":"EDD/UBR5 interacts with both HPV18 E6 and E6AP independently; EDD regulates E6AP expression levels independently of E6; loss of EDD stimulates the proteolytic activity of the E6/E6AP complex and enhances cell resistance to apoptotic stimuli.","method":"Mass spectrometry, co-immunoprecipitation, siRNA knockdown, protein stability/degradation assays","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS identification plus Co-IP and siRNA functional assays, single lab","pmids":["21228227"],"is_preprint":false},{"year":2011,"finding":"EDD/UBR5 physically interacts with p53 and this interaction blocks ATM-mediated phosphorylation of p53 at Ser15; EDD depletion induces p53 Ser15 phosphorylation and activates p53 target genes; EDD overexpression inhibits p53-Ser15 phosphorylation during DNA damage independently of E3 ligase activity; G1 arrest from EDD depletion is p53-dependent.","method":"Co-immunoprecipitation, siRNA knockdown, phospho-specific western blotting, co-depletion epistasis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus functional RNAi epistasis and phosphorylation assays, single lab","pmids":["21383020"],"is_preprint":false},{"year":2012,"finding":"TRIP12 and UBR5 control accumulation of RNF168 by targeting it for degradation; depletion of TRIP12 and UBR5 allows supraphysiological accumulation of RNF168, leading to massive spreading of ubiquitin conjugates (H2A-Ub) and hyperaccumulation of 53BP1 and BRCA1 beyond DNA lesions.","method":"siRNA depletion, immunofluorescence, DSB-induced ubiquitin spreading assay","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — RNAi epistasis with clear substrate (RNF168) and quantified chromatin ubiquitin spreading, high-tier journal","pmids":["22884692"],"is_preprint":false},{"year":2012,"finding":"Crystal structure of the C-lobe of the HECT domain of human UBR5 reveals a unique four-residue insert elongating helix 2, creating a protruding loop likely important for E2 specificity toward UBCH4; the C-lobe forms a thioester-linked E3-ubiquitin complex in ubiquitination assays.","method":"X-ray crystallography, ubiquitination thioester assay, NMR","journal":"Acta crystallographica Section F","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with biochemical validation of catalytic thioester intermediate","pmids":["23027739"],"is_preprint":false},{"year":2012,"finding":"EDD/UBR5 interacts with microspherule protein Msp58 (MCRS1); EDD depletion increases Msp58 protein levels and extends its half-life, demonstrating EDD negatively regulates Msp58 stability via the ubiquitin-proteasome pathway; knockdown of either protein affects cyclin B, D, E levels and cell cycle progression.","method":"Co-immunoprecipitation, in vitro binding, confocal co-localization, protein half-life assay, siRNA knockdown, flow cytometry","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP, protein stability, and cell cycle assays in single lab","pmids":["23069210"],"is_preprint":false},{"year":2013,"finding":"Dyrk2 phosphorylates TERT; phosphorylated TERT associates with the EDD-DDB1-VprBP E3 ligase complex, leading to ubiquitin-mediated TERT degradation at G2/M phase; Dyrk2 depletion disrupts cell cycle-dependent TERT regulation and causes constitutive telomerase activation.","method":"Co-immunoprecipitation, ubiquitination assay, telomerase activity assay (TRAP), kinase assay, siRNA knockdown, cell cycle synchronization","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — kinase assay plus ubiquitination assay plus telomerase functional readout, multiple orthogonal methods","pmids":["23362280"],"is_preprint":false},{"year":2013,"finding":"HIV-1 Vpr enhances interaction between TERT and the VPRBP substrate receptor of the DYRK2-associated EDD-DDB1-VPRBP E3 ligase, increasing TERT ubiquitination and degradation; a Vpr mutation found in long-term non-progressors failed to promote TERT destabilization.","method":"Co-immunoprecipitation, ubiquitination assay, telomerase activity assay, mutant Vpr analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assay with mutant controls, single lab","pmids":["23612978"],"is_preprint":false},{"year":2013,"finding":"VprBP/DCAF1 serves as substrate recognition subunit for both the RING-type CRL4 and HECT-type EDD/UBR5 E3 ligase complexes; VprBP assembles into the EDD complex with DYRK2 and DDB1.","method":"Review/biochemical analysis of prior co-IP and complex assembly data (cited from primary studies)","journal":"BMC molecular biology","confidence":"Low","confidence_rationale":"Tier 4 / Weak — review paper synthesizing prior findings without new primary experiments described","pmids":["24028781"],"is_preprint":false},{"year":2014,"finding":"UBR5-mediated ubiquitination of ATMIN at lysine 238 is stimulated by ionizing radiation (IR); this ubiquitination decreases ATMIN-ATM interaction, promotes MRN-mediated ATM signaling, and NBS1/ATM foci formation; UBR5 deficiency or ATMIN K238 mutation impairs ATM checkpoint activation and increases radiosensitivity.","method":"Co-immunoprecipitation, site-directed mutagenesis (K238), ubiquitination assay, IR treatment, ATM foci formation, checkpoint assays, siRNA knockdown","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — site-specific mutagenesis of ubiquitination site plus functional checkpoint and foci assays, mechanistic dissection of pathway","pmids":["25092319"],"is_preprint":false},{"year":2014,"finding":"UBR5 and DYRK2 regulate hPXR stability; UBR5 knockdown causes hPXR accumulation and increased hPXR transcriptional activity; DYRK2-dependent phosphorylation of hPXR facilitates its subsequent ubiquitination by UBR5.","method":"siRNA knockdown, MS analysis, kinome-wide siRNA screen, protein stability assay, reporter assay","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — kinome siRNA screen plus functional protein stability and activity assays, single lab","pmids":["24438055"],"is_preprint":false},{"year":2015,"finding":"The MLLE domain of UBR5 binds PAM2 peptides (from Paip1 and GW182) with defined network of hydrophobic and ionic interactions shown by crystal structure with Paip1 PAM2 peptide; a novel intramolecular interaction between MLLE domain and adjacent HECT domain via a PAM2-like sequence was identified, suggesting regulation of UBR5 ligase activity.","method":"X-ray crystallography (MLLE-PAM2 complex), NMR, ITC, domain interaction mapping","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with multiple biophysical validations plus intramolecular interaction discovery","pmids":["26224628"],"is_preprint":false},{"year":2015,"finding":"EDD1/UBR5 interacts with TIP60 acetyltransferase and negatively regulates TIP60 stability through the proteasome pathway; HPV E6 oncogene exploits EDD1 to destabilize TIP60; depletion of EDD1 or gain-of-function of TIP60 inhibits HPV-positive cervical cancer cell growth in vitro and in vivo.","method":"Proteomics, co-immunoprecipitation, ubiquitination/turnover assay, colony formation, soft agar, xenograft","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS identification plus Co-IP, ubiquitination assay, and in vivo tumor model, single lab","pmids":["26234678"],"is_preprint":false},{"year":2016,"finding":"UBR5 physically interacts with MOAP-1, ubiquitylates MOAP-1 in vitro, and inhibits MOAP-1 stability in cells; Dyrk2 kinase cooperates with UBR5 in MOAP-1 ubiquitylation; UBR5 knockdown increases MOAP-1 levels, enhances Bax activation, and sensitizes cisplatin-resistant ovarian cancer cells to apoptosis.","method":"Co-immunoprecipitation, in vitro ubiquitination assay, protein stability assay, siRNA knockdown, Bax activation assay, cisplatin sensitivity assay","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro ubiquitination plus Co-IP and functional apoptosis assays, mechanistic dissection with kinase cooperation","pmids":["27721409"],"is_preprint":false},{"year":2016,"finding":"UBR5 forms damage-inducible nuclear foci dependent on PRC1 components BMI1, RING1a, and RING1b; UBR5 associates with BMI1 and FACT components SPT16/SSRP1; UBR5 ubiquitinates SPT16; UBR5 and BMI1 repress RNA Pol II transcription elongation at UV-damaged chromatin by negatively regulating FACT-dependent Pol II elongation; UBR5/BMI1 KO cells are hypersensitive to UV.","method":"Mass spectrometry, co-immunoprecipitation, in vitro ubiquitination assay, transcription elongation assay, immunofluorescence foci analysis, CRISPR KO, UV sensitivity assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro ubiquitination, MS, Co-IP, functional transcription assay, and genetic KO, multiple orthogonal methods","pmids":["27647897"],"is_preprint":false},{"year":2016,"finding":"UBR5 depletion reduces primary cilia formation; CSPP1 (centrosomal/ciliary protein required for cilia formation) is a UBR5-interacting protein; UBR5 ubiquitylates CSPP1; UBR5 is required for cytoplasmic organization of CSPP1-comprising centriolar satellites in centrosomal periphery.","method":"Co-immunoprecipitation, in vitro ubiquitination assay, siRNA knockdown, ciliogenesis assay, centrosomal fractionation","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay and functional ciliogenesis readout, single lab","pmids":["29742019"],"is_preprint":false},{"year":2016,"finding":"C. elegans UBR-5 negatively regulates Notch-type (GLP-1/LIN-12) signaling; ubr-5 loss-of-function suppresses glp-1 and lin-12 loss-of-function defects; ubr-5 acts in germ cells (receiving cells) to limit GLP-1 signaling; UBR-5 acts redundantly with SEL-10 (SCF E3 ligase) to limit Notch signaling in certain tissues.","method":"C. elegans genetics, suppressor/enhancer epistasis, loss-of-function analysis","journal":"G3 (Bethesda)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic epistasis in C. elegans with multiple alleles and tissue-specific analysis, single lab","pmids":["27185398"],"is_preprint":false},{"year":2017,"finding":"Hyd/UBR5 is required for Wnt signal responses downstream of activated Armadillo/β-catenin in Drosophila and human cell lines; Groucho/TLE is a functionally relevant substrate whose ubiquitylation by UBR5 is induced by Wnt signaling and conferred by β-catenin; TLE inactivation by UBR5-dependent ubiquitylation also involves VCP/p97 AAA ATPase.","method":"Drosophila genetics, siRNA knockdown in human cells, co-immunoprecipitation, ubiquitination assay, epistasis","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — genetic epistasis in Drosophila plus human cell biochemistry (Co-IP, ubiquitination), replicated across species","pmids":["28689657"],"is_preprint":false},{"year":2017,"finding":"CRISPR/Cas9 deletion of UBR5 in murine TNBC model abrogates tumor growth and metastasis in vivo; reconstitution with wild-type UBR5 but not a catalytically inactive mutant rescues this phenotype, demonstrating E3 ligase activity is required for tumor growth and metastasis functions.","method":"CRISPR/Cas9 knockout, catalytically inactive mutant rescue, in vivo tumor model","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — CRISPR KO with catalytic mutant rescue in vivo, establishing enzymatic activity requirement","pmids":["28330927"],"is_preprint":false},{"year":2018,"finding":"UBR5 is highly expressed in iPSCs and is required for proteasomal degradation of both normal and polyQ-expanded mutant huntingtin (HTT); UBR5 loss increases HTT levels and triggers polyQ-expanded aggregation in HD-iPSCs; UBR5 overexpression induces polyubiquitination and degradation of mutant HTT, reducing aggregates.","method":"siRNA knockdown, overexpression, ubiquitination assay, aggregation assay, invertebrate model (C. elegans/Drosophila) knockdown","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — RNAi plus OE with ubiquitination assay, replicated in invertebrate models, multiple orthogonal methods","pmids":["30038412"],"is_preprint":false},{"year":2018,"finding":"UBR5 is required for cytoplasmic organization of CSPP1-comprising centriolar satellites; UBR5 ubiquitylates CSPP1 and is required for primary cilia formation.","method":"Co-immunoprecipitation, ubiquitination assay, ciliogenesis assay, siRNA knockdown","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, and ciliogenesis functional assay, single lab","pmids":["29742019"],"is_preprint":false},{"year":2019,"finding":"OTUD5 deubiquitinase stabilizes UBR5 E3 ligase; OTUD5 localizes to DSBs and interacts with UBR5; the OTUD5-UBR5 complex represses RNA Pol II elongation and RNA synthesis at DSBs; OTUD5 interacts with FACT component SPT16 via a separate region; both UBR5 stabilization (catalytic) and FACT binding (scaffolding) activities of OTUD5 are required for Pol II arrest.","method":"DUB RNAi screen, co-immunoprecipitation, transcription elongation assay, domain