{"gene":"UBE2K","run_date":"2026-04-28T21:43:00","timeline":{"discoveries":[{"year":1990,"finding":"Yeast UBC1 (ortholog of UBE2K) is a ubiquitin-conjugating enzyme that mediates selective degradation of short-lived and abnormal proteins, functioning redundantly with UBC4 and UBC5 in an essential subfamily required for cell growth and viability.","method":"Gene disruption, genetic epistasis, growth phenotype analysis","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — clean KO with defined cellular phenotype, replicated across multiple alleles and genetic backgrounds","pmids":["2265617"],"is_preprint":false},{"year":2004,"finding":"UBE2K/Ubc1 is a class II E2 enzyme with a C-terminal ubiquitin-associated (UBA) domain connected to the catalytic domain by a flexible 22-residue tether; the UBA domain interacts with ubiquitin in a regioselective manner and facilitates polyubiquitin chain formation.","method":"NMR solution structure, NMR chemical shift perturbation experiments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — NMR structure with functional validation of UBA-ubiquitin interaction","pmids":["15328341"],"is_preprint":false},{"year":2001,"finding":"UBE2K (Hip-2) physically interacts with RING finger protein RNF2, and this interaction requires the RING domain of RNF2; RNF2 exhibits E3 ubiquitin ligase activity in the presence of UBE2K, establishing UBE2K as the cognate E2 for a subset of RING finger E3 ligases.","method":"Yeast two-hybrid screen, in vitro and in vivo co-immunoprecipitation, RING domain mutagenesis, ubiquitin ligase activity assay","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP with in vitro activity assay and mutagenesis confirmation","pmids":["11513855"],"is_preprint":false},{"year":2015,"finding":"UBE2K preferentially catalyses formation of Lys48-linked ubiquitin chains via a monomeric mechanism; crystal structure of the UBE2K~ubiquitin conjugate reveals key features including acceptor ubiquitin positioning required for K48-specific chain synthesis.","method":"Crystal structure determination, site-directed mutagenesis, in vitro ubiquitin chain synthesis assay, structural modeling","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus mutagenesis plus in vitro reconstitution in single study","pmids":["26592444"],"is_preprint":false},{"year":2015,"finding":"The HIP2 (UBE2K)~ubiquitin thioester conjugate remains predominantly monomeric in solution and adopts an open/backbent conformation; the UBA domain enhances di-ubiquitin formation rate approximately 5-fold compared to UBE2K lacking the UBA domain.","method":"Analytical ultracentrifugation, SAXS, stable disulfide complex analysis, in vitro activity assays comparing full-length vs. UBA-deleted UBE2K","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 — multiple orthogonal biophysical methods plus functional activity assay","pmids":["25799589"],"is_preprint":false},{"year":2018,"finding":"Crystal structure of UBE2K/E2-25K in complex with K48-linked di-ubiquitin (at 2.47 Å) reveals that interaction between acceptor di-ubiquitin and auxiliary UBE2K promotes discharge reaction and K48-linked tri-ubiquitin synthesis without an E3 ligase.","method":"Crystal structure determination, biochemical ubiquitin chain synthesis assays","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with biochemical functional validation","pmids":["30336976"],"is_preprint":false},{"year":2021,"finding":"The UBA domain of UBE2K (and yeast Ubc1) preferentially binds K63-linked ubiquitin chains and facilitates assembly of K48/K63-branched ubiquitin conjugates; this branched chain topology is linked to maintenance of cellular proteostasis.","method":"NMR, structural modeling, in vitro ubiquitination assays, genetic epistasis in yeast, mass spectrometry proteomics","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods (NMR, in vitro reconstitution, genetics, proteomics) across two organisms","pmids":["33576509"],"is_preprint":false},{"year":2021,"finding":"Ubiquitin variants (UbVs) bind UBE2K at a hydrophobic cleft distinct from the active site and inhibit both ubiquitin charging of UBE2K and E3-catalyzed ubiquitin transfer, likely by steric