{"gene":"UBE2D3","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2011,"finding":"Crystal structure of the Bmi1/Ring1b RING-RING heterodimer in complex with UbcH5c revealed that UbcH5c interacts exclusively with Ring1b (not Bmi1) in a manner typical of E2-E3 interactions; the Bmi1/Ring1b dimer also contacts nucleosomal DNA via a basic surface patch unique to this heterodimer, enabling specific monoubiquitination of histone H2A at K119.","method":"X-ray crystallography, mutagenesis of DNA-binding surface residues, H2A ubiquitination activity assays, computational modelling of nucleosome interface","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional mutagenesis and in vitro ubiquitination assays in a single rigorous study","pmids":["21772249"],"is_preprint":false},{"year":2010,"finding":"The E4B U box domain (monomer) binds UbcH5c and Ubc4; structural and calorimetric/NMR binding assays indicate allosteric regulation of UbcH5c by the E4B U box, defining the molecular basis of E4B–UbcH5c assembly in polyubiquitin chain elongation.","method":"X-ray crystallography, NMR spectroscopy (scalar coupling measurements), isothermal titration calorimetry, NMR-based binding assays","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus NMR and calorimetry, multiple orthogonal methods in one study","pmids":["20696396"],"is_preprint":false},{"year":2017,"finding":"UBE2D3 (Ube2D3) was identified by chromatographic purification as an activator of RIG-I; together with the E3 ligase Riplet, UBE2D3 promotes covalent conjugation of polyubiquitin chains to RIG-I, which in turn induces MAVS prion-like aggregation on mitochondria to trigger innate immune signalling.","method":"Chromatographic purification, biochemical reconstitution, cell-based ubiquitination assays, MAVS aggregation assays, gene knockdown","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — chromatographic purification combined with reconstitution and multiple functional assays, replicated with genetic knockdown","pmids":["28469175"],"is_preprint":false},{"year":2007,"finding":"UBE2D3 is physically associated with cyclin D1 and mediates ATRA-induced cyclin D1 degradation; shRNA knockdown of UBE2D3 blocks ATRA-induced cyclin D1 degradation and cell-cycle arrest in APL NB4 cells.","method":"shRNA screen, co-immunoprecipitation, RNA interference knockdown, cell-cycle analysis","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and functional knockdown in a single lab with two orthogonal methods","pmids":["17420285"],"is_preprint":false},{"year":2020,"finding":"MDMX interacts with UbcH5c (but MDM2 does not), and this interaction is essential for MDMX to enable MDM2 E3 ligase activity toward p53 degradation in vivo; grafting MDMX C-terminal residues onto MDM2 restored UbcH5c binding and enhanced MDM2-mediated p53 degradation in the absence of MDMX.","method":"In vivo mouse models (inducible p53 allele with MDM2/MDMX deletions), co-immunoprecipitation, domain-swap mutagenesis, mouse embryonic fibroblast assays","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic models combined with Co-IP and domain-swap mutagenesis, multiple orthogonal approaches","pmids":["33277368"],"is_preprint":false},{"year":2013,"finding":"UBE2D3 interacts with hTERT (identified by yeast two-hybrid and validated biochemically); UBE2D3 knockdown causes accumulation of hTERT and cyclin D1, increases hTERT activity, and accelerates G1-S transition, indicating UBE2D3 regulates radiosensitivity by controlling hTERT and cyclin D1 levels.","method":"Yeast two-hybrid screen, co-immunoprecipitation validation, shRNA knockdown, cell-cycle analysis, telomerase activity assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Y2H with biochemical validation and functional knockdown, single lab","pmids":["23741361"],"is_preprint":false},{"year":2016,"finding":"UBE2D3 overexpression in EC109 esophageal cancer cells increases ubiquitinated hTERT species (detected after proteasome inhibition with MG132), decreases hTERT protein levels, and reduces telomerase activity, demonstrating UBE2D3 targets hTERT for proteasomal degradation via ubiquitination.","method":"UBE2D3 overexpression, proteasome inhibitor (MG132) treatment, detection of ubiquitinated hTERT by immunoprecipitation/western blot, telomerase activity assay, in vivo xenograft","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional overexpression with ubiquitination detection and proteasome inhibitor rescue, single lab","pmids":["27105523"],"is_preprint":false},{"year":2017,"finding":"Crystal structure of recombinant human UbcH5c was solved (space group P2₁2₁2₁, one molecule in asymmetric unit), providing structural basis for inhibitor design.","method":"X-ray crystallography of recombinant UbcH5c","journal":"Acta pharmaceutica Sinica. B","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — crystal structure determined but no functional validation accompanying the structure in this paper","pmids":["28540177"],"is_preprint":false},{"year":2017,"finding":"The α-santonin-derived compound 6d covalently modifies the active-site Cys85 of UbcH5c, inactivating the enzyme and suppressing NF-κB activation; confirmed by BIAcore, in-gel fluorescence, and immunoprecipitation assays.","method":"BIAcore binding assay, in-gel fluorescence imaging, immunoprecipitation, NF-κB luciferase reporter assay, western blotting","journal":"Journal of medicinal chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal biochemical methods identifying Cys85 as the active site target, single lab","pmids":["28696694"],"is_preprint":false},{"year":2021,"finding":"UBE2D3 interacts with SHP-2 and promotes its ubiquitination, which activates the STAT3 signalling pathway and promotes glioma cell proliferation and glycolysis; UBE2D3 knockdown suppressed STAT3 phosphorylation, proliferation, and glycolysis both in vitro and in vivo.","method":"Co-immunoprecipitation, ubiquitination assays, siRNA knockdown, xenograft experiments, flow cytometry, metabolic flux assays","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus functional knockdown with in vivo validation, single lab","pmids":["34195079"],"is_preprint":false},{"year":2021,"finding":"UBE2D3 promotes ubiquitination of p62/SQSTM1, thereby impairing autophagic flux in myocardial ischemia-reperfusion injury; UBE2D3 also negatively regulates mTOR independently of the mTOR-beclin1 pathway.","method":"siRNA knockdown in vitro and in vivo rat I/R model, proteasome inhibitor (MG132) treatment, western blotting for p62 ubiquitination, autophagic flux assays","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional knockdown with ubiquitination readout and in vivo model, single lab","pmids":["34391873"],"is_preprint":false},{"year":2021,"finding":"PP1 and PP2A phosphatases and UBE2D3 were identified as required for COP1-mediated c-Jun degradation upon Erk1/2 inactivation; the C-terminus of c-Jun (last four amino acids) is also required for its degradation in this pathway.","method":"Pharmacological inhibitors (PP1/PP2A), siRNA gene knockdown of UBE2D3, c-Jun C-terminal deletion/tag mutagenesis, western blotting for c-Jun levels","journal":"International journal of molecular sciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — siRNA knockdown with western blot readout, single lab, single method per component","pmids":["33918729"],"is_preprint":false},{"year":2020,"finding":"A conserved serine at position 138 (S138) in the C-terminal α-helical region of UBE2D3, present only in amniotes, is phosphorylated by Aurora B kinase; phosphorylation of S138 disrupts UBE2D3 structure and reduces its protein level in mouse ESCs. S138A substitution increases UBE2D3 levels, is an early embryonic lethal gain-of-function mutation in mice, reduces PDGFRα and FGFR1 levels, and increases UBE2D3 interaction with E3 ligase CBL.","method":"Protein sequence comparison, mouse genetics (S138A knock-in), proximity ligation assay, ESC differentiation assays, western blotting","journal":"Molecular biology and evolution","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo mouse genetics combined with proximity ligation and cell differentiation assays, multiple orthogonal methods","pmids":["32145025"],"is_preprint":false},{"year":2023,"finding":"UBE2D3 depletion by quantitative diGly ubiquitinomics revealed that RPS10 and RPS20 (ribosomal proteins critical for ribosome-associated protein quality control) are direct in vivo ubiquitination substrates of UBE2D3; catalytic activity of UBE2D3 is required for RPS10 ubiquitination in vivo. UBE2D3 also acts at multiple levels in autophagic protein quality control.","method":"SILAC-based diGly proteomics, label-free quantitative ubiquitinomics (UBE2D3 depletion), TULIP2 (Targets of Ubiquitin Ligases Identified by Proteomics) methodology, catalytic mutant UBE2D3","journal":"Molecular & cellular proteomics","confidence":"High","confidence_rationale":"Tier 1 / Strong — TULIP2 direct substrate identification combined with quantitative proteomics and catalytic mutant validation in a single rigorous study","pmids":["37059365"],"is_preprint":false},{"year":2023,"finding":"UbcH5c (UBE2D3) is required for DNA-PK activation specifically in response to one-ended DSBs caused by replication fork collapse (CPT-induced), but not two-ended DSBs (neocarzinostatin-induced); this activation is independent of DNA end resection, and UbcH5c loss reduced DNA-PK-dependent chromosomal aberrations and attenuated cell-cycle checkpoint activation after CPT.","method":"siRNA library screen against E2 enzymes, siRNA knockdown of UbcH5c, camptothecin and neocarzinostatin treatment, DNA-PK activation assay, chromosomal aberration analysis, checkpoint assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA screen followed by functional validation with two distinct DSB-inducing agents, single lab","pmids":["37244033"],"is_preprint":false},{"year":2024,"finding":"UBE2D3 promotes NHEJ at telomeres by acting as a multi-level regulator: it contributes to DDR-induced chromatin ubiquitination and 53BP1 recruitment mediated by RNF168 upon ATM activation, while also limiting RNF168 accumulation and facilitating ATM-mediated phosphorylation of KAP1-S824. UBE2D3 deficiency leads to RNF168 hyperaccumulation, aberrant PP2A phosphatase activity, and defective KAP1-S824 phosphorylation, revealing a negative regulatory circuit.","method":"UBE2D3 knockout/depletion, telomeric NHEJ assays, 53BP1 recruitment assays, RNF168 accumulation monitoring, KAP1 phosphorylation assays, PP2A activity assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal functional assays establishing UBE2D3 as a multi-level regulator of NHEJ and ATM signalling, single rigorous study","pmids":["38866770"],"is_preprint":false},{"year":2022,"finding":"The small-molecule DHPO directly binds UbcH5c (confirmed by SPR and CETSA) and inhibits UbcH5c-mediated IκBα ubiquitination and degradation, thereby blocking NF-κB activation in pancreatic cancer cells.","method":"Surface plasmon resonance (SPR), cellular thermal shift assay (CETSA), western blot for IκBα ubiquitination, NF-κB reporter assay, in vitro and in vivo pancreatic cancer models","journal":"Molecular cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding confirmed by two orthogonal biophysical methods and functional ubiquitination assay, single lab","pmids":["35272681"],"is_preprint":false},{"year":2023,"finding":"Arteannuin B (ATB) covalently binds the catalytic Cys85 of UBE2D3, inhibiting its function and preventing ubiquitination of RIP1 and NEMO, thereby blocking NF-κB activation; confirmed by CETSA, DARTS, MST, LC-MS/MS identification of the binding site, and UBE2D3 siRNA knockdown.","method":"CETSA, DARTS, microscale thermophoresis (MST), LC-MS/MS mass spectrometry, molecular docking, siRNA knockdown, western blotting for RIP1/NEMO ubiquitination","journal":"Phytomedicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal biophysical and chemical methods identifying Cys85 as the binding site, confirmed by genetic knockdown, single lab","pmids":["38181532"],"is_preprint":false},{"year":2025,"finding":"UBE2D3 binds the E3 ligase KLHL13 to mediate K63-linked polyubiquitination at K245 of TAP2, causing steric hindrance that blocks the TAP2 transporter; this impairs antigen presentation and enables pancreatic cancer cells to evade CD8+ T-cell surveillance in response to IFN-γ.","method":"Co-immunoprecipitation, ubiquitination site mapping (K245), genetic knockout/knockdown of UBE2D3, antigen presentation assays, CD8+ T-cell killing assays, mouse tumor models","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP with site-specific ubiquitination mapping, functional immune assays, and in vivo mouse models in a single study","pmids":["41315272"],"is_preprint":false},{"year":2025,"finding":"UBE2D3 is the most highly expressed E2 enzyme in mouse oocytes and is essential for meiotic division; depletion causes metaphase I arrest and Cyclin B1 accumulation, while overexpression reduces Cyclin B1 levels, causes kinetochore-microtubule mis-attachments, spindle assembly checkpoint dysfunction, and aneuploidy. Elevated UBE2D3 in aged oocytes contributes to age-related meiotic defects reversible by UBE2D3 knockdown or Cyclin B1 overexpression.","method":"UBE2D3 knockdown and overexpression in mouse oocytes, meiotic progression assays, Cyclin B1 level measurement, kinetochore-microtubule attachment analysis, spindle assembly checkpoint assays, aneuploidy scoring","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss- and gain-of-function in oocytes with multiple orthogonal cellular phenotype readouts, single lab","pmids":["39921465"],"is_preprint":false},{"year":2007,"finding":"Xenopus ube2d3.2 (ortholog of UBE2D3) interacts with xMLK2 (identified by yeast two-hybrid) and limits xMLK2 accumulation; ectopic ube2d3.2 expression inhibits pronephric tubule formation, phenocopying loss of xMLK2, implicating ube2d3.2 as an endogenous regulator of xMLK2 and JNK activity.","method":"Yeast two-hybrid screen, ectopic expression in Xenopus embryos, xMLK2 protein level assay, pronephros morphology analysis","journal":"Differentiation","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Y2H plus overexpression phenotype in Xenopus, mechanism inferred, single lab","pmids":["18021256"],"is_preprint":false},{"year":2024,"finding":"Cryo-EM structure of the RNF168/UbcH5c–Ub/H1.0–K63-Ub₃ chromatosome complex revealed the recruitment orientation between the RNF168 UDM1 domain and K63-linked ubiquitin chain on H1.0, providing structural basis for how K63-polyubiquitinated H1.0 stimulates RNF168-mediated H2A ubiquitination.","method":"Cryo-EM structural analysis, biochemical reconstitution with synthetic ubiquitylated H1.0, RNF168 ubiquitylation activity assays on chromatosomes","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — cryo-EM structure with reconstitution, but preprint not yet peer-reviewed","pmids":["bio_10.1101_2024.07.22.604500"],"is_preprint":true}],"current_model":"UBE2D3 (UbcH5c) is a promiscuous E2 ubiquitin-conjugating enzyme that transfers ubiquitin to diverse substrates—including histone H2A (via Ring1b/Bmi1), cyclin D1, hTERT, Cyclin B1, RIP1, NEMO, IκBα, SHP-2, p62, RPS10, RPS20, and TAP2—working with cognate E3 ligases (Riplet, MDM2/MDMX via MDMX recruitment, COP1, KLHL13, E4B, RNF168, CBL) to regulate NF-κB, p53, RIG-I/MAVS innate immune, ATM/NHEJ DNA damage, meiotic cell-cycle, and protein quality-control pathways; its catalytic active site Cys85 can be covalently inactivated by small molecules, and its activity is modulated by Aurora B-mediated phosphorylation of S138."},"narrative":{"mechanistic_narrative":"UBE2D3 (UbcH5c) is a promiscuous E2 ubiquitin-conjugating enzyme that partners with diverse RING and U-box E3 ligases to ubiquitinate substrates across chromatin regulation, innate immunity, DNA damage signalling, cell-cycle control, and protein quality control [PMID:21772249, PMID:28469175, PMID:38866770, PMID:37059365]. Its catalytic transfer activity depends on an active-site cysteine, Cys85, which can be covalently modified by small molecules to inactivate the enzyme [PMID:28696694, PMID:38181532]. In chromatin and DNA-damage contexts it cooperates with the Ring1b/Bmi1 RING heterodimer—interacting exclusively with Ring1b—to monoubiquitinate histone H2A at K119 [PMID:21772249], and works with RNF168 downstream of ATM to drive DDR-induced chromatin ubiquitination, 53BP1 recruitment, and KAP1-S824 phosphorylation while simultaneously limiting RNF168 accumulation, defining a negative feedback circuit that promotes telomeric NHEJ [PMID:38866770]; it is also selectively required for DNA-PK activation at one-ended replication-associated double-strand breaks [PMID:37244033]. In innate immunity UBE2D3 acts with the E3 ligase Riplet to polyubiquitinate RIG-I, driving MAVS aggregation and antiviral signalling [PMID:28469175], and its activity supports NF-κB activation through ubiquitination of pathway components such as IκBα, RIP1, and NEMO [PMID:35272681, PMID:38181532]. It directs substrate degradation in cell-cycle and p53 control, mediating ATRA-induced cyclin D1 turnover [PMID:17420285], targeting hTERT for proteasomal degradation [PMID:23741361, PMID:27105523], and enabling MDM2-driven p53 degradation through a UbcH5c-binding surface contributed by MDMX [PMID:33277368]. Direct ubiquitinomic profiling identified the ribosomal proteins RPS10 and RPS20 as in vivo catalytic substrates linking UBE2D3 to ribosome-associated and autophagic protein quality control [PMID:37059365]. Aurora B-mediated phosphorylation of a conserved amniote-specific Ser138 destabilizes UBE2D3 and modulates its interaction with the E3 ligase CBL, with this regulation being essential for early embryonic development [PMID:32145025].","teleology":[{"year":2007,"claim":"Establishing that UBE2D3 controls a specific cell-cycle regulator addressed whether this E2 has dedicated substrate-degradation roles beyond generic ubiquitin transfer.","evidence":"Co-IP and shRNA knockdown linking UBE2D3 to ATRA-induced cyclin D1 degradation in APL cells","pmids":["17420285"],"confidence":"Medium","gaps":["Cognate E3 ligase for cyclin D1 turnover not identified","Direct ubiquitination of cyclin D1 by UBE2D3 not reconstituted"]},{"year":2010,"claim":"Defining the structural basis of E4B U-box engagement clarified how an E3 module allosterically activates UBE2D3 for polyubiquitin chain elongation.","evidence":"Crystallography, NMR, and ITC of the E4B U-box bound to UbcH5c/Ubc4","pmids":["20696396"],"confidence":"High","gaps":["In vivo substrates of the E4B-UBE2D3 pair not defined","Chain linkage type not established in this study"]},{"year":2011,"claim":"The Bmi1/Ring1b–UbcH5c structure explained how E2 selectivity and nucleosome contacts achieve site-specific H2A monoubiquitination on chromatin.","evidence":"X-ray crystallography with DNA-binding mutagenesis and H2A ubiquitination assays","pmids":["21772249"],"confidence":"High","gaps":["Cellular consequences of H2A-K119 ubiquitination not addressed here","Regulation of complex assembly in vivo unknown"]},{"year":2013,"claim":"Identifying hTERT as a UBE2D3 interactor connected this E2 to telomerase control and radiosensitivity.","evidence":"Y2H, Co-IP, and knockdown with telomerase and cell-cycle assays","pmids":["23741361"],"confidence":"Medium","gaps":["E3 ligase mediating hTERT ubiquitination not identified","Direct versus indirect regulation of hTERT not resolved"]},{"year":2016,"claim":"Demonstrating proteasome-dependent hTERT turnover upon UBE2D3 overexpression confirmed a causal degradation role rather than mere association.","evidence":"Overexpression with MG132 rescue, ubiquitination detection, and xenografts in esophageal cancer cells","pmids":["27105523"],"confidence":"Medium","gaps":["Cognate E3 ligase still unidentified","Ubiquitin chain linkage on hTERT not characterized"]},{"year":2017,"claim":"Reconstituting RIG-I activation placed UBE2D3 at the heart of antiviral innate immune signalling.","evidence":"Chromatographic purification, reconstitution, and MAVS aggregation assays with Riplet","pmids":["28469175"],"confidence":"High","gaps":["Ubiquitin chain architecture on RIG-I not fully defined","Regulation of Riplet-UBE2D3 pairing unknown"]},{"year":2017,"claim":"Solving the apo UbcH5c structure and identifying a covalent Cys85 inhibitor established the active site as a druggable handle for NF-κB suppression.","evidence":"X-ray crystallography of recombinant UbcH5c, plus α-santonin compound 6d binding by BIAcore, in-gel fluorescence, and NF-κB reporter assays","pmids":["28540177","28696694"],"confidence":"Medium","gaps":["Selectivity of Cys85 inhibitors over other E2s not fully established","Functional structure determined without bound E3 in apo study"]},{"year":2020,"claim":"Genetic dissection of the MDM2/MDMX axis showed UBE2D3 binding is the limiting step that allows MDM2 to degrade p53.","evidence":"In vivo mouse p53 models, Co-IP, and MDMX-to-MDM2 domain-swap mutagenesis","pmids":["33277368"],"confidence":"High","gaps":["Structural detail of the MDMX C-terminus–UbcH5c interface not resolved","Chain type built on p53 not addressed"]},{"year":2020,"claim":"Identifying Aurora B phosphorylation of an amniote-specific Ser138 revealed post-translational control of UBE2D3 stability and E3 partner selection with developmental consequences.","evidence":"Mouse S138A knock-in genetics, proximity ligation, and ESC differentiation assays","pmids":["32145025"],"confidence":"High","gaps":["Mechanism by which phosphorylation