{"gene":"UBE2L6","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2024,"finding":"Cryo-EM structure of the UBA7•UBE2L6 disulfide complex reveals three factors governing complex formation: (1) strong binding affinity and specificity of UBA7 for UBE2L6 (distinct from UBE2L3); (2) conformational differences in the catalytic cysteine capping loop (CCL); and (3) increased thiolate/thiol ratios at catalytic cysteines. Modification of any of these factors impacts complex activation and ISG15 transfer cascade.","method":"Cryo-EM structure determination, biochemical reconstitution, mutagenesis","journal":"bioRxiv (preprint)","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — cryo-EM structure with mechanistic dissection, but preprint, single lab, not yet peer-reviewed","pmids":["bio_10.1101_2024.11.07.622398"],"is_preprint":true},{"year":2024,"finding":"UBE2L6/UbcH8 exists in a concentration-dependent monomer-dimer equilibrium in solution, with a backside dimerization interface identified by NMR. Dimer formation induces conformational dynamics at E1 and E3 interfaces.","method":"SAXS and NMR spectroscopy; GST-fusion construct used to dissociate dimer","journal":"ACS omega","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — two orthogonal structural methods (SAXS + NMR) in one study, peer-reviewed, single lab","pmids":["39346869"],"is_preprint":false},{"year":2020,"finding":"UBE2L6 is the E2 enzyme in the ISGylation cascade, functioning between E1 (UBA7) and E3 (HERC5). UBE2L6 depletion in NB4 APL cells attenuated ATRA-induced ISG15 conjugation and impeded ATRA-induced neutrophil differentiation, demonstrating a catalytic role in ISGylation that is functionally required for leukemic differentiation.","method":"shRNA-mediated UBE2L6 knockdown, Western blotting for ISG15 conjugates, differentiation assays","journal":"Molecular oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KD with defined cellular phenotype and biochemical readout, two cell line models tested","pmids":["31820845"],"is_preprint":false},{"year":2017,"finding":"UBE2L6 and ISG15 act as inhibitors of basal autophagic flux in esophageal cancer cells; siRNA depletion of UBE2L6 or ISG15 enhanced endogenous autophagy as measured by LC3II accumulation and Cyto-ID staining.","method":"siRNA knockdown, LC3II western blotting, Cyto-ID autophagy staining","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with specific biochemical and cellular readouts, multiple cell lines","pmids":["28186990"],"is_preprint":false},{"year":2023,"finding":"In high-pigmented melanoma cells, UBE2L6 (E2 conjugating enzyme) cooperates with UBR4 (E3 ligase) to ubiquitinate EZH2 at lysine 381, promoting EZH2 protein degradation. UHRF1-mediated CpG methylation downregulates UBE2L6 in low-pigmented melanoma cells, thereby stabilizing EZH2.","method":"Biochemical ubiquitination assays, siRNA knockdown, site-directed mutagenesis (K381), animal studies, proteasome inhibitor treatment","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — biochemical assays with mutagenesis identifying specific ubiquitination site, in vivo validation, multiple orthogonal methods","pmids":["36906655"],"is_preprint":false},{"year":2020,"finding":"UBE2L6 promotes ubiquitination of adipocyte triglyceride lipase (ATGL), reducing ATGL protein stability and lipolysis. Adipose-specific Ube2l6 knockout mice on a high-fat diet showed enhanced ATGL protein expression and increased ATGL-mediated lipolysis compared to controls.","method":"Adipose-specific knockout mouse model, Western blotting, lipolysis assays","journal":"Journal of pharmacological sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo knockout with biochemical substrate validation, single lab","pmids":["33712284"],"is_preprint":false},{"year":2013,"finding":"A hypomorphic loss-of-function allele of Ube2l6 (nonsynonymous SNP abolishing protein expression) in BALB/c mice prolongs ATGL half-life in adipose tissue and represses adipogenesis. Ube2l6 knockdown in 3T3-L1 adipocytes recapitulated repressed adipogenesis, indicating Ube2l6 promotes adipogenic differentiation.","method":"Congenic mouse strain generation, Ube2l6 siRNA knockdown in 3T3-L1 cells, adipogenesis assays, ATGL half-life measurement","journal":"Diabetes","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo genetic model plus in vitro knockdown, single lab, replicated across two experimental systems","pmids":["23557705"],"is_preprint":false},{"year":2023,"finding":"UBE2L6 promotes ISGylation of STAT1, leading to STAT1 activation and M1 macrophage polarization. Ube2l6 adipose-specific knockout mice showed reduced M1 macrophage polarization on high-fat diet, and co-immunoprecipitation confirmed UBE2L6 interaction with STAT1.","method":"Adipose-specific Ube2l6 knockout mice, Co-IP, flow cytometry, RT-qPCR, Western blotting, ELISA","journal":"Obesity facts","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo KO with Co-IP interaction validation and defined signaling phenotype, single lab","pmids":["37820603"],"is_preprint":false},{"year":2020,"finding":"UBE2L6 interacts with and ubiquitinates the Senecavirus A RNA-dependent RNA polymerase (3D protein) at lysines 169 and 321, inhibiting 3D degradation and promoting viral replication. This ubiquitination requires cysteine 86 in UBE2L6. Recombinant viruses with K169R and K321R mutations in 3D showed