{"gene":"UBE2E3","run_date":"2026-04-28T21:43:00","timeline":{"discoveries":[{"year":1999,"finding":"UBE2E3 (cloned from human cDNA) forms a thioester bond with ubiquitin in an E1-dependent manner; a C145S active-site mutant fails to form this thioester, establishing Cys-145 as the catalytic residue essential for E2 function.","method":"In vitro thioester formation assay with recombinant GST-UBE2E3 and active-site mutagenesis (C145S)","journal":"Cytogenetics and cell genetics","confidence":"High","confidence_rationale":"Tier 1 — direct in vitro enzymatic assay with mutagenesis confirming active-site residue","pmids":["10343118"],"is_preprint":false},{"year":2001,"finding":"UBE2E3 interacts with the RING-finger E3 ligases ARA54 and RNF8 through its UBC domain (and their RING domains), and supports their E2-dependent autoubiquitination activity in vitro and in cells.","method":"Yeast two-hybrid screen, deletion mutagenesis, in vitro ubiquitination assay with insect-cell-expressed proteins, co-IP in COS-7 cells","journal":"European journal of biochemistry","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods (Y2H, in vitro assay, cell-based Co-IP) in a single study","pmids":["11322894"],"is_preprint":false},{"year":2004,"finding":"UBE2E3 acts as the E2 enzyme partnered with the E3 ligase Nedd4-2 to regulate the epithelial Na+ channel (ENaC); UBE2E3 and Nedd4-2 co-immunoprecipitate, and catalytically inactive UBE2E3-CS increases ENaC surface expression, dependent on ENaC PY motifs and ubiquitination sites.","method":"Co-immunoprecipitation, Xenopus oocyte electrophysiology, dominant-negative (C→S) mutant expression, transepithelial Na+ transport assay in mpkCCD cells","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, functional electrophysiology, dominant-negative mutant, two cell systems","pmids":["14993279"],"is_preprint":false},{"year":2005,"finding":"UBE2E3 can serve as the E2 enzyme for Nedd4 as well as Nedd4-2, though less efficiently than UbcH5b, in in vitro ubiquitination assays.","method":"In vitro ubiquitination assay","journal":"The international journal of biochemistry & cell biology","confidence":"Medium","confidence_rationale":"Tier 1 — in vitro assay, single lab, single method","pmids":["16337426"],"is_preprint":false},{"year":2008,"finding":"UBE2E3 localizes to the nucleus of retinal pigment epithelial (RPE) cells and is essential for their proliferation; siRNA depletion causes cell-cycle exit, loss of Ki-67, accumulation of p27Kip1, and increased cell area. The mouse ortholog UbcM2 is transcriptionally downregulated during RPE maturation in vivo.","method":"siRNA knockdown, immunofluorescence localization, Ki-67/p27Kip1 immunolabeling, cell counting, rescue experiments, beta-galactosidase reporter mouse","journal":"Investigative ophthalmology & visual science","confidence":"High","confidence_rationale":"Tier 2 — clean KD with defined proliferation phenotype, rescue experiments, in vivo developmental data","pmids":["18614808"],"is_preprint":false},{"year":2011,"finding":"The N-terminal extension unique to UBE2E3 (among class III E2 enzymes) prevents it from functionally interacting with the Ro52 (TRIM21) RING-domain E3 ligase, whereas the N-terminal extensions of UBE2E1 and UBE2E2 permit this interaction.","method":"In vitro ubiquitination assay panel, NMR mapping of E2/E3 interface, Ro52 RING mutants","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro activity assay plus structural (NMR) mapping, multiple E2 variants compared","pmids":["21862588"],"is_preprint":false},{"year":2014,"finding":"UBE2E3 ubiquitinates TDP-43; catalytically inactive UBE2E3(C145S) is much less efficient, all three UBE2E members enhance TDP-43 ubiquitination, and siRNA silencing of UBE2E3 reduces TDP-43 ubiquitination and affects its solubility.","method":"Yeast two-hybrid screen (human brain library), co-immunoprecipitation, ubiquitination assays in HEK293E cells with proteasome inhibition, siRNA knockdown, active-site mutant (C145S)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — Y2H identification, Co-IP confirmation, cell-based ubiquitination assays, active-site mutagenesis, KD rescue experiments","pmids":["24825905"],"is_preprint":false},{"year":2014,"finding":"UBE2E3 (UbcM2) is intrinsically restricted to monoubiquitylation; this restriction is governed by multiple backside residues of the enzyme (remote from the active site) and by Lys48 of ubiquitin. Backside mutants gain the ability to synthesize K63-linked (and to a lesser extent K6- and K48-linked) polyubiquitin chains.","method":"Fully recombinant in vitro ubiquitylation assay, systematic backside-residue mutagenesis, ubiquitin chain-linkage analysis","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro system with systematic mutagenesis defining the mechanistic basis for monoubiquitylation","pmids":["24901938"],"is_preprint":false},{"year":2014,"finding":"UBE2E3 and its nuclear import receptor importin-11 regulate the subcellular distribution and transcriptional activity of the antioxidant transcription factor NRF2; knockdown of UBE2E3 reduces nuclear NRF2, decreases NRF2 target gene expression, and relocalizes NRF2 to a perinuclear cluster of mitochondria.","method":"siRNA knockdown, subcellular fractionation/immunofluorescence localization, target gene expression analysis (qPCR), complementary knockdown of importin-11","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — clean KD with defined localization and transcriptional phenotype, complementary experiments with binding partner","pmids":["25378586"],"is_preprint":false},{"year":2014,"finding":"UBE2E3 interacts with the mitochondrial outer membrane E3 ligase Mulan, and the Mulan–UBE2E3 complex recruits GABARAP (an autophagy/mitophagy regulator) via an LC3-interacting region (LIR) in Mulan's RING finger; this interaction requires the presence of UBE2E3.","method":"Modified