destabilizes the fold not structurally defined","Full set of CBL-dependent substrates affected unknown"]},{"year":2021,"claim":"A cluster of disease-context studies extended UBE2D3 substrate scope to SHP-2/STAT3 signalling, p62 autophagy regulation, and COP1-mediated c-Jun degradation.","evidence":"Co-IP, ubiquitination assays, and knockdown across glioma, cardiac I/R, and Erk-inactivation models","pmids":["34195079","34391873","33918729"],"confidence":"Medium","gaps":["c-Jun finding rests on single-method siRNA with western readout (Low confidence)","Direct ubiquitination versus indirect effects not always separated","Chain linkages on these substrates undefined"]},{"year":2022,"claim":"A second direct-binding inhibitor (DHPO) reinforced UBE2D3 as a tractable NF-κB target via blockade of IκBα ubiquitination.","evidence":"SPR and CETSA binding plus IκBα ubiquitination and NF-κB assays in pancreatic cancer models","pmids":["35272681"],"confidence":"Medium","gaps":["Binding site on UBE2D3 not mapped in this study","Off-target E2 inhibition not excluded"]},{"year":2023,"claim":"Quantitative ubiquitinomics provided the first direct in vivo substrate identification, linking UBE2D3 catalysis to ribosome-associated protein quality control.","evidence":"SILAC diGly proteomics and TULIP2 with catalytic-mutant validation identifying RPS10/RPS20","pmids":["37059365"],"confidence":"High","gaps":["E3 ligases pairing with UBE2D3 on RPS10/RPS20 not defined","Downstream fate of ubiquitinated ribosomal proteins not fully traced"]},{"year":2023,"claim":"An E2 siRNA screen pinpointed UBE2D3 as selectively required for DNA-PK activation at replication-associated one-ended breaks, distinguishing it from two-ended DSB responses.","evidence":"siRNA screen and knockdown with CPT versus neocarzinostatin, DNA-PK activation and chromosomal aberration assays","pmids":["37244033"],"confidence":"Medium","gaps":["Direct ubiquitination substrate driving DNA-PK activation not identified","Cognate E3 ligase in this pathway unknown"]},{"year":2024,"claim":"Functional and structural work placed UBE2D3 as a multi-level regulator of ATM/RNF168 signalling and telomeric NHEJ, including a negative feedback loop constraining RNF168.","evidence":"UBE2D3 knockout with 53BP1, RNF168, KAP1, and PP2A assays; cryo-EM of the RNF168/UbcH5c–H1.0 chromatosome complex (preprint)","pmids":["38866770","bio_10.1101_2024.07.22.604500"],"confidence":"High","gaps":["Mechanism by which UBE2D3 limits RNF168 accumulation not fully defined","Cryo-EM structure is a preprint"]},{"year":2025,"claim":"Two studies extended UBE2D3 into immune evasion via TAP2 ubiquitination and into meiotic cell-cycle control via Cyclin B1 turnover, demonstrating dosage-sensitive physiological roles.","evidence":"KLHL13-dependent K63 ubiquitination at TAP2-K245 with CD8+ T-cell and tumor assays; loss/gain-of-function in mouse oocytes with checkpoint and aneuploidy readouts","pmids":["41315272","39921465"],"confidence":"High","gaps":["Whether UBE2D3 directly ubiquitinates Cyclin B1 versus acting through an E3 not resolved","Regulation of UBE2D3 expression in aged oocytes not mechanistically explained"]},{"year":null,"claim":"How E3 ligase pairing, chain-linkage choice, and post-translational state combine to dictate UBE2D3 substrate selectivity across its many pathways remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying model linking E3 partner to linkage specificity","Cognate E3 ligases for several substrates (cyclin D1, hTERT, Cyclin B1) undefined","Tissue-specific regulation of UBE2D3 abundance poorly understood"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,13,18]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[1,13,18]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[4,15]}],"localization":[{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[0,15]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[2,16,18]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[14,15]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[13]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[3,4,19]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[0]}],"complexes":[],"partners":["RING1B","BMI1","RNF168","RIPLET","MDMX","KLHL13","CBL","E4B"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P61077","full_name":"Ubiquitin-conjugating enzyme E2 D3","aliases":["(E3-independent) E2 ubiquitin-conjugating enzyme D3","E2 ubiquitin-conjugating enzyme D3","Ubiquitin carrier protein D3","Ubiquitin-conjugating enzyme E2(17)KB 3","Ubiquitin-conjugating enzyme E2-17 kDa 3","Ubiquitin-protein ligase D3"],"length_aa":147,"mass_kda":16.7,"function":"Accepts ubiquitin from the E1 complex and catalyzes its covalent attachment to other proteins (PubMed:15247280, PubMed:15496420, PubMed:18284575, PubMed:20061386, PubMed:21532592, PubMed:28322253). In vitro catalyzes 'Lys-11'-, as well as 'Lys-48'-linked polyubiquitination (PubMed:15247280, PubMed:15496420, PubMed:18284575, PubMed:20061386, PubMed:21532592). Cooperates with the E2 CDC34 and the SCF(FBXW11) E3 ligase complex for the polyubiquitination of NFKBIA leading to its subsequent proteasomal degradation (PubMed:20347421). Acts as an initiator E2, priming the phosphorylated NFKBIA target at positions 'Lys-21' and/or 'Lys-22' with a monoubiquitin (PubMed:10329681). Ubiquitin chain elongation is then performed by CDC34, building ubiquitin chains from the UBE2D3-primed NFKBIA-linked ubiquitin (PubMed:10329681). Also acts as an initiator E2, in conjunction with RNF8, for the priming of PCNA (PubMed:18948756). Monoubiquitination of PCNA, and its subsequent polyubiquitination, are essential events in the operation of the DNA damage tolerance (DDT) pathway that is activated after DNA damage caused by UV or chemical agents during S-phase (PubMed:18948756). Associates with the BRCA1/BARD1 E3 ligase complex to perform ubiquitination at DNA damage sites following ionizing radiation leading to DNA repair (PubMed:16628214). Targets DAPK3 for ubiquitination which influences promyelocytic leukemia protein nuclear body (PML-NB) formation in the nucleus (PubMed:18515077). In conjunction with the MDM2 and TOPORS E3 ligases, functions ubiquitination of p53/TP53 (PubMed:12646252, PubMed:15280377). In conjunction with the CBL E3 ligase, targets EGFR for polyubiquitination at the plasma membrane as well as during its internalization and transport on endosomes (PubMed:18508924). In conjunction with the STUB1 E3 quality control E3 ligase, ubiquitinates unfolded proteins to catalyze their immediate destruction (PubMed:11743028). Together with RNF135, catalyzes the viral RNA-dependent 'Lys-63'-linked polyubiquitination of RIGI to activate the downstream signaling pathway that leads to interferon beta production (PubMed:28469175). Together with ZNF598, catalyzes ubiquitination of 40S ribosomal proteins in response to ribosome collisions (PubMed:28685749). In cooperation with the GATOR2 complex, catalyzes 'Lys-6'-linked ubiquitination of NPRL2 (PubMed:36528027)","subcellular_location":"Cell membrane; Endosome membrane","url":"https://www.uniprot.org/uniprotkb/P61077/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/UBE2D3","classification":"Common Essential","n_dependent_lines":938,"n_total_lines":1208,"dependency_fraction":0.7764900662251656},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/UBE2D3","total_profiled":1310},"omim":[{"mim_id":"621029","title":"RING FINGER PROTEIN 183; RNF183","url":"https://www.omim.org/entry/621029"},{"mim_id":"620307","title":"WD REPEAT-CONTAINING PROTEIN 24; WDR24","url":"https://www.omim.org/entry/620307"},{"mim_id":"620096","title":"RING FINGER PROTEIN 185; 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all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/UBE2D3"},"hgnc":{"alias_symbol":["UbcH5C"],"prev_symbol":[]},"alphafold":{"accession":"P61077","domains":[{"cath_id":"3.10.110.10","chopping":"1-147","consensus_level":"medium","plddt":96.4964,"start":1,"end":147}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P61077","model_url":"https://alphafold.ebi.ac.uk/files/AF-P61077-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P61077-F1-predicted_aligned_error_v6.png","plddt_mean":96.