mapping, co-localization","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNAi screen plus Co-IP and functional transcription assay, single lab","pmids":["30508113"],"is_preprint":false},{"year":2019,"finding":"AKT phosphorylates SOX2 at Thr116, which inhibits UBR5 interaction with SOX2; UBR5 ubiquitinates SOX2 at Lys115, promoting its degradation; AKT-mediated phosphorylation stabilizes SOX2 by blocking this UBR5-mediated ubiquitination.","method":"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (K115, T116), kinase assay, protein stability assay","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 / Strong — site-specific mutagenesis of ubiquitination site and phosphorylation site, in vitro ubiquitination, mechanistic dissection","pmids":["30894683"],"is_preprint":false},{"year":2019,"finding":"PPARγ promotes ATM signaling through interaction with MRN complex and UBR5; PPARγ is essential for UBR5 activity targeting ATMIN; PPARγ depletion increases ATMIN protein and suppresses DDR-induced ATM signaling; disrupted PPARγ-UBR5 interaction is observed in PAH patient endothelial cells.","method":"Proteomic interaction screen, co-immunoprecipitation, siRNA knockdown, ATM signaling assays","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomics plus Co-IP and functional ATM signaling assays, single lab","pmids":["30699358"],"is_preprint":false},{"year":2019,"finding":"UBR5 interacts with components of the replication fork including TLS polymerase polη; UBR5 depletion causes S-phase progression defects, ssDNA accumulation, and mis-regulation of H2A ubiquitination (UbiH2A); blocking H2A ubiquitination rescues replication problems in UBR5-depleted cells; polη is the main cause of replication defects when UBR5 is silenced; polη interacts with H2A, suggesting UbiH2A regulates polη recruitment.","method":"Co-immunoprecipitation, siRNA knockdown, DNA fiber assay (S-phase progression), ssDNA accumulation assay, epistasis with H2A modification","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus epistasis and replication fiber assay, single lab with multiple methods","pmids":["31586398"],"is_preprint":false},{"year":2019,"finding":"UBR5 interacts with H/ACA ribonucleoprotein complex components in ESCs; loss of UBR5 induces abnormal accumulation of rRNA processing intermediates, diminished ribosomal levels, increased p53 levels, and decreased cell proliferation.","method":"Protein interactome (MS), co-immunoprecipitation, rRNA processing assay, western blot, proliferation assay","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS interactome plus Co-IP and rRNA functional assay, single lab","pmids":["31365120"],"is_preprint":false},{"year":2020,"finding":"UBR5 ubiquitinates MYC and promotes its degradation independently of FBXW7; UBR5 silencing induces MYC protein accumulation; UBR5 and MYC are co-amplified in MYC-driven cancers; in p53-mutant MYC-amplified cells, UBR5 suppresses MYC-mediated apoptosis; Drosophila HYD suppresses dMYC-dependent overgrowth.","method":"CRISPR/Cas9 screen, co-immunoprecipitation, ubiquitination assay (K48-linked), siRNA knockdown, protein stability assay, Drosophila genetics","journal":"Cancer research / Scientific reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — CRISPR screen identification plus in-cell ubiquitination assay and Drosophila genetic validation, replicated across two independent papers (PMID 32029551, 33208877)","pmids":["32029551","33208877"],"is_preprint":false},{"year":2020,"finding":"Drosophila Hyd/UBR5 mediates Lys63-linked polyubiquitination of the NF-κB cofactor Akirin, which is required for efficient Akirin-Relish (NF-κB) interaction and transcription of immune-induced anti-microbial peptide genes; human UBR5 is also required for IL-6 transcription downstream of NF-κB signaling by LPS or IL-1β.","method":"RNAi screen, ubiquitination assay (K63 chain), co-immunoprecipitation, Drosophila survival assay, human cell siRNA knockdown, luciferase reporter","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1 / Strong — K63 ubiquitination assay plus genetic epistasis in Drosophila and human cell validation, mechanistic dissection","pmids":["32339205"],"is_preprint":false},{"year":2020,"finding":"Loss of UBR5 HECT domain in B cells causes a block in B-cell maturation in spleen and upregulation of spliceosome components; UBR5 is required for B-cell maturation by promoting degradation/destabilization of spliceosome components during B-cell development.","method":"Conditional knockout (HECT domain deletion), flow cytometry, western blot, gene expression analysis","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with defined B-cell maturation phenotype, single lab","pmids":["32325489"],"is_preprint":false},{"year":2020,"finding":"UBR5 controls β-catenin-mediated signaling and regulates p53 protein level in ovarian cancer; tumor-derived UBR5 promotes macrophage recruitment via chemokines/cytokines.","method":"siRNA knockdown, CRISPR KO, protein stability assay, cytokine/chemokine profiling","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO and siRNA with β-catenin/p53 mechanistic readouts, single lab","pmids":["33293516"],"is_preprint":false},{"year":2021,"finding":"UBR5 interacts with and promotes degradation of CAPZA1 (F-actin capping protein α subunit) via ubiquitin-proteasome system; UBR5 overexpression induces F-actin accumulation; CAPZA1 downregulation reverses UBR5-knockdown-mediated suppression of pancreatic cancer cell migration/invasion.","method":"Co-immunoprecipitation with mass spectrometry, ubiquitination assay, protein stability assay, siRNA knockdown, F-actin staining, migration/invasion assay, in vivo liver metastasis model","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS substrate identification plus Co-IP, ubiquitination assay, and functional rescue epistasis, single lab","pmids":["33777788"],"is_preprint":false},{"year":2022,"finding":"UBR5 promotes antiviral immunity by mediating Lys63-linked ubiquitination of TRIM28 (epigenetic repressor of RLRs); this modification prevents intramolecular SUMOylation of TRIM28, disengaging TRIM28-imposed transcriptional repression of RIG-I-like receptor genes; Ubr5 KO mice are more susceptible to RNA virus infection.","method":"CRISPR KO screen (375 E3 ligases), ubiquitination assay (K63-linked), SUMOylation assay, co-immunoprecipitation, Ubr5 KO mice, viral challenge","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — genome-wide CRISPR screen plus mechanistic K63-Ub and SUMO assays and in vivo mouse validation","pmids":["38278841"],"is_preprint":false},{"year":2022,"finding":"UBR5 polyubiquitinates CDC73 at Lys243, Lys247, and Lys257 in a non-canonical manner dependent on non-phosphorylated CDC73 (Ser465); CDC73 destabilization by UBR5 regulates β-catenin and E-cadherin expression and tumor cell apoptosis and CD8+ T cell infiltration in TNBC.","method":"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (K243/247/257, S465), protein stability assay, in vivo tumor model","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 1 / Strong — site-specific ubiquitination mapping by mutagenesis with phosphorylation regulation, functional in vivo validation","pmids":["35551175"],"is_preprint":false},{"year":2022,"finding":"UBR5 ubiquitinates BubR1, Bub3, and Cdc20 (MCC components); UBR5 promotes dissociation of the Bub3•BubR1 subcomplex from APC/C via ubiquitylation, facilitated by ATP hydrolysis; UBR5 immunodepletion slows MCC disassembly and prolongs lag period in APC/C activity recovery; a reconstituted system from purified components shows UBR5- and ubiquitylation-dependent Bub3•BubR1 dissociation.","method":"Co-immunoprecipitation from nocodazole-arrested HeLa cells, in vitro ubiquitination assay, immunodepletion, reconstitution from purified components, APC/C activity assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution from purified components plus in vitro ubiquitination and immunodepletion assays, mechanistic dissection of MCC disassembly","pmids":["35217622"],"is_preprint":false},{"year":2022,"finding":"UBR5 enhances IFN-γ-induced PD-L1 transcription through its PABC domain by upregulating PKR and downstream STAT1/IRF1, in an E3 ubiquitin activity-independent manner.","method":"RNA-seq, qPCR, ChIP-qPCR, luciferase reporter, siRNA/CRISPR knockdown and reconstitution, domain deletion analysis","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — transcriptomic plus ChIP and reporter assays with domain mutant, single lab","pmids":["35836797"],"is_preprint":false},{"year":2023,"finding":"Cryo-EM structure of full-length human UBR5 reveals an α-solenoid scaffold assembled into an antiparallel dimer with further oligomeric states; the catalytic HECT domain is dynamic; AKIRIN2 (proteasomal nuclear import factor) was identified as an interacting protein; UBR5 preferentially acts as a ubiquitin chain elongator on pre-ubiquitinated substrates.","method":"Cryo-EM (full-length human UBR5), negative stain EM (UBR5-RARA/RXRA complex), co-immunoprecipitation, in vitro ubiquitin chain elongation assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM structure with biochemical validation of chain elongation preference and new interacting protein","pmids":["37409633"],"is_preprint":false},{"year":2023,"finding":"UBR5 drives agonist-dependent degradation of multiple nuclear hormone receptors (RARA, RXRA, GR, ERα, LXR, PR, VDR); cryo-EM structure of full-length human UBR5 obtained; agonist ligands induce sequential, mutually exclusive recruitment of NCOAs and UBR5 to chromatin; SERDs degrade ERα through differential recruitment of UBR5 or RNF111.","method":"Cryo-EM structure, negative-stain EM with RARA/RXRA complex, co-immunoprecipitation, chromatin recruitment assays, siRNA knockdown, protein stability assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure plus chromatin recruitment and substrate degradation assays across multiple NRs, high-tier journal","pmids":["37478846"],"is_preprint":false},{"year":2024,"finding":"UBR5 is required for Rb protein concentration decrease during G1; UBR5 KO cells have increased Rb concentration in early G1, lower G1-S transition rate, and increased sensitivity to CDK4/6 inhibitors.","method":"UBR5 CRISPR KO, single-cell protein concentration measurement, cell cycle analysis, CDK4/6 inhibitor sensitivity assay","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO with defined cell cycle phenotype, single lab, no direct ubiquitination of Rb demonstrated in abstract","pmids":["39441926"],"is_preprint":false},{"year":2024,"finding":"UBR5 acts as an E3 ubiquitin ligase for OGA (O-GlcNAcase); UBR5 binds and promotes OGA ubiquitination and degradation, leading to increased O-GlcNAcylation; this promotes EMT-mediated gemcitabine resistance in pancreatic cancer.","method":"Co-immunoprecipitation, ubiquitination assay, protein stability assay, siRNA knockdown, OGA level rescue experiments","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay and functional epistasis, single lab","pmids":["38755129"],"is_preprint":false},{"year":2024,"finding":"UBR5 stabilizes oxaliplatin-activated Smad3 via Lys11-linked polyubiquitination (non-degradative), which facilitates transcriptional repression of ATF3, induction of SLC7A11, and inhibition of ferroptosis, conferring chemoresistance in colorectal cancer.","method":"Co-immunoprecipitation, ubiquitination assay (Lys11-linked chain), siRNA knockdown, ferroptosis assay, transcriptional reporter","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus K11 chain-specific ubiquitination and functional ferroptosis assays, single lab","pmids":["39260061"],"is_preprint":false},{"year":2015,"finding":"RanGTP promotes the dissociation of importin-β from BuGZ and Bub3 in metaphase, resulting in increased binding of BuGZ and Bub3 to Ubr5; this leads to Ubr5-dependent ubiquitination and turnover of BuGZ and Bub3, facilitating metaphase-to-anaphase transition (SAC silencing).","method":"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, cell cycle analysis","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, and functional metaphase-anaphase transition assay, single lab","pmids":["26438829"],"is_preprint":false},{"year":2017,"finding":"Cep78 localizes to mature centrioles and directly interacts with VprBP, a component of the EDD-DYRK2-DDB1VprBP E3 ligase; Cep78 binds specifically to EDD-DYRK2-DDB1VprBP (not CRL4VprBP) and inhibits its activity; EDD-DYRK2-DDB1VprBP ubiquitinates CP110 (novel centrosomal substrate) after DYRK2-dependent phosphorylation; Cep78 impedes ubiquitin transfer from EDD to CP110 without affecting CP110 phosphorylation.","method":"Co-immunoprecipitation, in vitro ubiquitination assay, centrosome fractionation, domain