{"gene":"UBE2E1","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":1996,"finding":"UbcH6 (UBE2E1) was cloned as a human E2 ubiquitin-conjugating enzyme that physically interacts with the HECT-domain E3 ligase E6-AP, but unlike UbcH5, UbcH6 is only weakly active in E6-AP-dependent ubiquitination of p53, and does not efficiently interact with the HECT protein RSP5.","method":"In vitro binding assays, ubiquitination assays with E1/E2/E3 reconstitution","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro reconstitution assay, single lab, single study","pmids":["8576257"],"is_preprint":false},{"year":2005,"finding":"UbcH6 (UBE2E1) is itself covalently modified by ISG15 at Lys136 (near the catalytic Cys131); ISG15-modified UbcH6 cannot form a thioester intermediate with ubiquitin, demonstrating that ISG15 conjugation suppresses UbcH6 ubiquitin E2 enzyme activity. UbcH6 can also form a thioester intermediate with ISG15 through Cys131.","method":"In vitro thioester formation assay, site-directed mutagenesis, biochemical fractionation/isolation of ISG15-modified vs. unmodified UbcH6","journal":"Journal of biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of thioester, mutagenesis of active site, direct biochemical demonstration with purified proteins","pmids":["16428300"],"is_preprint":false},{"year":2006,"finding":"UbcH6 (UBE2E1) functions as the E2 ubiquitin-conjugating enzyme in concert with the novel RING-finger E3 ligase RING105 to polyubiquitinate the tumor suppressor TSSC5, with the polyubiquitin target site on TSSC5 mapped to a region in its 6th hydrophilic loop.","method":"In vitro ubiquitination assay, mapping of ubiquitination site, ectopic expression in HeLa cells with RING105 wild-type vs. RING finger mutant","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro ubiquitination reconstitution plus cellular overexpression with RING mutant controls, single lab","pmids":["16314844"],"is_preprint":false},{"year":2008,"finding":"UbcH6 (UBE2E1) directly interacts with and ubiquitinates ataxin-1 (SCA1 gene product) independently of an E3 ligase; the interaction is mediated through the AXH domain of ataxin-1, and UbcH6 expression level regulates the rate of ataxin-1 degradation. UbcH6 co-immunoprecipitates and co-localizes with ataxin-1 in the nucleus.","method":"Yeast two-hybrid screen, co-immunoprecipitation, co-localization, in vitro ubiquitination assay (E3-free), pulse-chase degradation assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (Y2H, Co-IP, in vitro ubiquitination, degradation assay), single lab","pmids":["18439907"],"is_preprint":false},{"year":2008,"finding":"UbcH6 (UBE2E1) modulates the transcriptional repression activity of ataxin-1 by promoting its ubiquitin-proteasome-mediated degradation; overexpression of UbcH6 reduces ataxin-1 transcriptional repression and aggregate formation, while UbcH6 knockdown (shRNA) enhances repression activity. Normal-length ataxin-1(30Q) is more susceptible to UbcH6-mediated degradation than polyglutamine-expanded ataxin-1(82Q).","method":"Transcriptional reporter assay, shRNA knockdown, overexpression, half-life (pulse-chase) measurement","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple cellular assays (reporter, KD, OE, half-life), single lab, builds on prior interaction data","pmids":["18519031"],"is_preprint":false},{"year":2013,"finding":"USP7 (ubiquitin-specific protease 7) forms a complex with UBE2E1 in vitro and in vivo through the ASTS motif within UBE2E1's unique N-terminal extension; USP7 attenuates UBE2E1-mediated ubiquitination in a manner requiring both the N-terminal ASTS sequence and USP7 catalytic activity, and USP7 is critical for maintaining steady-state levels of UBE2E1 in cells.","method":"Co-immunoprecipitation, in vitro binding assay, ubiquitination assay, mutant analysis (ASTS motif deletion, catalytic USP7 mutant), cell-based steady-state level analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, in vitro binding, functional ubiquitination assay, mutagenesis of both partners, multiple orthogonal methods in single study","pmids":["23603909"],"is_preprint":false},{"year":2017,"finding":"UBE2E1 (UbcH6) is a critical in vivo E2 for the Polycomb repressive complex 1 (PRC1)-catalyzed monoubiquitination of histone H2A at Lys-119 (uH2AK119); UBE2E1 interacts with PRC1 subunits Ring1A and Ring1B, and its loss of function (knockdown or catalytically inactive C131A mutant) reduces uH2AK119 levels, relieves p16INK4a promoter repression, and induces growth inhibition in HCT116 cells. USP7 also regulates uH2AK119 levels through UBE2E1.","method":"Co-immunoprecipitation, overexpression of wild-type and catalytically inactive UBE2E1 (C131A), siRNA knockdown, chromatin immunoprecipitation (p16 promoter), cell growth assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, KD, dominant-negative mutant, ChIP, phenotypic readout), single lab with rigorous controls","pmids":["28073915"],"is_preprint":false},{"year":2018,"finding":"OTUB1 non-catalytically stabilizes UBE2E1 by binding to it and suppressing UBE2E1 autoubiquitination, thereby preventing UBE2E1 from being targeted for proteasomal degradation. OTUB1 knockout mice exhibit late-stage embryonic lethality, and OTUB1 depletion dramatically destabilizes UBE2E1 in mouse and human cell lines. The stabilizing effect depends on OTUB1's ability to bind UBE2E1, not its deubiquitinase catalytic activity.","method":"Otub1 knockout mice and derived cell lines, OTUB1-catalytic mutant analysis, in