significantly decreased replication.","method":"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis, recombinant virus rescue","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — Co-IP, biochemical ubiquitination assay, mutagenesis of specific residues in both enzyme and substrate validated by recombinant virus phenotype","pmids":["33104725"],"is_preprint":false},{"year":2025,"finding":"PRRSV infection upregulates UBE2L6, which promotes K48-linked ubiquitination and proteasomal degradation of RIG-I and MDA5 (in cooperation with viral NSP5), thereby inhibiting type I IFN production and ISGylation. UBE2L6 also interacts with and stabilizes PRRSV NSP5. Knockdown of UBE2L6 reduced viral replication.","method":"Co-immunoprecipitation, knockdown/overexpression, Western blotting, ubiquitination assay with K48 linkage-specific analysis","journal":"Veterinary research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, specific K48-linked ubiquitination, loss-of-function phenotype, single lab","pmids":["40611336"],"is_preprint":false},{"year":2024,"finding":"p53 promotes FoxO3a ubiquitination and proteasomal degradation by upregulating UBE2L6 expression in retinal endothelial cells, accelerating cellular senescence in diabetic retinopathy.","method":"p53 inhibition, siRNA knockdown, Western blotting, ubiquitination assay, scRNA-seq and bulk RNA-seq in db/db mice","journal":"Experimental gerontology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — mechanism proposed based on expression upregulation and knockdown, direct ubiquitination of FoxO3a by UBE2L6 not fully biochemically reconstituted per abstract","pmids":["38437929"],"is_preprint":false},{"year":2021,"finding":"UBE2L6 upregulation induces proteasome-mediated degradation of Mcl-1 in FLT3-ITD AML cells; UBE2L6 knockdown protected Mcl-1 from degradation via the ubiquitin-proteasome system.","method":"UBE2L6 knockdown (shRNA), Western blotting for Mcl-1, proteasome inhibitor rescue","journal":"Journal of oncology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — knockdown with protein level readout, single lab, no direct ubiquitination biochemistry demonstrated per abstract","pmids":["34594375"],"is_preprint":false},{"year":2025,"finding":"UBE2L6 mediates proteasomal degradation of FLT3-ITD protein; decursin increases UBE2L6 expression and UBE2L6 knockdown by shRNA reduces FLT3-ITD degradation and FLT3-ITD-linked apoptosis.","method":"shRNA knockdown, Western blotting, cell viability assays, patient-derived mouse model","journal":"Cell communication and signaling","confidence":"Low","confidence_rationale":"Tier 3 / Weak — knockdown with protein degradation readout, no direct ubiquitination biochemistry shown per abstract, single lab","pmids":["40176110"],"is_preprint":false},{"year":2020,"finding":"UBE2L6 silencing in cisplatin-resistant cells reduced ABCB6 mRNA and ABCB6 promoter activity, suggesting UBE2L6 regulates cisplatin resistance through transcriptional control of ABCB6.","method":"siRNA knockdown, gene expression profiling, ABCB6 promoter reporter assay","journal":"Anti-cancer agents in medicinal chemistry","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, indirect mechanism (transcriptional regulation not directly linked to E2 enzymatic activity), single method","pmids":["32329696"],"is_preprint":false},{"year":2000,"finding":"UBE2L6 maps to chromosome 11q12 and its genomic organization (intron/exon structure) is highly conserved with its paralog UBE2L3, consistent with functional redundancy analogous to yeast UBC4/UBC5.","method":"Genomic cloning, sequence analysis, chromosomal mapping","journal":"Cytogenetics and cell genetics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — structural genomic characterization without functional experiment, single lab","pmids":["10894956"],"is_preprint":false}],"current_model":"UBE2L6 (UbcH8) is the E2 ubiquitin/ISG15-conjugating enzyme that acts between E1 (UBA7) and E3 (HERC5) in the ISGylation cascade — a role structurally defined by a cryo-EM complex with UBA7 and a SAXS/NMR-characterized monomer-dimer equilibrium — and it additionally performs canonical K48-linked ubiquitination of diverse substrates (EZH2 at K381 via UBR4, ATGL, RIG-I, MDA5, Mcl-1, FLT3-ITD, and viral polymerases), thereby regulating protein stability, innate immune signaling, autophagy, and adipogenesis."},"narrative":{"mechanistic_narrative":"UBE2L6 (UbcH8) is a dual-specificity E2 conjugating enzyme that bridges activating E1 and substrate-targeting E3 enzymes to drive both ISG15 conjugation (ISGylation) and canonical K48-linked ubiquitination, thereby controlling protein stability across innate immune signaling, lipid metabolism, and cell differentiation [PMID:31820845, PMID:36906655]. In the ISGylation cascade it operates as the E2 between E1 (UBA7) and E3 (HERC5), and its catalytic activity is required for ATRA-induced ISG15 conjugation and leukemic neutrophil differentiation [PMID:31820845]. Structurally, UBA7 binds UBE2L6 with specificity distinct from its paralog UBE2L3 through a disulfide-linked complex whose activation depends on the catalytic cysteine capping loop and catalytic-cysteine thiolate state [PMID:bio_10.1101_2024.11.07.622398], and UBE2L6 itself populates a concentration-dependent monomer-dimer equilibrium via a backside interface that modulates its E1 and E3 contacts [PMID:39346869]. As a ubiquitin-conjugating enzyme it cooperates with the E3 ligase UBR4 to ubiquitinate EZH2 at K381 and target it for degradation [PMID:36906655], promotes K48-linked ubiquitination of the innate sensors RIG-I and MDA5 to dampen type I interferon responses [PMID:40611336], and ubiquitinates the lipase ATGL to restrain lipolysis [PMID:33712284]. Beyond degradative targeting, UBE2L6 promotes ISGylation and activation of STAT1 to drive M1 macrophage polarization [PMID:37820603], and is exploited by viruses such as Senecavirus A, whose RNA-dependent RNA polymerase it ubiquitinates at K169/K321 (requiring cysteine 86) to stabilize the polymerase and enhance replication [PMID:33104725]. Through its control of ATGL stability it also acts as a positive regulator of adipogenic differentiation [PMID:23557705]. Lower-confidence reports extend its substrate range to FoxO3a, Mcl-1, and FLT3-ITD.","teleology":[{"year":2000,"claim":"Established the gene's identity and locus, framing UBE2L6 as a UBE2L3 paralog with conserved genomic architecture before any enzymatic function was assigned.","evidence":"Genomic cloning, sequence analysis, and chromosomal mapping to 11q12","pmids":["10894956"],"confidence":"Low","gaps":["No functional experiment; redundancy with UBE2L3 inferred from sequence, not tested","No substrate or pathway assignment"]},{"year":2013,"claim":"Defined a physiological role in metabolism by showing a loss-of-function Ube2l6 allele prolongs ATGL half-life and represses adipogenesis, establishing UBE2L6 as a positive regulator of adipogenic differentiation.","evidence":"Congenic mouse strain plus siRNA knockdown in 3T3-L1 adipocytes with ATGL half-life and adipogenesis assays","pmids":["23557705"],"confidence":"Medium","gaps":["Direct ubiquitination of ATGL by UBE2L6 not biochemically reconstituted here","E3 partner for ATGL not identified"]},{"year":2017,"claim":"Linked UBE2L6 to autophagy regulation, showing it acts with ISG15 to restrain basal autophagic flux.","evidence":"siRNA knockdown with LC3II western blotting and Cyto-ID staining in esophageal cancer cells","pmids":["28186990"],"confidence":"Medium","gaps":["Molecular target of ISGylation/ubiquitination controlling autophagy not defined","Mechanism linking ISG15 conjugation to flux unresolved"]},{"year":2020,"claim":"Cemented UBE2L6 as the catalytic E2 of the ISGylation cascade between UBA7 and HERC5 and tied this activity to a defined differentiation phenotype.","evidence":"shRNA knockdown in NB4 APL cells with ISG15-conjugate western blots and ATRA differentiation assays","pmids":["31820845"],"confidence":"Medium","gaps":["Specific ISGylation substrates driving differentiation not enumerated","Distinction between ISGylation and ubiquitination contributions not separated"]},{"year":2020,"claim":"Provided the first residue-level demonstration that UBE2L6 directly ubiquitinates a substrate, showing it modifies the Senecavirus A 3D polymerase at K169/K321 via catalytic Cys86 to stabilize it and promote viral replication.","evidence":"Co-IP, ubiquitination assay, enzyme and substrate mutagenesis, and recombinant virus rescue","pmids":["33104725"],"confidence":"High","gaps":["E3 ligase cooperating with UBE2L6 on the polymerase not identified","Ubiquitin linkage type not specified"]},{"year":2020,"claim":"Tested UBE2L6 in drug resistance, indicating it influences cisplatin resistance through transcriptional control of ABCB6.","evidence":"siRNA knockdown, expression profiling, and ABCB6 promoter reporter assay","pmids":["32329696"],"confidence":"Low","gaps":["Transcriptional effect not connected to E2 enzymatic activity","Single method, single lab; mechanism indirect"]},{"year":2021,"claim":"Extended the substrate repertoire toward leukemia survival proteins by linking UBE2L6 to proteasomal degradation of Mcl-1 in FLT3-ITD AML.","evidence":"shRNA knockdown with Mcl-1 western blotting and proteasome inhibitor rescue","pmids":["34594375"],"confidence":"Low","gaps":["No direct ubiquitination biochemistry shown","Single lab, protein-level readout only"]},{"year":2023,"claim":"Identified a specific E2-E3 pairing and ubiquitination site by showing UBE2L6 cooperates with UBR4 to ubiquitinate EZH2 at K381 for degradation, with epigenetic silencing of UBE2L6 stabilizing EZH2 in melanoma.","evidence":"Biochemical ubiquitination assays, K381 mutagenesis, siRNA, proteasome inhibition, and animal studies","pmids":["36906655"],"confidence":"High","gaps":["Ubiquitin linkage type on EZH2 not specified","Generality beyond melanoma context untested"]},{"year":2023,"claim":"Demonstrated a signaling-activation role distinct from degradation, showing UBE2L6 ISGylates and activates STAT1 to promote M1 macrophage polarization in adipose tissue.","evidence":"Adipose-specific Ube2l6 knockout mice, Co-IP with STAT1, flow cytometry, and expression analyses","pmids":["37820603"],"confidence":"Medium","gaps":["STAT1 ISGylation site not mapped","E3 ligase for STAT1 ISGylation not identified"]},{"year":2024,"claim":"Resolved the structural basis for E1-E2 selectivity, showing UBA7 binds UBE2L6 (not UBE2L3) through a disulfide complex governed by the catalytic cysteine capping loop and thiolate state.","evidence":"Cryo-EM of the UBA7•UBE2L6 complex with biochemical reconstitution and mutagenesis (preprint)","pmids":["bio_10.1101_2024.11.07.622398"],"confidence":"Medium","gaps":["Preprint, single