interaction mapping, centriole length measurement, cilia assay","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro ubiquitination reconstitution with step-specific inhibitor analysis (Cep78 blocks transfer step), multiple orthogonal methods","pmids":["28242748"],"is_preprint":false},{"year":2018,"finding":"UBR5 interacts with HTLV-1 HBZ protein; UBR5 knockdown enhances HBZ steady-state levels by stabilizing HBZ; Co-IP assays confirmed HBZ ubiquitination that is reduced upon UBR5 knockdown; MS/MS identified seven ubiquitinated lysines in HBZ.","method":"Affinity-tagged protein pulldown, shotgun proteomics, co-immunoprecipitation, shRNA knockdown, protein stability assay, MS/MS ubiquitin site mapping","journal":"Frontiers in microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS identification plus Co-IP, ubiquitination assay, and stability assay, single lab","pmids":["29441057"],"is_preprint":false},{"year":2022,"finding":"UBR5 ubiquitinates MERS-CoV ORF4b at Lys36, promoting its degradation; UBR5 can translocate into the nucleus via its NLS to regulate ORF4b stability in both cytoplasm and nucleus; UBR5 knockdown enhances ORF4b anti-immune activity and increases MERS-CoV replication.","method":"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (K36), siRNA knockdown, viral replication assay, subcellular localization assay","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay with mutagenesis of ubiquitination site, functional viral assay, single lab","pmids":["35980206"],"is_preprint":false},{"year":2020,"finding":"UBR5 regulates FBP1 expression in pancreatic cancer by binding to C/EBPα transcription factor and promoting its ubiquitination and degradation; UBR5-induced aerobic glycolysis is dependent on this FBP1-C/EBPα axis.","method":"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, protein stability assay, glycolysis assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay and functional metabolic readout, single lab","pmids":["33122826"],"is_preprint":false},{"year":2013,"finding":"EDD/UBR5 physically interacts with alpha4 phosphoprotein; Co-IP confirmed EDD-alpha4 interaction; EDD knockdown leads to decreased PP2Ac polyubiquitination and accumulation of PP2Ac protein, identifying PP2Ac as an EDD substrate regulated via alpha4 as scaffold.","method":"Co-immunoprecipitation using deletion mutants, siRNA knockdown, proteasome inhibitor treatment, polyubiquitination assay","journal":"Molecular and cellular endocrinology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP with deletion analysis plus siRNA functional assay, single lab","pmids":["24145130"],"is_preprint":false}],"current_model":"UBR5 is a nuclear HECT-domain E3 ubiquitin ligase that forms an antiparallel dimer (cryo-EM resolved) and functions primarily as a ubiquitin chain elongator; it ubiquitinates a broad range of substrates—including PEPCK1 (acetylation-triggered), RNF168, TLE/Groucho (Wnt-dependent), ATMIN (IR-stimulated, K238), MYC, SOX2 (AKT-phosphorylation regulated, K115), TERT (Dyrk2-phosphorylation gated), CDC73 (K243/247/257), CP110, BubR1/Bub3/Cdc20 (MCC disassembly), MOAP-1, Rb, OGA, Smad3 (K11-non-degradative), CAPZA1, and viral proteins—using distinct chain linkages (K11, K29, K48, K63) depending on context; it additionally acts in a ligase-independent manner via its PABC/MLLE domain to scaffold miRNA silencing complexes (interacting with GW182/DDX6/Tob) and to regulate transcription (myocardin coactivation, PD-L1 via PKR-STAT1); it participates in DNA damage response (CHK2 activation, ATM pathway selection via ATMIN ubiquitination, replication fork protection via H2A ubiquitination control, transcriptional silencing at lesions via BMI1-FACT axis), Wnt signaling (TLE ubiquitylation), Notch pathway suppression, antiviral immunity (K63-ubiquitination of TRIM28 to de-repress RLR transcription), mitotic checkpoint disassembly, ciliogenesis (CSPP1 ubiquitylation), and nuclear hormone receptor turnover (agonist-dependent degradation of RARA, ERα, and other NRs on chromatin)."},"narrative":{"mechanistic_narrative":"UBR5 (EDD/HYD) is a nuclear HECT-domain E3 ubiquitin ligase that integrates protein quality control, transcriptional regulation, the DNA damage response, and cell-cycle progression by selectively modifying substrates with distinct ubiquitin chain linkages [PMID:12011095, PMID:28330927, PMID:37409633]. Structurally, it is built from an α-solenoid scaffold that assembles into an antiparallel dimer with higher-order oligomers and a dynamic catalytic HECT domain; biochemically it preferentially acts as a ubiquitin chain elongator on pre-ubiquitinated substrates, and its activity is tuned by an intramolecular contact between the MLLE/PABC domain and the HECT domain [PMID:26224628, PMID:37409633]. Its modular architecture combines a ubiquitin-binding UBA domain, a UBR1 zinc finger, a HECT catalytic lobe whose unique C-lobe insert dictates E2 (UBCH4) specificity, and a PAM2-recognizing MLLE/PABC domain [PMID:12011095, PMID:17897937, PMID:23027739, PMID:26224628]. Catalytic substrates span cell-cycle and growth regulators (MYC degraded independently of FBXW7, SOX2 at Lys115 gated by AKT phosphorylation, CDC73, Rb, and the mitotic checkpoint complex components BubR1/Bub3/Cdc20 whose ubiquitylation drives MCC disassembly and APC/C reactivation), metabolic and stress factors (acetylated PEPCK1, OGA, MOAP-1), and the centriolar substrate CP110 and ciliary protein CSPP1, with several substrates handed to UBR5 only after priming phosphorylation by DYRK2 within an EDD-DDB1-VprBP/DYRK2 complex [PMID:21726808, PMID:23362280, PMID:27721409, PMID:29742019, PMID:30894683, PMID:32029551, PMID:33208877, PMID:35551175, PMID:35217622, PMID:39441926, PMID:28242748]. In the DNA damage response UBR5 restrains RNF168-driven ubiquitin spreading, ubiquitylates ATMIN at Lys238 to bias ATM toward MRN-dependent signaling, and cooperates with PRC1/BMI1 and the FACT subunit SPT16 to silence RNA Pol II elongation at lesions [PMID:22884692, PMID:25092319, PMID:27647897, PMID:31586398]. Beyond degradative chemistry, UBR5 builds non-degradative K11 chains on Smad3 and K63 chains on TRIM28 and Akirin to control transcription, ferroptosis resistance, and NF-κB- and RLR-driven immunity [PMID:32339205, PMID:38278841, PMID:39260061]. UBR5 also acts independently of its ligase activity: through the MLLE/PABC domain it scaffolds miRNA silencing by recruiting GW182, DDX6, and Tob1/2, stabilizes the transcription factor myocardin, and drives IFN-γ-induced PD-L1 transcription via PKR-STAT1 [PMID:21726813, PMID:20167605, PMID:35836797]. It additionally executes agonist-dependent, chromatin-coupled degradation of nuclear hormone receptors including RARA, ERα, and others through sequential exchange of coactivators and UBR5 [PMID:37478846]. Genetic loss is embryonic lethal in mouse with failed extraembryonic vascular development, and UBR5 catalytic activity is required for breast tumor growth and metastasis [PMID:15282321, PMID:28330927].","teleology":[{"year":2002,"claim":"Established UBR5 as a nuclear, multidomain protein physically coupled to hormone-receptor transcription, defining the cellular compartment and an early functional context.","evidence":"Co-IP, NLS-driven nuclear import assay, and progesterone-receptor transactivation reporter","pmids":["12011095"],"confidence":"Medium","gaps":["No catalytic activity demonstrated","PR not shown to be a ubiquitination substrate"]},{"year":2004,"claim":"Demonstrated UBR5 is essential in vivo, establishing a non-redundant developmental requirement.","evidence":"Murine Edd knockout with embryo phenotyping","pmids":["15282321"],"confidence":"High","gaps":["Molecular substrate underlying vascular defect unidentified","Catalytic dependence not tested in this model"]},{"year":2006,"claim":"Defined the biochemistry of the MLLE/PABC domain as a PAM2-peptide recognition module linking UBR5 to translation/anti-proliferative regulators, the structural basis for its later ligase-independent scaffolding roles.","evidence":"ITC, SPR, NMR, GST pulldown, Co-IP with Tob2","pmids":["16554297"],"confidence":"High","gaps":["Functional consequence of Tob2 binding not resolved","Did not connect to a defined complex"]},{"year":2006,"claim":"Placed UBR5 upstream of CHK2 activation, implicating it in DNA-damage checkpoint signaling.","evidence":"FHA-dependent Co-IP, RNAi epistasis, kinase activity assay after IR","pmids":["17074762"],"confidence":"Medium","gaps":["Whether regulation is catalytic or scaffolding unresolved","No direct substrate in the CHK2 axis defined"]},{"year":2007,"claim":"Resolved how UBR5 reads ubiquitin (UBA domain) and validated catalytic competence and E2 specificity of its HECT C-lobe, anchoring its mechanism as a HECT ligase.","evidence":"1.85 Å UBA crystal structure with mutagenesis and SPR; HECT C-lobe crystal structure with thioester ubiquitination assay","pmids":["17897937","23027739"],"confidence":"High","gaps":["No full-length structure at this stage","Chain-linkage preferences not defined"]},{"year":2007,"claim":"Showed UBR5 is required for multiple cell-cycle checkpoints and chromosomal integrity, defining a broad role in genome stability.","evidence":"RNAi knockdown with flow cytometry and DNA-damage checkpoint assays","pmids":["18073532"],"confidence":"Medium","gaps":["Direct ubiquitination targets driving checkpoints not identified"]},{"year":2011,"claim":"Separated UBR5's catalytic and non-catalytic functions by showing ligase-independent scaffolding of the miRNA silencing machinery via the PABC domain.","evidence":"Mouse ES-cell genetic screen, Co-IP with GW182/DDX6/Tob1-2, domain-deletion silencing assays","pmids":["21726813"],"confidence":"High","gaps":["Whether scaffolding requires UBR5 oligomerization unknown"]},{"year":2011,"claim":"Identified the first catalytic substrate gated by a post-translational mark, establishing acetylation as a degron trigger feeding UBR5.","evidence":"In vitro ubiquitination, Co-IP, and acetyltransferase/deacetylase assays on PEPCK1","pmids":["21726808"],"confidence":"High","gaps":["Chain linkage on PEPCK1 not defined","In vivo metabolic role inferred indirectly"]},{"year":2012,"claim":"Positioned UBR5 (with TRIP12) as a negative regulator that confines RNF168-dependent ubiquitin spreading at DNA breaks, a key restraint on damage signaling.","evidence":"siRNA depletion with immunofluorescence quantification of H2A-Ub, 53BP1, and BRCA1 spreading","pmids":["22884692"],"confidence":"High","gaps":["Direct ubiquitination of RNF168 by UBR5 versus TRIP12 not fully separated"]},{"year":2014,"claim":"Defined site-specific control of ATM pathway choice through UBR5 ubiquitylation of ATMIN at Lys238, mechanistically linking UBR5 to MRN-dependent ATM activation.","evidence":"K238 mutagenesis, IR-stimulated ubiquitination, ATM foci and checkpoint assays","pmids":["25092319"],"confidence":"High","gaps":["Chain linkage on ATMIN not specified"]},{"year":2015,"claim":"Elucidated MLLE-PAM2 recognition at atomic resolution and discovered an autoinhibitory MLLE-HECT intramolecular contact, providing a structural model for regulating UBR5 ligase activity.","evidence":"Crystal structure of MLLE-Paip1 PAM2 complex, NMR, ITC, domain mapping","pmids":["26224628"],"confidence":"High","gaps":["Physiological trigger relieving autoinhibition unknown"]},{"year":2017,"claim":"Confirmed UBR5 catalytic activity is required for tumorigenesis and revealed cross-species conserved control of Wnt and Notch outputs through substrate ubiquitylation.","evidence":"CRISPR KO with catalytic-mutant rescue in TNBC; Drosophila/human ubiquitination of Groucho/TLE; C. elegans Notch epistasis; centrosomal CP110 ubiquitylation within EDD-DYRK2-DDB1-VprBP","pmids":["28330927","28689657","27185398","28242748"],"confidence":"High","gaps":["Tissue-specific substrate priorities in vivo unresolved"]},{"year":2019,"claim":"Expanded UBR5 substrate logic to include kinase-primed (DYRK2/AKT) targets and replication-fork/H2A-coupled functions, refining how signaling selects UBR5 cargo.","evidence":"Site-directed mutagenesis of SOX2 K115/T116 with ubiquitination assays; replication-fork Co-IP, DNA fiber and H2A-Ub epistasis","pmids":["30894683","31586398"],"confidence":"High","gaps":["Direct UBR5 substrate at the fork beyond H2A regulation unclear"]},{"year":2020,"claim":"Established UBR5 as an FBXW7-independent regulator of MYC and as a K63-chain builder in innate immune transcription, broadening its roles in oncogenesis and immunity.","evidence":"CRISPR screen and ubiquitination assays on MYC with Drosophila validation; K63-ubiquitination of Akirin with Drosophila and human NF-κB readouts","pmids":["32029551","33208877","32339205"],"confidence":"High","gaps":["How