clash with E1 and allosteric disruption of E3 interactions.","method":"Crystal structure of UbV-UBE2K complex, in vitro charging and transfer assays, mutagenesis","journal":"ACS chemical biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus in vitro functional assays demonstrating inhibitory mechanism","pmids":["34397214"],"is_preprint":false},{"year":2020,"finding":"UBE2K in human embryonic stem cells binds histone H3 and induces its K48-linked polyubiquitination and proteasomal degradation; loss of UBE2K upregulates SETDB1, causing H3K9 trimethylation and repression of neurogenic genes during differentiation. The worm ortholog ubc-20 similarly regulates H3 levels and H3K9me3 in C. elegans germ cells.","method":"RNAi/shRNA knockdown, co-immunoprecipitation, ubiquitination assays, Western blot, differentiation assays in hESCs, C. elegans genetics","journal":"Communications biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (Co-IP, ubiquitination assay, KD phenotype) replicated in two organisms","pmids":["32451438"],"is_preprint":false},{"year":2013,"finding":"Hip2 (UBE2K) overcomes radiation-induced G2/M arrest by interacting with p53 and targeting it for ubiquitin-proteasome-mediated degradation, thereby activating cdc2-cyclin B1 kinase to promote mitotic entry.","method":"Bimolecular fluorescence complementation, co-immunoprecipitation, flow cytometry, live cell fluorescence imaging, overexpression studies","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 — BiFC plus Co-IP with defined cellular phenotype, single lab","pmids":["23933584"],"is_preprint":false},{"year":2010,"finding":"Hip2 (UBE2K) interacts with cyclin B1 and promotes its degradation through the ubiquitin proteasome pathway, thereby blocking cyclin B1-mediated cell death.","method":"Co-immunoprecipitation, ubiquitin proteasome degradation assay, cell death assay","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP plus degradation assay, single lab","pmids":["20965177"],"is_preprint":false},{"year":2010,"finding":"Hip2 (UBE2K) interacts with and promotes proteasomal degradation of the pro-apoptotic protein Smac/DIABLO, blocking Smac-mediated cell death.","method":"Co-immunoprecipitation (in vivo and in vitro), ubiquitin proteasome degradation assay, cell death assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2-3 — reciprocal in vivo/in vitro Co-IP plus degradation assay, single lab","pmids":["20537984"],"is_preprint":false},{"year":2018,"finding":"Hip2 (UBE2K) is phosphorylated after UV radiation, and this phosphorylation inhibits p53 to suppress p21 expression and allow cell cycle re-entry from G1/S arrest.","method":"pIMAGO phosphorylation assay, thymidine synchronization, Western blot, flow cytometry","journal":"Genes & genomics","confidence":"Low","confidence_rationale":"Tier 3 — single lab, phosphorylation detection without identified kinase","pmids":["30264212"],"is_preprint":false},{"year":2021,"finding":"HIP2 (UBE2K) interacts specifically with mutant (not wild-type) SOD1, is upregulated in response to mutant SOD1 expression, and modifies mutant SOD1 via K48-linked polyubiquitination leading to UPS-mediated degradation, protecting cells from mutant SOD1 proteotoxicity.","method":"Co-immunoprecipitation, ubiquitination assay with linkage-specific antibodies, Western blot in transgenic mouse spinal cord, cell viability assay","journal":"Biochimica et biophysica acta. Molecular basis of disease","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP plus in cellulo ubiquitination assay plus in vivo mouse model, single lab","pmids":["34856358"],"is_preprint":false},{"year":2015,"finding":"UbcD4, the Drosophila ortholog of UBE2K/E2-25K, is required for activation of the IMD (NF-κB) immune deficiency pathway; RNAi knockdown of UBE2K in human cells similarly inhibited TNFα- and LPS-mediated NF-κB pathway activation.","method":"RNAi knockdown in Drosophila, immunoblot-based pathway activity assay, RNAi knockdown in human cells","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — loss-of-function with defined pathway phenotype confirmed in two organisms","pmids":["26707646"],"is_preprint":false},{"year":2016,"finding":"The