lab, not peer-reviewed","Structure of E2-E3 (HERC5) transfer step not resolved"]},{"year":2024,"claim":"Revealed an intrinsic regulatory feature, a concentration-dependent monomer-dimer equilibrium with a backside interface that reshapes E1 and E3 contact surfaces.","evidence":"SAXS and NMR spectroscopy with GST-fusion dimer dissociation","pmids":["39346869"],"confidence":"Medium","gaps":["Functional consequence of dimerization in cells untested","Physiological concentrations relative to equilibrium unknown"]},{"year":2024,"claim":"Placed UBE2L6 downstream of p53 in a senescence circuit, indicating p53-induced UBE2L6 promotes FoxO3a degradation in diabetic retinopathy.","evidence":"p53 inhibition, siRNA, ubiquitination assay, and scRNA-seq/bulk RNA-seq in db/db mice","pmids":["38437929"],"confidence":"Low","gaps":["Direct ubiquitination of FoxO3a by UBE2L6 not fully reconstituted","Largely expression- and knockdown-based"]},{"year":2025,"claim":"Defined a viral-hijack mechanism in which PRRSV upregulates UBE2L6 to drive K48-linked degradation of RIG-I and MDA5, suppressing type I IFN, while UBE2L6 stabilizes viral NSP5.","evidence":"Co-IP, knockdown/overexpression, and K48 linkage-specific ubiquitination assays","pmids":["40611336"],"confidence":"Medium","gaps":["E3 ligase mediating RIG-I/MDA5 degradation not pinpointed","Direct versus NSP5-bridged ubiquitination contributions not separated"]},{"year":2025,"claim":"Reinforced a therapeutic angle in AML by showing UBE2L6 mediates FLT3-ITD degradation, sensitizing cells to decursin-induced apoptosis.","evidence":"shRNA knockdown, western blotting, viability assays, and patient-derived mouse model","pmids":["40176110"],"confidence":"Low","gaps":["No direct ubiquitination biochemistry on FLT3-ITD","Single lab; degradation readout only"]},{"year":null,"claim":"How UBE2L6 partitions between ISG15- and ubiquitin-conjugation, selects among its many reported substrates, and which E3 ligases pair with it in each context remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["Determinants of ISG15 versus ubiquitin charging in cells unknown","E3 partners for most substrates (ATGL, STAT1, RIG-I/MDA5, FLT3-ITD) unidentified","Structure of the productive E2-E3 transfer complex not solved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[2,4,8,9]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[4,8]},{"term_id":"GO:0031386","term_label":"protein tag activity","supporting_discovery_ids":[2,7]}],"localization":[],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[2,7,9]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[4,5,8]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[3]}],"complexes":[],"partners":["UBA7","HERC5","UBR4","STAT1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O14933","full_name":"Ubiquitin/ISG15-conjugating enzyme E2 L6","aliases":["E2 ubiquitin-conjugating enzyme L6","Retinoic acid-induced gene B protein","RIG-B","UbcH8","Ubiquitin carrier protein L6","Ubiquitin-protein ligase L6"],"length_aa":153,"mass_kda":17.8,"function":"Catalyzes the covalent attachment of ubiquitin or ISG15 to other proteins. Functions in the E6/E6-AP-induced ubiquitination of p53/TP53. Promotes ubiquitination and subsequent proteasomal degradation of FLT3","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/O14933/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/UBE2L6","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/UBE2L6","total_profiled":1310},"omim":[{"mim_id":"618869","title":"RING FINGER PROTEIN 144B; RNF144B","url":"https://www.omim.org/entry/618869"},{"mim_id":"612548","title":"TRIPARTITE MOTIF-CONTAINING PROTEIN 50; TRIM50","url":"https://www.omim.org/entry/612548"},{"mim_id":"610872","title":"RING FINGER PROTEIN 19B; RNF19B","url":"https://www.omim.org/entry/610872"},{"mim_id":"610432","title":"RING FINGER PROTEIN 125; RNF125","url":"https://www.omim.org/entry/610432"},{"mim_id":"609631","title":"RNA SENSOR RIGI; RIGI","url":"https://www.omim.org/entry/609631"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/UBE2L6"},"hgnc":{"alias_symbol":["UBCH8"],"prev_symbol":[]},"alphafold":{"accession":"O14933","domains":[{"cath_id":"3.10.110.10","chopping":"1-153","consensus_level":"medium","plddt":95.8541,"start":1,"end":153}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O14933","model_url":"https://alphafold.ebi.ac.uk/files/AF-O14933-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O14933-F1-predicted_aligned_error_v6.png","plddt_mean":95.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=UBE2L6","jax_strain_url":"https://www.jax.org/strain/search?query=UBE2L6"},"sequence":{"accession":"O14933","fasta_url":"https://rest.uniprot.org/uniprotkb/O14933.