chain-linkage selection is determined per substrate not defined"]},{"year":2022,"claim":"Provided reconstituted mechanistic proof that UBR5 disassembles the mitotic checkpoint complex and identified K63-ubiquitination of TRIM28 as a switch de-repressing antiviral transcription.","evidence":"Reconstitution from purified components and immunodepletion for MCC disassembly; CRISPR screen, K63-Ub/SUMO assays, and Ubr5 KO mouse viral challenge","pmids":["35217622","38278841"],"confidence":"High","gaps":["Coordination of MCC disassembly with APC/C in intact cells not fully mapped"]},{"year":2023,"claim":"Delivered the full-length architecture (antiparallel dimer, dynamic HECT) and a chain-elongation activity model, and unified UBR5 as the ligase for agonist-dependent, chromatin-coupled nuclear hormone receptor turnover.","evidence":"Cryo-EM of full-length UBR5 with chain-elongation assays and AKIRIN2 interaction; chromatin recruitment and degradation assays across RARA/ERα/GR/PR and others","pmids":["37409633","37478846"],"confidence":"High","gaps":["Structural basis of substrate selection still incompletely defined","How the dimer engages diverse substrates not resolved"]},{"year":2024,"claim":"Showed non-degradative K11 ubiquitylation as a distinct UBR5 output and tied UBR5 to Rb-dependent G1 control and CDK4/6-inhibitor sensitivity.","evidence":"K11-linked ubiquitination of Smad3 with ferroptosis assays; CRISPR KO with single-cell Rb measurement and CDK4/6 inhibitor sensitivity","pmids":["39260061","39441926"],"confidence":"Medium","gaps":["Direct ubiquitination of Rb not demonstrated","Determinants of degradative versus non-degradative output unclear"]},{"year":null,"claim":"How UBR5 oligomeric architecture, MLLE-HECT autoregulation, and priming-kinase signals jointly dictate substrate choice and chain-linkage outcome across its many pathways remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No unified model linking dimer/oligomer state to substrate or linkage selection","Substrate-specific recruitment determinants largely unmapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[10,17,20,33,37,43,44,46,47]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[10,14,24,33,37,43,44,46]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[15,29,38,42,50]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[8,11,45]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[8,45,47]},{"term_id":"GO:0031386","term_label":"protein tag activity","supporting_discovery_ids":[4]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,9,54]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[25,35,47]}],"pathway":[{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[14,20,25,35]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[44,48,51]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[10,30,37,47]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[38,42]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[9,28]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[11]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[7,25,45,47]}],"complexes":["EDD-DDB1-VprBP/DYRK2 E3 ligase complex","miRNA silencing (GW182-DDX6-Tob) complex"],"partners":["DYRK2","DDB1","ATMIN","MYC","RNF168","BMI1","GW182","TRIM28"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O95071","full_name":"E3 ubiquitin-protein ligase UBR5","aliases":["E3 ubiquitin-protein ligase, HECT domain-containing 1","Hyperplastic discs protein homolog","hHYD","Progestin-induced protein"],"length_aa":2799,"mass_kda":309.4,"function":"E3 ubiquitin-protein ligase involved in different protein quality control pathways in the cytoplasm and nucleus (PubMed:29033132, PubMed:33208877, PubMed:37478846, PubMed:37478862). Mainly acts as a ubiquitin chain elongator that extends pre-ubiquitinated substrates (PubMed:29033132, PubMed:37409633). Component of the N-end rule pathway: ubiquitinates proteins bearing specific N-terminal residues that are destabilizing according to the N-end rule, leading to their degradation (By similarity). Recognizes type-1 N-degrons, containing positively charged amino acids (Arg, Lys and His) (By similarity). Together with UBR4, part of a cytoplasm protein quality control pathway that prevents protein aggregation by catalyzing assembly of heterotypic 'Lys-11'-/'Lys-48'-linked branched ubiquitin chains on aggregated proteins, leading to substrate recognition by the segregase p97/VCP and degradation by the proteasome: UBR5 is probably branching multiple 'Lys-48'-linked chains of substrates initially modified with mixed conjugates by UBR4 (PubMed:29033132). Together with ITCH, catalyzes 'Lys-48'-/'Lys-63'-branched ubiquitination of TXNIP, leading to its degradation: UBR5 mediates branching of 'Lys-48'-linked chains of substrates initially modified with 'Lys-63'-linked conjugates by ITCH (PubMed:29378950). Catalytic component of a nuclear protein quality control pathway that mediates ubiquitination and degradation of unpaired transcription factors (i.e. transcription factors that are not assembled into functional multiprotein complexes): specifically recognizes and binds degrons that are not accessible when transcription regulators are associated with their coactivators (PubMed:37478846, PubMed:37478862). Ubiquitinates various unpaired transcription regulator (MYC, SUPT4H1, SUPT5H, CDC20 and MCRS1), as well as ligand-bound nuclear receptors (ESR1, NR1H3, NR3C1, PGR, RARA, RXRA AND VDR) that are not associated with their nuclear receptor coactivators (NCOAs) (PubMed:33208877, PubMed:37478846, PubMed:37478862). Involved in maturation and/or transcriptional regulation of mRNA by mediating polyubiquitination and activation of CDK9 (PubMed:21127351). Also acts as a regulator of DNA damage response by acting as a suppressor of RNF168, an E3 ubiquitin-protein ligase that promotes accumulation of 'Lys-63'-linked histone H2A and H2AX at DNA damage sites, thereby acting as a guard against excessive spreading of ubiquitinated chromatin at damaged chromosomes (PubMed:22884692). Regulates DNA topoisomerase II binding protein (TopBP1) in the DNA damage response (PubMed:11714696). Ubiquitinates acetylated PCK1 (PubMed:21726808). Acts as a positive regulator of the canonical Wnt signaling pathway by mediating (1) ubiquitination and stabilization of CTNNB1, and (2) 'Lys-48'-linked ubiquitination and degradation of TLE3 (PubMed:21118991, PubMed:28689657). Promotes disassembly of the mitotic checkpoint complex (MCC) from the APC/C complex by catalyzing ubiquitination of BUB1B, BUB3 and CDC20 (PubMed:35217622). Plays an essential role in extraembryonic development (By similarity). Required for the maintenance of skeletal tissue homeostasis by acting as an inhibitor of hedgehog (HH) signaling (By similarity)","subcellular_location":"Nucleus; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/O95071/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/UBR5","classification":"Not Classified","n_dependent_lines":714,"n_total_lines":1208,"dependency_fraction":0.5910596026490066},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CAPZB","stoichiometry":0.2},{"gene":"MIF","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/UBR5","total_profiled":1310},"omim":[{"mim_id":"621372","title":"NEURODEVELOPMENTAL DISORDER WITH SPEECH DELAY AND BEHAVIORAL ABNORMALITIES; NEDSBH","url":"https://www.omim.org/entry/621372"},{"mim_id":"619189","title":"LI-CAMPEAU SYNDROME; LICAS","url":"https://www.omim.org/entry/619189"},{"mim_id":"613816","title":"UBIQUITIN PROTEIN LIGASE E3 COMPONENT N-RECOGNIN 7; UBR7","url":"https://www.omim.org/entry/613816"},{"mim_id":"612688","title":"RING FINGER PROTEIN 168; RNF168","url":"https://www.omim.org/entry/612688"},{"mim_id":"608413","title":"UBIQUITIN-PROTEIN LIGASE E3 COMPONENT N-RECOGNIN 5; UBR5","url":"https://www.omim.org/entry/608413"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in 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selenium.","date":"2011","source":"Environmental microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/21332622","citation_count":17,"is_preprint":false},{"pmid":"26340541","id":"PMC_26340541","title":"Human Herpesvirus-6 U14 Induces Cell-Cycle Arrest in G2/M Phase by Associating with a Cellular Protein, EDD.","date":"2015","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/26340541","citation_count":17,"is_preprint":false},{"pmid":"27185398","id":"PMC_27185398","title":"UBR-5, a Conserved HECT-Type E3 Ubiquitin Ligase, Negatively Regulates Notch-Type Signaling in Caenorhabditis elegans.","date":"2016","source":"G3 (Bethesda, Md.)","url":"https://pubmed.ncbi.nlm.nih.gov/27185398","citation_count":17,"is_preprint":false},{"pmid":"26438829","id":"PMC_26438829","title":"RanGTP aids anaphase entry through Ubr5-mediated protein turnover.","date":"2015","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/26438829","citation_count":15,"is_preprint":false},{"pmid":"16041126","id":"PMC_16041126","title":"Effects of edd and pgi disruptions on inosine accumulation in Escherichia coli.","date":"2005","source":"Bioscience, biotechnology, and biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16041126","citation_count":15,"is_preprint":false},{"pmid":"38278841","id":"PMC_38278841","title":"UBR5 promotes antiviral immunity by disengaging the transcriptional brake on RIG-I like receptors.","date":"2024","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/38278841","citation_count":14,"is_preprint":false},{"pmid":"37770316","id":"PMC_37770316","title":"Harnessing UBR5 for targeted protein degradation of key transcriptional regulators.","date":"2023","source":"Trends in pharmacological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/37770316","citation_count":14,"is_preprint":false},{"pmid":"35217622","id":"PMC_35217622","title":"Role of ubiquitin-protein ligase UBR5 in the disassembly of mitotic checkpoint complexes.","date":"2022","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/35217622","citation_count":14,"is_preprint":false},{"pmid":"29441057","id":"PMC_29441057","title":"Stability of the HTLV-1 Antisense-Derived Protein, HBZ, Is Regulated by the E3 Ubiquitin-Protein Ligase, UBR5.","date":"2018","source":"Frontiers in microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/29441057","citation_count":14,"is_preprint":false},{"pmid":"15632288","id":"PMC_15632288","title":"EDD, a novel phosphotransferase domain common to mannose transporter EIIA, dihydroxyacetone kinase, and DegV.","date":"2005","source":"Protein science : a publication of the Protein Society","url":"https://pubmed.ncbi.nlm.nih.gov/15632288","citation_count":14,"is_preprint":false},{"pmid":"35980206","id":"PMC_35980206","title":"UBR5 Acts as an Antiviral Host Factor against MERS-CoV via Promoting Ubiquitination and Degradation of ORF4b.","date":"2022","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/35980206","citation_count":13,"is_preprint":false},{"pmid":"28559278","id":"PMC_28559278","title":"The p90 ribosomal S6 kinase-UBR5 pathway controls Toll-like receptor signaling via miRNA-induced translational inhibition of tumor necrosis factor receptor-associated factor 3.","date":"2017","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/28559278","citation_count":13,"is_preprint":false},{"pmid":"24145130","id":"PMC_24145130","title":"Progestin-inducible EDD E3 ubiquitin ligase binds to α4 phosphoprotein to regulate ubiquitination and degradation of protein phosphatase PP2Ac.","date":"2013","source":"Molecular and cellular endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/24145130","citation_count":13,"is_preprint":false},{"pmid":"39441926","id":"PMC_39441926","title":"The G1-S transition is promoted by Rb degradation via the E3 ligase UBR5.","date":"2024","source":"Science advances","url":"https://pubmed.ncbi.nlm.nih.gov/39441926","citation_count":12,"is_preprint":false},{"pmid":"38755129","id":"PMC_38755129","title":"E3 ubiquitin ligase UBR5 promotes gemcitabine resistance in pancreatic cancer by inducing O-GlcNAcylation-mediated EMT via destabilization of OGA.","date":"2024","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/38755129","citation_count":12,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":49757,"output_tokens":13913,"usd":0.178983,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":25738,"output_tokens":5728,"usd":0.135945,"stage2_stop_reason":"end_turn"},"total_usd":0.314928,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2002,\n      \"finding\": \"EDD/UBR5 contains a UBA domain, UBR1 zinc finger motif, and HECT domain; localizes to cell nuclei via importin alpha5 through consensus NLS; binds progesterone receptor (PR) and potentiates progestin-mediated gene transactivation; also binds CIB/DNAPK-interacting protein with altered association after DNA damage.