yeast two-hybrid screen using Mulan RING fused to E2 enzymes, co-immunoprecipitation","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 3 — Y2H plus Co-IP, functional consequence of complex inferred but not directly tested","pmids":["25224329"],"is_preprint":false},{"year":2018,"finding":"Loss of UBE2E3 induces cellular senescence independently of overt DNA damage; this senescence is partially suppressed by co-depletion of p53, p21CIP1/WAF1, or p16INK4a, and is accompanied by increased mitochondrial and lysosomal mass and elevated autophagic flux.","method":"siRNA knockdown, senescence assays (SA-β-Gal, SASP markers), organelle mass staining, autophagic flux measurement, co-depletion epistasis experiments","journal":"Redox biology","confidence":"High","confidence_rationale":"Tier 2 — clean KD with multiple phenotypic readouts and epistasis to p53/p21/p16 pathway","pmids":["29879550"],"is_preprint":false},{"year":2020,"finding":"The E3 ligase Praja1 (PJA1) binds to TDP-43 C-terminal fragments and to UBE2E3, and suppresses TDP-43 phosphorylation and cytoplasmic aggregate formation; PJA1 is one of the principal E3 ubiquitin ligases acting with UBE2E3 on TDP-43.","method":"Co-immunoprecipitation, adenoviral overexpression in neural stem cell-derived neurons, in vivo mouse facial motor neuron model, DNA microarray/candidate screen","journal":"Neuropathology","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP identifying E2–E3–substrate complex, supported by in vivo aggregate suppression data","pmids":["32686212"],"is_preprint":false},{"year":2020,"finding":"OTUB1, a K48-specific deubiquitinase, noncatalytically inhibits the polyubiquitination activity of UBE2E3 (as well as UBE2E1 and UBE2E2), but uniquely cannot suppress UBE2E3's autoubiquitination activity, revealing a functional distinction between UBE2E3 and its paralogs in OTUB1-mediated regulation.","method":"Quantitative kinetic/thermodynamic in vitro ubiquitination assays, DUB activity assays with purified components","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — fully reconstituted in vitro system with quantitative biochemistry distinguishing UBE2E3 from paralogs","pmids":["32049508"],"is_preprint":false},{"year":2021,"finding":"UBE2E3 knockdown in bone marrow mesenchymal stem cells (BMSCs) accelerates cellular senescence and inhibits osteogenic differentiation, while overexpression of UBE2E3 in old BMSCs attenuates senescence and enhances osteogenic differentiation; this is mechanistically linked to regulation of Nrf2.","method":"siRNA knockdown, lentiviral overexpression, senescence assays, osteogenic differentiation assays, Nrf2 pathway analysis in BMSCs","journal":"PeerJ","confidence":"Medium","confidence_rationale":"Tier 2–3 — gain- and loss-of-function in primary cells with defined phenotypes, Nrf2 pathway link inferred but not fully reconstituted","pmids":["34820159"],"is_preprint":false},{"year":2023,"finding":"RCBTB1 co-immunoprecipitates with UBE2E3 and CUL3 from RPE cell lysates, placing RCBTB1 as a component of a UBE2E3/CUL3-containing complex involved in the antioxidant (NRF2) response in retinal pigment epithelial cells.","method":"Co-immunoprecipitation from iPSC-derived RPE cells","journal":"Cells","confidence":"Low","confidence_rationale":"Tier 3 — single Co-IP, no functional reconstitution of the complex","pmids":["37408192"],"is_preprint":false},{"year":2023,"finding":"miR-143-3p directly targets UBE2E3 in ovarian granulosa cells; overexpression of miR-143-3p reduces UBE2E3 protein, inhibits granulosa cell proliferation, induces senescence-associated secretory phenotype, and impedes steroid hormone synthesis.","method":"miR-143-3p mimic transfection, luciferase 3'UTR reporter assay, proliferation/senescence/SASP assays in granulosa cells","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2–3 — direct 3'UTR reporter validation, cell-based phenotype with defined target (UBE2E3), single lab","pmids":["37628741"],"is_preprint":false},{"year":2024,"finding":"miR-143-3p targets UBE2E3 in Sertoli cells; knockdown of UBE2E3 limits Sertoli cell proliferation, promotes senescence, and causes blood-testis barrier dysfunction.","method":"miR-143-3p mimic/inhibitor transfection, luciferase reporter, siRNA knockdown of UBE2E3, BTB permeability assay, proliferation/senescence assays","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2–3 — reporter validation, direct UBE2E3 KD with defined barrier and senescence phenotype, single lab","pmids":["38391926"],"is_preprint":false},{"year":2024,"finding":"miR-379-5p directly targets UBE2E3 mRNA via its 3'UTR; miR-379-5p mimic reduces UBE2E3 protein levels and decreases breast cell viability and proliferation, while siRNA silencing of UBE2E3 paradoxically increases both proliferation and apoptosis.","method":"miR-379-5p mimic transfection, luciferase 3'UTR reporter assay (WT vs. scrambled binding site), siRNA knockdown, resazurin viability assay, BrdU proliferation assay, caspase 3/7 apoptosis assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 — direct 3'UTR reporter validation, multiple orthogonal functional assays, single lab","pmids":["39621672"],"is_preprint":false}],"current_model":"UBE2E3 is a metazoan ubiquitin-conjugating enzyme (E2) that uses its catalytic Cys-145 to form a thioester with ubiquitin and is intrinsically restricted to monoubiquitylation by backside residues in its UBC domain and Lys48 of ubiquitin; it partners with HECT-type E3s (Nedd4-2, Nedd4) to regulate ENaC surface expression, with RING-type E3s (ARA54, RNF8, Mulan, PJA1) to ubiquitinate nuclear and mitochondrial substrates including TDP-43, and with importin-11 to promote nuclear NRF2 localization and antioxidant transcription; its nuclear localization is essential for RPE cell proliferation, and its loss triggers a p53/p21/p16-dependent senescence program."