E3 ligase San1 functions preferentially with yeast Ubc1 (ortholog of UBE2K) over Cdc34 for ubiquitylation of misfolded protein substrates during nuclear protein quality control, as demonstrated by reconstituted in vitro ubiquitylation kinetics.","method":"In vitro reconstitution of San1-mediated ubiquitylation, kinetic measurements with synthetic misfolded substrate peptide, competition assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with quantitative kinetics, rigorous controls","pmids":["27405755"],"is_preprint":false},{"year":2025,"finding":"A microsporidial otubain deubiquitinase (ehOTUB1) selectively binds UBE2K and inhibits its ubiquitin conjugation by sterically blocking UBE2K docking onto the E1 adenylation site, preventing ubiquitin transfer to UBE2K independent of DUB catalytic activity.","method":"In vitro binding assays, ubiquitin conjugation/charging assays, structural modeling, mutagenesis","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — multiple in vitro functional assays revealing novel mechanism, preprint not yet peer-reviewed","pmids":["40766534"],"is_preprint":true},{"year":2025,"finding":"UBE2K promotes ubiquitination and proteasomal degradation of STUB1 (CHIP) in breast cancer cells, activating the PKA/CREB1 signaling pathway; CREB1 in turn transcriptionally activates UBE2K, forming a positive feedback loop.","method":"Co-immunoprecipitation, ubiquitination assay, ChIP assay, dual-luciferase reporter assay, Western blot, functional rescue experiments","journal":"Biochimica et biophysica acta. Molecular basis of disease","confidence":"Medium","confidence_rationale":"Tier 2-3 — multiple orthogonal methods (Co-IP, ChIP, reporter assay, ubiquitination assay), single lab","pmids":["41435988"],"is_preprint":false},{"year":2025,"finding":"Amuc_1409 (an Akkermansia muciniphila protein) interacts with UBE2K to reduce ubiquitination-mediated degradation of Foxp3 in regulatory T cells, promoting Treg differentiation and IL-10 production to suppress inflammatory responses.","method":"Co-immunoprecipitation, ubiquitination assay, Foxp3-DTR and IL-10-KO mouse models, cell differentiation assays","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP plus ubiquitination assay plus in vivo mouse models, identifies UBE2K as ubiquitin writer for Foxp3","pmids":["40464424"],"is_preprint":false},{"year":2024,"finding":"Circular RNA circ-UBE2K binds HNRNPU to form a complex that upregulates expression of the parental gene UBE2K, leading to abnormal microglial activation and neuroinflammation promoting major depressive disorder.","method":"Pull-down mass spectrometry, RNA immunoprecipitation (RIP), FISH, lentiviral/AAV overexpression and knockdown in mouse hippocampus, behavioral tests","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2-3 — RIP and pulldown-MS identify HNRNPU as circ-UBE2K binding partner; in vivo functional validation links to UBE2K upregulation","pmids":["38994030"],"is_preprint":false}],"current_model":"UBE2K is a class II E2 ubiquitin-conjugating enzyme that autonomously synthesizes K48-linked polyubiquitin chains (and K48/K63-branched chains facilitated by its UBA domain's preferential binding to K63-linked ubiquitin) to target substrates—including histone H3, p53, cyclin B1, Smac/DIABLO, mutant SOD1, Foxp3, and STUB1—for proteasomal degradation, acting with diverse RING-finger E3 ligases (e.g., RNF2, San1) to regulate protein quality control, cell cycle progression, apoptosis suppression, neurogenic potential, and immune signaling."},"narrative":{"teleology":[{"year":1990,"claim":"Before characterization, it was unknown whether UBC1 contributed a specific degradation function; gene disruption in yeast established that UBC1 (the UBE2K ortholog) mediates selective turnover of short-lived and abnormal proteins and is essential for viability redundantly with UBC4/UBC5.","evidence":"Gene disruption and genetic epistasis in S. cerevisiae","pmids":["2265617"],"confidence":"High","gaps":["Mammalian substrate specificity unknown","Mechanism of chain type selectivity not addressed"]},{"year":2001,"claim":"The identity of E3 partners for UBE2K was unknown; yeast two-hybrid and Co-IP