{"gene":"UBE2O","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":1996,"finding":"E2-230K (UBE2O) functions as an E2-E3 hybrid enzyme. Phenylarsenoxide inhibition studies demonstrated that two cysteine residues sequentially form thiol esters with ubiquitin (a 'thiol relay'), consistent with the enzyme possessing both E2 and intrinsic E3 activities without requiring a separate E3 ligase.","method":"In vitro biochemical assay with phenylarsenoxide and NEM inhibitors; E2~ubiquitin adduct formation assay; active-site cysteine protection experiments","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro mechanistic reconstitution with active-site probes and multiple orthogonal inhibitor experiments in a single rigorous study","pmids":["8634298"],"is_preprint":false},{"year":2013,"finding":"UBE2O functions as an E2-E3 hybrid to monoubiquitinate SMAD6 at lysine 174; the catalytic cysteine C885 of UBE2O is essential for this activity. Monoubiquitination of SMAD6 impairs its binding to the BMP type I receptor, thereby potentiating BMP7/SMAD1 signaling and adipogenesis.","method":"Proteomic interaction screen, co-IP, in vitro ubiquitination assay, site-directed mutagenesis (K174R on SMAD6; C885A on UBE2O), SMAD1 phosphorylation reporter assays, luciferase transcriptional assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro ubiquitination with active-site mutagenesis, substrate lysine mapping, and functional receptor-binding consequence demonstrated in a single study with multiple orthogonal methods","pmids":["23455153"],"is_preprint":false},{"year":2013,"finding":"UBE2O binds TRAF6 and inhibits its K63-polyubiquitination, preventing TAB2/3-TAK1 recruitment and NF-κB activation downstream of IL-1R/TLR. The inhibitory effect is independent of the UBC domain of UBE2O and instead operates by disrupting the IL-1β-induced TRAF6–MyD88 association.","method":"Co-IP, in vitro ubiquitination assay, NF-κB luciferase reporter assay, domain-deletion constructs, IL-1β/LPS stimulation experiments","journal":"Cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP plus functional NF-κB reporter and domain-mapping in a single lab with multiple methods","pmids":["23381138"],"is_preprint":false},{"year":2014,"finding":"UBE2O multi-monoubiquitinates the nuclear localization signal (NLS) of BAP1, inducing its cytoplasmic sequestration. BAP1 autodeubiquitination via intramolecular interactions counteracts this, maintaining nuclear localization. Cancer-derived BAP1 mutations that abrogate autodeubiquitination promote cytoplasmic retention. UBE2O also promotes cytoplasmic localization of BAP1 during adipogenesis.","method":"Co-IP, in vitro ubiquitination assay, subcellular fractionation/immunofluorescence, site-directed mutagenesis of BAP1 NLS and catalytic residues, cancer mutation analysis, adipogenesis differentiation assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (in vitro ubiquitination, fractionation, mutagenesis, cancer mutations) with functional localization consequence; replicated across cell types and differentiation contexts","pmids":["24703950"],"is_preprint":false},{"year":2017,"finding":"UBE2O directly recognizes juxtaposed basic and hydrophobic patches on unassembled (orphan) proteins and mediates their ubiquitination without a separate E3 ligase. In reticulocytes, unassembled α-globin that fails to pair with β-globin is selectively ubiquitinated by UBE2O. In non-reticulocytes, ribosomal proteins that do not engage nuclear import factors are UBE2O targets, directing them to proteasomal degradation.","method":"In vitro ubiquitination reconstitution, proteomic substrate identification, cell-based ubiquitination assays, substrate feature mapping (basic/hydrophobic patches)","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct biochemical reconstitution of substrate recognition and ubiquitination without E3; replicated across multiple substrates and cell types; published simultaneously with independent corroborating paper (PMID 28774900)","pmids":["28774922"],"is_preprint":false},{"year":2017,"finding":"UBE2O is a broad-spectrum ubiquitinating enzyme that remodels the erythroid proteome during reticulocyte differentiation. A loss-of-function mutation in murine Ube2o causes anemia. UBE2O directly recognizes ribosomal proteins and other substrates, targeting them for proteasomal degradation, and ribosome elimination is defective in Ube2o mutants.","method":"Mouse genetic mutant (Ube2o loss-of-function), quantitative proteomics of reticulocyte proteome, in vivo and in vitro ubiquitination assays, ribosome content analysis","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 2 / Strong — mouse genetic KO with defined anemia phenotype, quantitative proteomics, and direct substrate ubiquitination assays; corroborated by independent simultaneous publication (PMID 28774922)","pmids":["28774900"],"is_preprint":false},{"year":2017,"finding":"UBE2O specifically targets AMPKα2 (but not AMPKα1) for ubiquitination and proteasomal degradation, thereby activating the mTOR-HIF1α pathway and promoting tumor initiation and progression. Genetic deletion of Ube2o in mouse cancer models impairs tumorigenesis, and inactivation of AMPKα2 (but not AMPKα1) abrogates the tumor-attenuating effect of UBE2O loss.","method":"Ube2o knockout mouse cancer models (MMTV-PyVT, TRAMP), in vitro ubiquitination assay, epistasis via AMPKα1/α2 isoform-selective inactivation, rapamycin treatment rescue, HIF1α inhibitor rescue","journal":"Cancer cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo mouse genetic epistasis with isoform-selective AMPKα2 rescue, in vitro ubiquitination confirmation, and pharmacological validation via rapamycin/HIF1α inhibition","pmids":["28162974"],"is_preprint":false},{"year":2017,"finding":"UBE2O interacts with and mediates polyubiquitination and proteasomal degradation of c-Maf, a transcription factor critical in multiple myeloma, thereby reducing c-Maf transcriptional activity and cyclin D2 expression.","method":"Affinity purification/tandem MS, co-IP, in vitro polyubiquitination assay, immunoblotting for c-Maf stability, luciferase transcriptional assay, lentiviral re-expression, xenograft mouse model","journal":"Journal of hematology & oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP plus in vitro ubiquitination and functional transcriptional assays, single lab, multiple methods","pmids":["28673317"],"is_preprint":false},{"year":2018,"finding":"UBE2O interacts with and promotes ubiquitination and proteasomal degradation of BMAL1, the core circadian clock transcription factor. The conserved region 2 (CR2) domain of UBE2O is required for BMAL1 ubiquitination, and a catalytic Cys-to-Ser substitution in CR2 abolishes this activity. UBE2O knockdown elevates circadian clock amplitude in U2OS cells.","method":"Co-IP and MS, immunoblotting for BMAL1 stability, in vitro ubiquitination assay, domain-mapping and Cys→Ser mutagenesis, bioluminescence circadian reporter assay in U2OS cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — active-site mutagenesis with domain mapping, in vitro ubiquitination, and functional circadian phenotype readout; multiple orthogonal methods in single study","pmids":["29871923"],"is_preprint":false},{"year":2019,"finding":"In skeletal muscle, UBE2O acts as a ubiquitin ligase that targets AMPKα2 for ubiquitin-dependent degradation. Muscle-specific knockout of Ube2o improves insulin sensitivity and energy expenditure, and muscle-specific heterozygous knockout of Prkaa2 (AMPKα2) abrogates the metabolic benefits of Ube2o loss, establishing UBE2O→AMPKα2 as the causal axis in skeletal muscle metabolic regulation.","method":"Tissue-specific (muscle, fat, liver) Ube2o knockout mice, hyperinsulinemic-euglycemic clamp, Prkaa2 heterozygous genetic epistasis, in vitro ubiquitination assay, metabolic phenotyping","journal":"JCI insight","confidence":"High","confidence_rationale":"Tier 2 / Strong — tissue-specific genetic KO with rigorous metabolic phenotyping, in vivo epistasis using AMPKα2 haploinsufficiency, and in vitro ubiquitination; multiple orthogonal methods across multiple mouse models","pmids":["31292296"],"is_preprint":false},{"year":2020,"finding":"UBE2O interacts with and targets Mxi1 for ubiquitination and degradation at the K46 residue of Mxi1. This promotes lung cancer progression and radioresistance; these effects are reversed by Mxi1 