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O14933/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O14933"}},"corpus_meta":[{"pmid":"28186990","id":"PMC_28186990","title":"UBE2L6/UBCH8 and ISG15 attenuate autophagy in esophageal cancer cells.","date":"2017","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/28186990","citation_count":35,"is_preprint":false},{"pmid":"33104725","id":"PMC_33104725","title":"E2 ubiquitin-conjugating enzyme UBE2L6 promotes Senecavirus A proliferation by stabilizing the viral RNA polymerase.","date":"2020","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/33104725","citation_count":34,"is_preprint":false},{"pmid":"15670748","id":"PMC_15670748","title":"Down-regulation of UCRP and UBE2L6 in BRCA2 knocked-down human breast cells.","date":"2005","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/15670748","citation_count":20,"is_preprint":false},{"pmid":"38437929","id":"PMC_38437929","title":"p53 accelerates endothelial cell senescence in diabetic retinopathy by enhancing FoxO3a ubiquitylation and degradation via UBE2L6.","date":"2024","source":"Experimental 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biology","url":"https://pubmed.ncbi.nlm.nih.gov/37090523","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.11.07.622398","title":"Elucidating the Mechanism Underlying UBA7•UBE2L6 Disulfide Complex Formation","date":"2024-11-08","source":"bioRxiv","url":"https://doi.org/10.1101/2024.11.07.622398","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":12559,"output_tokens":3556,"usd":0.045509,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11076,"output_tokens":4531,"usd":0.084327,"stage2_stop_reason":"end_turn"},"total_usd":0.129836,"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\": 2024,\n      \"finding\": \"Cryo-EM structure of the UBA7•UBE2L6 disulfide complex reveals three factors governing complex formation: (1) strong binding affinity and specificity of UBA7 for UBE2L6 (distinct from UBE2L3); (2) conformational differences in the catalytic cysteine capping loop (CCL); and (3) increased thiolate/thiol ratios at catalytic cysteines. Modification of any of these factors impacts complex activation and ISG15 transfer cascade.\",\n      \"method\": \"Cryo-EM structure determination, biochemical reconstitution, mutagenesis\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — cryo-EM structure with mechanistic dissection, but preprint, single lab, not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2024.11.07.622398\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"UBE2L6/UbcH8 exists in a concentration-dependent monomer-dimer equilibrium in solution, with a backside dimerization interface identified by NMR. Dimer formation induces conformational dynamics at E1 and E3 interfaces.\",\n      \"method\": \"SAXS and NMR spectroscopy; GST-fusion construct used to dissociate dimer\",\n      \"journal\": \"ACS omega\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — two orthogonal structural methods (SAXS + NMR) in one study, peer-reviewed, single lab\",\n      \"pmids\": [\"39346869\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"UBE2L6 is the E2 enzyme in the ISGylation cascade, functioning between E1 (UBA7) and E3 (HERC5). UBE2L6 depletion in NB4 APL cells attenuated ATRA-induced ISG15 conjugation and impeded ATRA-induced neutrophil differentiation, demonstrating a catalytic role in ISGylation that is functionally required for leukemic differentiation.\",\n      \"method\": \"shRNA-mediated UBE2L6 knockdown, Western blotting for ISG15 conjugates, differentiation assays\",\n      \"journal\": \"Molecular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KD with defined cellular phenotype and biochemical readout, two cell line models tested\",\n      \"pmids\": [\"31820845\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"UBE2L6 and ISG15 act as inhibitors of basal autophagic flux in esophageal cancer cells; siRNA depletion of UBE2L6 or ISG15 enhanced endogenous autophagy as measured by LC3II accumulation and Cyto-ID staining.\",\n      \"method\": \"siRNA knockdown, LC3II western blotting, Cyto-ID autophagy staining\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with specific biochemical and cellular readouts, multiple cell lines\",\n      \"pmids\": [\"28186990\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In high-pigmented melanoma cells, UBE2L6 (E2 conjugating enzyme) cooperates with UBR4 (E3 ligase) to ubiquitinate EZH2 at lysine 381, promoting EZH2 protein degradation. UHRF1-mediated CpG methylation downregulates UBE2L6 in low-pigmented melanoma cells, thereby stabilizing EZH2.\",\n      \"method\": \"Biochemical ubiquitination assays, siRNA knockdown, site-directed mutagenesis (K381), animal studies, proteasome inhibitor treatment\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — biochemical assays with mutagenesis identifying specific ubiquitination site, in vivo validation, multiple orthogonal methods\",\n      \"pmids\": [\"36906655\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"UBE2L6 promotes ubiquitination of adipocyte triglyceride lipase (ATGL), reducing ATGL protein stability and lipolysis. Adipose-specific Ube2l6 knockout mice on a high-fat diet showed enhanced ATGL protein expression and increased ATGL-mediated lipolysis compared to controls.\",\n      \"method\": \"Adipose-specific knockout mouse model, Western blotting, lipolysis assays\",\n      \"journal\": \"Journal of pharmacological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo knockout with biochemical substrate validation, single lab\",\n      \"pmids\": [\"33712284\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"A hypomorphic loss-of-function allele of Ube2l6 (nonsynonymous SNP abolishing protein expression) in BALB/c mice prolongs ATGL half-life in adipose tissue and represses adipogenesis. Ube2l6 knockdown in 3T3-L1 adipocytes recapitulated repressed adipogenesis, indicating Ube2l6 promotes adipogenic differentiation.