vitro autoubiquitination assay, protein stability/degradation assay, proteasome inhibitor rescue","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro ubiquitination assay, genetic KO in mice and cell lines, catalytic mutant of OTUB1, multiple orthogonal methods, independently validated in two species","pmids":["30282802"],"is_preprint":false},{"year":2018,"finding":"UBE2E1 (E2E1) is preferentially expressed in slow-twitch (type I and IIA) muscle fibers and is absent in type IIB fibers; its knockdown in C2C12 myotubes and mouse tibialis anterior muscle aggravates dexamethasone-induced atrophy, indicating a protective role on muscle mass under catabolic conditions. UBE2E1 interacts with the E3 ligase MuRF1 in a substrate (telethonin)-dependent manner.","method":"Immunofluorescence fiber-type analysis, siRNA knockdown in C2C12 myotubes, in vivo muscle knockdown in mice, measurement of muscle cross-sectional area/protein content","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro and in vivo KD with phenotypic readout, fiber-type localization by immunofluorescence, single lab, multiple systems tested","pmids":["30453501"],"is_preprint":false},{"year":2019,"finding":"Crystal structure (2.82 Å) of the human TRIM21 RING domain in complex with UBE2E1 revealed that a ubiquitin-targeted substrate lysine (from TRIM21) is captured in the UBE2E1 active site; the direction of lysine entry resembles that for PCNA ubiquitination. Key UBE2E1 active-site residues involved in lysine capture are conserved in ubiquitin-conjugating E2s, and coordination of the acceptor lysine triggers remodeling of side-chain interactions at the E2-E3 interface including the 'linchpin' residue, suggesting an allosteric mechanism coupling substrate lysine activation to catalytic activity.","method":"X-ray crystallography (2.82 Å), structure-function analysis, mutagenesis of active-site residues, in vitro ubiquitination assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure at 2.82 Å with functional mutagenesis, single study but high method quality","pmids":["31160341"],"is_preprint":false},{"year":2022,"finding":"UBE2E1 interacts with the ciliopathy protein MKS1 and the E3 ligase RNF34; UBE2E1 polyubiquitinates β-catenin and MKS1 (both regulatory and degradative ubiquitination of MKS1); UBE2E1 and MKS1 co-localize at the ciliary base, and loss of UBE2E1 recapitulates ciliary and Wnt signaling phenotypes seen with MKS1 loss. UBE2E1 and MKS1 protein levels are co-dependent.","method":"Co-immunoprecipitation, co-localization by immunofluorescence, siRNA knockdown, in vitro/cellular ubiquitination assay, Mks1 knockout mouse model","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, genetic KO mouse, in vitro ubiquitination, co-localization, multiple orthogonal methods in single study","pmids":["35170427"],"is_preprint":false},{"year":2024,"finding":"UBE2E1 can perform E3-independent substrate ubiquitination in a sequence-dependent manner; a crystal structure of UBE2E1 in complex with a SETDB1-derived substrate peptide revealed the mechanism of peptide sequence-dependent ubiquitin transfer. This mechanism was exploited to engineer an E3-free enzymatic ubiquitination strategy (SUE1) capable of generating site-specific ubiquitinated proteins, customized polyubiquitin chain linkages, branched ubiquitin chains, and NEDD8-modified proteins.","method":"X-ray crystallography (structure of UBE2E1-substrate peptide complex), in vitro E3-free ubiquitination assay, structure-guided mutagenesis, biochemical reconstitution","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure combined with in vitro reconstitution and structure-guided engineering, single study with multiple orthogonal validations","pmids":["38341401"],"is_preprint":false}],"current_model":"UBE2E1 (UbcH6) is a human E2 ubiquitin-conjugating enzyme with a unique N-terminal extension containing an ASTS motif; it can ubiquitinate substrates either with E3 partners (TRIM21/PRC1 Ring1A/B for H2AK119 monoubiquitination, RING105 for TSSC5, RNF34/MKS1 for β-catenin) or independently of an E3 in a substrate sequence-dependent manner (ataxin-1, SETDB1-derived peptides); its activity is regulated by USP7 (which binds the N-terminal ASTS motif to attenuate ubiquitination) and OTUB1 (which non-catalytically suppresses UBE2E1 autoubiquitination to prevent its proteasomal degradation); ISG15 conjugation to Lys136 suppresses UBE2E1 ubiquitin-thioester activity; crystal structures of UBE2E1 in complex with TRIM21 RING and with substrate peptide have revealed how substrate lysine capture in the active site allosterically activates the E2-E3 interface for catalysis."},"narrative":{"mechanistic_narrative":"UBE2E1 (UbcH6) is a human E2 ubiquitin-conjugating enzyme that catalyzes substrate ubiquitination both with dedicated RING/HECT E3 partners and, distinctively, in an E3-independent, substrate-sequence-dependent manner [PMID:8576257, PMID:38341401]. Through its catalytic cysteine (Cys131) it forms ubiquitin thioester intermediates and transfers ubiquitin to a captured acceptor lysine; crystallographic analysis of UBE2E1 with the TRIM21 RING shows that coordination of the substrate lysine in the active site allosterically remodels the E2-E3 'linchpin' interface to couple substrate engagement to catalysis [PMID:31160341], while a complex with a SETDB1-derived peptide explains how UBE2E1 reads substrate sequence to transfer ubiquitin without an E3 [PMID:38341401]. It serves as the in vivo E2 for PRC1-catalyzed monoubiquitination