inhibition.","method":"Co-IP, in vitro ubiquitination assay, site-directed mutagenesis (Mxi1 K46R), genetic/pharmacological UBE2O blockade with Mxi1 rescue, in vitro and in vivo tumor models","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus substrate lysine-site mutagenesis and in vivo rescue epistasis, single lab with multiple methods","pmids":["32901121"],"is_preprint":false},{"year":2020,"finding":"UBE2O decreases SMAD6 protein levels, thereby activating BMP2 signaling to promote angiogenesis in wound healing; saliva exosome-delivered UBE2O mRNA reproduces this effect in endothelial cells.","method":"In vitro HUVEC proliferation/migration/angiogenesis assays, in vivo wound healing model, siRNA knockdown and overexpression, western blot for SMAD6 and BMP2 pathway","journal":"Journal of nanobiotechnology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, phenotypic assays with pathway inference; no direct in vitro ubiquitination reconstitution of SMAD6 by UBE2O in this paper","pmids":["32375794"],"is_preprint":false},{"year":2022,"finding":"UBE2O client selection is regulated by two mechanisms: (1) a feed-forward mechanism where a single ubiquitin added to a client enhances UBE2O binding and promotes multi-mono-ubiquitylation via a distinct SH3-like ubiquitin-binding domain; (2) the histone chaperone NAP1L1 acts as a cofactor/adapter to recruit a subset of UBE2O clients. Cryo-EM structures of human UBE2O with NAP1L1 reveal a malleable client recruitment interface autoinhibited by the reactive UBC domain.","method":"Cryo-EM structure determination of human UBE2O–NAP1L1 complex, biochemical ubiquitylation assays, identification of SH3-like ubiquitin-binding domain by mutagenesis, client ubiquitylation reconstitution","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure plus biochemical reconstitution and mutagenesis of client-binding and ubiquitin-binding domains; multiple orthogonal methods in one rigorous study","pmids":["35915257"],"is_preprint":false},{"year":2022,"finding":"UBE2O physically interacts with RECQL4 and mediates its multi-monoubiquitinylation, leading to proteasomal degradation. This inhibits homologous recombination (HR)-mediated DSB repair by attenuating RECQL4 interactions with MRE11-RAD50-NBS1 and CtIP. The deubiquitinase USP7 antagonizes UBE2O by interacting with both UBE2O and RECQL4 to stabilize RECQL4.","method":"Co-IP, in vitro ubiquitination assay, HR reporter assay, UBE2O catalytic mutant (E2 activity-dead), rescue experiments with RECQL4 overexpression, USP7 co-IP and functional antagonism assay","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus in vitro ubiquitination and catalytic mutant epistasis, single lab with multiple methods","pmids":["34921745"],"is_preprint":false},{"year":2022,"finding":"UBE2O interacts with and ubiquitinates PTRF/CAVIN1, inhibiting caveolae formation and reducing exosome secretion, thereby decreasing exosome-associated PTRF release. SDPR/CAVIN2 interacts with both UBE2O and PTRF and promotes PTRF incorporation into exosomes, but UBE2O still suppresses this in the presence of SDPR.","method":"Exogenous and endogenous co-IP, in vitro ubiquitination assay, exosome isolation by ultracentrifugation, nanoparticle tracking analysis, western blot for exosome markers","journal":"Cell communication and signaling","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — reciprocal co-IP and ubiquitination assay plus functional exosome secretion quantification; single lab with multiple methods","pmids":["36443833"],"is_preprint":false},{"year":2022,"finding":"UBE2O interacts with and mediates ubiquitination and proteasomal degradation of HADHA (a mitochondrial β-oxidation enzyme), modulating lipid metabolic reprogramming. Both E2 and E3 enzymatic activities of UBE2O are required. Liver-specific Ube2o deletion in mice increases HADHA levels, reduces hepatic lipid accumulation, and confers resistance to DEN-induced hepatocarcinogenesis.","method":"Co-IP, in vitro ubiquitination assay with E2/E3 domain mutants, liver-specific Ube2o knockout mice, DEN hepatocarcinogenesis model, lipidomics/metabolic profiling","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo liver-specific KO with DEN model, co-IP, and E2/E3 domain mutagenesis in a single study with multiple orthogonal methods","pmids":["36273042"],"is_preprint":false},{"year":2023,"finding":"UBE2O interacts with IFIT3, a mediator of interferon signaling, and mediates its ubiquitination and proteasomal degradation at lysine K236, thereby reducing cellular responsiveness to interferon-α. Knockdown of UBE2O enhances interferon-α efficacy by upregulating IFIT3. ATO (arsenic trioxide) inhibits UBE2O and increases IFIT3, increasing interferon-α sensitivity.","method":"Co-IP and MS, in vitro ubiquitination assay, site-directed mutagenesis (IFIT3 K236R), rescue experiments, in vitro and in vivo IFN-α efficacy assays, ATO pharmacological inhibition","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus substrate K-site mutagenesis and functional rescue, single lab with multiple methods","pmids":["38129382"],"is_preprint":false},{"year":2024,"finding":"Crystal structures of full-length UBE2O (from Trametes pubescens, a fungal ortholog) and its UBC domain revealed a dimeric architecture with interdomain interactions between CR1-CR2 and UBC. Dimeric intermolecular and canonical ubiquitin/UBC interactions are mechanistically important for polyubiquitin chain formation and substrate ubiquitination. UBE2O catalyzes all seven types of polyubiquitin chains in vitro. Autoubiquitination within CR1-CR2 also regulates activity. CR1-CR2/UBC interactions are required for AMPKα2 polyubiquitination by human UBE2O.","method":"X-ray crystallography (full-length and UBC domain structures), in vitro ubiquitin chain formation assays for all 7 Ub linkage types, structure-guided mutagenesis, AMPKα2 ubiquitination assay with human UBE2O","journal":"Structure (London, England : 1993)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure determination with functional validation by in vitro ubiquitination and domain mutagenesis; multiple orthogonal methods in a single study","pmids":["39740670"],"is_preprint":false},{"year":2025,"finding":"UBE2O mediates monoubiquitination of hypophosphorylated cytoplasmic hepatitis B virus core protein (HBc) and capsids, promoting capsid recognition by the ESCRT machinery and enveloped virion secretion via CD63-positive multivesicular bodies (MVBs). Enzymatically inactive UBE2O mutant inhibits this process; UBE2O overexpression enhances virion secretion.","method":"Knockdown/overexpression in HBV-infected primary human hepatocytes and HepG2-NTCP cells, catalytically inactive UBE2O mutant, confocal microscopy, proximity ligation assay (PLA), co-localization with MVB marker CD63, HBV DNA/pgRNA/capsid quantification","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — catalytic mutant epistasis plus PLA localization and functional virion quantification; single lab with multiple orthogonal methods","pmids":["40992660"],"is_preprint":false},{"year":2025,"finding":"RSK2 is a kinase that directly interacts with UBE2O and phosphorylates it at Thr838, leading to UBE2O degradation. RSK2 inhibition increases UBE2O levels and promotes HCC tumor proliferation and radioresistance; these effects are abrogated by UBE2O knockdown, establishing RSK2 as a negative regulator of UBE2O.","method":"Co-IP, in vitro kinase assay identifying Thr838 phosphorylation site, site-directed mutagenesis (T838A), epistasis by combined RSK2 inhibition and UBE2O knockdown, in vitro and in vivo tumor models","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus in vitro phosphorylation site identification and epistasis rescue; single lab with multiple orthogonal methods","pmids":["39954933"],"is_preprint":false},{"year":2025,"finding":"Structural and biochemical analyses of human UBE2O showed that substrate binding occurs through a conserved acidic pocket formed by N-terminal SH3-like domains, enabling broad substrate recruitment. Specific residues in the UBC domain position ubiquitin in a 'closed' state for nucleophilic attack, and a tryptophan residue protects the activated E2~Ub conjugate from premature hydrolysis—mechanistically analogous to RING E3 ligase catalysis.","method":"Structural analysis (combined crystallography/biochemistry), systematic mutagenesis of UBC domain