},"narrative":{"teleology":[{"year":1999,"claim":"Establishing UBE2E3 as a bona fide E2 enzyme resolved whether this gene product possessed ubiquitin-conjugating activity and identified Cys-145 as the catalytic residue.","evidence":"In vitro thioester formation assay with recombinant GST-UBE2E3 and C145S mutagenesis","pmids":["10343118"],"confidence":"High","gaps":["No E3 partner or substrate identified at this stage","No chain-linkage specificity determined"]},{"year":2001,"claim":"Demonstrating that UBE2E3 physically and functionally pairs with RING-finger E3 ligases ARA54 and RNF8 established it as a RING E3-compatible E2 and mapped the interaction to its UBC domain.","evidence":"Yeast two-hybrid, deletion mutagenesis, in vitro ubiquitination, and co-IP in COS-7 cells","pmids":["11322894"],"confidence":"High","gaps":["No physiological substrate identified","Autoubiquitination shown but substrate ubiquitination not tested"]},{"year":2004,"claim":"Identifying UBE2E3 as the E2 for Nedd4-2 in ENaC regulation provided the first physiological context, linking it to HECT-type E3s and ion channel trafficking.","evidence":"Co-IP, Xenopus oocyte electrophysiology, dominant-negative C→S mutant, transepithelial transport in mpkCCD cells","pmids":["14993279"],"confidence":"High","gaps":["Direct ubiquitination of ENaC subunits by UBE2E3 not reconstituted","Relative contribution versus other E2s in vivo unclear"]},{"year":2008,"claim":"Showing that UBE2E3 localizes to the nucleus and is essential for RPE cell proliferation connected its enzymatic activity to a specific cell-biological role and revealed a proliferation-to-quiescence switch upon its loss.","evidence":"siRNA knockdown in RPE cells, Ki-67/p27 immunolabeling, rescue experiments, beta-galactosidase reporter mouse for developmental downregulation","pmids":["18614808"],"confidence":"High","gaps":["Nuclear substrates responsible for proliferation phenotype unknown","Whether quiescence versus senescence was not distinguished at this point"]},{"year":2011,"claim":"Revealing that UBE2E3's unique N-terminal extension prevents it from partnering with TRIM21/Ro52, unlike its paralogs UBE2E1/E2, established paralog-specific E3 selectivity within the UBE2E family.","evidence":"In vitro ubiquitination panel and NMR mapping of E2–E3 interface with multiple UBE2E variants","pmids":["21862588"],"confidence":"High","gaps":["Structural basis of why UBE2E3's N-terminus is uniquely incompatible not resolved at atomic detail"]},{"year":2014,"claim":"Four contemporaneous studies defined UBE2E3's substrate (TDP-43), its intrinsic monoubiquitylation restriction mechanism, its role in NRF2 nuclear import via importin-11, and its partnership with mitochondrial E3 Mulan, collectively establishing UBE2E3 as a versatile but product-constrained E2 operating in nuclear, cytoplasmic, and mitochondrial compartments.","evidence":"Y2H/Co-IP/ubiquitination assays for TDP-43 (PMID:24825905); reconstituted ubiquitylation with systematic backside mutagenesis (PMID:24901938); siRNA/fractionation/qPCR for NRF2 pathway (PMID:25378586); modified Y2H/Co-IP for Mulan–GABARAP interaction (PMID:25224329)","pmids":["24825905","24901938","25378586","25224329"],"confidence":"High","gaps":["Structural basis for backside-residue constraint not solved","Whether monoubiquitylation restriction holds in all E3 contexts in vivo untested","Direct ubiquitination site on TDP-43 not mapped","Functional consequence of Mulan–UBE2E3–GABARAP complex on mitophagy not demonstrated"]},{"year":2018,"claim":"Demonstrating that UBE2E3 loss triggers senescence via a p53/p21/p16-dependent program—with accompanying organelle remodeling—unified earlier proliferation and NRF2 findings into a coherent anti-senescence mechanism.","evidence":"siRNA knockdown, SA-β-Gal, SASP markers, organelle mass staining, autophagic flux, co-depletion epistasis with p53/p21/p16","pmids":["29879550"],"confidence":"High","gaps":["Direct ubiquitination target(s) linking UBE2E3 to p53/p21/p16 pathway unknown","Whether NRF2 loss fully explains the senescence phenotype not resolved"]},{"year":2020,"claim":"Identifying PJA1 as an E3 ligase partnering with UBE2E3 to suppress TDP-43 aggregation in neurons, and showing that OTUB1 noncatalytically inhibits UBE2E3 polyubiquitination but uniquely cannot suppress its autoubiquitination, refined the regulatory landscape of UBE2E3 activity.","evidence":"Co-IP and in vivo motor neuron model for PJA1–TDP-43 (PMID:32686212); quantitative kinetic in vitro assays for OTUB1 regulation (PMID:32049508)","pmids":["32686212","32049508"],"confidence":"High","gaps":["Whether PJA1 is the predominant E3 for TDP-43 ubiquitination in disease-relevant neurons unclear","Structural basis of OTUB1's failure to suppress UBE2E3 autoubiquitination not determined"]},{"year":2021,"claim":"Extending the anti-senescence role of UBE2E3 to bone marrow mesenchymal stem cells and linking it to NRF2-dependent osteogenic differentiation broadened the tissue relevance of the UBE2E3–NRF2 axis.","evidence":"Gain- and loss-of-function experiments in BMSCs with senescence and osteogenic differentiation assays, Nrf2 pathway analysis","pmids":["34820159"],"confidence":"Medium","gaps":["Whether UBE2E3 directly ubiquitinates an NRF2 pathway component in BMSCs not shown","Mechanism linking UBE2E3 to NRF2 not reconstituted biochemically in this system"]},{"year":2023,"claim":"Identification of miR-143-3p as a direct UBE2E3-targeting microRNA in granulosa cells, and of RCBTB1 as a co-immunoprecipitating partner within a potential CUL3-containing complex in RPE cells, expanded the regulatory and interaction network around UBE2E3.","evidence":"3′UTR luciferase reporter for miR-143-3p (PMID:37628741); Co-IP from iPSC-derived RPE cells for RCBTB1/CUL3 (PMID:37408192)","pmids":["37628741","37408192"],"confidence":"Medium","gaps":["RCBTB1–CUL3–UBE2E3 complex not functionally reconstituted","Single Co-IP for