of histone H2A at Lys119, interacting with Ring1A/Ring1B to maintain Polycomb repression of the p16INK4a promoter and restrain cell growth [PMID:28073915], and partners with E3 ligases to ubiquitinate diverse substrates including TSSC5 (with RING105) [PMID:16314844] and β-catenin and the ciliopathy protein MKS1 (with RNF34), localizing with MKS1 at the ciliary base to control ciliary and Wnt signaling [PMID:35170427]. UBE2E1 also directly ubiquitinates and drives degradation of ataxin-1, modulating its transcriptional repression and aggregation [PMID:18439907, PMID:18519031]. Its abundance and activity are tightly regulated: USP7 binds the N-terminal ASTS motif to attenuate ubiquitination [PMID:23603909], OTUB1 non-catalytically suppresses UBE2E1 autoubiquitination to prevent its proteasomal degradation [PMID:30282802], and ISG15 conjugation at Lys136 blocks ubiquitin-thioester formation [PMID:16428300]. UBE2E1 is enriched in slow-twitch muscle fibers where it protects against catabolic atrophy and engages the E3 MuRF1 in a substrate-dependent fashion [PMID:30453501].","teleology":[{"year":1996,"claim":"Established UBE2E1 as a bona fide human E2 enzyme by showing it engages a HECT-domain E3, while revealing it is functionally distinct from the better-known UbcH5.","evidence":"In vitro binding and reconstituted E1/E2/E3 ubiquitination assays with E6-AP and p53","pmids":["8576257"],"confidence":"Medium","gaps":["Did not identify physiological substrates or cognate E3 partners","Weak activity with E6-AP left its productive E3 partnerships unknown"]},{"year":2005,"claim":"Showed that UBE2E1 activity is switched off by post-translational modification, defining ISG15 conjugation as a regulatory brake on its enzymatic cycle.","evidence":"In vitro thioester formation assays with active-site mutagenesis and isolation of ISG15-modified UbcH6","pmids":["16428300"],"confidence":"High","gaps":["The cellular ISG15 E3 ligase and trigger for UBE2E1 ISGylation not defined","Physiological consequences of suppressed activity not tested in vivo"]},{"year":2006,"claim":"Identified a specific E3 partner (RING105) and substrate (TSSC5), demonstrating UBE2E1 builds polyubiquitin chains in a defined E2-E3 pairing.","evidence":"In vitro ubiquitination with site mapping and cellular expression with RING-finger mutant controls","pmids":["16314844"],"confidence":"Medium","gaps":["Chain linkage type not characterized","Fate of ubiquitinated TSSC5 not established"]},{"year":2008,"claim":"Revealed an E3-independent mode of action, showing UBE2E1 can bind and ubiquitinate a substrate directly and control its degradation.","evidence":"Yeast two-hybrid, Co-IP, co-localization, E3-free in vitro ubiquitination, and pulse-chase degradation of ataxin-1","pmids":["18439907","18519031"],"confidence":"Medium","gaps":["Molecular basis of E3-free substrate recognition unresolved at the time","Polyglutamine-length-dependent resistance to degradation mechanism unclear"]},{"year":2013,"claim":"Defined the N-terminal ASTS motif as a regulatory docking site, showing USP7 both binds and dampens UBE2E1 activity and sets its steady-state level.","evidence":"Reciprocal Co-IP, in vitro binding, ubiquitination assays, ASTS-deletion and USP7 catalytic mutants, cellular level analysis","pmids":["23603909"],"confidence":"High","gaps":["Whether USP7 deubiquitinates UBE2E1 itself or its substrates not fully separated","Substrate-specific consequences of attenuation not mapped"]},{"year":2017,"claim":"Placed UBE2E1 in a major chromatin pathway as the in vivo E2 for PRC1-mediated H2AK119 monoubiquitination, linking it to Polycomb repression and growth control.","evidence":"Co-IP with Ring1A/B, C131A dominant-negative and siRNA, p16 promoter ChIP, and growth assays in HCT116 cells","pmids":["28073915"],"confidence":"High","gaps":["Genome-wide extent of UBE2E1-dependent H2AK119ub not defined","How UBE2E1 is selectively recruited to PRC1 versus other E3s unclear"]},{"year":2018,"claim":"Identified OTUB1 as a non-catalytic stabilizer that prevents UBE2E1 self-destruction, establishing autoubiquitination as the route controlling its abundance.","evidence":"Otub1 knockout mice and cell lines, OTUB1 catalytic mutant, in vitro autoubiquitination, and proteasome-inhibitor rescue","pmids":["30282802"],"confidence":"High","gaps":["Which downstream UBE2E1 functions are most sensitive to OTUB1 loss not resolved","Relationship between OTUB1 and USP7 control of UBE2E1 not integrated"]},{"year":2018,"claim":"Assigned a tissue-specific physiological role, showing UBE2E1 is a slow-fiber-enriched protector against muscle atrophy that engages MuRF1.","evidence":"Fiber-type immunofluorescence, siRNA in C2C12 and in vivo muscle knockdown with cross-sectional area readouts","pmids":["30453501"],"confidence":"Medium","gaps":["Direct ubiquitination substrates in muscle not identified","Mechanism by which UBE2E1 opposes rather than promotes atrophy unresolved"]},{"year":2019,"claim":"Provided structural mechanism for E2-E3 catalysis, showing acceptor-lysine capture in the UBE2E1 active site allosterically activates the E3 interface.","evidence":"2.82 Å crystal structure of TRIM21 RING-UBE2E1 with active-site mutagenesis and in vitro ubiquitination","pmids":["31160341"],"confidence":"High","gaps":["Generality of the allosteric model across other E3 partners not tested structurally","Dynamics of the catalytic cycle beyond the captured-lysine state not resolved"]},{"year":2022,"claim":"Connected UBE2E1 to ciliary