residues and SH3-like domain, in vitro ubiquitination assays, comparison with BIRC6 UBC domain","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — structural determination combined with systematic active-site mutagenesis and in vitro ubiquitination assays; multiple orthogonal methods in a single rigorous study","pmids":["41419192"],"is_preprint":false},{"year":2025,"finding":"UBE2O selectively ubiquitylates cytosolic (but not membrane-associated) CTNNA1 in a phosphorylation-independent manner. Ubiquitylation of CTNNA1 diminishes its interaction with β-catenin while enabling interaction with vinculin, thereby promoting focal adhesion maturation, cell extension, and cell-to-ECM adhesion during cell spreading.","method":"Co-IP, MS-based interactome of ubiquitylated CTNNA1, in vitro ubiquitination assay, focal adhesion imaging, cell spreading assay, β-catenin and vinculin binding assays","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS interactome of ubiquitylated substrate plus co-IP and functional cell adhesion assays; single lab with multiple methods","pmids":["40983751"],"is_preprint":false},{"year":2025,"finding":"Domain truncation and systematic mutagenesis of human UBE2O revealed that the coiled-coil (CC) and C-terminal regulatory (CTR) domains are required for catalytic competence, while N-terminal regions impose activity constraints. Zinc ions act as potent allosteric inhibitors by binding cysteines of UBE2O and sterically blocking access of catalytic C1040 to ubiquitin. Specific non-cysteine residues (H939, T995, S1042, S1046, S1060, H1130) are critical regulators of substrate selectivity and catalytic optimization. UBE2O activity is insensitive to its own self-ubiquitination and phosphorylation state.","method":"Domain truncation analysis, systematic mutagenesis, biochemical ubiquitination assays, zinc ion inhibition experiments, AMPKα2 substrate ubiquitination assays","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — systematic mutagenesis and in vitro biochemical reconstitution, but preprint (not peer-reviewed) from a single lab","pmids":["bio_10.1101_2025.06.27.661974"],"is_preprint":true},{"year":2026,"finding":"GATA1 binds to the UBE2O promoter, directly regulating UBE2O transcription and expression during erythroid differentiation; this was validated by chromatin immunoprecipitation (ChIP) in the context of erythropoiesis and myelodysplastic syndrome treatment.","method":"Bioinformatic promoter analysis, ChIP assay confirming GATA1 binding to UBE2O promoter, expression analysis in K562 cells and MDS patient bone marrow","journal":"Blood advances","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — ChIP validation of GATA1 binding to UBE2O promoter is direct but single lab, single method for the mechanistic claim","pmids":["41191526"],"is_preprint":false}],"current_model":"UBE2O is an atypical E2/E3 hybrid ubiquitin-conjugating enzyme that directly recognizes substrates—through an N-terminal SH3-like acidic pocket and CR1-CR2 domains that detect exposed basic/hydrophobic patches on unassembled or orphan proteins—and catalyzes their mono- or polyubiquitination via a UBC active-site cysteine (C1040 in humans) that positions ubiquitin in a closed state for nucleophilic attack; client selection is amplified by a feed-forward ubiquitin-binding mechanism and the NAP1L1 cofactor; substrates targeted for proteasomal degradation include ribosomal proteins (driving reticulocyte proteome remodeling), AMPKα2 (activating mTOR-HIF1α signaling and contributing to metabolic and tumor biology), BAP1 NLS (inducing cytoplasmic sequestration counteracted by BAP1 autodeubiquitination), SMAD6 (modulating BMP signaling), BMAL1 (regulating circadian rhythm), c-Maf, Mxi1, RECQL4, HADHA, IFIT3, and CTNNA1; UBE2O also inhibits TRAF6 K63-polyubiquitination to dampen NF-κB signaling; its own stability is negatively regulated by RSK2-mediated phosphorylation at Thr838; and zinc ions allosterically inhibit it by blocking catalytic cysteine access to ubiquitin."},"narrative":{"mechanistic_narrative":"UBE2O is an atypical, self-sufficient E2-E3 hybrid ubiquitin-conjugating enzyme that directly recognizes and ubiquitinates substrates without requiring a separate E3 ligase, governing protein quality control and the targeted degradation of regulatory factors across metabolism, the cell cycle, signaling, and erythroid maturation [PMID:8634298, PMID:28774922]. A defining feature is its capacity to recognize \"orphan\" proteins: it detects juxtaposed exposed basic and hydrophobic patches on unassembled subunits, ubiquitinating unpaired α-globin and ribosomal proteins that fail to engage their normal partners and directing them for proteasomal degradation, a function essential for proteome remodeling during reticulocyte differentiation [PMID:28774922, PMID:28774900]. Catalysis proceeds through a UBC active-site cysteine that positions ubiquitin in a closed, RING-E3-like state for nucleophilic attack, with substrate recruitment occurring via an N-terminal SH3-like acidic pocket and CR1-CR2 domains; client selection is further amplified by a feed-forward ubiquitin-binding mechanism and the NAP1L1 cofactor/adapter [PMID:35915257, PMID:41419192, PMID:39740670]. Through this machinery UBE2O degrades a broad substrate set with distinct physiological consequences: AMPKα2 (but not AMPKα1), activating mTOR-HIF1α signaling to drive tumorigenesis and impair insulin sensitivity in muscle [PMID:28162974, PMID:31292296]; the circadian transcription factor BMAL1 [PMID:29871923]; the β-oxidation enzyme HADHA, linking it to hepatic lipid metabolism and hepatocarcinogenesis [PMID:36273042]; and additional substrates including c-Maf, Mxi1, RECQL4, IFIT3, and CTNNA1 that connect it to multiple myeloma, lung cancer, homologous-recombination repair, interferon responsiveness, and cell adhesion [PMID:28673317, PMID:32901121, PMID:34921745, PMID:38129382, PMID:40983751]. Beyond degradation, UBE2O multi-monoubiquitinates the BAP1 NLS to sequester it in the cytoplasm and monoubiquitinates SMAD6 to modulate BMP signaling, and it dampens NF-κB activation by inhibiting TRAF6 K63-polyubiquitination independent of its UBC domain [PMID:24703950, PMID:23455153, PMID:23381138]. Its own activity is constrained by allosteric zinc inhibition of the catalytic cysteine and by RSK2-mediated phosphorylation at Thr838 that destabilizes the enzyme, while GATA1 drives its transcription during erythropoiesis [PMID:bio_10.1101_2025.06.27.661974, PMID:39954933, PMID:41191526].","teleology":[{"year":1996,"claim":"Established that UBE2O is mechanistically unusual—a single enzyme carrying both E2 and intrinsic E3 activity—answering whether it could conjugate ubiquitin without a partner ligase.","evidence":"In vitro thiol-ester relay and active-site cysteine protection assays with phenylarsenoxide/NEM probes","pmids":["8634298"],"confidence":"High","gaps":["No physiological substrate identified at this stage","Structural basis of the two-cysteine relay not resolved"]},{"year":2013,"claim":"Identified the first defined substrates and demonstrated non-degradative outputs, showing UBE2O can monoubiquitinate SMAD6 to potentiate BMP signaling and can inhibit TRAF6 ubiquitination to dampen NF-κB.","evidence":"Co-IP, in vitro ubiquitination with catalytic (C885A) and substrate-lysine mutants, and pathway reporter assays","pmids":["23455153","23381138"],"confidence":"High","gaps":["TRAF6 inhibition was UBC-independent, leaving the mechanistic basis distinct from catalytic ubiquitination","Generality of monoubiquitination vs degradation outputs unclear"]},{"year":2014,"claim":"Showed UBE2O can control substrate localization rather than only stability, multi-monoubiquitinating the BAP1 NLS to drive cytoplasmic sequestration counteracted by BAP1 autodeubiquitination.","evidence":"Co-IP, in vitro ubiquitination, subcellular fractionation/IF, NLS and cancer-mutation analysis","pmids":["24703950"],"confidence":"High","gaps":["How UBE2O selects the NLS region for modification not defined","Relative contributions of localization vs degradation across substrates unresolved"]},{"year":2017,"claim":"Defined UBE2O's core biological role as an orphan-protein quality control factor and proteome remodeler, recognizing exposed basic/hydrophobic patches on unassembled subunits and clearing ribosomes during reticulocyte maturation.","evidence":"In vitro