RCBTB1 without reciprocal validation","Whether miR-143-3p regulation of UBE2E3 operates in vivo not established"]},{"year":2024,"claim":"Additional miRNA-mediated regulation of UBE2E3 was confirmed in Sertoli cells (miR-143-3p) and breast cells (miR-379-5p), with UBE2E3 depletion consistently linked to senescence and proliferative defects across diverse tissues.","evidence":"miRNA mimic/inhibitor transfection, 3′UTR reporters, siRNA knockdown, BTB permeability assay (PMID:38391926); resazurin/BrdU/caspase assays (PMID:39621672)","pmids":["38391926","39621672"],"confidence":"Medium","gaps":["Paradoxical increase in both proliferation and apoptosis upon UBE2E3 knockdown in breast cells unexplained mechanistically","Whether blood-testis barrier dysfunction is a direct UBE2E3 substrate effect or secondary to senescence unknown"]},{"year":null,"claim":"Critical open questions include the identity of UBE2E3's direct ubiquitination substrate(s) that trigger the p53/p21/p16 senescence cascade, the structural basis of its monoubiquitylation restriction at atomic resolution, and whether its NRF2-regulatory function is catalytic (ubiquitin-dependent) or noncatalytic.","evidence":"","pmids":[],"confidence":"High","gaps":["No substrate linking UBE2E3 catalysis to p53/p21/p16 activation identified","No high-resolution structure of UBE2E3 in complex with any E3 partner","Catalytic vs. noncatalytic role in NRF2 nuclear import not distinguished"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,2,6,7]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[6,7,11]},{"term_id":"GO:0031386","term_label":"protein tag activity","supporting_discovery_ids":[0,7]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[4,8]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[9]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,6,7,12]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[8,10,13]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[9,10]},{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[2]}],"complexes":[],"partners":["NEDD4L","NEDD4","ARA54","RNF8","PJA1","MUL1","IPO11","OTUB1"],"other_free_text":[]},"mechanistic_narrative":"UBE2E3 is a class III ubiquitin-conjugating enzyme (E2) that functions as a monoubiquitylation-restricted catalyst whose product specificity is governed by backside residues of its UBC domain and Lys48 of ubiquitin [PMID:24901938]. It forms a Cys-145-dependent thioester with ubiquitin and partners with both HECT-type E3 ligases (Nedd4-2, Nedd4) to regulate epithelial Na+ channel surface expression and RING-type E3 ligases (ARA54, RNF8, PJA1, Mulan) to ubiquitinate substrates including TDP-43 [PMID:10343118, PMID:14993279, PMID:11322894, PMID:24825905, PMID:32686212]. Through its importin-11-mediated nuclear localization, UBE2E3 promotes NRF2 nuclear accumulation and antioxidant gene transcription, and its depletion triggers a p53/p21/p16-dependent cellular senescence program characterized by increased mitochondrial and lysosomal mass and elevated autophagic flux [PMID:25378586, PMID:29879550, PMID:18614808]. Loss of UBE2E3 consistently impairs proliferation and induces senescence across multiple cell types including retinal pigment epithelial cells, bone marrow mesenchymal stem cells, granulosa cells, and Sertoli cells [PMID:18614808, PMID:34820159, PMID:37628741, PMID:38391926]."},"prefetch_data":{"uniprot":{"accession":"Q969T4","full_name":"Ubiquitin-conjugating enzyme E2 E3","aliases":["E2 ubiquitin-conjugating enzyme E3","UbcH9","Ubiquitin carrier protein E3","Ubiquitin-conjugating enzyme E2-23 kDa","Ubiquitin-protein ligase E3"],"length_aa":207,"mass_kda":22.9,"function":"Accepts ubiquitin from the E1 complex and catalyzes its covalent attachment to other proteins. In vitro catalyzes 'Lys-11'- and 'Lys-48'-, as well as 'Lys-63'-linked polyubiquitination. Participates in the regulation of transepithelial sodium transport in renal cells","subcellular_location":"Nucleus; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q969T4/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/UBE2E3","classification":"Not Classified","n_dependent_lines":22,"n_total_lines":1208,"dependency_fraction":0.018211920529801324},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/UBE2E3","total_profiled":1310},"omim":[{"mim_id":"616014","title":"RING FINGER PROTEIN 25; RNF25","url":"https://www.omim.org/entry/616014"},{"mim_id":"612548","title":"TRIPARTITE MOTIF-CONTAINING PROTEIN 50; TRIM50","url":"https://www.omim.org/entry/612548"},{"mim_id":"610889","title":"IMPORTIN 11; IPO11","url":"https://www.omim.org/entry/610889"},{"mim_id":"610637","title":"MEMBRANE-ASSOCIATED RING-CH FINGER PROTEIN 5; MARCHF5","url":"https://www.omim.org/entry/610637"},{"mim_id":"607867","title":"RCC1 DOMAIN- AND BTB DOMAIN-CONTAINING PROTEIN 1; RCBTB1","url":"https://www.omim.org/entry/607867"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/UBE2E3"},"hgnc":{"alias_symbol":["UbcH9"],"prev_symbol":[]},"alphafold":{"accession":"Q969T4","domains":[{"cath_id":"3.10.110.10","chopping":"60-205","consensus_level":"high","plddt":97.6604,"start":60,"end":205}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q969T4","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q969T4-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q969T4-F1-predicted_aligned_error_v6.png","plddt_mean":85.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=UBE2E3","jax_strain_url":"https://www.jax.org/strain/search?query=UBE2E3"},"sequence":{"accession":"Q969T4","fasta_url":"https://rest.uniprot.org/uniprotkb/Q969T4.