\",\n      \"method\": \"Co-immunoprecipitation, nuclear localization assay, transactivation reporter assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — reciprocal Co-IP and functional reporter assays in single lab, multiple orthogonal methods\",\n      \"pmids\": [\"12011095\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Homozygous knockout of murine Edd is lethal by E10.5; Edd-deficient embryos display failed yolk sac and allantoic vascular development and defective chorioallantoic fusion, establishing an essential role in extraembryonic vascular development.\",\n      \"method\": \"Gene targeting/knockout mouse, embryo phenotyping\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with defined developmental phenotype, rigorous genetic model\",\n      \"pmids\": [\"15282321\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"EDD/UBR5 interacts with CHK2 via a phospho-dependent interaction involving the CHK2 Forkhead-associated (FHA) domain and EDD's FHA-binding threonines; EDD is required upstream of CHK2 for efficient activating phosphorylation of CHK2 after ionizing radiation or radiomimetic treatment.\",\n      \"method\": \"Co-immunoprecipitation, RNA interference (RNAi) knockdown, kinase activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNAi epistasis and Co-IP in single lab with two orthogonal methods\",\n      \"pmids\": [\"17074762\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The PABC/MLLE domain of UBR5/HYD binds PAM2 peptide motifs with micromolar affinity, similar to PABP's PABC domain; UBR5 PABC domain interacts with anti-proliferative Tob2 protein, linking UBR5 to translation regulation and cell cycle control.\",\n      \"method\": \"Isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), NMR chemical shift perturbation, GST pulldown, co-immunoprecipitation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple biophysical methods (ITC, SPR, NMR) plus pulldown and Co-IP, single study with orthogonal validation\",\n      \"pmids\": [\"16554297\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The UBA domain of EDD/UBR5 binds ubiquitin; crystal structure at 1.85 Å reveals recognition via UBA helices α1 and α3 with hydrogen bonds including ordered water molecules; site-directed mutagenesis confirmed functional importance of interface residues; SPR showed no strong preference for polyubiquitin chains over monoubiquitin.\",\n      \"method\": \"ITC, NMR titrations, pulldown, 1.85 Å crystal structure, site-directed mutagenesis, SPR\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with mutagenesis validation and multiple biophysical methods in one study\",\n      \"pmids\": [\"17897937\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"EDD/UBR5 interacts with APC tumor suppressor protein; EDD overexpression increases APC and Axin protein levels and inhibits β-catenin/LEF1 Wnt signaling; EDD knockdown reduces APC protein level without altering its mRNA, increasing β-catenin.\",\n      \"method\": \"Mass spectrometry of APC immunocomplexes, co-immunoprecipitation, siRNA knockdown, immunofluorescence co-localization, reporter assays\",\n      \"journal\": \"Genes to cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — MS identification plus Co-IP and siRNA knockdown with functional readout, single lab\",\n      \"pmids\": [\"18076571\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"EDD/UBR5 is necessary for G1/S and intra-S phase DNA damage checkpoint activation and for maintenance of G2/M arrest after DSBs; EDD depletion leads to radioresistant DNA synthesis, premature mitotic entry, polyploidy, and mitotic catastrophe.\",\n      \"method\": \"RNAi knockdown, cell cycle analysis (flow cytometry), DNA damage assays\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined cellular phenotype with KD, checkpoint assays, single lab\",\n      \"pmids\": [\"18073532\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"UBR5 associates with CDK9 subunit of P-TEFb and mediates its polyubiquitination; TFIIS binds UBR5 and stimulates CDK9 polyubiquitination; UBR5, CDK9, and TFIIS co-localize along the γ-fibrinogen gene; TFIIS overexpression increases CDK9 association with gene regions and enhances RNAPII Ser2-CTD phosphorylation.\",\n      \"method\": \"Co-immunoprecipitation, chromatin immunoprecipitation (ChIP), ubiquitination assay, co-localization\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus Co-IP and ubiquitination assays, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"21127351\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"UBR5 localizes to smooth muscle cell nuclei and forms a complex with myocardin both in vivo and in vitro; UBR5 enhances transactivation of smooth muscle-specific promoters by myocardin family proteins and stabilizes myocardin protein (attenuates its degradation) independently of E3 ligase activity, requiring only HECT and UBR1 domains.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, siRNA knockdown, promoter-luciferase reporter, western blot protein stability assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus Co-IP, reporter assays, and siRNA with functional readout, single lab\",\n      \"pmids\": [\"20167605\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"EDD/UBR5 interacts with GSK-3β and β-catenin; EDD expression promotes nuclear accumulation of both proteins and enhances β-catenin stability and activity by ubiquitinating β-catenin through Lys29- or Lys11-linked ubiquitin chains, leading to increased β-catenin protein levels.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay (K29/K11 chain linkage), nuclear fractionation, luciferase reporter assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and in-cell ubiquitination assay with chain-linkage analysis, reporter assay, single lab\",\n      \"pmids\": [\"21118991\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PEPCK1 is acetylated by P300 acetyltransferase; acetylated PEPCK1 interacts with UBR5 HECT E3 ligase, promoting PEPCK1 ubiquitination and degradation; SIRT2 deacetylates PEPCK1, stabilizing it; high glucose destabilizes PEPCK1 via this acetylation-UBR5 axis.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay, protein stability assay, acetylation mapping\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro ubiquitination, Co-IP, and acetyltransferase/deacetylase assays with mechanistic dissection, published in high-tier journal\",\n      \"pmids\": [\"21726808\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"EDD/UBR5 is a key component of the miRNA silencing pathway; EDD interacts with GW182 proteins in Argonaute-miRNA complexes; EDD E3 ligase activity is dispensable for miRNA silencing; the PABC domain of EDD is essential for silencing function by recruiting DDX6 and Tob1/2.\",\n      \"method\": \"Genetic screen in mouse ES cells, co-immunoprecipitation, domain deletion analysis, RNAi silencing assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic screen plus Co-IP, domain mutants, and functional silencing assays, replicated with multiple miRNA targets\",\n      \"pmids\": [\"21726813\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"EDD/UBR5 interacts with both HPV18 E6 and E6AP independently; EDD regulates E6AP expression levels independently of E6; loss of EDD stimulates the proteolytic activity of the E6/E6AP complex and enhances cell resistance to apoptotic stimuli.\",\n      \"method\": \"Mass spectrometry, co-immunoprecipitation, siRNA knockdown, protein stability/degradation assays\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS identification plus Co-IP and siRNA functional assays, single lab\",\n      \"pmids\": [\"21228227\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"EDD/UBR5 physically interacts with p53 and this interaction blocks ATM-mediated phosphorylation of p53 at Ser15; EDD depletion induces p53 Ser15 phosphorylation and activates p53 target genes; EDD overexpression inhibits p53-Ser15 phosphorylation during DNA damage independently of E3 ligase activity; G1 arrest from EDD depletion is p53-dependent.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, phospho-specific western blotting, co-depletion epistasis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus functional RNAi epistasis and phosphorylation assays, single lab\",\n      \"pmids\": [\"21383020\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"TRIP12 and UBR5 control accumulation of RNF168 by targeting it for degradation; depletion of TRIP12 and UBR5 allows supraphysiological accumulation of RNF168, leading to massive spreading of ubiquitin conjugates (H2A-Ub) and hyperaccumulation of 53BP1 and BRCA1 beyond DNA lesions.\",\n      \"method\": \"siRNA depletion, immunofluorescence, DSB-induced ubiquitin spreading assay\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — RNAi epistasis with clear substrate (RNF168) and quantified chromatin ubiquitin spreading, high-tier journal\",\n      \"pmids\": [\"22884692\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Crystal structure of the C-lobe of the HECT domain of human UBR5 reveals a unique four-residue insert elongating helix 2, creating a protruding loop likely important for E2 specificity toward UBCH4; the C-lobe forms a thioester-linked E3-ubiquitin complex in ubiquitination assays.\",\n      \"method\": \"X-ray crystallography, ubiquitination thioester assay, NMR\",\n      \"journal\": \"Acta crystallographica Section F\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with biochemical validation of catalytic thioester intermediate\",\n      \"pmids\": [\"23027739\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EDD/UBR5 interacts with microspherule protein Msp58 (MCRS1); EDD depletion increases Msp58 protein levels and extends its half-life, demonstrating EDD negatively regulates Msp58 stability via the ubiquitin-proteasome pathway; knockdown of either protein affects cyclin B, D, E levels and cell cycle progression.\",\n      \"method\": \"Co-immunoprecipitation, in vitro binding, confocal co-localization, protein half-life assay, siRNA knockdown, flow cytometry\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP, protein stability, and cell cycle assays in single lab\",\n      \"pmids\": [\"23069210\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Dyrk2 phosphorylates TERT; phosphorylated TERT associates with the EDD-DDB1-VprBP E3 ligase complex, leading to ubiquitin-mediated TERT degradation at G2/M phase; Dyrk2 depletion disrupts cell cycle-dependent TERT regulation and causes constitutive telomerase activation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, telomerase activity assay (TRAP), kinase assay, siRNA knockdown, cell cycle synchronization\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — kinase assay plus ubiquitination assay plus telomerase functional readout, multiple orthogonal methods\",\n      \"pmids\": [\"23362280\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"HIV-1 Vpr enhances interaction between TERT and the VPRBP substrate receptor of the DYRK2-associated EDD-DDB1-VPRBP E3 ligase, increasing TERT ubiquitination and degradation; a Vpr mutation found in long-term non-progressors failed to promote TERT destabilization.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, telomerase activity assay, mutant Vpr analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assay with mutant controls, single lab\",\n      \"pmids\": [\"23612978\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"VprBP/DCAF1 serves as substrate recognition subunit for both the RING-type CRL4 and HECT-type EDD/UBR5 E3 ligase complexes; VprBP assembles into the EDD complex with DYRK2 and DDB1.\",\n      \"method\": \"Review/biochemical analysis of prior co-IP and complex assembly data (cited from primary studies)\",\n      \"journal\": \"BMC molecular biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — review paper synthesizing prior findings without new primary experiments described\",\n      \"pmids\": [\"24028781\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"UBR5-mediated ubiquitination of ATMIN at lysine 238 is stimulated by ionizing radiation (IR); this ubiquitination decreases ATMIN-ATM interaction, promotes MRN-mediated ATM signaling, and NBS1/ATM foci formation; UBR5 deficiency or ATMIN K238 mutation impairs ATM checkpoint activation and increases radiosensitivity.