experiments demonstrated that UBE2K physically interacts with the RING domain of RNF2, establishing it as a cognate E2 for RING-finger E3 ligases.","evidence":"Yeast two-hybrid, reciprocal Co-IP, RING mutagenesis, in vitro ubiquitin ligase assay","pmids":["11513855"],"confidence":"High","gaps":["Breadth of E3 partners beyond RNF2 not known","In vivo substrates of the UBE2K-RNF2 pair unidentified"]},{"year":2004,"claim":"It was unclear how the unusual UBA domain of UBE2K contributed to chain assembly; NMR structural analysis revealed the UBA domain connected by a flexible tether interacts with ubiquitin in a regioselective manner and facilitates polyubiquitin chain formation.","evidence":"NMR solution structure and chemical shift perturbation experiments","pmids":["15328341"],"confidence":"High","gaps":["Chain linkage specificity not yet defined","Structural basis of acceptor ubiquitin positioning unresolved"]},{"year":2010,"claim":"Whether UBE2K had anti-apoptotic roles in mammalian cells was unknown; Co-IP and degradation assays showed UBE2K interacts with cyclin B1 and Smac/DIABLO, promoting their proteasomal degradation and blocking cell death.","evidence":"Co-immunoprecipitation and proteasome-dependent degradation assays with cell death readouts","pmids":["20965177","20537984"],"confidence":"Medium","gaps":["E3 ligase(s) mediating cyclin B1 and Smac degradation not identified","Single-lab studies without independent replication"]},{"year":2013,"claim":"The mechanism by which cells overcome radiation-induced G2/M arrest was unclear with respect to UBE2K; experiments showed UBE2K interacts with p53 and targets it for proteasomal degradation, thereby activating cdc2-cyclin B1 kinase and promoting mitotic re-entry.","evidence":"BiFC, Co-IP, flow cytometry, live-cell imaging in irradiated cells","pmids":["23933584"],"confidence":"Medium","gaps":["E3 partner for p53 ubiquitination by UBE2K not defined","Relationship to canonical MDM2-mediated p53 degradation unclear"]},{"year":2015,"claim":"The structural basis for K48 linkage specificity was unresolved; crystal structures of UBE2K~ubiquitin conjugates and biophysical analyses established that UBE2K operates as a monomer in an open/backbent conformation, with the UBA domain enhancing di-ubiquitin synthesis ~5-fold and positioning the acceptor ubiquitin for K48-specific chain elongation.","evidence":"Crystal structures, AUC, SAXS, mutagenesis, in vitro chain synthesis comparing full-length vs. UBA-deleted UBE2K","pmids":["26592444","25799589"],"confidence":"High","gaps":["How E3 ligases modulate the monomeric chain-building mechanism not addressed","No in vivo confirmation of UBA-dependent chain enhancement"]},{"year":2015,"claim":"Whether UBE2K had roles in innate immunity was unknown; RNAi of the Drosophila ortholog UbcD4 and of UBE2K in human cells demonstrated requirement for NF-κB pathway activation downstream of IMD/TNFα/LPS signaling.","evidence":"RNAi knockdown in Drosophila and human cells, immunoblot pathway readouts","pmids":["26707646"],"confidence":"Medium","gaps":["Direct ubiquitination target within NF-κB cascade not identified","Mechanism of action in NF-κB signaling unclear"]},{"year":2016,"claim":"It was unknown which E2 partnered with the nuclear protein quality control E3 San1; reconstituted ubiquitylation kinetics showed San1 preferentially uses Ubc1/UBE2K over Cdc34 for misfolded protein substrates.","evidence":"In vitro reconstitution with synthetic misfolded substrates, kinetic measurements","pmids":["27405755"],"confidence":"High","gaps":["Whether this preference extends to mammalian San1 orthologs untested","In vivo validation limited"]},{"year":2018,"claim":"How UBE2K elongates chains beyond di-ubiquitin without an E3 was structurally unresolved; a crystal structure of UBE2K bound to K48-linked di-ubiquitin revealed that an auxiliary UBE2K molecule promotes discharge to synthesize tri-ubiquitin.","evidence":"Crystal structure at 2.47 Å, biochemical chain synthesis assays","pmids":["30336976"],"confidence":"High","gaps":["Physiological relevance of E3-independent chain elongation in vivo not established"]},{"year":2020,"claim":"Whether