biology and Wnt signaling through MKS1 and RNF34, with co-dependent stability between UBE2E1 and MKS1.","evidence":"Reciprocal Co-IP, ciliary-base co-localization, siRNA, in vitro/cellular ubiquitination of β-catenin and MKS1, and Mks1 knockout mouse","pmids":["35170427"],"confidence":"High","gaps":["Distinction between regulatory and degradative MKS1 ubiquitination outcomes not fully mapped","How UBE2E1 is targeted to the ciliary base unknown"]},{"year":2024,"claim":"Resolved the basis of E3-free ubiquitination, showing UBE2E1 reads substrate peptide sequence to transfer ubiquitin and enabling engineered ubiquitination chemistry.","evidence":"Crystal structure of UBE2E1 with a SETDB1-derived peptide, E3-free in vitro ubiquitination, and structure-guided engineering (SUE1)","pmids":["38341401"],"confidence":"High","gaps":["Endogenous repertoire of sequence-dependent substrates not catalogued","Physiological prevalence of E3-free versus E3-dependent activity not quantified"]},{"year":null,"claim":"How the multiple regulators (USP7, OTUB1, ISG15) and dual E3-dependent/E3-free modes are integrated to determine which substrates UBE2E1 ubiquitinates in a given cell remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model of substrate selection across contexts","Linkage-type specificity in vivo not systematically defined","Tissue-specific substrate maps (e.g. muscle) absent"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,6,9,10,11]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[1,9,11]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[5,7]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[3,6]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[10]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,2,5,7,11]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[6]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[10]}],"complexes":["PRC1"],"partners":["RING1","RNBP2","USP7","OTUB1","TRIM21","MKS1","RNF34","TRIM37"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P51965","full_name":"Ubiquitin-conjugating enzyme E2 E1","aliases":["(E3-independent) E2 ubiquitin-conjugating enzyme E1","E2 ubiquitin-conjugating enzyme E1","UbcH6","Ubiquitin carrier protein E1","Ubiquitin-protein ligase E1"],"length_aa":193,"mass_kda":21.4,"function":"Accepts ubiquitin from the E1 complex and catalyzes its covalent attachment to other proteins. Catalyzes the covalent attachment of ISG15 to other proteins. Mediates the selective degradation of short-lived and abnormal proteins. In vitro also catalyzes 'Lys-48'-linked polyubiquitination. Catalyzes monoubiquitination of other proteins in both an E3-dependent and E3-independent manner (PubMed:27237050)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/P51965/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/UBE2E1","classification":"Not Classified","n_dependent_lines":5,"n_total_lines":1208,"dependency_fraction":0.0041390728476821195},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/UBE2E1","total_profiled":1310},"omim":[{"mim_id":"617233","title":"WD REPEAT-CONTAINING PROTEIN 70; WDR70","url":"https://www.omim.org/entry/617233"},{"mim_id":"617206","title":"NEURALIZED E3 UBIQUITIN PROTEIN LIGASE 3; NEURL3","url":"https://www.omim.org/entry/617206"},{"mim_id":"616017","title":"TRIPARTITE MOTIF-CONTAINING PROTEIN 69; TRIM69","url":"https://www.omim.org/entry/616017"},{"mim_id":"616015","title":"RING FINGER PROTEIN 180; RNF180","url":"https://www.omim.org/entry/616015"},{"mim_id":"616014","title":"RING FINGER PROTEIN 25; RNF25","url":"https://www.omim.org/entry/616014"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/UBE2E1"},"hgnc":{"alias_symbol":["UbcH6"],"prev_symbol":[]},"alphafold":{"accession":"P51965","domains":[{"cath_id":"3.10.110.10","chopping":"45-191","consensus_level":"high","plddt":97.1087,"start":45,"end":191}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P51965","model_url":"https://alphafold.ebi.ac.uk/files/AF-P51965-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P51965-F1-predicted_aligned_error_v6.png","plddt_mean":86.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=UBE2E1","jax_strain_url":"https://www.jax.org/strain/search?query=UBE2E1"},"sequence":{"accession":"P51965","fasta_url":"https://rest.uniprot.org/uniprotkb/P51965.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P51965/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P51965"}},"corpus_meta":[{"pmid":"8576257","id":"PMC_8576257","title":"Cloning of human ubiquitin-conjugating enzymes UbcH6 and UbcH7 (E2-F1) and characterization of their interaction with E6-AP and RSP5.","date":"1996","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8576257","citation_count":137,"is_preprint":false},{"pmid":"16428300","id":"PMC_16428300","title":"Link between the ubiquitin conjugation system and the ISG15 conjugation system: ISG15 conjugation to the UbcH6 ubiquitin E2 enzyme.","date":"2005","source":"Journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16428300","citation_count":48,"is_preprint":false},{"pmid":"23603909","id":"PMC_23603909","title":"Ubiquitin-specific protease 7 is a regulator of ubiquitin-conjugating enzyme UbE2E1.","date":"2013","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23603909","citation_count":42,"is_preprint":false},{"pmid":"16314844","id":"PMC_16314844","title":"Tumor suppressor candidate TSSC5 is regulated by UbcH6 and a novel ubiquitin ligase RING105.","date":"2006","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/16314844","citation_count":39,"is_preprint":false},{"pmid":"30282802","id":"PMC_30282802","title":"OTUB1 