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q969T4/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q969T4"}},"corpus_meta":[{"pmid":"9734360","id":"PMC_9734360","title":"SUMO-1 modification of IkappaBalpha inhibits NF-kappaB activation.","date":"1998","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/9734360","citation_count":925,"is_preprint":false},{"pmid":"9409737","id":"PMC_9409737","title":"Ubch9 conjugates SUMO but not ubiquitin.","date":"1997","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/9409737","citation_count":314,"is_preprint":false},{"pmid":"10187858","id":"PMC_10187858","title":"Identification of the enzyme required for activation of the small ubiquitin-like protein SUMO-1.","date":"1999","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10187858","citation_count":304,"is_preprint":false},{"pmid":"22526258","id":"PMC_22526258","title":"The circadian protein period 1 contributes to blood pressure control and coordinately regulates renal sodium transport genes.","date":"2012","source":"Hypertension (Dallas, Tex. : 1979)","url":"https://pubmed.ncbi.nlm.nih.gov/22526258","citation_count":120,"is_preprint":false},{"pmid":"22965876","id":"PMC_22965876","title":"Selective histone deacetylase (HDAC) inhibition imparts beneficial effects in Huntington's disease mice: implications for the ubiquitin-proteasomal and autophagy systems.","date":"2012","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/22965876","citation_count":99,"is_preprint":false},{"pmid":"10582246","id":"PMC_10582246","title":"Control of NF-kappa B transcriptional activation by signal induced proteolysis of I kappa B alpha.","date":"1999","source":"Philosophical transactions of the Royal Society of London. 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a C145S active-site mutant fails to form this thioester, establishing Cys-145 as the catalytic residue essential for E2 function.\",\n      \"method\": \"In vitro thioester formation assay with recombinant GST-UBE2E3 and active-site mutagenesis (C145S)\",\n      \"journal\": \"Cytogenetics and cell genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct in vitro enzymatic assay with mutagenesis confirming active-site residue\",\n      \"pmids\": [\"10343118\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"UBE2E3 interacts with the RING-finger E3 ligases ARA54 and RNF8 through its UBC domain (and their RING domains), and supports their E2-dependent autoubiquitination activity in vitro and in cells.\",\n      \"method\": \"Yeast two-hybrid screen, deletion mutagenesis, in vitro ubiquitination assay with insect-cell-expressed proteins, co-IP in COS-7 cells\",\n      \"journal\": \"European journal of biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods (Y2H, in vitro assay, cell-based Co-IP) in a single study\",\n      \"pmids\": [\"11322894\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"UBE2E3 acts as the E2 enzyme partnered with the E3 ligase Nedd4-2 to regulate the epithelial Na+ channel (ENaC); UBE2E3 and Nedd4-2 co-immunoprecipitate, and catalytically inactive UBE2E3-CS increases ENaC surface expression, dependent on ENaC PY motifs and ubiquitination sites.\",\n      \"method\": \"Co-immunoprecipitation, Xenopus oocyte electrophysiology, dominant-negative (C→S) mutant expression, transepithelial Na+ transport assay in mpkCCD cells\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, functional electrophysiology, dominant-negative mutant, two cell systems\",\n      \"pmids\": [\"14993279\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"UBE2E3 can serve as the E2 enzyme for Nedd4 as well as Nedd4-2, though less efficiently than UbcH5b, in in vitro ubiquitination assays.\",\n      \"method\": \"In vitro ubiquitination assay\",\n      \"journal\": \"The international journal of biochemistry & cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — in vitro assay, single lab, single method\",\n      \"pmids\": [\"16337426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"UBE2E3 localizes to the nucleus of retinal pigment epithelial (RPE) cells and is essential for their proliferation; siRNA depletion causes cell-cycle exit, loss of Ki-67, accumulation of p27Kip1, and increased cell area. The mouse ortholog UbcM2 is transcriptionally downregulated during RPE maturation in vivo.\",\n      \"method\": \"siRNA knockdown, immunofluorescence localization, Ki-67/p27Kip1 immunolabeling, cell counting, rescue experiments, beta-galactosidase reporter mouse\",\n      \"journal\": \"Investigative ophthalmology & visual science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KD with defined proliferation phenotype, rescue experiments, in vivo developmental data\",\n      \"pmids\": [\"18614808\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The N-terminal extension unique to UBE2E3 (among class III E2 enzymes) prevents it from functionally interacting with the Ro52 (TRIM21) RING-domain E3 ligase, whereas the N-terminal extensions of UBE2E1 and UBE2E2 permit this interaction.\",\n      \"method\": \"In vitro ubiquitination assay panel, NMR mapping of E2/E3 interface, Ro52 RING mutants\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro activity assay plus structural (NMR) mapping, multiple E2 variants compared\",\n      \"pmids\": [\"21862588\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"UBE2E3 ubiquitinates TDP-43; catalytically inactive UBE2E3(C145S) is much less efficient, all three UBE2E members enhance TDP-43 ubiquitination, and siRNA silencing of UBE2E3 reduces TDP-43 ubiquitination and affects its solubility.\",\n      \"method\": \"Yeast two-hybrid screen (human brain library), co-immunoprecipitation, ubiquitination assays in HEK293E cells with proteasome inhibition, siRNA knockdown, active-site mutant (C145S)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Y2H identification, Co-IP confirmation, cell-based ubiquitination assays, active-site mutagenesis, KD rescue experiments\",\n      \"pmids\": [\"24825905\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"UBE2E3 (UbcM2) is intrinsically restricted to monoubiquitylation; this restriction is governed by multiple backside residues of the enzyme (remote from the active site) and by Lys48 of ubiquitin. Backside mutants gain the ability to synthesize K63-linked (and to a lesser extent K6- and K48-linked) polyubiquitin chains.