UBE2K regulated chromatin during differentiation was unknown; knockdown in human ESCs and C. elegans showed UBE2K ubiquitinates histone H3 for K48-linked proteasomal degradation, and its loss upregulates SETDB1, causing H3K9me3-mediated repression of neurogenic genes.","evidence":"shRNA/RNAi knockdown, Co-IP, ubiquitination assays, hESC differentiation, C. elegans genetics","pmids":["32451438"],"confidence":"High","gaps":["How UBE2K selectively targets histone H3 over other histones is unexplained","E3 ligase for H3 ubiquitination not identified"]},{"year":2021,"claim":"The chain topology produced by UBE2K in the context of mixed-linkage ubiquitin was unclear; NMR, proteomics, and genetics demonstrated the UBA domain preferentially binds K63-linked chains and facilitates K48/K63-branched conjugate assembly, linking this topology to proteostasis.","evidence":"NMR, in vitro ubiquitination, yeast genetic epistasis, mass spectrometry","pmids":["33576509"],"confidence":"High","gaps":["Identity of in vivo substrates receiving branched chains from UBE2K unknown","Quantitative contribution to total branched ubiquitin pool not measured"]},{"year":2021,"claim":"No selective small-molecule or protein-based inhibitors of UBE2K existed; engineered ubiquitin variants were shown to bind a hydrophobic cleft on UBE2K and inhibit both E1 charging and E3-mediated transfer by steric and allosteric mechanisms.","evidence":"Crystal structure of UbV-UBE2K complex, mutagenesis, in vitro charging and transfer assays","pmids":["34397214"],"confidence":"High","gaps":["In vivo efficacy and selectivity of UbV inhibitors not tested"]},{"year":2021,"claim":"Whether UBE2K contributed to clearance of ALS-linked mutant SOD1 was unknown; Co-IP and linkage-specific ubiquitination assays showed UBE2K selectively binds and K48-polyubiquitinates mutant (not wild-type) SOD1, promoting its proteasomal degradation and protecting against proteotoxicity.","evidence":"Co-IP, K48-specific ubiquitination assay, transgenic mouse spinal cord, cell viability assay","pmids":["34856358"],"confidence":"Medium","gaps":["E3 ligase mediating mutant SOD1 ubiquitination with UBE2K not identified","Single-lab finding awaiting independent replication"]},{"year":2025,"claim":"New substrate-level roles and regulatory inputs for UBE2K continue to emerge: UBE2K ubiquitinates STUB1/CHIP in breast cancer cells within a CREB1-driven positive feedback loop, and UBE2K-mediated Foxp3 ubiquitination in Tregs is modulated by the gut microbial protein Amuc_1409.","evidence":"Co-IP, ubiquitination assays, ChIP, dual-luciferase reporter, mouse Treg models","pmids":["41435988","40464424"],"confidence":"Medium","gaps":["Whether UBE2K acts directly or through an E3 for Foxp3 ubiquitination is unresolved","Physiological relevance of UBE2K-STUB1 axis beyond breast cancer cell lines not established","Feedback loop dynamics not modeled quantitatively"]},{"year":null,"claim":"A systematic map of E3 ligase partners and substrates of UBE2K in vivo, the structural basis of E3-dependent substrate selection, and the relative contributions of linear vs. branched chain building to UBE2K's physiological functions remain to be established.","evidence":"","pmids":[],"confidence":"High","gaps":["No comprehensive E3 partner catalog exists","No structural model of UBE2K within an E3-substrate ternary complex","In vivo phenotypes in mammalian knockout models not systematically characterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,3,5,6,8,13,17,18]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[3,5,6]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[8,15]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[4,9]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,3,5,6,8,13,15,17,18]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[6,15]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[14,18]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[9,10]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[10,11]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[8]}],"complexes":[],"partners":["RNF2","STUB1","H3","TP53","CCNB1","DIABLO","SOD1","FOXP3"],"other_free_text":[]},"mechanistic_narrative":"UBE2K