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=UBE2D3","jax_strain_url":"https://www.jax.org/strain/search?query=UBE2D3"},"sequence":{"accession":"P61077","fasta_url":"https://rest.uniprot.org/uniprotkb/P61077.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P61077/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P61077"}},"corpus_meta":[{"pmid":"21772249","id":"PMC_21772249","title":"Recognition of UbcH5c and the 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the Bmi1/Ring1b dimer also contacts nucleosomal DNA via a basic surface patch unique to this heterodimer, enabling specific monoubiquitination of histone H2A at K119.\",\n      \"method\": \"X-ray crystallography, mutagenesis of DNA-binding surface residues, H2A ubiquitination activity assays, computational modelling of nucleosome interface\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional mutagenesis and in vitro ubiquitination assays in a single rigorous study\",\n      \"pmids\": [\"21772249\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The E4B U box domain (monomer) binds UbcH5c and Ubc4; structural and calorimetric/NMR binding assays indicate allosteric regulation of UbcH5c by the E4B U box, defining the molecular basis of E4B–UbcH5c assembly in polyubiquitin chain elongation.\",\n      \"method\": \"X-ray crystallography, NMR spectroscopy (scalar coupling measurements), isothermal titration calorimetry, NMR-based binding assays\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus NMR and calorimetry, multiple orthogonal methods in one study\",\n      \"pmids\": [\"20696396\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"UBE2D3 (Ube2D3) was identified by chromatographic purification as an activator of RIG-I; together with the E3 ligase Riplet, UBE2D3 promotes covalent conjugation of polyubiquitin chains to RIG-I, which in turn induces MAVS prion-like aggregation on mitochondria to trigger innate immune signalling.\",\n      \"method\": \"Chromatographic purification, biochemical reconstitution, cell-based ubiquitination assays, MAVS aggregation assays, gene knockdown\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — chromatographic purification combined with reconstitution and multiple functional assays, replicated with genetic knockdown\",\n      \"pmids\": [\"28469175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"UBE2D3 is physically associated with cyclin D1 and mediates ATRA-induced cyclin D1 degradation; shRNA knockdown of UBE2D3 blocks ATRA-induced cyclin D1 degradation and cell-cycle arrest in APL NB4 cells.\",\n      \"method\": \"shRNA screen, co-immunoprecipitation, RNA interference knockdown, cell-cycle analysis\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and functional knockdown in a single lab with two orthogonal methods\",\n      \"pmids\": [\"17420285\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MDMX interacts with UbcH5c (but MDM2 does not), and this interaction is essential for MDMX to enable MDM2 E3 ligase activity toward p53 degradation in vivo; grafting MDMX C-terminal residues onto MDM2 restored UbcH5c binding and enhanced MDM2-mediated p53 degradation in the absence of MDMX.\",\n      \"method\": \"In vivo mouse models (inducible p53 allele with MDM2/MDMX deletions), co-immunoprecipitation, domain-swap mutagenesis, mouse embryonic fibroblast assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic models combined with Co-IP and domain-swap mutagenesis, multiple orthogonal approaches\",\n      \"pmids\": [\"33277368\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"UBE2D3 interacts with hTERT (identified by yeast two-hybrid and validated biochemically); UBE2D3 knockdown causes accumulation of hTERT and cyclin D1, increases hTERT activity, and accelerates G1-S transition, indicating UBE2D3 regulates radiosensitivity by controlling hTERT and cyclin D1 levels.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation validation, shRNA knockdown, cell-cycle analysis, telomerase activity assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Y2H with biochemical validation and functional knockdown, single lab\",\n      \"pmids\": [\"23741361\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"UBE2D3 overexpression in EC109 esophageal cancer cells increases ubiquitinated hTERT species (detected after proteasome inhibition with MG132), decreases hTERT protein levels, and reduces telomerase activity, demonstrating UBE2D3 targets hTERT for proteasomal degradation via ubiquitination.\",\n      \"method\": \"UBE2D3 overexpression, proteasome inhibitor (MG132) treatment, detection of ubiquitinated hTERT by immunoprecipitation/western blot, telomerase activity assay, in vivo xenograft\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional overexpression with ubiquitination detection and proteasome inhibitor rescue, single lab\",\n      \"pmids\": [\"27105523\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Crystal structure of recombinant human UbcH5c was solved (space group P2₁2₁2₁, one molecule in asymmetric unit), providing structural basis for inhibitor design.\",\n      \"method\": \"X-ray crystallography of recombinant UbcH5c\",\n      \"journal\": \"Acta pharmaceutica Sinica. B\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — crystal structure determined but no functional validation accompanying the structure in this paper\",\n      \"pmids\": [\"28540177\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The α-santonin-derived compound 6d covalently modifies the active-site Cys85 of UbcH5c, inactivating the enzyme and suppressing NF-κB activation; confirmed by BIAcore, in-gel fluorescence, and immunoprecipitation assays.\",\n      \"method\": \"BIAcore binding assay, in-gel fluorescence imaging, immunoprecipitation, NF-κB luciferase reporter assay, western blotting\",\n      \"journal\": \"Journal of medicinal chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal biochemical methods identifying Cys85 as the active site target, single lab\",\n      \"pmids\": [\"28696694\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"UBE2D3 interacts with SHP-2 and promotes its ubiquitination, which activates the STAT3 signalling pathway and promotes glioma cell proliferation and glycolysis; UBE2D3 knockdown suppressed STAT3 phosphorylation, proliferation, and glycolysis both in vitro and in vivo.