\",\n      \"method\": \"Fully recombinant in vitro ubiquitylation assay, systematic backside-residue mutagenesis, ubiquitin chain-linkage analysis\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro system with systematic mutagenesis defining the mechanistic basis for monoubiquitylation\",\n      \"pmids\": [\"24901938\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"UBE2E3 and its nuclear import receptor importin-11 regulate the subcellular distribution and transcriptional activity of the antioxidant transcription factor NRF2; knockdown of UBE2E3 reduces nuclear NRF2, decreases NRF2 target gene expression, and relocalizes NRF2 to a perinuclear cluster of mitochondria.\",\n      \"method\": \"siRNA knockdown, subcellular fractionation/immunofluorescence localization, target gene expression analysis (qPCR), complementary knockdown of importin-11\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KD with defined localization and transcriptional phenotype, complementary experiments with binding partner\",\n      \"pmids\": [\"25378586\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"UBE2E3 interacts with the mitochondrial outer membrane E3 ligase Mulan, and the Mulan–UBE2E3 complex recruits GABARAP (an autophagy/mitophagy regulator) via an LC3-interacting region (LIR) in Mulan's RING finger; this interaction requires the presence of UBE2E3.\",\n      \"method\": \"Modified yeast two-hybrid screen using Mulan RING fused to E2 enzymes, co-immunoprecipitation\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Y2H plus Co-IP, functional consequence of complex inferred but not directly tested\",\n      \"pmids\": [\"25224329\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Loss of UBE2E3 induces cellular senescence independently of overt DNA damage; this senescence is partially suppressed by co-depletion of p53, p21CIP1/WAF1, or p16INK4a, and is accompanied by increased mitochondrial and lysosomal mass and elevated autophagic flux.\",\n      \"method\": \"siRNA knockdown, senescence assays (SA-β-Gal, SASP markers), organelle mass staining, autophagic flux measurement, co-depletion epistasis experiments\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KD with multiple phenotypic readouts and epistasis to p53/p21/p16 pathway\",\n      \"pmids\": [\"29879550\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The E3 ligase Praja1 (PJA1) binds to TDP-43 C-terminal fragments and to UBE2E3, and suppresses TDP-43 phosphorylation and cytoplasmic aggregate formation; PJA1 is one of the principal E3 ubiquitin ligases acting with UBE2E3 on TDP-43.\",\n      \"method\": \"Co-immunoprecipitation, adenoviral overexpression in neural stem cell-derived neurons, in vivo mouse facial motor neuron model, DNA microarray/candidate screen\",\n      \"journal\": \"Neuropathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP identifying E2–E3–substrate complex, supported by in vivo aggregate suppression data\",\n      \"pmids\": [\"32686212\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"OTUB1, a K48-specific deubiquitinase, noncatalytically inhibits the polyubiquitination activity of UBE2E3 (as well as UBE2E1 and UBE2E2), but uniquely cannot suppress UBE2E3's autoubiquitination activity, revealing a functional distinction between UBE2E3 and its paralogs in OTUB1-mediated regulation.\",\n      \"method\": \"Quantitative kinetic/thermodynamic in vitro ubiquitination assays, DUB activity assays with purified components\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — fully reconstituted in vitro system with quantitative biochemistry distinguishing UBE2E3 from paralogs\",\n      \"pmids\": [\"32049508\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"UBE2E3 knockdown in bone marrow mesenchymal stem cells (BMSCs) accelerates cellular senescence and inhibits osteogenic differentiation, while overexpression of UBE2E3 in old BMSCs attenuates senescence and enhances osteogenic differentiation; this is mechanistically linked to regulation of Nrf2.\",\n      \"method\": \"siRNA knockdown, lentiviral overexpression, senescence assays, osteogenic differentiation assays, Nrf2 pathway analysis in BMSCs\",\n      \"journal\": \"PeerJ\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — gain- and loss-of-function in primary cells with defined phenotypes, Nrf2 pathway link inferred but not fully reconstituted\",\n      \"pmids\": [\"34820159\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RCBTB1 co-immunoprecipitates with UBE2E3 and CUL3 from RPE cell lysates, placing RCBTB1 as a component of a UBE2E3/CUL3-containing complex involved in the antioxidant (NRF2) response in retinal pigment epithelial cells.\",\n      \"method\": \"Co-immunoprecipitation from iPSC-derived RPE cells\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP, no functional reconstitution of the complex\",\n      \"pmids\": [\"37408192\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"miR-143-3p directly targets UBE2E3 in ovarian granulosa cells; overexpression of miR-143-3p reduces UBE2E3 protein, inhibits granulosa cell proliferation, induces senescence-associated secretory phenotype, and impedes steroid hormone synthesis.\",\n      \"method\": \"miR-143-3p mimic transfection, luciferase 3'UTR reporter assay, proliferation/senescence/SASP assays in granulosa cells\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — direct 3'UTR reporter validation, cell-based phenotype with defined target (UBE2E3), single lab\",\n      \"pmids\": [\"37628741\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"miR-143-3p targets UBE2E3 in Sertoli cells; knockdown of UBE2E3 limits Sertoli cell proliferation, promotes senescence, and causes blood-testis barrier dysfunction.