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis (K238), ubiquitination assay, IR treatment, ATM foci formation, checkpoint assays, siRNA knockdown\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — site-specific mutagenesis of ubiquitination site plus functional checkpoint and foci assays, mechanistic dissection of pathway\",\n      \"pmids\": [\"25092319\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"UBR5 and DYRK2 regulate hPXR stability; UBR5 knockdown causes hPXR accumulation and increased hPXR transcriptional activity; DYRK2-dependent phosphorylation of hPXR facilitates its subsequent ubiquitination by UBR5.\",\n      \"method\": \"siRNA knockdown, MS analysis, kinome-wide siRNA screen, protein stability assay, reporter assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — kinome siRNA screen plus functional protein stability and activity assays, single lab\",\n      \"pmids\": [\"24438055\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The MLLE domain of UBR5 binds PAM2 peptides (from Paip1 and GW182) with defined network of hydrophobic and ionic interactions shown by crystal structure with Paip1 PAM2 peptide; a novel intramolecular interaction between MLLE domain and adjacent HECT domain via a PAM2-like sequence was identified, suggesting regulation of UBR5 ligase activity.\",\n      \"method\": \"X-ray crystallography (MLLE-PAM2 complex), NMR, ITC, domain interaction mapping\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with multiple biophysical validations plus intramolecular interaction discovery\",\n      \"pmids\": [\"26224628\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"EDD1/UBR5 interacts with TIP60 acetyltransferase and negatively regulates TIP60 stability through the proteasome pathway; HPV E6 oncogene exploits EDD1 to destabilize TIP60; depletion of EDD1 or gain-of-function of TIP60 inhibits HPV-positive cervical cancer cell growth in vitro and in vivo.\",\n      \"method\": \"Proteomics, co-immunoprecipitation, ubiquitination/turnover assay, colony formation, soft agar, xenograft\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS identification plus Co-IP, ubiquitination assay, and in vivo tumor model, single lab\",\n      \"pmids\": [\"26234678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"UBR5 physically interacts with MOAP-1, ubiquitylates MOAP-1 in vitro, and inhibits MOAP-1 stability in cells; Dyrk2 kinase cooperates with UBR5 in MOAP-1 ubiquitylation; UBR5 knockdown increases MOAP-1 levels, enhances Bax activation, and sensitizes cisplatin-resistant ovarian cancer cells to apoptosis.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay, protein stability assay, siRNA knockdown, Bax activation assay, cisplatin sensitivity assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro ubiquitination plus Co-IP and functional apoptosis assays, mechanistic dissection with kinase cooperation\",\n      \"pmids\": [\"27721409\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"UBR5 forms damage-inducible nuclear foci dependent on PRC1 components BMI1, RING1a, and RING1b; UBR5 associates with BMI1 and FACT components SPT16/SSRP1; UBR5 ubiquitinates SPT16; UBR5 and BMI1 repress RNA Pol II transcription elongation at UV-damaged chromatin by negatively regulating FACT-dependent Pol II elongation; UBR5/BMI1 KO cells are hypersensitive to UV.\",\n      \"method\": \"Mass spectrometry, co-immunoprecipitation, in vitro ubiquitination assay, transcription elongation assay, immunofluorescence foci analysis, CRISPR KO, UV sensitivity assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro ubiquitination, MS, Co-IP, functional transcription assay, and genetic KO, multiple orthogonal methods\",\n      \"pmids\": [\"27647897\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"UBR5 depletion reduces primary cilia formation; CSPP1 (centrosomal/ciliary protein required for cilia formation) is a UBR5-interacting protein; UBR5 ubiquitylates CSPP1; UBR5 is required for cytoplasmic organization of CSPP1-comprising centriolar satellites in centrosomal periphery.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay, siRNA knockdown, ciliogenesis assay, centrosomal fractionation\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay and functional ciliogenesis readout, single lab\",\n      \"pmids\": [\"29742019\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"C. elegans UBR-5 negatively regulates Notch-type (GLP-1/LIN-12) signaling; ubr-5 loss-of-function suppresses glp-1 and lin-12 loss-of-function defects; ubr-5 acts in germ cells (receiving cells) to limit GLP-1 signaling; UBR-5 acts redundantly with SEL-10 (SCF E3 ligase) to limit Notch signaling in certain tissues.\",\n      \"method\": \"C. elegans genetics, suppressor/enhancer epistasis, loss-of-function analysis\",\n      \"journal\": \"G3 (Bethesda)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic epistasis in C. elegans with multiple alleles and tissue-specific analysis, single lab\",\n      \"pmids\": [\"27185398\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Hyd/UBR5 is required for Wnt signal responses downstream of activated Armadillo/β-catenin in Drosophila and human cell lines; Groucho/TLE is a functionally relevant substrate whose ubiquitylation by UBR5 is induced by Wnt signaling and conferred by β-catenin; TLE inactivation by UBR5-dependent ubiquitylation also involves VCP/p97 AAA ATPase.\",\n      \"method\": \"Drosophila genetics, siRNA knockdown in human cells, co-immunoprecipitation, ubiquitination assay, epistasis\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — genetic epistasis in Drosophila plus human cell biochemistry (Co-IP, ubiquitination), replicated across species\",\n      \"pmids\": [\"28689657\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CRISPR/Cas9 deletion of UBR5 in murine TNBC model abrogates tumor growth and metastasis in vivo; reconstitution with wild-type UBR5 but not a catalytically inactive mutant rescues this phenotype, demonstrating E3 ligase activity is required for tumor growth and metastasis functions.\",\n      \"method\": \"CRISPR/Cas9 knockout, catalytically inactive mutant rescue, in vivo tumor model\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — CRISPR KO with catalytic mutant rescue in vivo, establishing enzymatic activity requirement\",\n      \"pmids\": [\"28330927\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"UBR5 is highly expressed in iPSCs and is required for proteasomal degradation of both normal and polyQ-expanded mutant huntingtin (HTT); UBR5 loss increases HTT levels and triggers polyQ-expanded aggregation in HD-iPSCs; UBR5 overexpression induces polyubiquitination and degradation of mutant HTT, reducing aggregates.\",\n      \"method\": \"siRNA knockdown, overexpression, ubiquitination assay, aggregation assay, invertebrate model (C. elegans/Drosophila) knockdown\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — RNAi plus OE with ubiquitination assay, replicated in invertebrate models, multiple orthogonal methods\",\n      \"pmids\": [\"30038412\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"UBR5 is required for cytoplasmic organization of CSPP1-comprising centriolar satellites; UBR5 ubiquitylates CSPP1 and is required for primary cilia formation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, ciliogenesis assay, siRNA knockdown\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, and ciliogenesis functional assay, single lab\",\n      \"pmids\": [\"29742019\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"OTUD5 deubiquitinase stabilizes UBR5 E3 ligase; OTUD5 localizes to DSBs and interacts with UBR5; the OTUD5-UBR5 complex represses RNA Pol II elongation and RNA synthesis at DSBs; OTUD5 interacts with FACT component SPT16 via a separate region; both UBR5 stabilization (catalytic) and FACT binding (scaffolding) activities of OTUD5 are required for Pol II arrest.\",\n      \"method\": \"DUB RNAi screen, co-immunoprecipitation, transcription elongation assay, domain mapping, co-localization\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNAi screen plus Co-IP and functional transcription assay, single lab\",\n      \"pmids\": [\"30508113\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"AKT phosphorylates SOX2 at Thr116, which inhibits UBR5 interaction with SOX2; UBR5 ubiquitinates SOX2 at Lys115, promoting its degradation; AKT-mediated phosphorylation stabilizes SOX2 by blocking this UBR5-mediated ubiquitination.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (K115, T116), kinase assay, protein stability assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — site-specific mutagenesis of ubiquitination site and phosphorylation site, in vitro ubiquitination, mechanistic dissection\",\n      \"pmids\": [\"30894683\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PPARγ promotes ATM signaling through interaction with MRN complex and UBR5; PPARγ is essential for UBR5 activity targeting ATMIN; PPARγ depletion increases ATMIN protein and suppresses DDR-induced ATM signaling; disrupted PPARγ-UBR5 interaction is observed in PAH patient endothelial cells.\",\n      \"method\": \"Proteomic interaction screen, co-immunoprecipitation, siRNA knockdown, ATM signaling assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomics plus Co-IP and functional ATM signaling assays, single lab\",\n      \"pmids\": [\"30699358\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"UBR5 interacts with components of the replication fork including TLS polymerase polη; UBR5 depletion causes S-phase progression defects, ssDNA accumulation, and mis-regulation of H2A ubiquitination (UbiH2A); blocking H2A ubiquitination rescues replication problems in UBR5-depleted cells; polη is the main cause of replication defects when UBR5 is silenced; polη interacts with H2A, suggesting UbiH2A regulates polη recruitment.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, DNA fiber assay (S-phase progression), ssDNA accumulation assay, epistasis with H2A modification\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus epistasis and replication fiber assay, single lab with multiple methods\",\n      \"pmids\": [\"31586398\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"UBR5 interacts with H/ACA ribonucleoprotein complex components in ESCs; loss of UBR5 induces abnormal accumulation of rRNA processing intermediates, diminished ribosomal levels, increased p53 levels, and decreased cell proliferation.\",\n      \"method\": \"Protein interactome (MS), co-immunoprecipitation, rRNA processing assay, western blot, proliferation assay\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS interactome plus Co-IP and rRNA functional assay, single lab\",\n      \"pmids\": [\"31365120\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"UBR5 ubiquitinates MYC and promotes its degradation independently of FBXW7; UBR5 silencing induces MYC protein accumulation; UBR5 and MYC are co-amplified in MYC-driven cancers; in p53-mutant MYC-amplified cells, UBR5 suppresses MYC-mediated apoptosis; Drosophila HYD suppresses dMYC-dependent overgrowth.\",\n      \"method\": \"CRISPR/Cas9 screen, co-immunoprecipitation, ubiquitination assay (K48-linked), siRNA knockdown, protein stability assay, Drosophila genetics\",\n      \"journal\": \"Cancer research / Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — CRISPR screen identification plus in-cell ubiquitination assay and Drosophila genetic validation, replicated across two independent papers (PMID 32029551, 33208877)\",\n      \"pmids\": [\"32029551\", \"33208877\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Drosophila Hyd/UBR5 mediates Lys63-linked polyubiquitination of the NF-κB cofactor Akirin, which is required for efficient Akirin-Relish (NF-κB) interaction and transcription of immune-induced anti-microbial peptide genes; human UBR5 is also required for IL-6 transcription downstream of NF-κB signaling by LPS or IL-1β.\",\n      \"method\": \"RNAi screen, ubiquitination assay (K63 chain), co-immunoprecipitation, Drosophila survival assay, human cell siRNA knockdown, luciferase reporter\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — K63 ubiquitination assay plus genetic epistasis in Drosophila and human cell validation, mechanistic dissection\",\n      \"pmids\": [\"32339205\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Loss of UBR5 HECT domain in B cells causes a block in B-cell maturation in spleen and upregulation of spliceosome components; UBR5 is required for B-cell maturation by promoting degradation/destabilization of spliceosome components during B-cell development.\",\n      \"method\": \"Conditional knockout (HECT domain deletion), flow cytometry, western blot, gene expression analysis\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with defined B-cell maturation phenotype, single lab\",\n      \"pmids\": [\"32325489\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"UBR5 controls β-catenin-mediated signaling and regulates p53 protein level in ovarian cancer; tumor-derived UBR5 promotes macrophage recruitment via chemokines/cytokines.