\",\n      \"method\": \"Congenic mouse strain generation, Ube2l6 siRNA knockdown in 3T3-L1 cells, adipogenesis assays, ATGL half-life measurement\",\n      \"journal\": \"Diabetes\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo genetic model plus in vitro knockdown, single lab, replicated across two experimental systems\",\n      \"pmids\": [\"23557705\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"UBE2L6 promotes ISGylation of STAT1, leading to STAT1 activation and M1 macrophage polarization. Ube2l6 adipose-specific knockout mice showed reduced M1 macrophage polarization on high-fat diet, and co-immunoprecipitation confirmed UBE2L6 interaction with STAT1.\",\n      \"method\": \"Adipose-specific Ube2l6 knockout mice, Co-IP, flow cytometry, RT-qPCR, Western blotting, ELISA\",\n      \"journal\": \"Obesity facts\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo KO with Co-IP interaction validation and defined signaling phenotype, single lab\",\n      \"pmids\": [\"37820603\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"UBE2L6 interacts with and ubiquitinates the Senecavirus A RNA-dependent RNA polymerase (3D protein) at lysines 169 and 321, inhibiting 3D degradation and promoting viral replication. This ubiquitination requires cysteine 86 in UBE2L6. Recombinant viruses with K169R and K321R mutations in 3D showed significantly decreased replication.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis, recombinant virus rescue\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — Co-IP, biochemical ubiquitination assay, mutagenesis of specific residues in both enzyme and substrate validated by recombinant virus phenotype\",\n      \"pmids\": [\"33104725\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PRRSV infection upregulates UBE2L6, which promotes K48-linked ubiquitination and proteasomal degradation of RIG-I and MDA5 (in cooperation with viral NSP5), thereby inhibiting type I IFN production and ISGylation. UBE2L6 also interacts with and stabilizes PRRSV NSP5. Knockdown of UBE2L6 reduced viral replication.\",\n      \"method\": \"Co-immunoprecipitation, knockdown/overexpression, Western blotting, ubiquitination assay with K48 linkage-specific analysis\",\n      \"journal\": \"Veterinary research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, specific K48-linked ubiquitination, loss-of-function phenotype, single lab\",\n      \"pmids\": [\"40611336\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"p53 promotes FoxO3a ubiquitination and proteasomal degradation by upregulating UBE2L6 expression in retinal endothelial cells, accelerating cellular senescence in diabetic retinopathy.\",\n      \"method\": \"p53 inhibition, siRNA knockdown, Western blotting, ubiquitination assay, scRNA-seq and bulk RNA-seq in db/db mice\",\n      \"journal\": \"Experimental gerontology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — mechanism proposed based on expression upregulation and knockdown, direct ubiquitination of FoxO3a by UBE2L6 not fully biochemically reconstituted per abstract\",\n      \"pmids\": [\"38437929\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"UBE2L6 upregulation induces proteasome-mediated degradation of Mcl-1 in FLT3-ITD AML cells; UBE2L6 knockdown protected Mcl-1 from degradation via the ubiquitin-proteasome system.\",\n      \"method\": \"UBE2L6 knockdown (shRNA), Western blotting for Mcl-1, proteasome inhibitor rescue\",\n      \"journal\": \"Journal of oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — knockdown with protein level readout, single lab, no direct ubiquitination biochemistry demonstrated per abstract\",\n      \"pmids\": [\"34594375\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"UBE2L6 mediates proteasomal degradation of FLT3-ITD protein; decursin increases UBE2L6 expression and UBE2L6 knockdown by shRNA reduces FLT3-ITD degradation and FLT3-ITD-linked apoptosis.\",\n      \"method\": \"shRNA knockdown, Western blotting, cell viability assays, patient-derived mouse model\",\n      \"journal\": \"Cell communication and signaling\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — knockdown with protein degradation readout, no direct ubiquitination biochemistry shown per abstract, single lab\",\n      \"pmids\": [\"40176110\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"UBE2L6 silencing in cisplatin-resistant cells reduced ABCB6 mRNA and ABCB6 promoter activity, suggesting UBE2L6 regulates cisplatin resistance through transcriptional control of ABCB6.\",\n      \"method\": \"siRNA knockdown, gene expression profiling, ABCB6 promoter reporter assay\",\n      \"journal\": \"Anti-cancer agents in medicinal chemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, indirect mechanism (transcriptional regulation not directly linked to E2 enzymatic activity), single method\",\n      \"pmids\": [\"32329696\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"UBE2L6 maps to chromosome 11q12 and its genomic organization (intron/exon structure) is highly conserved with its paralog UBE2L3, consistent with functional redundancy analogous to yeast UBC4/UBC5.