non-catalytically stabilizes the E2 ubiquitin-conjugating enzyme UBE2E1 by preventing its autoubiquitination.","date":"2018","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/30282802","citation_count":36,"is_preprint":false},{"pmid":"31160341","id":"PMC_31160341","title":"E3 ubiquitin-protein ligase TRIM21-mediated lysine capture by UBE2E1 reveals substrate-targeting mode of a ubiquitin-conjugating E2.","date":"2019","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/31160341","citation_count":27,"is_preprint":false},{"pmid":"28073915","id":"PMC_28073915","title":"UbE2E1/UBCH6 Is a Critical in Vivo E2 for the PRC1-catalyzed Ubiquitination of H2A at Lys-119.","date":"2017","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/28073915","citation_count":24,"is_preprint":false},{"pmid":"38341401","id":"PMC_38341401","title":"Structure-guided engineering enables E3 ligase-free and versatile protein ubiquitination via UBE2E1.","date":"2024","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/38341401","citation_count":23,"is_preprint":false},{"pmid":"18439907","id":"PMC_18439907","title":"UbcH6 interacts with and ubiquitinates the SCA1 gene product ataxin-1.","date":"2008","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/18439907","citation_count":19,"is_preprint":false},{"pmid":"18519031","id":"PMC_18519031","title":"The ubiquitin-conjugating enzyme UbcH6 regulates the transcriptional repression activity of the SCA1 gene product ataxin-1.","date":"2008","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/18519031","citation_count":13,"is_preprint":false},{"pmid":"35170427","id":"PMC_35170427","title":"Regulation of canonical Wnt signalling by the ciliopathy protein MKS1 and the E2 ubiquitin-conjugating enzyme UBE2E1.","date":"2022","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/35170427","citation_count":12,"is_preprint":false},{"pmid":"30453501","id":"PMC_30453501","title":"UBE2E1 Is Preferentially Expressed in the Cytoplasm of Slow-Twitch Fibers and Protects Skeletal Muscles from Exacerbated Atrophy upon Dexamethasone Treatment.","date":"2018","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/30453501","citation_count":11,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8090,"output_tokens":3772,"usd":0.040425,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11259,"output_tokens":4107,"usd":0.079485,"stage2_stop_reason":"end_turn"},"total_usd":0.11991,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1996,\n      \"finding\": \"UbcH6 (UBE2E1) was cloned as a human E2 ubiquitin-conjugating enzyme that physically interacts with the HECT-domain E3 ligase E6-AP, but unlike UbcH5, UbcH6 is only weakly active in E6-AP-dependent ubiquitination of p53, and does not efficiently interact with the HECT protein RSP5.\",\n      \"method\": \"In vitro binding assays, ubiquitination assays with E1/E2/E3 reconstitution\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro reconstitution assay, single lab, single study\",\n      \"pmids\": [\"8576257\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"UbcH6 (UBE2E1) is itself covalently modified by ISG15 at Lys136 (near the catalytic Cys131); ISG15-modified UbcH6 cannot form a thioester intermediate with ubiquitin, demonstrating that ISG15 conjugation suppresses UbcH6 ubiquitin E2 enzyme activity. UbcH6 can also form a thioester intermediate with ISG15 through Cys131.\",\n      \"method\": \"In vitro thioester formation assay, site-directed mutagenesis, biochemical fractionation/isolation of ISG15-modified vs. unmodified UbcH6\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of thioester, mutagenesis of active site, direct biochemical demonstration with purified proteins\",\n      \"pmids\": [\"16428300\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"UbcH6 (UBE2E1) functions as the E2 ubiquitin-conjugating enzyme in concert with the novel RING-finger E3 ligase RING105 to polyubiquitinate the tumor suppressor TSSC5, with the polyubiquitin target site on TSSC5 mapped to a region in its 6th hydrophilic loop.\",\n      \"method\": \"In vitro ubiquitination assay, mapping of ubiquitination site, ectopic expression in HeLa cells with RING105 wild-type vs. RING finger mutant\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro ubiquitination reconstitution plus cellular overexpression with RING mutant controls, single lab\",\n      \"pmids\": [\"16314844\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"UbcH6 (UBE2E1) directly interacts with and ubiquitinates ataxin-1 (SCA1 gene product) independently of an E3 ligase; the interaction is mediated through the AXH domain of ataxin-1, and UbcH6 expression level regulates the rate of ataxin-1 degradation. UbcH6 co-immunoprecipitates and co-localizes with ataxin-1 in the nucleus.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation, co-localization, in vitro ubiquitination assay (E3-free), pulse-chase degradation assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (Y2H, Co-IP, in vitro ubiquitination, degradation assay), single lab\",\n      \"pmids\": [\"18439907\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"UbcH6 (UBE2E1) modulates the transcriptional repression activity of ataxin-1 by promoting its ubiquitin-proteasome-mediated degradation; overexpression of UbcH6 reduces ataxin-1 transcriptional repression and aggregate formation, while UbcH6 knockdown (shRNA) enhances repression activity. Normal-length ataxin-1(30Q) is more susceptible to UbcH6-mediated degradation than polyglutamine-expanded ataxin-1(82Q).