\",\n      \"method\": \"miR-143-3p mimic/inhibitor transfection, luciferase reporter, siRNA knockdown of UBE2E3, BTB permeability assay, proliferation/senescence assays\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — reporter validation, direct UBE2E3 KD with defined barrier and senescence phenotype, single lab\",\n      \"pmids\": [\"38391926\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"miR-379-5p directly targets UBE2E3 mRNA via its 3'UTR; miR-379-5p mimic reduces UBE2E3 protein levels and decreases breast cell viability and proliferation, while siRNA silencing of UBE2E3 paradoxically increases both proliferation and apoptosis.\",\n      \"method\": \"miR-379-5p mimic transfection, luciferase 3'UTR reporter assay (WT vs. scrambled binding site), siRNA knockdown, resazurin viability assay, BrdU proliferation assay, caspase 3/7 apoptosis assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct 3'UTR reporter validation, multiple orthogonal functional assays, single lab\",\n      \"pmids\": [\"39621672\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"UBE2E3 is a metazoan ubiquitin-conjugating enzyme (E2) that uses its catalytic Cys-145 to form a thioester with ubiquitin and is intrinsically restricted to monoubiquitylation by backside residues in its UBC domain and Lys48 of ubiquitin; it partners with HECT-type E3s (Nedd4-2, Nedd4) to regulate ENaC surface expression, with RING-type E3s (ARA54, RNF8, Mulan, PJA1) to ubiquitinate nuclear and mitochondrial substrates including TDP-43, and with importin-11 to promote nuclear NRF2 localization and antioxidant transcription; its nuclear localization is essential for RPE cell proliferation, and its loss triggers a p53/p21/p16-dependent senescence program.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"UBE2E3 is a class III ubiquitin-conjugating enzyme (E2) that functions as a monoubiquitylation-restricted catalyst whose product specificity is governed by backside residues of its UBC domain and Lys48 of ubiquitin [PMID:24901938]. It forms a Cys-145-dependent thioester with ubiquitin and partners with both HECT-type E3 ligases (Nedd4-2, Nedd4) to regulate epithelial Na+ channel surface expression and RING-type E3 ligases (ARA54, RNF8, PJA1, Mulan) to ubiquitinate substrates including TDP-43 [PMID:10343118, PMID:14993279, PMID:11322894, PMID:24825905, PMID:32686212]. Through its importin-11-mediated nuclear localization, UBE2E3 promotes NRF2 nuclear accumulation and antioxidant gene transcription, and its depletion triggers a p53/p21/p16-dependent cellular senescence program characterized by increased mitochondrial and lysosomal mass and elevated autophagic flux [PMID:25378586, PMID:29879550, PMID:18614808]. Loss of UBE2E3 consistently impairs proliferation and induces senescence across multiple cell types including retinal pigment epithelial cells, bone marrow mesenchymal stem cells, granulosa cells, and Sertoli cells [PMID:18614808, PMID:34820159, PMID:37628741, PMID:38391926].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Establishing UBE2E3 as a bona fide E2 enzyme resolved whether this gene product possessed ubiquitin-conjugating activity and identified Cys-145 as the catalytic residue.\",\n      \"evidence\": \"In vitro thioester formation assay with recombinant GST-UBE2E3 and C145S mutagenesis\",\n      \"pmids\": [\"10343118\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No E3 partner or substrate identified at this stage\", \"No chain-linkage specificity determined\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Demonstrating that UBE2E3 physically and functionally pairs with RING-finger E3 ligases ARA54 and RNF8 established it as a RING E3-compatible E2 and mapped the interaction to its UBC domain.\",\n      \"evidence\": \"Yeast two-hybrid, deletion mutagenesis, in vitro ubiquitination, and co-IP in COS-7 cells\",\n      \"pmids\": [\"11322894\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No physiological substrate identified\", \"Autoubiquitination shown but substrate ubiquitination not tested\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identifying UBE2E3 as the E2 for Nedd4-2 in ENaC regulation provided the first physiological context, linking it to HECT-type E3s and ion channel trafficking.\",\n      \"evidence\": \"Co-IP, Xenopus oocyte electrophysiology, dominant-negative C→S mutant, transepithelial transport in mpkCCD cells\",\n      \"pmids\": [\"14993279\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct ubiquitination of ENaC subunits by UBE2E3 not reconstituted\", \"Relative contribution versus other E2s in vivo unclear\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Showing that UBE2E3 localizes to the nucleus and is essential for RPE cell proliferation connected its enzymatic activity to a specific cell-biological role and revealed a proliferation-to-quiescence switch upon its loss.\",\n      \"evidence\": \"siRNA knockdown in RPE cells, Ki-67/p27 immunolabeling, rescue experiments, beta-galactosidase reporter mouse for developmental downregulation\",\n      \"pmids\": [\"18614808\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nuclear substrates responsible for proliferation phenotype unknown\", \"Whether quiescence versus senescence was not distinguished at this point\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Revealing that UBE2E3's unique N-terminal extension prevents it from partnering with TRIM21/Ro52, unlike its paralogs UBE2E1/E2, established paralog-specific E3 selectivity within the UBE2E family.\",\n      \"evidence\": \"In vitro ubiquitination panel and NMR mapping of E2–E3 interface with multiple UBE2E variants\",\n      \"pmids\": [\"21862588\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of why UBE2E3's N-terminus is uniquely incompatible not resolved at atomic detail\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Four contemporaneous studies defined UBE2E3's substrate (TDP-43), its intrinsic monoubiquitylation restriction mechanism, its role in NRF2 nuclear import via importin-11, and its partnership with mitochondrial E3 Mulan, collectively establishing UBE2E3 as a versatile but product-constrained E2 operating in nuclear, cytoplasmic, and mitochondrial compartments.