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, siRNA knockdown, xenograft experiments, flow cytometry, metabolic flux assays\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus functional knockdown with in vivo validation, single lab\",\n      \"pmids\": [\"34195079\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"UBE2D3 promotes ubiquitination of p62/SQSTM1, thereby impairing autophagic flux in myocardial ischemia-reperfusion injury; UBE2D3 also negatively regulates mTOR independently of the mTOR-beclin1 pathway.\",\n      \"method\": \"siRNA knockdown in vitro and in vivo rat I/R model, proteasome inhibitor (MG132) treatment, western blotting for p62 ubiquitination, autophagic flux assays\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional knockdown with ubiquitination readout and in vivo model, single lab\",\n      \"pmids\": [\"34391873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PP1 and PP2A phosphatases and UBE2D3 were identified as required for COP1-mediated c-Jun degradation upon Erk1/2 inactivation; the C-terminus of c-Jun (last four amino acids) is also required for its degradation in this pathway.\",\n      \"method\": \"Pharmacological inhibitors (PP1/PP2A), siRNA gene knockdown of UBE2D3, c-Jun C-terminal deletion/tag mutagenesis, western blotting for c-Jun levels\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — siRNA knockdown with western blot readout, single lab, single method per component\",\n      \"pmids\": [\"33918729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"A conserved serine at position 138 (S138) in the C-terminal α-helical region of UBE2D3, present only in amniotes, is phosphorylated by Aurora B kinase; phosphorylation of S138 disrupts UBE2D3 structure and reduces its protein level in mouse ESCs. S138A substitution increases UBE2D3 levels, is an early embryonic lethal gain-of-function mutation in mice, reduces PDGFRα and FGFR1 levels, and increases UBE2D3 interaction with E3 ligase CBL.\",\n      \"method\": \"Protein sequence comparison, mouse genetics (S138A knock-in), proximity ligation assay, ESC differentiation assays, western blotting\",\n      \"journal\": \"Molecular biology and evolution\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo mouse genetics combined with proximity ligation and cell differentiation assays, multiple orthogonal methods\",\n      \"pmids\": [\"32145025\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"UBE2D3 depletion by quantitative diGly ubiquitinomics revealed that RPS10 and RPS20 (ribosomal proteins critical for ribosome-associated protein quality control) are direct in vivo ubiquitination substrates of UBE2D3; catalytic activity of UBE2D3 is required for RPS10 ubiquitination in vivo. UBE2D3 also acts at multiple levels in autophagic protein quality control.\",\n      \"method\": \"SILAC-based diGly proteomics, label-free quantitative ubiquitinomics (UBE2D3 depletion), TULIP2 (Targets of Ubiquitin Ligases Identified by Proteomics) methodology, catalytic mutant UBE2D3\",\n      \"journal\": \"Molecular & cellular proteomics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — TULIP2 direct substrate identification combined with quantitative proteomics and catalytic mutant validation in a single rigorous study\",\n      \"pmids\": [\"37059365\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"UbcH5c (UBE2D3) is required for DNA-PK activation specifically in response to one-ended DSBs caused by replication fork collapse (CPT-induced), but not two-ended DSBs (neocarzinostatin-induced); this activation is independent of DNA end resection, and UbcH5c loss reduced DNA-PK-dependent chromosomal aberrations and attenuated cell-cycle checkpoint activation after CPT.\",\n      \"method\": \"siRNA library screen against E2 enzymes, siRNA knockdown of UbcH5c, camptothecin and neocarzinostatin treatment, DNA-PK activation assay, chromosomal aberration analysis, checkpoint assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA screen followed by functional validation with two distinct DSB-inducing agents, single lab\",\n      \"pmids\": [\"37244033\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"UBE2D3 promotes NHEJ at telomeres by acting as a multi-level regulator: it contributes to DDR-induced chromatin ubiquitination and 53BP1 recruitment mediated by RNF168 upon ATM activation, while also limiting RNF168 accumulation and facilitating ATM-mediated phosphorylation of KAP1-S824. UBE2D3 deficiency leads to RNF168 hyperaccumulation, aberrant PP2A phosphatase activity, and defective KAP1-S824 phosphorylation, revealing a negative regulatory circuit.\",\n      \"method\": \"UBE2D3 knockout/depletion, telomeric NHEJ assays, 53BP1 recruitment assays, RNF168 accumulation monitoring, KAP1 phosphorylation assays, PP2A activity assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal functional assays establishing UBE2D3 as a multi-level regulator of NHEJ and ATM signalling, single rigorous study\",\n      \"pmids\": [\"38866770\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The small-molecule DHPO directly binds UbcH5c (confirmed by SPR and CETSA) and inhibits UbcH5c-mediated IκBα ubiquitination and degradation, thereby blocking NF-κB activation in pancreatic cancer cells.\",\n      \"method\": \"Surface plasmon resonance (SPR), cellular thermal shift assay (CETSA), western blot for IκBα ubiquitination, NF-κB reporter assay, in vitro and in vivo pancreatic cancer models\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding confirmed by two orthogonal biophysical methods and functional ubiquitination assay, single lab\",\n      \"pmids\": [\"35272681\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Arteannuin B (ATB) covalently binds the catalytic Cys85 of UBE2D3, inhibiting its function and preventing ubiquitination of RIP1 and NEMO, thereby blocking NF-κB activation; confirmed by CETSA, DARTS, MST, LC-MS/MS identification of the binding site, and UBE2D3 siRNA knockdown.\",\n      \"method\": \"CETSA, DARTS, microscale thermophoresis (MST), LC-MS/MS mass spectrometry, molecular docking, siRNA knockdown, western blotting for RIP1/NEMO ubiquitination\",\n      \"journal\": \"Phytomedicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal biophysical and chemical methods identifying Cys85 as the binding site, confirmed by genetic knockdown, single lab\",\n      \"pmids\": [\"38181532\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"UBE2D3 binds the E3 ligase KLHL13 to mediate K63-linked polyubiquitination at K245 of TAP2, causing steric hindrance that blocks the TAP2 transporter; this impairs antigen presentation and enables pancreatic cancer cells to evade CD8+ T-cell surveillance in response to IFN-γ.