\",\n      \"method\": \"Transcriptional reporter assay, shRNA knockdown, overexpression, half-life (pulse-chase) measurement\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple cellular assays (reporter, KD, OE, half-life), single lab, builds on prior interaction data\",\n      \"pmids\": [\"18519031\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"USP7 (ubiquitin-specific protease 7) forms a complex with UBE2E1 in vitro and in vivo through the ASTS motif within UBE2E1's unique N-terminal extension; USP7 attenuates UBE2E1-mediated ubiquitination in a manner requiring both the N-terminal ASTS sequence and USP7 catalytic activity, and USP7 is critical for maintaining steady-state levels of UBE2E1 in cells.\",\n      \"method\": \"Co-immunoprecipitation, in vitro binding assay, ubiquitination assay, mutant analysis (ASTS motif deletion, catalytic USP7 mutant), cell-based steady-state level analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, in vitro binding, functional ubiquitination assay, mutagenesis of both partners, multiple orthogonal methods in single study\",\n      \"pmids\": [\"23603909\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"UBE2E1 (UbcH6) is a critical in vivo E2 for the Polycomb repressive complex 1 (PRC1)-catalyzed monoubiquitination of histone H2A at Lys-119 (uH2AK119); UBE2E1 interacts with PRC1 subunits Ring1A and Ring1B, and its loss of function (knockdown or catalytically inactive C131A mutant) reduces uH2AK119 levels, relieves p16INK4a promoter repression, and induces growth inhibition in HCT116 cells. USP7 also regulates uH2AK119 levels through UBE2E1.\",\n      \"method\": \"Co-immunoprecipitation, overexpression of wild-type and catalytically inactive UBE2E1 (C131A), siRNA knockdown, chromatin immunoprecipitation (p16 promoter), cell growth assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, KD, dominant-negative mutant, ChIP, phenotypic readout), single lab with rigorous controls\",\n      \"pmids\": [\"28073915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"OTUB1 non-catalytically stabilizes UBE2E1 by binding to it and suppressing UBE2E1 autoubiquitination, thereby preventing UBE2E1 from being targeted for proteasomal degradation. OTUB1 knockout mice exhibit late-stage embryonic lethality, and OTUB1 depletion dramatically destabilizes UBE2E1 in mouse and human cell lines. The stabilizing effect depends on OTUB1's ability to bind UBE2E1, not its deubiquitinase catalytic activity.\",\n      \"method\": \"Otub1 knockout mice and derived cell lines, OTUB1-catalytic mutant analysis, in vitro autoubiquitination assay, protein stability/degradation assay, proteasome inhibitor rescue\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro ubiquitination assay, genetic KO in mice and cell lines, catalytic mutant of OTUB1, multiple orthogonal methods, independently validated in two species\",\n      \"pmids\": [\"30282802\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"UBE2E1 (E2E1) is preferentially expressed in slow-twitch (type I and IIA) muscle fibers and is absent in type IIB fibers; its knockdown in C2C12 myotubes and mouse tibialis anterior muscle aggravates dexamethasone-induced atrophy, indicating a protective role on muscle mass under catabolic conditions. UBE2E1 interacts with the E3 ligase MuRF1 in a substrate (telethonin)-dependent manner.\",\n      \"method\": \"Immunofluorescence fiber-type analysis, siRNA knockdown in C2C12 myotubes, in vivo muscle knockdown in mice, measurement of muscle cross-sectional area/protein content\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro and in vivo KD with phenotypic readout, fiber-type localization by immunofluorescence, single lab, multiple systems tested\",\n      \"pmids\": [\"30453501\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Crystal structure (2.82 Å) of the human TRIM21 RING domain in complex with UBE2E1 revealed that a ubiquitin-targeted substrate lysine (from TRIM21) is captured in the UBE2E1 active site; the direction of lysine entry resembles that for PCNA ubiquitination. Key UBE2E1 active-site residues involved in lysine capture are conserved in ubiquitin-conjugating E2s, and coordination of the acceptor lysine triggers remodeling of side-chain interactions at the E2-E3 interface including the 'linchpin' residue, suggesting an allosteric mechanism coupling substrate lysine activation to catalytic activity.\",\n      \"method\": \"X-ray crystallography (2.82 Å), structure-function analysis, mutagenesis of active-site residues, in vitro ubiquitination assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure at 2.82 Å with functional mutagenesis, single study but high method quality\",\n      \"pmids\": [\"31160341\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"UBE2E1 interacts with the ciliopathy protein MKS1 and the E3 ligase RNF34; UBE2E1 polyubiquitinates β-catenin and MKS1 (both regulatory and degradative ubiquitination of MKS1); UBE2E1 and MKS1 co-localize at the ciliary base, and loss of UBE2E1 recapitulates ciliary and Wnt signaling phenotypes seen with MKS1 loss. UBE2E1 and MKS1 protein levels are co-dependent.