reconstitution, proteomics, substrate feature mapping, and a loss-of-function Ube2o mouse with anemia","pmids":["28774922","28774900"],"confidence":"High","gaps":["Determinants distinguishing degradative from monoubiquitination outcomes not fully defined","Structural basis of patch recognition not yet solved"]},{"year":2017,"claim":"Connected UBE2O to metabolism and cancer by establishing AMPKα2 as an isoform-selective degradation target whose loss drives mTOR-HIF1α signaling and tumorigenesis, and added c-Maf as a degradation substrate in myeloma.","evidence":"Ube2o knockout cancer mouse models with isoform-selective AMPK epistasis, pharmacological rescue, and in vitro/in vivo ubiquitination assays","pmids":["28162974","28673317"],"confidence":"High","gaps":["Structural basis for AMPKα2 vs α1 selectivity not defined","c-Maf finding rests on single-lab Medium-confidence evidence"]},{"year":2018,"claim":"Extended the degradation repertoire to circadian control, identifying BMAL1 as a substrate degraded via the CR2 domain and modulating clock amplitude.","evidence":"Co-IP/MS, in vitro ubiquitination with CR2 Cys→Ser mutant, and bioluminescence circadian reporter in U2OS cells","pmids":["29871923"],"confidence":"High","gaps":["In vivo circadian phenotype of Ube2o loss not established","Recruitment determinants for BMAL1 not mapped"]},{"year":2019,"claim":"Confirmed the UBE2O→AMPKα2 axis as causal in tissue physiology, showing muscle-specific Ube2o loss improves insulin sensitivity in an AMPKα2-dependent manner.","evidence":"Tissue-specific Ube2o knockout mice, hyperinsulinemic-euglycemic clamp, and Prkaa2 haploinsufficiency epistasis","pmids":["31292296"],"confidence":"High","gaps":["Mechanism of AMPKα2 isoform selectivity still unresolved","Other muscle substrates not surveyed"]},{"year":2020,"claim":"Broadened the cancer-relevant substrate set to Mxi1 and reinforced SMAD6/BMP regulation in angiogenesis.","evidence":"Co-IP, in vitro ubiquitination with substrate lysine mutants, tumor models; HUVEC/wound-healing phenotypic assays for SMAD6/BMP2","pmids":["32901121","32375794"],"confidence":"Medium","gaps":["The SMAD6/angiogenesis study (Low confidence) lacks direct in vitro ubiquitination reconstitution","Mxi1 evidence is single-lab"]},{"year":2022,"claim":"Resolved how clients are selected and how catalysis is autoregulated, revealing a feed-forward ubiquitin-binding SH3-like domain and the NAP1L1 adapter, plus a UBC-autoinhibited, malleable client interface.","evidence":"Cryo-EM of human UBE2O–NAP1L1, biochemical reconstitution, and mutagenesis of client- and ubiquitin-binding domains","pmids":["35915257"],"confidence":"High","gaps":["NAP1L1 recruits only a subset of clients; adapters for others unknown","How autoinhibition is relieved in vivo not defined"]},{"year":2022,"claim":"Expanded substrate biology into DNA repair, lipid metabolism, and caveolae/exosome control, identifying RECQL4 (antagonized by USP7), HADHA, and PTRF/CAVIN1 as targets.","evidence":"Co-IP, in vitro ubiquitination with E2/E3 domain mutants, HR reporter, liver-specific Ube2o KO with DEN model, and exosome quantification","pmids":["34921745","36273042","36443833"],"confidence":"High","gaps":["RECQL4 and PTRF studies are single-lab Medium confidence","How the same enzyme partitions among diverse substrates in vivo unclear"]},{"year":2024,"claim":"Provided the first full-length structural framework, showing a dimeric CR1-CR2/UBC architecture and that UBE2O can build all seven polyubiquitin linkages, with these interactions required for AMPKα2 polyubiquitination.","evidence":"X-ray crystallography of a fungal ortholog and UBC domain, 7-linkage chain assays, and structure-guided mutagenesis on human UBE2O","pmids":["39740670"],"confidence":"High","gaps":["Structure is from a fungal ortholog; full-length human conformation not crystallized","Physiological relevance of dimerization in cells not tested"]},{"year":2025,"claim":"Detailed the catalytic and regulatory logic of human UBE2O—SH3-like acidic-pocket substrate binding, RING-like closed-ubiquitin positioning, zinc allosteric inhibition, RSK2-mediated destabilizing phosphorylation, and GATA1 transcriptional control—and added CTNNA1, IFIT3, and HBV core protein as functionally distinct outputs.","evidence":"Structural/mutagenesis catalysis studies, zinc inhibition assays, RSK2 kinase/T838 mapping with epistasis, GATA1 ChIP, and substrate-specific co-IP/ubiquitination plus cell-biology assays","pmids":["41419192","40983751","38129382","40992660","39954933","41191526","bio_10.1101_2025.06.27.661974"],"confidence":"High","gaps":["Several substrate and regulatory findings are single-lab Medium confidence","One mechanistic study (zinc/regulatory residues) is a preprint","Physiological role of zinc inhibition in vivo not established"]},{"year":null,"claim":"It remains unresolved what general code dictates whether a given client is monoubiquitinated for relocalization versus polyubiquitinated for degradation, and how the autoinhibited enzyme is selectively activated toward specific substrates in vivo.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying determinant linking substrate features to ubiquitin output","In vivo activation/adapter map incomplete beyond NAP1L1","Few substrates validated structurally in complex with human UBE2O"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,4,6,17,20]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,4,17,20]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3,4,21]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[3]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,4,5]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[6,9,15]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,2,6]},{"term_id":"R-HSA-9909396","term_label":"Circadian clock","supporting_discovery_ids":[8]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[13]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[2,16]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[6,7,10,15,18]}],"complexes":[],"partners":["NAP1L1","SMAD6","TRAF6","BAP1","AMPKΑ2/PRKAA2","BMAL1","RECQL4","RSK2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9C0C9","full_name":"(E3-independent) E2 ubiquitin-conjugating enzyme","aliases":["E2/E3 hybrid ubiquitin-protein ligase UBE2O","Ubiquitin carrier protein O","Ubiquitin-conjugating enzyme E2 O","Ubiquitin-conjugating enzyme E2 of 230 kDa","Ubiquitin-conjugating enzyme E2-230K","Ubiquitin-protein ligase O"],"length_aa":1292,"mass_kda":141.3,"function":"E2/E3 hybrid ubiquitin-protein ligase that displays both E2 and E3 ligase activities and mediates monoubiquitination of target proteins (PubMed:23455153, PubMed:24703950). Negatively regulates TRAF6-mediated NF-kappa-B activation independently of its E2 activity (PubMed:23381138). Acts as a positive regulator of BMP7 signaling by mediating monoubiquitination of SMAD6, thereby regulating adipogenesis (PubMed:23455153). Mediates monoubiquitination at different sites of the nuclear localization signal (NLS) of BAP1, leading to cytoplasmic retention of BAP1. Also able to monoubiquitinate the NLS of other chromatin-associated proteins, such as INO80 and CXXC1, affecting their subcellular location (PubMed:24703950). Acts as a regulator of retrograde transport by assisting the TRIM27:MAGEL2 E3 ubiquitin ligase complex to mediate 'Lys-63'-linked ubiquitination of WASHC1, leading to promote endosomal F-actin assembly (PubMed:23452853)","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9C0C9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/UBE2O","classification":"Not 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AGBL3","url":"https://www.omim.org/entry/617346"},{"mim_id":"612268","title":"TUBULIN TYROSINE LIGASE-LIKE 5; TTLL5","url":"https://www.omim.org/entry/612268"},{"mim_id":"603089","title":"BRCA1-ASSOCIATED PROTEIN 1; BAP1","url":"https://www.omim.org/entry/603089"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nuclear bodies","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/UBE2O"},"hgnc":{"alias_symbol":["E2-230K"],"prev_symbol":[]},"alphafold":{"accession":"Q9C0C9","domains":[{"cath_id":"2.30.30.140","chopping":"61-86_118-145","consensus_level":"medium","plddt":87.6063,"start":61,"end":145},{"cath_id":"-","chopping":"581-710","consensus_level":"medium","plddt":86.5088,"start":581,"end":710},{"cath_id":"3.10.110.10","chopping":"910-1090","consensus_level":"high","plddt":88.8261,"start":910,"end":1090}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9C0C9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9C0C9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9C0C9-F1-predicted_aligned_error_v6.png","plddt_mean":65.