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination site mapping (K245), genetic knockout/knockdown of UBE2D3, antigen presentation assays, CD8+ T-cell killing assays, mouse tumor models\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP with site-specific ubiquitination mapping, functional immune assays, and in vivo mouse models in a single study\",\n      \"pmids\": [\"41315272\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"UBE2D3 is the most highly expressed E2 enzyme in mouse oocytes and is essential for meiotic division; depletion causes metaphase I arrest and Cyclin B1 accumulation, while overexpression reduces Cyclin B1 levels, causes kinetochore-microtubule mis-attachments, spindle assembly checkpoint dysfunction, and aneuploidy. Elevated UBE2D3 in aged oocytes contributes to age-related meiotic defects reversible by UBE2D3 knockdown or Cyclin B1 overexpression.\",\n      \"method\": \"UBE2D3 knockdown and overexpression in mouse oocytes, meiotic progression assays, Cyclin B1 level measurement, kinetochore-microtubule attachment analysis, spindle assembly checkpoint assays, aneuploidy scoring\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss- and gain-of-function in oocytes with multiple orthogonal cellular phenotype readouts, single lab\",\n      \"pmids\": [\"39921465\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Xenopus ube2d3.2 (ortholog of UBE2D3) interacts with xMLK2 (identified by yeast two-hybrid) and limits xMLK2 accumulation; ectopic ube2d3.2 expression inhibits pronephric tubule formation, phenocopying loss of xMLK2, implicating ube2d3.2 as an endogenous regulator of xMLK2 and JNK activity.\",\n      \"method\": \"Yeast two-hybrid screen, ectopic expression in Xenopus embryos, xMLK2 protein level assay, pronephros morphology analysis\",\n      \"journal\": \"Differentiation\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Y2H plus overexpression phenotype in Xenopus, mechanism inferred, single lab\",\n      \"pmids\": [\"18021256\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Cryo-EM structure of the RNF168/UbcH5c–Ub/H1.0–K63-Ub₃ chromatosome complex revealed the recruitment orientation between the RNF168 UDM1 domain and K63-linked ubiquitin chain on H1.0, providing structural basis for how K63-polyubiquitinated H1.0 stimulates RNF168-mediated H2A ubiquitination.\",\n      \"method\": \"Cryo-EM structural analysis, biochemical reconstitution with synthetic ubiquitylated H1.0, RNF168 ubiquitylation activity assays on chromatosomes\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — cryo-EM structure with reconstitution, but preprint not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2024.07.22.604500\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"UBE2D3 (UbcH5c) is a promiscuous E2 ubiquitin-conjugating enzyme that transfers ubiquitin to diverse substrates—including histone H2A (via Ring1b/Bmi1), cyclin D1, hTERT, Cyclin B1, RIP1, NEMO, IκBα, SHP-2, p62, RPS10, RPS20, and TAP2—working with cognate E3 ligases (Riplet, MDM2/MDMX via MDMX recruitment, COP1, KLHL13, E4B, RNF168, CBL) to regulate NF-κB, p53, RIG-I/MAVS innate immune, ATM/NHEJ DNA damage, meiotic cell-cycle, and protein quality-control pathways; its catalytic active site Cys85 can be covalently inactivated by small molecules, and its activity is modulated by Aurora B-mediated phosphorylation of S138.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"UBE2D3 (UbcH5c) is a promiscuous E2 ubiquitin-conjugating enzyme that partners with diverse RING and U-box E3 ligases to ubiquitinate substrates across chromatin regulation, innate immunity, DNA damage signalling, cell-cycle control, and protein quality control [#0, #2, #15, #13]. Its catalytic transfer activity depends on an active-site cysteine, Cys85, which can be covalently modified by small molecules to inactivate the enzyme [#8, #17]. In chromatin and DNA-damage contexts it cooperates with the Ring1b/Bmi1 RING heterodimer\\u2014interacting exclusively with Ring1b\\u2014to monoubiquitinate histone H2A at K119 [#0], and works with RNF168 downstream of ATM to drive DDR-induced chromatin ubiquitination, 53BP1 recruitment, and KAP1-S824 phosphorylation while simultaneously limiting RNF168 accumulation, defining a negative feedback circuit that promotes telomeric NHEJ [#15]; it is also selectively required for DNA-PK activation at one-ended replication-associated double-strand breaks [#14]. In innate immunity UBE2D3 acts with the E3 ligase Riplet to polyubiquitinate RIG-I, driving MAVS aggregation and antiviral signalling [#2], and its activity supports NF-\\u03baB activation through ubiquitination of pathway components such as I\\u03baB\\u03b1, RIP1, and NEMO [#16, #17]. It directs substrate degradation in cell-cycle and p53 control, mediating ATRA-induced cyclin D1 turnover [#3], targeting hTERT for proteasomal degradation [#5, #6], and enabling MDM2-driven p53 degradation through a UbcH5c-binding surface contributed by MDMX [#4]. Direct ubiquitinomic profiling identified the ribosomal proteins RPS10 and RPS20 as in vivo catalytic substrates linking UBE2D3 to ribosome-associated and autophagic protein quality control [#13]. Aurora B-mediated phosphorylation of a conserved amniote-specific Ser138 destabilizes UBE2D3 and modulates its interaction with the E3 ligase CBL, with this regulation being essential for early embryonic development [#12].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Establishing that UBE2D3 controls a specific cell-cycle regulator addressed whether this E2 has dedicated substrate-degradation roles beyond generic ubiquitin transfer.\",\n      \"evidence\": \"Co-IP and shRNA knockdown linking UBE2D3 to ATRA-induced cyclin D1 degradation in APL cells\",\n      \"pmids\": [\"17420285\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cognate E3 ligase for cyclin D1 turnover not identified\", \"Direct ubiquitination of cyclin D1 by UBE2D3 not reconstituted\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defining the structural basis of E4B U-box engagement clarified how an E3 module allosterically activates UBE2D3 for polyubiquitin chain elongation.\",\n      \"evidence\": \"Crystallography, NMR, and ITC of the E4B U-box bound to UbcH5c/Ubc4\",\n      \"pmids\": [\"20696396\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo substrates of the E4B-UBE2D3 pair not defined\", \"Chain linkage type not established in this study\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"The Bmi1/Ring1b\\u2013UbcH5c structure explained how E2 selectivity and nucleosome contacts achieve site-specific H2A monoubiquitination on chromatin.\",\n      \"evidence\": \"X-ray crystallography with DNA-binding mutagenesis and H2A ubiquitination assays\",\n      \"pmids\": [\"21772249\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular consequences of H2A-K119 ubiquitination not addressed here\", \"Regulation of complex assembly in vivo unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identifying hTERT as a UBE2D3 interactor connected this E2 to telomerase control and radiosensitivity.\",\n      \"evidence\": \"Y2H, Co-IP, and knockdown with telomerase and cell-cycle assays\",\n      \"pmids\": [\"23741361\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E3 ligase mediating hTERT ubiquitination not identified\", \"Direct versus indirect regulation of hTERT not resolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Demonstrating proteasome-dependent hTERT turnover upon UBE2D3 overexpression confirmed a causal degradation role rather than mere association.