\",\n      \"method\": \"Genomic cloning, sequence analysis, chromosomal mapping\",\n      \"journal\": \"Cytogenetics and cell genetics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — structural genomic characterization without functional experiment, single lab\",\n      \"pmids\": [\"10894956\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"UBE2L6 (UbcH8) is the E2 ubiquitin/ISG15-conjugating enzyme that acts between E1 (UBA7) and E3 (HERC5) in the ISGylation cascade — a role structurally defined by a cryo-EM complex with UBA7 and a SAXS/NMR-characterized monomer-dimer equilibrium — and it additionally performs canonical K48-linked ubiquitination of diverse substrates (EZH2 at K381 via UBR4, ATGL, RIG-I, MDA5, Mcl-1, FLT3-ITD, and viral polymerases), thereby regulating protein stability, innate immune signaling, autophagy, and adipogenesis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"UBE2L6 (UbcH8) is a dual-specificity E2 conjugating enzyme that bridges activating E1 and substrate-targeting E3 enzymes to drive both ISG15 conjugation (ISGylation) and canonical K48-linked ubiquitination, thereby controlling protein stability across innate immune signaling, lipid metabolism, and cell differentiation [#2, #4]. In the ISGylation cascade it operates as the E2 between E1 (UBA7) and E3 (HERC5), and its catalytic activity is required for ATRA-induced ISG15 conjugation and leukemic neutrophil differentiation [#2]. Structurally, UBA7 binds UBE2L6 with specificity distinct from its paralog UBE2L3 through a disulfide-linked complex whose activation depends on the catalytic cysteine capping loop and catalytic-cysteine thiolate state [#0], and UBE2L6 itself populates a concentration-dependent monomer-dimer equilibrium via a backside interface that modulates its E1 and E3 contacts [#1]. As a ubiquitin-conjugating enzyme it cooperates with the E3 ligase UBR4 to ubiquitinate EZH2 at K381 and target it for degradation [#4], promotes K48-linked ubiquitination of the innate sensors RIG-I and MDA5 to dampen type I interferon responses [#9], and ubiquitinates the lipase ATGL to restrain lipolysis [#5]. Beyond degradative targeting, UBE2L6 promotes ISGylation and activation of STAT1 to drive M1 macrophage polarization [#7], and is exploited by viruses such as Senecavirus A, whose RNA-dependent RNA polymerase it ubiquitinates at K169/K321 (requiring cysteine 86) to stabilize the polymerase and enhance replication [#8]. Through its control of ATGL stability it also acts as a positive regulator of adipogenic differentiation [#6]. Lower-confidence reports extend its substrate range to FoxO3a, Mcl-1, and FLT3-ITD.\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established the gene's identity and locus, framing UBE2L6 as a UBE2L3 paralog with conserved genomic architecture before any enzymatic function was assigned.\",\n      \"evidence\": \"Genomic cloning, sequence analysis, and chromosomal mapping to 11q12\",\n      \"pmids\": [\"10894956\"],\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No functional experiment; redundancy with UBE2L3 inferred from sequence, not tested\", \"No substrate or pathway assignment\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined a physiological role in metabolism by showing a loss-of-function Ube2l6 allele prolongs ATGL half-life and represses adipogenesis, establishing UBE2L6 as a positive regulator of adipogenic differentiation.\",\n      \"evidence\": \"Congenic mouse strain plus siRNA knockdown in 3T3-L1 adipocytes with ATGL half-life and adipogenesis assays\",\n      \"pmids\": [\"23557705\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Direct ubiquitination of ATGL by UBE2L6 not biochemically reconstituted here\", \"E3 partner for ATGL not identified\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Linked UBE2L6 to autophagy regulation, showing it acts with ISG15 to restrain basal autophagic flux.\",\n      \"evidence\": \"siRNA knockdown with LC3II western blotting and Cyto-ID staining in esophageal cancer cells\",\n      \"pmids\": [\"28186990\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Molecular target of ISGylation/ubiquitination controlling autophagy not defined\", \"Mechanism linking ISG15 conjugation to flux unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Cemented UBE2L6 as the catalytic E2 of the ISGylation cascade between UBA7 and HERC5 and tied this activity to a defined differentiation phenotype.\",\n      \"evidence\": \"shRNA knockdown in NB4 APL cells with ISG15-conjugate western blots and ATRA differentiation assays\",\n      \"pmids\": [\"31820845\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Specific ISGylation substrates driving differentiation not enumerated\", \"Distinction between ISGylation and ubiquitination contributions not separated\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Provided the first residue-level demonstration that UBE2L6 directly ubiquitinates a substrate, showing it modifies the Senecavirus A 3D polymerase at K169/K321 via catalytic Cys86 to stabilize it and promote viral replication.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, enzyme and substrate mutagenesis, and recombinant virus rescue\",\n      \"pmids\": [\"33104725\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"E3 ligase cooperating with UBE2L6 on the polymerase not identified\", \"Ubiquitin linkage type not specified\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Tested UBE2L6 in drug resistance, indicating it influences cisplatin resistance through transcriptional control of ABCB6.