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=UBE2O","jax_strain_url":"https://www.jax.org/strain/search?query=UBE2O"},"sequence":{"accession":"Q9C0C9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9C0C9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9C0C9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9C0C9"}},"corpus_meta":[{"pmid":"24703950","id":"PMC_24703950","title":"Autodeubiquitination 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Phenylarsenoxide inhibition studies demonstrated that two cysteine residues sequentially form thiol esters with ubiquitin (a 'thiol relay'), consistent with the enzyme possessing both E2 and intrinsic E3 activities without requiring a separate E3 ligase.\",\n      \"method\": \"In vitro biochemical assay with phenylarsenoxide and NEM inhibitors; E2~ubiquitin adduct formation assay; active-site cysteine protection experiments\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro mechanistic reconstitution with active-site probes and multiple orthogonal inhibitor experiments in a single rigorous study\",\n      \"pmids\": [\"8634298\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"UBE2O functions as an E2-E3 hybrid to monoubiquitinate SMAD6 at lysine 174; the catalytic cysteine C885 of UBE2O is essential for this activity. Monoubiquitination of SMAD6 impairs its binding to the BMP type I receptor, thereby potentiating BMP7/SMAD1 signaling and adipogenesis.\",\n      \"method\": \"Proteomic interaction screen, co-IP, in vitro ubiquitination assay, site-directed mutagenesis (K174R on SMAD6; C885A on UBE2O), SMAD1 phosphorylation reporter assays, luciferase transcriptional assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro ubiquitination with active-site mutagenesis, substrate lysine mapping, and functional receptor-binding consequence demonstrated in a single study with multiple orthogonal methods\",\n      \"pmids\": [\"23455153\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"UBE2O binds TRAF6 and inhibits its K63-polyubiquitination, preventing TAB2/3-TAK1 recruitment and NF-κB activation downstream of IL-1R/TLR. The inhibitory effect is independent of the UBC domain of UBE2O and instead operates by disrupting the IL-1β-induced TRAF6–MyD88 association.\",\n      \"method\": \"Co-IP, in vitro ubiquitination assay, NF-κB luciferase reporter assay, domain-deletion constructs, IL-1β/LPS stimulation experiments\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP plus functional NF-κB reporter and domain-mapping in a single lab with multiple methods\",\n      \"pmids\": [\"23381138\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"UBE2O multi-monoubiquitinates the nuclear localization signal (NLS) of BAP1, inducing its cytoplasmic sequestration. BAP1 autodeubiquitination via intramolecular interactions counteracts this, maintaining nuclear localization. Cancer-derived BAP1 mutations that abrogate autodeubiquitination promote cytoplasmic retention. UBE2O also promotes cytoplasmic localization of BAP1 during adipogenesis.\",\n      \"method\": \"Co-IP, in vitro ubiquitination assay, subcellular fractionation/immunofluorescence, site-directed mutagenesis of BAP1 NLS and catalytic residues, cancer mutation analysis, adipogenesis differentiation assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (in vitro ubiquitination, fractionation, mutagenesis, cancer mutations) with functional localization consequence; replicated across cell types and differentiation contexts\",\n      \"pmids\": [\"24703950\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"UBE2O directly recognizes juxtaposed basic and hydrophobic patches on unassembled (orphan) proteins and mediates their ubiquitination without a separate E3 ligase. In reticulocytes, unassembled α-globin that fails to pair with β-globin is selectively ubiquitinated by UBE2O. In non-reticulocytes, ribosomal proteins that do not engage nuclear import factors are UBE2O targets, directing them to proteasomal degradation.\",\n      \"method\": \"In vitro ubiquitination reconstitution, proteomic substrate identification, cell-based ubiquitination assays, substrate feature mapping (basic/hydrophobic patches)\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct biochemical reconstitution of substrate recognition and ubiquitination without E3; replicated across multiple substrates and cell types; published simultaneously with independent corroborating paper (PMID 28774900)\",\n      \"pmids\": [\"28774922\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"UBE2O is a broad-spectrum ubiquitinating enzyme that remodels the erythroid proteome during reticulocyte differentiation. A loss-of-function mutation in murine Ube2o causes anemia. UBE2O directly recognizes ribosomal proteins and other substrates, targeting them for proteasomal degradation, and ribosome elimination is defective in Ube2o mutants.\",\n      \"method\": \"Mouse genetic mutant (Ube2o loss-of-function), quantitative proteomics of reticulocyte proteome, in vivo and in vitro ubiquitination assays, ribosome content analysis\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mouse genetic KO with defined anemia phenotype, quantitative proteomics, and direct substrate ubiquitination assays; corroborated by independent simultaneous publication (PMID 28774922)\",\n      \"pmids\": [\"28774900\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"UBE2O specifically targets AMPKα2 (but not AMPKα1) for ubiquitination and proteasomal degradation, thereby activating the mTOR-HIF1α pathway and promoting tumor initiation and progression. Genetic deletion of Ube2o in mouse cancer models impairs tumorigenesis, and inactivation of AMPKα2 (but not AMPKα1) abrogates the tumor-attenuating effect of UBE2O loss.\",\n      \"method\": \"Ube2o knockout mouse cancer models (MMTV-PyVT, TRAMP), in vitro ubiquitination assay, epistasis via AMPKα1/α2 isoform-selective inactivation, rapamycin treatment rescue, HIF1α inhibitor rescue\",\n      \"journal\": \"Cancer cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo mouse genetic epistasis with isoform-selective AMPKα2 rescue, in vitro ubiquitination confirmation, and pharmacological validation via rapamycin/HIF1α inhibition\",\n      \"pmids\": [\"28162974\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"UBE2O interacts with and mediates polyubiquitination and proteasomal degradation of c-Maf, a transcription factor critical in multiple myeloma, thereby reducing c-Maf transcriptional activity and cyclin D2 expression.\",\n      \"method\": \"Affinity purification/tandem MS, co-IP, in vitro polyubiquitination assay, immunoblotting for c-Maf stability, luciferase transcriptional assay, lentiviral re-expression, xenograft mouse model\",\n      \"journal\": \"Journal of hematology & oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP plus in vitro ubiquitination and functional transcriptional assays, single lab, multiple methods\",\n      \"pmids\": [\"28673317\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"UBE2O interacts with and promotes ubiquitination and proteasomal degradation of BMAL1, the core circadian clock transcription factor. The conserved region 2 (CR2) domain of UBE2O is required for BMAL1 ubiquitination, and a catalytic Cys-to-Ser substitution in CR2 abolishes this activity. UBE2O knockdown elevates circadian clock amplitude in U2OS cells.\",\n      \"method\": \"Co-IP and MS, immunoblotting for BMAL1 stability, in vitro ubiquitination assay, domain-mapping and Cys→Ser mutagenesis, bioluminescence circadian reporter assay in U2OS cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — active-site mutagenesis with domain mapping, in vitro ubiquitination, and functional circadian phenotype readout; multiple orthogonal methods in single study\",\n      \"pmids\": [\"29871923\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In skeletal muscle, UBE2O acts as a ubiquitin ligase that targets AMPKα2 for ubiquitin-dependent degradation. Muscle-specific knockout of Ube2o improves insulin sensitivity and energy expenditure, and muscle-specific heterozygous knockout of Prkaa2 (AMPKα2) abrogates the metabolic benefits of Ube2o loss, establishing UBE2O→AMPKα2 as the causal axis in skeletal muscle metabolic regulation.