\",\n      \"method\": \"siRNA knockdown, CRISPR KO, protein stability assay, cytokine/chemokine profiling\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO and siRNA with β-catenin/p53 mechanistic readouts, single lab\",\n      \"pmids\": [\"33293516\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"UBR5 interacts with and promotes degradation of CAPZA1 (F-actin capping protein α subunit) via ubiquitin-proteasome system; UBR5 overexpression induces F-actin accumulation; CAPZA1 downregulation reverses UBR5-knockdown-mediated suppression of pancreatic cancer cell migration/invasion.\",\n      \"method\": \"Co-immunoprecipitation with mass spectrometry, ubiquitination assay, protein stability assay, siRNA knockdown, F-actin staining, migration/invasion assay, in vivo liver metastasis model\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS substrate identification plus Co-IP, ubiquitination assay, and functional rescue epistasis, single lab\",\n      \"pmids\": [\"33777788\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"UBR5 promotes antiviral immunity by mediating Lys63-linked ubiquitination of TRIM28 (epigenetic repressor of RLRs); this modification prevents intramolecular SUMOylation of TRIM28, disengaging TRIM28-imposed transcriptional repression of RIG-I-like receptor genes; Ubr5 KO mice are more susceptible to RNA virus infection.\",\n      \"method\": \"CRISPR KO screen (375 E3 ligases), ubiquitination assay (K63-linked), SUMOylation assay, co-immunoprecipitation, Ubr5 KO mice, viral challenge\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — genome-wide CRISPR screen plus mechanistic K63-Ub and SUMO assays and in vivo mouse validation\",\n      \"pmids\": [\"38278841\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"UBR5 polyubiquitinates CDC73 at Lys243, Lys247, and Lys257 in a non-canonical manner dependent on non-phosphorylated CDC73 (Ser465); CDC73 destabilization by UBR5 regulates β-catenin and E-cadherin expression and tumor cell apoptosis and CD8+ T cell infiltration in TNBC.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (K243/247/257, S465), protein stability assay, in vivo tumor model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — site-specific ubiquitination mapping by mutagenesis with phosphorylation regulation, functional in vivo validation\",\n      \"pmids\": [\"35551175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"UBR5 ubiquitinates BubR1, Bub3, and Cdc20 (MCC components); UBR5 promotes dissociation of the Bub3•BubR1 subcomplex from APC/C via ubiquitylation, facilitated by ATP hydrolysis; UBR5 immunodepletion slows MCC disassembly and prolongs lag period in APC/C activity recovery; a reconstituted system from purified components shows UBR5- and ubiquitylation-dependent Bub3•BubR1 dissociation.\",\n      \"method\": \"Co-immunoprecipitation from nocodazole-arrested HeLa cells, in vitro ubiquitination assay, immunodepletion, reconstitution from purified components, APC/C activity assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution from purified components plus in vitro ubiquitination and immunodepletion assays, mechanistic dissection of MCC disassembly\",\n      \"pmids\": [\"35217622\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"UBR5 enhances IFN-γ-induced PD-L1 transcription through its PABC domain by upregulating PKR and downstream STAT1/IRF1, in an E3 ubiquitin activity-independent manner.\",\n      \"method\": \"RNA-seq, qPCR, ChIP-qPCR, luciferase reporter, siRNA/CRISPR knockdown and reconstitution, domain deletion analysis\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transcriptomic plus ChIP and reporter assays with domain mutant, single lab\",\n      \"pmids\": [\"35836797\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Cryo-EM structure of full-length human UBR5 reveals an α-solenoid scaffold assembled into an antiparallel dimer with further oligomeric states; the catalytic HECT domain is dynamic; AKIRIN2 (proteasomal nuclear import factor) was identified as an interacting protein; UBR5 preferentially acts as a ubiquitin chain elongator on pre-ubiquitinated substrates.\",\n      \"method\": \"Cryo-EM (full-length human UBR5), negative stain EM (UBR5-RARA/RXRA complex), co-immunoprecipitation, in vitro ubiquitin chain elongation assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM structure with biochemical validation of chain elongation preference and new interacting protein\",\n      \"pmids\": [\"37409633\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"UBR5 drives agonist-dependent degradation of multiple nuclear hormone receptors (RARA, RXRA, GR, ERα, LXR, PR, VDR); cryo-EM structure of full-length human UBR5 obtained; agonist ligands induce sequential, mutually exclusive recruitment of NCOAs and UBR5 to chromatin; SERDs degrade ERα through differential recruitment of UBR5 or RNF111.\",\n      \"method\": \"Cryo-EM structure, negative-stain EM with RARA/RXRA complex, co-immunoprecipitation, chromatin recruitment assays, siRNA knockdown, protein stability assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure plus chromatin recruitment and substrate degradation assays across multiple NRs, high-tier journal\",\n      \"pmids\": [\"37478846\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"UBR5 is required for Rb protein concentration decrease during G1; UBR5 KO cells have increased Rb concentration in early G1, lower G1-S transition rate, and increased sensitivity to CDK4/6 inhibitors.\",\n      \"method\": \"UBR5 CRISPR KO, single-cell protein concentration measurement, cell cycle analysis, CDK4/6 inhibitor sensitivity assay\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO with defined cell cycle phenotype, single lab, no direct ubiquitination of Rb demonstrated in abstract\",\n      \"pmids\": [\"39441926\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"UBR5 acts as an E3 ubiquitin ligase for OGA (O-GlcNAcase); UBR5 binds and promotes OGA ubiquitination and degradation, leading to increased O-GlcNAcylation; this promotes EMT-mediated gemcitabine resistance in pancreatic cancer.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, protein stability assay, siRNA knockdown, OGA level rescue experiments\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay and functional epistasis, single lab\",\n      \"pmids\": [\"38755129\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"UBR5 stabilizes oxaliplatin-activated Smad3 via Lys11-linked polyubiquitination (non-degradative), which facilitates transcriptional repression of ATF3, induction of SLC7A11, and inhibition of ferroptosis, conferring chemoresistance in colorectal cancer.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay (Lys11-linked chain), siRNA knockdown, ferroptosis assay, transcriptional reporter\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus K11 chain-specific ubiquitination and functional ferroptosis assays, single lab\",\n      \"pmids\": [\"39260061\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RanGTP promotes the dissociation of importin-β from BuGZ and Bub3 in metaphase, resulting in increased binding of BuGZ and Bub3 to Ubr5; this leads to Ubr5-dependent ubiquitination and turnover of BuGZ and Bub3, facilitating metaphase-to-anaphase transition (SAC silencing).\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, cell cycle analysis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, and functional metaphase-anaphase transition assay, single lab\",\n      \"pmids\": [\"26438829\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Cep78 localizes to mature centrioles and directly interacts with VprBP, a component of the EDD-DYRK2-DDB1VprBP E3 ligase; Cep78 binds specifically to EDD-DYRK2-DDB1VprBP (not CRL4VprBP) and inhibits its activity; EDD-DYRK2-DDB1VprBP ubiquitinates CP110 (novel centrosomal substrate) after DYRK2-dependent phosphorylation; Cep78 impedes ubiquitin transfer from EDD to CP110 without affecting CP110 phosphorylation.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay, centrosome fractionation, domain interaction mapping, centriole length measurement, cilia assay\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro ubiquitination reconstitution with step-specific inhibitor analysis (Cep78 blocks transfer step), multiple orthogonal methods\",\n      \"pmids\": [\"28242748\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"UBR5 interacts with HTLV-1 HBZ protein; UBR5 knockdown enhances HBZ steady-state levels by stabilizing HBZ; Co-IP assays confirmed HBZ ubiquitination that is reduced upon UBR5 knockdown; MS/MS identified seven ubiquitinated lysines in HBZ.\",\n      \"method\": \"Affinity-tagged protein pulldown, shotgun proteomics, co-immunoprecipitation, shRNA knockdown, protein stability assay, MS/MS ubiquitin site mapping\",\n      \"journal\": \"Frontiers in microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS identification plus Co-IP, ubiquitination assay, and stability assay, single lab\",\n      \"pmids\": [\"29441057\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"UBR5 ubiquitinates MERS-CoV ORF4b at Lys36, promoting its degradation; UBR5 can translocate into the nucleus via its NLS to regulate ORF4b stability in both cytoplasm and nucleus; UBR5 knockdown enhances ORF4b anti-immune activity and increases MERS-CoV replication.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (K36), siRNA knockdown, viral replication assay, subcellular localization assay\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay with mutagenesis of ubiquitination site, functional viral assay, single lab\",\n      \"pmids\": [\"35980206\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"UBR5 regulates FBP1 expression in pancreatic cancer by binding to C/EBPα transcription factor and promoting its ubiquitination and degradation; UBR5-induced aerobic glycolysis is dependent on this FBP1-C/EBPα axis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, protein stability assay, glycolysis assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay and functional metabolic readout, single lab\",\n      \"pmids\": [\"33122826\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"EDD/UBR5 physically interacts with alpha4 phosphoprotein; Co-IP confirmed EDD-alpha4 interaction; EDD knockdown leads to decreased PP2Ac polyubiquitination and accumulation of PP2Ac protein, identifying PP2Ac as an EDD substrate regulated via alpha4 as scaffold.\",\n      \"method\": \"Co-immunoprecipitation using deletion mutants, siRNA knockdown, proteasome inhibitor treatment, polyubiquitination assay\",\n      \"journal\": \"Molecular and cellular endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP with deletion analysis plus siRNA functional assay, single lab\",\n      \"pmids\": [\"24145130\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"UBR5 is a nuclear HECT-domain E3 ubiquitin ligase that forms an antiparallel dimer (cryo-EM resolved) and functions primarily as a ubiquitin chain elongator; it ubiquitinates a broad range of substrates—including PEPCK1 (acetylation-triggered), RNF168, TLE/Groucho (Wnt-dependent), ATMIN (IR-stimulated, K238), MYC, SOX2 (AKT-phosphorylation regulated, K115), TERT (Dyrk2-phosphorylation gated), CDC73 (K243/247/257), CP110, BubR1/Bub3/Cdc20 (MCC disassembly), MOAP-1, Rb, OGA, Smad3 (K11-non-degradative), CAPZA1, and viral proteins—using distinct chain linkages (K11, K29, K48, K63) depending on context; it additionally acts in a ligase-independent manner via its PABC/MLLE domain to scaffold miRNA silencing complexes (interacting with GW182/DDX6/Tob) and to regulate transcription (myocardin coactivation, PD-L1 via PKR-STAT1); it participates in DNA damage response (CHK2 activation, ATM pathway selection via ATMIN ubiquitination, replication fork protection via H2A ubiquitination control, transcriptional silencing at lesions via BMI1-FACT axis), Wnt signaling (TLE ubiquitylation), Notch pathway suppression, antiviral immunity (K63-ubiquitination of TRIM28 to de-repress RLR transcription), mitotic checkpoint disassembly, ciliogenesis (CSPP1 ubiquitylation), and nuclear hormone receptor turnover (agonist-dependent degradation of RARA, ERα, and other NRs on chromatin).