\",\n      \"evidence\": \"siRNA knockdown, expression profiling, and ABCB6 promoter reporter assay\",\n      \"pmids\": [\"32329696\"],\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Transcriptional effect not connected to E2 enzymatic activity\", \"Single method, single lab; mechanism indirect\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Extended the substrate repertoire toward leukemia survival proteins by linking UBE2L6 to proteasomal degradation of Mcl-1 in FLT3-ITD AML.\",\n      \"evidence\": \"shRNA knockdown with Mcl-1 western blotting and proteasome inhibitor rescue\",\n      \"pmids\": [\"34594375\"],\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No direct ubiquitination biochemistry shown\", \"Single lab, protein-level readout only\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified a specific E2-E3 pairing and ubiquitination site by showing UBE2L6 cooperates with UBR4 to ubiquitinate EZH2 at K381 for degradation, with epigenetic silencing of UBE2L6 stabilizing EZH2 in melanoma.\",\n      \"evidence\": \"Biochemical ubiquitination assays, K381 mutagenesis, siRNA, proteasome inhibition, and animal studies\",\n      \"pmids\": [\"36906655\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Ubiquitin linkage type on EZH2 not specified\", \"Generality beyond melanoma context untested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrated a signaling-activation role distinct from degradation, showing UBE2L6 ISGylates and activates STAT1 to promote M1 macrophage polarization in adipose tissue.\",\n      \"evidence\": \"Adipose-specific Ube2l6 knockout mice, Co-IP with STAT1, flow cytometry, and expression analyses\",\n      \"pmids\": [\"37820603\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"STAT1 ISGylation site not mapped\", \"E3 ligase for STAT1 ISGylation not identified\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Resolved the structural basis for E1-E2 selectivity, showing UBA7 binds UBE2L6 (not UBE2L3) through a disulfide complex governed by the catalytic cysteine capping loop and thiolate state.\",\n      \"evidence\": \"Cryo-EM of the UBA7•UBE2L6 complex with biochemical reconstitution and mutagenesis (preprint)\",\n      \"pmids\": [\"bio_10.1101_2024.11.07.622398\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Preprint, single lab, not peer-reviewed\", \"Structure of E2-E3 (HERC5) transfer step not resolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealed an intrinsic regulatory feature, a concentration-dependent monomer-dimer equilibrium with a backside interface that reshapes E1 and E3 contact surfaces.\",\n      \"evidence\": \"SAXS and NMR spectroscopy with GST-fusion dimer dissociation\",\n      \"pmids\": [\"39346869\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Functional consequence of dimerization in cells untested\", \"Physiological concentrations relative to equilibrium unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Placed UBE2L6 downstream of p53 in a senescence circuit, indicating p53-induced UBE2L6 promotes FoxO3a degradation in diabetic retinopathy.\",\n      \"evidence\": \"p53 inhibition, siRNA, ubiquitination assay, and scRNA-seq/bulk RNA-seq in db/db mice\",\n      \"pmids\": [\"38437929\"],\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Direct ubiquitination of FoxO3a by UBE2L6 not fully reconstituted\", \"Largely expression- and knockdown-based\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined a viral-hijack mechanism in which PRRSV upregulates UBE2L6 to drive K48-linked degradation of RIG-I and MDA5, suppressing type I IFN, while UBE2L6 stabilizes viral NSP5.\",\n      \"evidence\": \"Co-IP, knockdown/overexpression, and K48 linkage-specific ubiquitination assays\",\n      \"pmids\": [\"40611336\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"E3 ligase mediating RIG-I/MDA5 degradation not pinpointed\", \"Direct versus NSP5-bridged ubiquitination contributions not separated\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Reinforced a therapeutic angle in AML by showing UBE2L6 mediates FLT3-ITD degradation, sensitizing cells to decursin-induced apoptosis.\",\n      \"evidence\": \"shRNA knockdown, western blotting, viability assays, and patient-derived mouse model\",\n      \"pmids\": [\"40176110\"],\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No direct ubiquitination biochemistry on FLT3-ITD\", \"Single lab; degradation readout only\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How UBE2L6 partitions between ISG15- and ubiquitin-conjugation, selects among its many reported substrates, and which E3 ligases pair with it in each context remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Determinants of ISG15 versus ubiquitin charging in cells unknown\", \"E3 partners for most substrates (ATGL, STAT1, RIG-I/MDA5, FLT3-ITD) unidentified\", \"Structure of the productive E2-E3 transfer complex not solved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [2, 4, 8, 9]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [4, 8]},\n      {\"term_id\": \"GO:0031386\", \"supporting_discovery_ids\": [2, 7]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [2, 7, 9]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [4, 5, 8]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"UBA7\", \"HERC5\", \"UBR4\", \"STAT1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}