\",\n      \"evidence\": \"Y2H/Co-IP/ubiquitination assays for TDP-43 (PMID:24825905); reconstituted ubiquitylation with systematic backside mutagenesis (PMID:24901938); siRNA/fractionation/qPCR for NRF2 pathway (PMID:25378586); modified Y2H/Co-IP for Mulan–GABARAP interaction (PMID:25224329)\",\n      \"pmids\": [\"24825905\", \"24901938\", \"25378586\", \"25224329\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for backside-residue constraint not solved\", \"Whether monoubiquitylation restriction holds in all E3 contexts in vivo untested\", \"Direct ubiquitination site on TDP-43 not mapped\", \"Functional consequence of Mulan–UBE2E3–GABARAP complex on mitophagy not demonstrated\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrating that UBE2E3 loss triggers senescence via a p53/p21/p16-dependent program—with accompanying organelle remodeling—unified earlier proliferation and NRF2 findings into a coherent anti-senescence mechanism.\",\n      \"evidence\": \"siRNA knockdown, SA-β-Gal, SASP markers, organelle mass staining, autophagic flux, co-depletion epistasis with p53/p21/p16\",\n      \"pmids\": [\"29879550\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct ubiquitination target(s) linking UBE2E3 to p53/p21/p16 pathway unknown\", \"Whether NRF2 loss fully explains the senescence phenotype not resolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identifying PJA1 as an E3 ligase partnering with UBE2E3 to suppress TDP-43 aggregation in neurons, and showing that OTUB1 noncatalytically inhibits UBE2E3 polyubiquitination but uniquely cannot suppress its autoubiquitination, refined the regulatory landscape of UBE2E3 activity.\",\n      \"evidence\": \"Co-IP and in vivo motor neuron model for PJA1–TDP-43 (PMID:32686212); quantitative kinetic in vitro assays for OTUB1 regulation (PMID:32049508)\",\n      \"pmids\": [\"32686212\", \"32049508\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PJA1 is the predominant E3 for TDP-43 ubiquitination in disease-relevant neurons unclear\", \"Structural basis of OTUB1's failure to suppress UBE2E3 autoubiquitination not determined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Extending the anti-senescence role of UBE2E3 to bone marrow mesenchymal stem cells and linking it to NRF2-dependent osteogenic differentiation broadened the tissue relevance of the UBE2E3–NRF2 axis.\",\n      \"evidence\": \"Gain- and loss-of-function experiments in BMSCs with senescence and osteogenic differentiation assays, Nrf2 pathway analysis\",\n      \"pmids\": [\"34820159\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether UBE2E3 directly ubiquitinates an NRF2 pathway component in BMSCs not shown\", \"Mechanism linking UBE2E3 to NRF2 not reconstituted biochemically in this system\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identification of miR-143-3p as a direct UBE2E3-targeting microRNA in granulosa cells, and of RCBTB1 as a co-immunoprecipitating partner within a potential CUL3-containing complex in RPE cells, expanded the regulatory and interaction network around UBE2E3.\",\n      \"evidence\": \"3′UTR luciferase reporter for miR-143-3p (PMID:37628741); Co-IP from iPSC-derived RPE cells for RCBTB1/CUL3 (PMID:37408192)\",\n      \"pmids\": [\"37628741\", \"37408192\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"RCBTB1–CUL3–UBE2E3 complex not functionally reconstituted\", \"Single Co-IP for RCBTB1 without reciprocal validation\", \"Whether miR-143-3p regulation of UBE2E3 operates in vivo not established\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Additional miRNA-mediated regulation of UBE2E3 was confirmed in Sertoli cells (miR-143-3p) and breast cells (miR-379-5p), with UBE2E3 depletion consistently linked to senescence and proliferative defects across diverse tissues.\",\n      \"evidence\": \"miRNA mimic/inhibitor transfection, 3′UTR reporters, siRNA knockdown, BTB permeability assay (PMID:38391926); resazurin/BrdU/caspase assays (PMID:39621672)\",\n      \"pmids\": [\"38391926\", \"39621672\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Paradoxical increase in both proliferation and apoptosis upon UBE2E3 knockdown in breast cells unexplained mechanistically\", \"Whether blood-testis barrier dysfunction is a direct UBE2E3 substrate effect or secondary to senescence unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Critical open questions include the identity of UBE2E3's direct ubiquitination substrate(s) that trigger the p53/p21/p16 senescence cascade, the structural basis of its monoubiquitylation restriction at atomic resolution, and whether its NRF2-regulatory function is catalytic (ubiquitin-dependent) or noncatalytic.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No substrate linking UBE2E3 catalysis to p53/p21/p16 activation identified\", \"No high-resolution structure of UBE2E3 in complex with any E3 partner\", \"Catalytic vs. noncatalytic role in NRF2 nuclear import not distinguished\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 2, 6, 7]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [6, 7, 11]},\n      {\"term_id\": \"GO:0031386\", \"supporting_discovery_ids\": [0, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [4, 8]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 6, 7, 12]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [8, 10, 13]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [9, 10]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"NEDD4L\", \"NEDD4\", \"ARA54\", \"RNF8\", \"PJA1\", \"MUL1\", \"IPO11\", \"OTUB1\"],\n    \"other_free_text\": []\n  }\n}\n```"}