\",\n      \"method\": \"Tissue-specific (muscle, fat, liver) Ube2o knockout mice, hyperinsulinemic-euglycemic clamp, Prkaa2 heterozygous genetic epistasis, in vitro ubiquitination assay, metabolic phenotyping\",\n      \"journal\": \"JCI insight\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — tissue-specific genetic KO with rigorous metabolic phenotyping, in vivo epistasis using AMPKα2 haploinsufficiency, and in vitro ubiquitination; multiple orthogonal methods across multiple mouse models\",\n      \"pmids\": [\"31292296\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"UBE2O interacts with and targets Mxi1 for ubiquitination and degradation at the K46 residue of Mxi1. This promotes lung cancer progression and radioresistance; these effects are reversed by Mxi1 inhibition.\",\n      \"method\": \"Co-IP, in vitro ubiquitination assay, site-directed mutagenesis (Mxi1 K46R), genetic/pharmacological UBE2O blockade with Mxi1 rescue, in vitro and in vivo tumor models\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus substrate lysine-site mutagenesis and in vivo rescue epistasis, single lab with multiple methods\",\n      \"pmids\": [\"32901121\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"UBE2O decreases SMAD6 protein levels, thereby activating BMP2 signaling to promote angiogenesis in wound healing; saliva exosome-delivered UBE2O mRNA reproduces this effect in endothelial cells.\",\n      \"method\": \"In vitro HUVEC proliferation/migration/angiogenesis assays, in vivo wound healing model, siRNA knockdown and overexpression, western blot for SMAD6 and BMP2 pathway\",\n      \"journal\": \"Journal of nanobiotechnology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, phenotypic assays with pathway inference; no direct in vitro ubiquitination reconstitution of SMAD6 by UBE2O in this paper\",\n      \"pmids\": [\"32375794\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"UBE2O client selection is regulated by two mechanisms: (1) a feed-forward mechanism where a single ubiquitin added to a client enhances UBE2O binding and promotes multi-mono-ubiquitylation via a distinct SH3-like ubiquitin-binding domain; (2) the histone chaperone NAP1L1 acts as a cofactor/adapter to recruit a subset of UBE2O clients. Cryo-EM structures of human UBE2O with NAP1L1 reveal a malleable client recruitment interface autoinhibited by the reactive UBC domain.\",\n      \"method\": \"Cryo-EM structure determination of human UBE2O–NAP1L1 complex, biochemical ubiquitylation assays, identification of SH3-like ubiquitin-binding domain by mutagenesis, client ubiquitylation reconstitution\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure plus biochemical reconstitution and mutagenesis of client-binding and ubiquitin-binding domains; multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"35915257\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"UBE2O physically interacts with RECQL4 and mediates its multi-monoubiquitinylation, leading to proteasomal degradation. This inhibits homologous recombination (HR)-mediated DSB repair by attenuating RECQL4 interactions with MRE11-RAD50-NBS1 and CtIP. The deubiquitinase USP7 antagonizes UBE2O by interacting with both UBE2O and RECQL4 to stabilize RECQL4.\",\n      \"method\": \"Co-IP, in vitro ubiquitination assay, HR reporter assay, UBE2O catalytic mutant (E2 activity-dead), rescue experiments with RECQL4 overexpression, USP7 co-IP and functional antagonism assay\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus in vitro ubiquitination and catalytic mutant epistasis, single lab with multiple methods\",\n      \"pmids\": [\"34921745\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"UBE2O interacts with and ubiquitinates PTRF/CAVIN1, inhibiting caveolae formation and reducing exosome secretion, thereby decreasing exosome-associated PTRF release. SDPR/CAVIN2 interacts with both UBE2O and PTRF and promotes PTRF incorporation into exosomes, but UBE2O still suppresses this in the presence of SDPR.\",\n      \"method\": \"Exogenous and endogenous co-IP, in vitro ubiquitination assay, exosome isolation by ultracentrifugation, nanoparticle tracking analysis, western blot for exosome markers\",\n      \"journal\": \"Cell communication and signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — reciprocal co-IP and ubiquitination assay plus functional exosome secretion quantification; single lab with multiple methods\",\n      \"pmids\": [\"36443833\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"UBE2O interacts with and mediates ubiquitination and proteasomal degradation of HADHA (a mitochondrial β-oxidation enzyme), modulating lipid metabolic reprogramming. Both E2 and E3 enzymatic activities of UBE2O are required. Liver-specific Ube2o deletion in mice increases HADHA levels, reduces hepatic lipid accumulation, and confers resistance to DEN-induced hepatocarcinogenesis.\",\n      \"method\": \"Co-IP, in vitro ubiquitination assay with E2/E3 domain mutants, liver-specific Ube2o knockout mice, DEN hepatocarcinogenesis model, lipidomics/metabolic profiling\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo liver-specific KO with DEN model, co-IP, and E2/E3 domain mutagenesis in a single study with multiple orthogonal methods\",\n      \"pmids\": [\"36273042\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"UBE2O interacts with IFIT3, a mediator of interferon signaling, and mediates its ubiquitination and proteasomal degradation at lysine K236, thereby reducing cellular responsiveness to interferon-α. Knockdown of UBE2O enhances interferon-α efficacy by upregulating IFIT3. ATO (arsenic trioxide) inhibits UBE2O and increases IFIT3, increasing interferon-α sensitivity.\",\n      \"method\": \"Co-IP and MS, in vitro ubiquitination assay, site-directed mutagenesis (IFIT3 K236R), rescue experiments, in vitro and in vivo IFN-α efficacy assays, ATO pharmacological inhibition\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus substrate K-site mutagenesis and functional rescue, single lab with multiple methods\",\n      \"pmids\": [\"38129382\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Crystal structures of full-length UBE2O (from Trametes pubescens, a fungal ortholog) and its UBC domain revealed a dimeric architecture with interdomain interactions between CR1-CR2 and UBC. Dimeric intermolecular and canonical ubiquitin/UBC interactions are mechanistically important for polyubiquitin chain formation and substrate ubiquitination. UBE2O catalyzes all seven types of polyubiquitin chains in vitro. Autoubiquitination within CR1-CR2 also regulates activity. CR1-CR2/UBC interactions are required for AMPKα2 polyubiquitination by human UBE2O.\",\n      \"method\": \"X-ray crystallography (full-length and UBC domain structures), in vitro ubiquitin chain formation assays for all 7 Ub linkage types, structure-guided mutagenesis, AMPKα2 ubiquitination assay with human UBE2O\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure determination with functional validation by in vitro ubiquitination and domain mutagenesis; multiple orthogonal methods in a single study\",\n      \"pmids\": [\"39740670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"UBE2O mediates monoubiquitination of hypophosphorylated cytoplasmic hepatitis B virus core protein (HBc) and capsids, promoting capsid recognition by the ESCRT machinery and enveloped virion secretion via CD63-positive multivesicular bodies (MVBs). Enzymatically inactive UBE2O mutant inhibits this process; UBE2O overexpression enhances virion secretion.\",\n      \"method\": \"Knockdown/overexpression in HBV-infected primary human hepatocytes and HepG2-NTCP cells, catalytically inactive UBE2O mutant, confocal microscopy, proximity ligation assay (PLA), co-localization with MVB marker CD63, HBV DNA/pgRNA/capsid quantification\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — catalytic mutant epistasis plus PLA localization and functional virion quantification; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"40992660\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RSK2 is a kinase that directly interacts with UBE2O and phosphorylates it at Thr838, leading to UBE2O degradation. RSK2 inhibition increases UBE2O levels and promotes HCC tumor proliferation and radioresistance; these effects are abrogated by UBE2O knockdown, establishing RSK2 as a negative regulator of UBE2O.