\",\n      \"evidence\": \"Overexpression with MG132 rescue, ubiquitination detection, and xenografts in esophageal cancer cells\",\n      \"pmids\": [\"27105523\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cognate E3 ligase still unidentified\", \"Ubiquitin chain linkage on hTERT not characterized\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Reconstituting RIG-I activation placed UBE2D3 at the heart of antiviral innate immune signalling.\",\n      \"evidence\": \"Chromatographic purification, reconstitution, and MAVS aggregation assays with Riplet\",\n      \"pmids\": [\"28469175\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin chain architecture on RIG-I not fully defined\", \"Regulation of Riplet-UBE2D3 pairing unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Solving the apo UbcH5c structure and identifying a covalent Cys85 inhibitor established the active site as a druggable handle for NF-\\u03baB suppression.\",\n      \"evidence\": \"X-ray crystallography of recombinant UbcH5c, plus \\u03b1-santonin compound 6d binding by BIAcore, in-gel fluorescence, and NF-\\u03baB reporter assays\",\n      \"pmids\": [\"28540177\", \"28696694\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Selectivity of Cys85 inhibitors over other E2s not fully established\", \"Functional structure determined without bound E3 in apo study\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Genetic dissection of the MDM2/MDMX axis showed UBE2D3 binding is the limiting step that allows MDM2 to degrade p53.\",\n      \"evidence\": \"In vivo mouse p53 models, Co-IP, and MDMX-to-MDM2 domain-swap mutagenesis\",\n      \"pmids\": [\"33277368\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural detail of the MDMX C-terminus\\u2013UbcH5c interface not resolved\", \"Chain type built on p53 not addressed\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identifying Aurora B phosphorylation of an amniote-specific Ser138 revealed post-translational control of UBE2D3 stability and E3 partner selection with developmental consequences.\",\n      \"evidence\": \"Mouse S138A knock-in genetics, proximity ligation, and ESC differentiation assays\",\n      \"pmids\": [\"32145025\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which phosphorylation destabilizes the fold not structurally defined\", \"Full set of CBL-dependent substrates affected unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"A cluster of disease-context studies extended UBE2D3 substrate scope to SHP-2/STAT3 signalling, p62 autophagy regulation, and COP1-mediated c-Jun degradation.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, and knockdown across glioma, cardiac I/R, and Erk-inactivation models\",\n      \"pmids\": [\"34195079\", \"34391873\", \"33918729\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"c-Jun finding rests on single-method siRNA with western readout (Low confidence)\", \"Direct ubiquitination versus indirect effects not always separated\", \"Chain linkages on these substrates undefined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"A second direct-binding inhibitor (DHPO) reinforced UBE2D3 as a tractable NF-\\u03baB target via blockade of I\\u03baB\\u03b1 ubiquitination.\",\n      \"evidence\": \"SPR and CETSA binding plus I\\u03baB\\u03b1 ubiquitination and NF-\\u03baB assays in pancreatic cancer models\",\n      \"pmids\": [\"35272681\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Binding site on UBE2D3 not mapped in this study\", \"Off-target E2 inhibition not excluded\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Quantitative ubiquitinomics provided the first direct in vivo substrate identification, linking UBE2D3 catalysis to ribosome-associated protein quality control.\",\n      \"evidence\": \"SILAC diGly proteomics and TULIP2 with catalytic-mutant validation identifying RPS10/RPS20\",\n      \"pmids\": [\"37059365\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"E3 ligases pairing with UBE2D3 on RPS10/RPS20 not defined\", \"Downstream fate of ubiquitinated ribosomal proteins not fully traced\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"An E2 siRNA screen pinpointed UBE2D3 as selectively required for DNA-PK activation at replication-associated one-ended breaks, distinguishing it from two-ended DSB responses.\",\n      \"evidence\": \"siRNA screen and knockdown with CPT versus neocarzinostatin, DNA-PK activation and chromosomal aberration assays\",\n      \"pmids\": [\"37244033\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ubiquitination substrate driving DNA-PK activation not identified\", \"Cognate E3 ligase in this pathway unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Functional and structural work placed UBE2D3 as a multi-level regulator of ATM/RNF168 signalling and telomeric NHEJ, including a negative feedback loop constraining RNF168.\",\n      \"evidence\": \"UBE2D3 knockout with 53BP1, RNF168, KAP1, and PP2A assays; cryo-EM of the RNF168/UbcH5c\\u2013H1.0 chromatosome complex (preprint)\",\n      \"pmids\": [\"38866770\", \"bio_10.1101_2024.07.22.604500\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which UBE2D3 limits RNF168 accumulation not fully defined\", \"Cryo-EM structure is a preprint\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Two studies extended UBE2D3 into immune evasion via TAP2 ubiquitination and into meiotic cell-cycle control via Cyclin B1 turnover, demonstrating dosage-sensitive physiological roles.\",\n      \"evidence\": \"KLHL13-dependent K63 ubiquitination at TAP2-K245 with CD8+ T-cell and tumor assays; loss/gain-of-function in mouse oocytes with checkpoint and aneuploidy readouts\",\n      \"pmids\": [\"41315272\", \"39921465\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether UBE2D3 directly ubiquitinates Cyclin B1 versus acting through an E3 not resolved\", \"Regulation of UBE2D3 expression in aged oocytes not mechanistically explained\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How E3 ligase pairing, chain-linkage choice, and post-translational state combine to dictate UBE2D3 substrate selectivity across its many pathways remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying model linking E3 partner to linkage specificity\", \"Cognate E3 ligases for several substrates (cyclin D1, hTERT, Cyclin B1) undefined\", \"Tissue-specific regulation of UBE2D3 abundance poorly understood\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 13, 18]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [1, 13, 18]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [4, 15]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [0, 15]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [2, 16, 18]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [14, 15]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [13]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [3, 4, 19]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"RING1B\", \"BMI1\", \"RNF168\", \"RIPLET\", \"MDMX\", \"KLHL13\", \"CBL\", \"E4B\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}