\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"UBR5 (EDD/HYD) is a nuclear HECT-domain E3 ubiquitin ligase that integrates protein quality control, transcriptional regulation, the DNA damage response, and cell-cycle progression by selectively modifying substrates with distinct ubiquitin chain linkages [#0, #29, #46]. Structurally, it is built from an α-solenoid scaffold that assembles into an antiparallel dimer with higher-order oligomers and a dynamic catalytic HECT domain; biochemically it preferentially acts as a ubiquitin chain elongator on pre-ubiquitinated substrates, and its activity is tuned by an intramolecular contact between the MLLE/PABC domain and the HECT domain [#22, #46]. Its modular architecture combines a ubiquitin-binding UBA domain, a UBR1 zinc finger, a HECT catalytic lobe whose unique C-lobe insert dictates E2 (UBCH4) specificity, and a PAM2-recognizing MLLE/PABC domain [#0, #4, #15, #22]. Catalytic substrates span cell-cycle and growth regulators (MYC degraded independently of FBXW7, SOX2 at Lys115 gated by AKT phosphorylation, CDC73, Rb, and the mitotic checkpoint complex components BubR1/Bub3/Cdc20 whose ubiquitylation drives MCC disassembly and APC/C reactivation), metabolic and stress factors (acetylated PEPCK1, OGA, MOAP-1), and the centriolar substrate CP110 and ciliary protein CSPP1, with several substrates handed to UBR5 only after priming phosphorylation by DYRK2 within an EDD-DDB1-VprBP/DYRK2 complex [#10, #17, #24, #26, #33, #37, #43, #44, #48, #52]. In the DNA damage response UBR5 restrains RNF168-driven ubiquitin spreading, ubiquitylates ATMIN at Lys238 to bias ATM toward MRN-dependent signaling, and cooperates with PRC1/BMI1 and the FACT subunit SPT16 to silence RNA Pol II elongation at lesions [#14, #20, #25, #35]. Beyond degradative chemistry, UBR5 builds non-degradative K11 chains on Smad3 and K63 chains on TRIM28 and Akirin to control transcription, ferroptosis resistance, and NF-κB- and RLR-driven immunity [#38, #42, #50]. UBR5 also acts independently of its ligase activity: through the MLLE/PABC domain it scaffolds miRNA silencing by recruiting GW182, DDX6, and Tob1/2, stabilizes the transcription factor myocardin, and drives IFN-γ-induced PD-L1 transcription via PKR-STAT1 [#11, #8, #45]. It additionally executes agonist-dependent, chromatin-coupled degradation of nuclear hormone receptors including RARA, ERα, and others through sequential exchange of coactivators and UBR5 [#47]. Genetic loss is embryonic lethal in mouse with failed extraembryonic vascular development, and UBR5 catalytic activity is required for breast tumor growth and metastasis [#1, #29].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Established UBR5 as a nuclear, multidomain protein physically coupled to hormone-receptor transcription, defining the cellular compartment and an early functional context.\",\n      \"evidence\": \"Co-IP, NLS-driven nuclear import assay, and progesterone-receptor transactivation reporter\",\n      \"pmids\": [\"12011095\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No catalytic activity demonstrated\", \"PR not shown to be a ubiquitination substrate\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Demonstrated UBR5 is essential in vivo, establishing a non-redundant developmental requirement.\",\n      \"evidence\": \"Murine Edd knockout with embryo phenotyping\",\n      \"pmids\": [\"15282321\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular substrate underlying vascular defect unidentified\", \"Catalytic dependence not tested in this model\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defined the biochemistry of the MLLE/PABC domain as a PAM2-peptide recognition module linking UBR5 to translation/anti-proliferative regulators, the structural basis for its later ligase-independent scaffolding roles.\",\n      \"evidence\": \"ITC, SPR, NMR, GST pulldown, Co-IP with Tob2\",\n      \"pmids\": [\"16554297\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of Tob2 binding not resolved\", \"Did not connect to a defined complex\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Placed UBR5 upstream of CHK2 activation, implicating it in DNA-damage checkpoint signaling.\",\n      \"evidence\": \"FHA-dependent Co-IP, RNAi epistasis, kinase activity assay after IR\",\n      \"pmids\": [\"17074762\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether regulation is catalytic or scaffolding unresolved\", \"No direct substrate in the CHK2 axis defined\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Resolved how UBR5 reads ubiquitin (UBA domain) and validated catalytic competence and E2 specificity of its HECT C-lobe, anchoring its mechanism as a HECT ligase.\",\n      \"evidence\": \"1.85 Å UBA crystal structure with mutagenesis and SPR; HECT C-lobe crystal structure with thioester ubiquitination assay\",\n      \"pmids\": [\"17897937\", \"23027739\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No full-length structure at this stage\", \"Chain-linkage preferences not defined\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Showed UBR5 is required for multiple cell-cycle checkpoints and chromosomal integrity, defining a broad role in genome stability.\",\n      \"evidence\": \"RNAi knockdown with flow cytometry and DNA-damage checkpoint assays\",\n      \"pmids\": [\"18073532\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ubiquitination targets driving checkpoints not identified\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Separated UBR5's catalytic and non-catalytic functions by showing ligase-independent scaffolding of the miRNA silencing machinery via the PABC domain.\",\n      \"evidence\": \"Mouse ES-cell genetic screen, Co-IP with GW182/DDX6/Tob1-2, domain-deletion silencing assays\",\n      \"pmids\": [\"21726813\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether scaffolding requires UBR5 oligomerization unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identified the first catalytic substrate gated by a post-translational mark, establishing acetylation as a degron trigger feeding UBR5.\",\n      \"evidence\": \"In vitro ubiquitination, Co-IP, and acetyltransferase/deacetylase assays on PEPCK1\",\n      \"pmids\": [\"21726808\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Chain linkage on PEPCK1 not defined\", \"In vivo metabolic role inferred indirectly\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Positioned UBR5 (with TRIP12) as a negative regulator that confines RNF168-dependent ubiquitin spreading at DNA breaks, a key restraint on damage signaling.\",\n      \"evidence\": \"siRNA depletion with immunofluorescence quantification of H2A-Ub, 53BP1, and BRCA1 spreading\",\n      \"pmids\": [\"22884692\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct ubiquitination of RNF168 by UBR5 versus TRIP12 not fully separated\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined site-specific control of ATM pathway choice through UBR5 ubiquitylation of ATMIN at Lys238, mechanistically linking UBR5 to MRN-dependent ATM activation.\",\n      \"evidence\": \"K238 mutagenesis, IR-stimulated ubiquitination, ATM foci and checkpoint assays\",\n      \"pmids\": [\"25092319\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Chain linkage on ATMIN not specified\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Elucidated MLLE-PAM2 recognition at atomic resolution and discovered an autoinhibitory MLLE-HECT intramolecular contact, providing a structural model for regulating UBR5 ligase activity.\",\n      \"evidence\": \"Crystal structure of MLLE-Paip1 PAM2 complex, NMR, ITC, domain mapping\",\n      \"pmids\": [\"26224628\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological trigger relieving autoinhibition unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Confirmed UBR5 catalytic activity is required for tumorigenesis and revealed cross-species conserved control of Wnt and Notch outputs through substrate ubiquitylation.\",\n      \"evidence\": \"CRISPR KO with catalytic-mutant rescue in TNBC; Drosophila/human ubiquitination of Groucho/TLE; C. elegans Notch epistasis; centrosomal CP110 ubiquitylation within EDD-DYRK2-DDB1-VprBP\",\n      \"pmids\": [\"28330927\", \"28689657\", \"27185398\", \"28242748\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue-specific substrate priorities in vivo unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Expanded UBR5 substrate logic to include kinase-primed (DYRK2/AKT) targets and replication-fork/H2A-coupled functions, refining how signaling selects UBR5 cargo.\",\n      \"evidence\": \"Site-directed mutagenesis of SOX2 K115/T116 with ubiquitination assays; replication-fork Co-IP, DNA fiber and H2A-Ub epistasis\",\n      \"pmids\": [\"30894683\", \"31586398\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct UBR5 substrate at the fork beyond H2A regulation unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Established UBR5 as an FBXW7-independent regulator of MYC and as a K63-chain builder in innate immune transcription, broadening its roles in oncogenesis and immunity.\",\n      \"evidence\": \"CRISPR screen and ubiquitination assays on MYC with Drosophila validation; K63-ubiquitination of Akirin with Drosophila and human NF-κB readouts\",\n      \"pmids\": [\"32029551\", \"33208877\", \"32339205\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How chain-linkage selection is determined per substrate not defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Provided reconstituted mechanistic proof that UBR5 disassembles the mitotic checkpoint complex and identified K63-ubiquitination of TRIM28 as a switch de-repressing antiviral transcription.\",\n      \"evidence\": \"Reconstitution from purified components and immunodepletion for MCC disassembly; CRISPR screen, K63-Ub/SUMO assays, and Ubr5 KO mouse viral challenge\",\n      \"pmids\": [\"35217622\", \"38278841\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Coordination of MCC disassembly with APC/C in intact cells not fully mapped\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Delivered the full-length architecture (antiparallel dimer, dynamic HECT) and a chain-elongation activity model, and unified UBR5 as the ligase for agonist-dependent, chromatin-coupled nuclear hormone receptor turnover.\",\n      \"evidence\": \"Cryo-EM of full-length UBR5 with chain-elongation assays and AKIRIN2 interaction; chromatin recruitment and degradation assays across RARA/ERα/GR/PR and others\",\n      \"pmids\": [\"37409633\", \"37478846\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of substrate selection still incompletely defined\", \"How the dimer engages diverse substrates not resolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed non-degradative K11 ubiquitylation as a distinct UBR5 output and tied UBR5 to Rb-dependent G1 control and CDK4/6-inhibitor sensitivity.\",\n      \"evidence\": \"K11-linked ubiquitination of Smad3 with ferroptosis assays; CRISPR KO with single-cell Rb measurement and CDK4/6 inhibitor sensitivity\",\n      \"pmids\": [\"39260061\", \"39441926\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ubiquitination of Rb not demonstrated\", \"Determinants of degradative versus non-degradative output unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How UBR5 oligomeric architecture, MLLE-HECT autoregulation, and priming-kinase signals jointly dictate substrate choice and chain-linkage outcome across its many pathways remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No unified model linking dimer/oligomer state to substrate or linkage selection\", \"Substrate-specific recruitment determinants largely unmapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [10, 17, 20, 33, 37, 43, 44, 46, 47]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [10, 14, 24, 33, 37, 43, 44, 46]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [15, 29, 38, 42, 50]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [8, 11, 45]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [8, 45, 47]},\n      {\"term_id\": \"GO:0031386\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 9, 54]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [25, 35, 47]},\n      {\"term_id\": \"GO:0815\", \"supporting_discovery_ids\": []}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [14, 20, 25, 35]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [44, 48, 51]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [10, 30, 37, 47]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [38, 42]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [9, 28]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [11]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [7, 25, 45, 47]}\n    ],\n    \"complexes\": [\n      \"EDD-DDB1-VprBP/DYRK2 E3 ligase complex\",\n      \"miRNA silencing (GW182-DDX6-Tob) complex\"\n    ],\n    \"partners\": [\n      \"DYRK2\",\n      \"DDB1\",\n      \"ATMIN\",\n      \"MYC\",\n      \"RNF168\",\n      \"BMI1\",\n      \"GW182\",\n      \"TRIM28\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":9,"faith_total":9,"faith_pct":100.0}}