\",\n      \"method\": \"Co-immunoprecipitation, co-localization by immunofluorescence, siRNA knockdown, in vitro/cellular ubiquitination assay, Mks1 knockout mouse model\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, genetic KO mouse, in vitro ubiquitination, co-localization, multiple orthogonal methods in single study\",\n      \"pmids\": [\"35170427\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"UBE2E1 can perform E3-independent substrate ubiquitination in a sequence-dependent manner; a crystal structure of UBE2E1 in complex with a SETDB1-derived substrate peptide revealed the mechanism of peptide sequence-dependent ubiquitin transfer. This mechanism was exploited to engineer an E3-free enzymatic ubiquitination strategy (SUE1) capable of generating site-specific ubiquitinated proteins, customized polyubiquitin chain linkages, branched ubiquitin chains, and NEDD8-modified proteins.\",\n      \"method\": \"X-ray crystallography (structure of UBE2E1-substrate peptide complex), in vitro E3-free ubiquitination assay, structure-guided mutagenesis, biochemical reconstitution\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure combined with in vitro reconstitution and structure-guided engineering, single study with multiple orthogonal validations\",\n      \"pmids\": [\"38341401\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"UBE2E1 (UbcH6) is a human E2 ubiquitin-conjugating enzyme with a unique N-terminal extension containing an ASTS motif; it can ubiquitinate substrates either with E3 partners (TRIM21/PRC1 Ring1A/B for H2AK119 monoubiquitination, RING105 for TSSC5, RNF34/MKS1 for β-catenin) or independently of an E3 in a substrate sequence-dependent manner (ataxin-1, SETDB1-derived peptides); its activity is regulated by USP7 (which binds the N-terminal ASTS motif to attenuate ubiquitination) and OTUB1 (which non-catalytically suppresses UBE2E1 autoubiquitination to prevent its proteasomal degradation); ISG15 conjugation to Lys136 suppresses UBE2E1 ubiquitin-thioester activity; crystal structures of UBE2E1 in complex with TRIM21 RING and with substrate peptide have revealed how substrate lysine capture in the active site allosterically activates the E2-E3 interface for catalysis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"UBE2E1 (UbcH6) is a human E2 ubiquitin-conjugating enzyme that catalyzes substrate ubiquitination both with dedicated RING/HECT E3 partners and, distinctively, in an E3-independent, substrate-sequence-dependent manner [#0, #11]. Through its catalytic cysteine (Cys131) it forms ubiquitin thioester intermediates and transfers ubiquitin to a captured acceptor lysine; crystallographic analysis of UBE2E1 with the TRIM21 RING shows that coordination of the substrate lysine in the active site allosterically remodels the E2-E3 'linchpin' interface to couple substrate engagement to catalysis [#9], while a complex with a SETDB1-derived peptide explains how UBE2E1 reads substrate sequence to transfer ubiquitin without an E3 [#11]. It serves as the in vivo E2 for PRC1-catalyzed monoubiquitination of histone H2A at Lys119, interacting with Ring1A/Ring1B to maintain Polycomb repression of the p16INK4a promoter and restrain cell growth [#6], and partners with E3 ligases to ubiquitinate diverse substrates including TSSC5 (with RING105) [#2] and β-catenin and the ciliopathy protein MKS1 (with RNF34), localizing with MKS1 at the ciliary base to control ciliary and Wnt signaling [#10]. UBE2E1 also directly ubiquitinates and drives degradation of ataxin-1, modulating its transcriptional repression and aggregation [#3, #4]. Its abundance and activity are tightly regulated: USP7 binds the N-terminal ASTS motif to attenuate ubiquitination [#5], OTUB1 non-catalytically suppresses UBE2E1 autoubiquitination to prevent its proteasomal degradation [#7], and ISG15 conjugation at Lys136 blocks ubiquitin-thioester formation [#1]. UBE2E1 is enriched in slow-twitch muscle fibers where it protects against catabolic atrophy and engages the E3 MuRF1 in a substrate-dependent fashion [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Established UBE2E1 as a bona fide human E2 enzyme by showing it engages a HECT-domain E3, while revealing it is functionally distinct from the better-known UbcH5.\",\n      \"evidence\": \"In vitro binding and reconstituted E1/E2/E3 ubiquitination assays with E6-AP and p53\",\n      \"pmids\": [\"8576257\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Did not identify physiological substrates or cognate E3 partners\", \"Weak activity with E6-AP left its productive E3 partnerships unknown\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Showed that UBE2E1 activity is switched off by post-translational modification, defining ISG15 conjugation as a regulatory brake on its enzymatic cycle.\",\n      \"evidence\": \"In vitro thioester formation assays with active-site mutagenesis and isolation of ISG15-modified UbcH6\",\n      \"pmids\": [\"16428300\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"The cellular ISG15 E3 ligase and trigger for UBE2E1 ISGylation not defined\", \"Physiological consequences of suppressed activity not tested in vivo\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Identified a specific E3 partner (RING105) and substrate (TSSC5), demonstrating UBE2E1 builds polyubiquitin chains in a defined E2-E3 pairing.\",\n      \"evidence\": \"In vitro ubiquitination with site mapping and cellular expression with RING-finger mutant controls\",\n      \"pmids\": [\"16314844\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Chain linkage type not characterized\", \"Fate of ubiquitinated TSSC5 not established\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Revealed an E3-independent mode of action, showing UBE2E1 can bind and ubiquitinate a substrate directly and control its degradation.