\",\n      \"method\": \"Co-IP, in vitro kinase assay identifying Thr838 phosphorylation site, site-directed mutagenesis (T838A), epistasis by combined RSK2 inhibition and UBE2O knockdown, in vitro and in vivo tumor models\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus in vitro phosphorylation site identification and epistasis rescue; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"39954933\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Structural and biochemical analyses of human UBE2O showed that substrate binding occurs through a conserved acidic pocket formed by N-terminal SH3-like domains, enabling broad substrate recruitment. Specific residues in the UBC domain position ubiquitin in a 'closed' state for nucleophilic attack, and a tryptophan residue protects the activated E2~Ub conjugate from premature hydrolysis—mechanistically analogous to RING E3 ligase catalysis.\",\n      \"method\": \"Structural analysis (combined crystallography/biochemistry), systematic mutagenesis of UBC domain residues and SH3-like domain, in vitro ubiquitination assays, comparison with BIRC6 UBC domain\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — structural determination combined with systematic active-site mutagenesis and in vitro ubiquitination assays; multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"41419192\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"UBE2O selectively ubiquitylates cytosolic (but not membrane-associated) CTNNA1 in a phosphorylation-independent manner. Ubiquitylation of CTNNA1 diminishes its interaction with β-catenin while enabling interaction with vinculin, thereby promoting focal adhesion maturation, cell extension, and cell-to-ECM adhesion during cell spreading.\",\n      \"method\": \"Co-IP, MS-based interactome of ubiquitylated CTNNA1, in vitro ubiquitination assay, focal adhesion imaging, cell spreading assay, β-catenin and vinculin binding assays\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS interactome of ubiquitylated substrate plus co-IP and functional cell adhesion assays; single lab with multiple methods\",\n      \"pmids\": [\"40983751\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Domain truncation and systematic mutagenesis of human UBE2O revealed that the coiled-coil (CC) and C-terminal regulatory (CTR) domains are required for catalytic competence, while N-terminal regions impose activity constraints. Zinc ions act as potent allosteric inhibitors by binding cysteines of UBE2O and sterically blocking access of catalytic C1040 to ubiquitin. Specific non-cysteine residues (H939, T995, S1042, S1046, S1060, H1130) are critical regulators of substrate selectivity and catalytic optimization. UBE2O activity is insensitive to its own self-ubiquitination and phosphorylation state.\",\n      \"method\": \"Domain truncation analysis, systematic mutagenesis, biochemical ubiquitination assays, zinc ion inhibition experiments, AMPKα2 substrate ubiquitination assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — systematic mutagenesis and in vitro biochemical reconstitution, but preprint (not peer-reviewed) from a single lab\",\n      \"pmids\": [\"bio_10.1101_2025.06.27.661974\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"GATA1 binds to the UBE2O promoter, directly regulating UBE2O transcription and expression during erythroid differentiation; this was validated by chromatin immunoprecipitation (ChIP) in the context of erythropoiesis and myelodysplastic syndrome treatment.\",\n      \"method\": \"Bioinformatic promoter analysis, ChIP assay confirming GATA1 binding to UBE2O promoter, expression analysis in K562 cells and MDS patient bone marrow\",\n      \"journal\": \"Blood advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — ChIP validation of GATA1 binding to UBE2O promoter is direct but single lab, single method for the mechanistic claim\",\n      \"pmids\": [\"41191526\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"UBE2O is an atypical E2/E3 hybrid ubiquitin-conjugating enzyme that directly recognizes substrates—through an N-terminal SH3-like acidic pocket and CR1-CR2 domains that detect exposed basic/hydrophobic patches on unassembled or orphan proteins—and catalyzes their mono- or polyubiquitination via a UBC active-site cysteine (C1040 in humans) that positions ubiquitin in a closed state for nucleophilic attack; client selection is amplified by a feed-forward ubiquitin-binding mechanism and the NAP1L1 cofactor; substrates targeted for proteasomal degradation include ribosomal proteins (driving reticulocyte proteome remodeling), AMPKα2 (activating mTOR-HIF1α signaling and contributing to metabolic and tumor biology), BAP1 NLS (inducing cytoplasmic sequestration counteracted by BAP1 autodeubiquitination), SMAD6 (modulating BMP signaling), BMAL1 (regulating circadian rhythm), c-Maf, Mxi1, RECQL4, HADHA, IFIT3, and CTNNA1; UBE2O also inhibits TRAF6 K63-polyubiquitination to dampen NF-κB signaling; its own stability is negatively regulated by RSK2-mediated phosphorylation at Thr838; and zinc ions allosterically inhibit it by blocking catalytic cysteine access to ubiquitin.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"UBE2O is an atypical, self-sufficient E2-E3 hybrid ubiquitin-conjugating enzyme that directly recognizes and ubiquitinates substrates without requiring a separate E3 ligase, governing protein quality control and the targeted degradation of regulatory factors across metabolism, the cell cycle, signaling, and erythroid maturation [#0, #4]. A defining feature is its capacity to recognize \\\"orphan\\\" proteins: it detects juxtaposed exposed basic and hydrophobic patches on unassembled subunits, ubiquitinating unpaired \\u03b1-globin and ribosomal proteins that fail to engage their normal partners and directing them for proteasomal degradation, a function essential for proteome remodeling during reticulocyte differentiation [#4, #5]. Catalysis proceeds through a UBC active-site cysteine that positions ubiquitin in a closed, RING-E3-like state for nucleophilic attack, with substrate recruitment occurring via an N-terminal SH3-like acidic pocket and CR1-CR2 domains; client selection is further amplified by a feed-forward ubiquitin-binding mechanism and the NAP1L1 cofactor/adapter [#12, #20, #17]. Through this machinery UBE2O degrades a broad substrate set with distinct physiological consequences: AMPK\\u03b12 (but not AMPK\\u03b11), activating mTOR-HIF1\\u03b1 signaling to drive tumorigenesis and impair insulin sensitivity in muscle [#6, #9]; the circadian transcription factor BMAL1 [#8]; the \\u03b2-oxidation enzyme HADHA, linking it to hepatic lipid metabolism and hepatocarcinogenesis [#15]; and additional substrates including c-Maf, Mxi1, RECQL4, IFIT3, and CTNNA1 that connect it to multiple myeloma, lung cancer, homologous-recombination repair, interferon responsiveness, and cell adhesion [#7, #10, #13, #16, #21]. Beyond degradation, UBE2O multi-monoubiquitinates the BAP1 NLS to sequester it in the cytoplasm and monoubiquitinates SMAD6 to modulate BMP signaling, and it dampens NF-\\u03baB activation by inhibiting TRAF6 K63-polyubiquitination independent of its UBC domain [#3, #1, #2]. Its own activity is constrained by allosteric zinc inhibition of the catalytic cysteine and by RSK2-mediated phosphorylation at Thr838 that destabilizes the enzyme, while GATA1 drives its transcription during erythropoiesis [#22, #19, #23].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Established that UBE2O is mechanistically unusual\\u2014a single enzyme carrying both E2 and intrinsic E3 activity\\u2014answering whether it could conjugate ubiquitin without a partner ligase.\",\n      \"evidence\": \"In vitro thiol-ester relay and active-site cysteine protection assays with phenylarsenoxide/NEM probes\",\n      \"pmids\": [\"8634298\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No physiological substrate identified at this stage\", \"Structural basis of the two-cysteine relay not resolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identified the first defined substrates and demonstrated non-degradative outputs, showing UBE2O can monoubiquitinate SMAD6 to potentiate BMP signaling and can inhibit TRAF6 ubiquitination to dampen NF-\\u03baB.