\",\n      \"evidence\": \"Yeast two-hybrid, Co-IP, co-localization, E3-free in vitro ubiquitination, and pulse-chase degradation of ataxin-1\",\n      \"pmids\": [\"18439907\", \"18519031\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Molecular basis of E3-free substrate recognition unresolved at the time\", \"Polyglutamine-length-dependent resistance to degradation mechanism unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined the N-terminal ASTS motif as a regulatory docking site, showing USP7 both binds and dampens UBE2E1 activity and sets its steady-state level.\",\n      \"evidence\": \"Reciprocal Co-IP, in vitro binding, ubiquitination assays, ASTS-deletion and USP7 catalytic mutants, cellular level analysis\",\n      \"pmids\": [\"23603909\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Whether USP7 deubiquitinates UBE2E1 itself or its substrates not fully separated\", \"Substrate-specific consequences of attenuation not mapped\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Placed UBE2E1 in a major chromatin pathway as the in vivo E2 for PRC1-mediated H2AK119 monoubiquitination, linking it to Polycomb repression and growth control.\",\n      \"evidence\": \"Co-IP with Ring1A/B, C131A dominant-negative and siRNA, p16 promoter ChIP, and growth assays in HCT116 cells\",\n      \"pmids\": [\"28073915\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Genome-wide extent of UBE2E1-dependent H2AK119ub not defined\", \"How UBE2E1 is selectively recruited to PRC1 versus other E3s unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified OTUB1 as a non-catalytic stabilizer that prevents UBE2E1 self-destruction, establishing autoubiquitination as the route controlling its abundance.\",\n      \"evidence\": \"Otub1 knockout mice and cell lines, OTUB1 catalytic mutant, in vitro autoubiquitination, and proteasome-inhibitor rescue\",\n      \"pmids\": [\"30282802\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Which downstream UBE2E1 functions are most sensitive to OTUB1 loss not resolved\", \"Relationship between OTUB1 and USP7 control of UBE2E1 not integrated\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Assigned a tissue-specific physiological role, showing UBE2E1 is a slow-fiber-enriched protector against muscle atrophy that engages MuRF1.\",\n      \"evidence\": \"Fiber-type immunofluorescence, siRNA in C2C12 and in vivo muscle knockdown with cross-sectional area readouts\",\n      \"pmids\": [\"30453501\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Direct ubiquitination substrates in muscle not identified\", \"Mechanism by which UBE2E1 opposes rather than promotes atrophy unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Provided structural mechanism for E2-E3 catalysis, showing acceptor-lysine capture in the UBE2E1 active site allosterically activates the E3 interface.\",\n      \"evidence\": \"2.82 Å crystal structure of TRIM21 RING-UBE2E1 with active-site mutagenesis and in vitro ubiquitination\",\n      \"pmids\": [\"31160341\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Generality of the allosteric model across other E3 partners not tested structurally\", \"Dynamics of the catalytic cycle beyond the captured-lysine state not resolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected UBE2E1 to ciliary biology and Wnt signaling through MKS1 and RNF34, with co-dependent stability between UBE2E1 and MKS1.\",\n      \"evidence\": \"Reciprocal Co-IP, ciliary-base co-localization, siRNA, in vitro/cellular ubiquitination of β-catenin and MKS1, and Mks1 knockout mouse\",\n      \"pmids\": [\"35170427\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Distinction between regulatory and degradative MKS1 ubiquitination outcomes not fully mapped\", \"How UBE2E1 is targeted to the ciliary base unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Resolved the basis of E3-free ubiquitination, showing UBE2E1 reads substrate peptide sequence to transfer ubiquitin and enabling engineered ubiquitination chemistry.\",\n      \"evidence\": \"Crystal structure of UBE2E1 with a SETDB1-derived peptide, E3-free in vitro ubiquitination, and structure-guided engineering (SUE1)\",\n      \"pmids\": [\"38341401\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Endogenous repertoire of sequence-dependent substrates not catalogued\", \"Physiological prevalence of E3-free versus E3-dependent activity not quantified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the multiple regulators (USP7, OTUB1, ISG15) and dual E3-dependent/E3-free modes are integrated to determine which substrates UBE2E1 ubiquitinates in a given cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No unified model of substrate selection across contexts\", \"Linkage-type specificity in vivo not systematically defined\", \"Tissue-specific substrate maps (e.g. muscle) absent\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 6, 9, 10, 11]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [1, 9, 11]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [5, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [3, 6]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 2, 5, 7, 11]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [10]}\n    ],\n    \"complexes\": [\"PRC1\"],\n    \"partners\": [\"RING1\", \"RNBP2\", \"USP7\", \"OTUB1\", \"TRIM21\", \"MKS1\", \"RNF34\", \"TRIM37\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}