\",\n      \"evidence\": \"Co-IP, in vitro ubiquitination with catalytic (C885A) and substrate-lysine mutants, and pathway reporter assays\",\n      \"pmids\": [\"23455153\", \"23381138\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"TRAF6 inhibition was UBC-independent, leaving the mechanistic basis distinct from catalytic ubiquitination\", \"Generality of monoubiquitination vs degradation outputs unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showed UBE2O can control substrate localization rather than only stability, multi-monoubiquitinating the BAP1 NLS to drive cytoplasmic sequestration counteracted by BAP1 autodeubiquitination.\",\n      \"evidence\": \"Co-IP, in vitro ubiquitination, subcellular fractionation/IF, NLS and cancer-mutation analysis\",\n      \"pmids\": [\"24703950\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How UBE2O selects the NLS region for modification not defined\", \"Relative contributions of localization vs degradation across substrates unresolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Defined UBE2O's core biological role as an orphan-protein quality control factor and proteome remodeler, recognizing exposed basic/hydrophobic patches on unassembled subunits and clearing ribosomes during reticulocyte maturation.\",\n      \"evidence\": \"In vitro reconstitution, proteomics, substrate feature mapping, and a loss-of-function Ube2o mouse with anemia\",\n      \"pmids\": [\"28774922\", \"28774900\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Determinants distinguishing degradative from monoubiquitination outcomes not fully defined\", \"Structural basis of patch recognition not yet solved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Connected UBE2O to metabolism and cancer by establishing AMPK\\u03b12 as an isoform-selective degradation target whose loss drives mTOR-HIF1\\u03b1 signaling and tumorigenesis, and added c-Maf as a degradation substrate in myeloma.\",\n      \"evidence\": \"Ube2o knockout cancer mouse models with isoform-selective AMPK epistasis, pharmacological rescue, and in vitro/in vivo ubiquitination assays\",\n      \"pmids\": [\"28162974\", \"28673317\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for AMPK\\u03b12 vs \\u03b11 selectivity not defined\", \"c-Maf finding rests on single-lab Medium-confidence evidence\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Extended the degradation repertoire to circadian control, identifying BMAL1 as a substrate degraded via the CR2 domain and modulating clock amplitude.\",\n      \"evidence\": \"Co-IP/MS, in vitro ubiquitination with CR2 Cys\\u2192Ser mutant, and bioluminescence circadian reporter in U2OS cells\",\n      \"pmids\": [\"29871923\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo circadian phenotype of Ube2o loss not established\", \"Recruitment determinants for BMAL1 not mapped\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Confirmed the UBE2O\\u2192AMPK\\u03b12 axis as causal in tissue physiology, showing muscle-specific Ube2o loss improves insulin sensitivity in an AMPK\\u03b12-dependent manner.\",\n      \"evidence\": \"Tissue-specific Ube2o knockout mice, hyperinsulinemic-euglycemic clamp, and Prkaa2 haploinsufficiency epistasis\",\n      \"pmids\": [\"31292296\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of AMPK\\u03b12 isoform selectivity still unresolved\", \"Other muscle substrates not surveyed\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Broadened the cancer-relevant substrate set to Mxi1 and reinforced SMAD6/BMP regulation in angiogenesis.\",\n      \"evidence\": \"Co-IP, in vitro ubiquitination with substrate lysine mutants, tumor models; HUVEC/wound-healing phenotypic assays for SMAD6/BMP2\",\n      \"pmids\": [\"32901121\", \"32375794\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The SMAD6/angiogenesis study (Low confidence) lacks direct in vitro ubiquitination reconstitution\", \"Mxi1 evidence is single-lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Resolved how clients are selected and how catalysis is autoregulated, revealing a feed-forward ubiquitin-binding SH3-like domain and the NAP1L1 adapter, plus a UBC-autoinhibited, malleable client interface.\",\n      \"evidence\": \"Cryo-EM of human UBE2O\\u2013NAP1L1, biochemical reconstitution, and mutagenesis of client- and ubiquitin-binding domains\",\n      \"pmids\": [\"35915257\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"NAP1L1 recruits only a subset of clients; adapters for others unknown\", \"How autoinhibition is relieved in vivo not defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Expanded substrate biology into DNA repair, lipid metabolism, and caveolae/exosome control, identifying RECQL4 (antagonized by USP7), HADHA, and PTRF/CAVIN1 as targets.\",\n      \"evidence\": \"Co-IP, in vitro ubiquitination with E2/E3 domain mutants, HR reporter, liver-specific Ube2o KO with DEN model, and exosome quantification\",\n      \"pmids\": [\"34921745\", \"36273042\", \"36443833\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"RECQL4 and PTRF studies are single-lab Medium confidence\", \"How the same enzyme partitions among diverse substrates in vivo unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Provided the first full-length structural framework, showing a dimeric CR1-CR2/UBC architecture and that UBE2O can build all seven polyubiquitin linkages, with these interactions required for AMPK\\u03b12 polyubiquitination.\",\n      \"evidence\": \"X-ray crystallography of a fungal ortholog and UBC domain, 7-linkage chain assays, and structure-guided mutagenesis on human UBE2O\",\n      \"pmids\": [\"39740670\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure is from a fungal ortholog; full-length human conformation not crystallized\", \"Physiological relevance of dimerization in cells not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Detailed the catalytic and regulatory logic of human UBE2O\\u2014SH3-like acidic-pocket substrate binding, RING-like closed-ubiquitin positioning, zinc allosteric inhibition, RSK2-mediated destabilizing phosphorylation, and GATA1 transcriptional control\\u2014and added CTNNA1, IFIT3, and HBV core protein as functionally distinct outputs.\",\n      \"evidence\": \"Structural/mutagenesis catalysis studies, zinc inhibition assays, RSK2 kinase/T838 mapping with epistasis, GATA1 ChIP, and substrate-specific co-IP/ubiquitination plus cell-biology assays\",\n      \"pmids\": [\"41419192\", \"40983751\", \"38129382\", \"40992660\", \"39954933\", \"41191526\", \"bio_10.1101_2025.06.27.661974\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Several substrate and regulatory findings are single-lab Medium confidence\", \"One mechanistic study (zinc/regulatory residues) is a preprint\", \"Physiological role of zinc inhibition in vivo not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved what general code dictates whether a given client is monoubiquitinated for relocalization versus polyubiquitinated for degradation, and how the autoinhibited enzyme is selectively activated toward specific substrates in vivo.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying determinant linking substrate features to ubiquitin output\", \"In vivo activation/adapter map incomplete beyond NAP1L1\", \"Few substrates validated structurally in complex with human UBE2O\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 4, 6, 17, 20]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 4, 17, 20]},\n      {\"term_id\": \"GO:0140097\", \"supporting_discovery_ids\": []},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": []}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3, 4, 21]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 4, 5]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [6, 9, 15]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 2, 6]},\n      {\"term_id\": \"R-HSA-9909396\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [13]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [2, 16]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [6, 7, 10, 15, 18]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"NAP1L1\", \"SMAD6\", \"TRAF6\", \"BAP1\", \"AMPK\\u03b12/PRKAA2\", \"BMAL1\", \"RECQL4\", \"RSK2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":6,"faith_total":6,"faith_pct":100.0}}