is a class II E2 ubiquitin-conjugating enzyme that autonomously synthesizes K48-linked polyubiquitin chains through a monomeric mechanism, with its C-terminal UBA domain enhancing chain formation rate and conferring preferential binding to K63-linked ubiquitin to facilitate assembly of K48/K63-branched chains important for proteostasis [PMID:26592444, PMID:33576509, PMID:25799589]. UBE2K partners with diverse RING-finger E3 ligases, including RNF2 and yeast San1, to ubiquitinate substrates such as histone H3, p53, cyclin B1, Smac/DIABLO, mutant SOD1, Foxp3, and STUB1, targeting them for proteasomal degradation [PMID:11513855, PMID:27405755, PMID:32451438, PMID:34856358, PMID:40464424, PMID:41435988]. Through these substrate-directed activities, UBE2K regulates nuclear protein quality control, neurogenic differentiation via histone H3 turnover and H3K9me3 modulation, cell cycle checkpoint recovery, apoptosis suppression, NF-κB immune signaling, and regulatory T cell homeostasis [PMID:32451438, PMID:23933584, PMID:20965177, PMID:26707646, PMID:40464424]. In the original yeast system, the ortholog UBC1 is essential redundantly with UBC4/UBC5 for degradation of short-lived and abnormal proteins and for cell growth [PMID:2265617]."},"prefetch_data":{"uniprot":{"accession":"P61086","full_name":"Ubiquitin-conjugating enzyme E2 K","aliases":["E2 ubiquitin-conjugating enzyme K","Huntingtin-interacting protein 2","HIP-2","Ubiquitin carrier protein","Ubiquitin-conjugating enzyme E2-25 kDa","Ubiquitin-conjugating enzyme E2(25K)","Ubiquitin-conjugating enzyme E2-25K","Ubiquitin-protein ligase"],"length_aa":200,"mass_kda":22.4,"function":"Accepts ubiquitin from the E1 complex and catalyzes its covalent attachment to other proteins. In vitro, in the presence or in the absence of BRCA1-BARD1 E3 ubiquitin-protein ligase complex, catalyzes the synthesis of 'Lys-48'-linked polyubiquitin chains. Does not transfer ubiquitin directly to but elongates monoubiquitinated substrate protein. Mediates the selective degradation of short-lived and abnormal proteins, such as the endoplasmic reticulum-associated degradation (ERAD) of misfolded lumenal proteins. Ubiquitinates huntingtin. May mediate foam cell formation by the suppression of apoptosis of lipid-bearing macrophages through ubiquitination and subsequence degradation of p53/TP53. Proposed to be involved in ubiquitination and proteolytic processing of NF-kappa-B; in vitro supports ubiquitination of NFKB1. In case of infection by cytomegaloviruses may be involved in the US11-dependent degradation of MHC class I heavy chains following their export from the ER to the cytosol. In case of viral infections may be involved in the HPV E7 protein-dependent degradation of RB1","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/P61086/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/UBE2K","classification":"Not Classified","n_dependent_lines":129,"n_total_lines":1208,"dependency_fraction":0.10678807947019868},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"UBA1","stoichiometry":0.2},{"gene":"UBA52","stoichiometry":0.2},{"gene":"UBC","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/UBE2K","total_profiled":1310},"omim":[{"mim_id":"620096","title":"RING FINGER PROTEIN 185; RNF185","url":"https://www.omim.org/entry/620096"},{"mim_id":"616319","title":"RING FINGER PROTEIN 138; RNF138","url":"https://www.omim.org/entry/616319"},{"mim_id":"609476","title":"NEMO-LIKE KINASE; NLK","url":"https://www.omim.org/entry/609476"},{"mim_id":"602846","title":"UBIQUITIN-CONJUGATING ENZYME E2 K; UBE2K","url":"https://www.omim.org/entry/602846"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"},{"location":"Plasma membrane","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/UBE2K"},"hgnc":{"alias_symbol":["HYPG","UBC1"],"prev_symbol":["HIP2"]},"alphafold":{"accession":"P61086","domains":[{"cath_id":"3.10.110.10","chopping":"2-117","consensus_level":"high","plddt":96.6383,"start":2,"end":117},{"cath_id":"1.10.8.10","chopping":"128-198","consensus_level":"medium","plddt":97.3699,"start":128,"end":198}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P61086","model_url":"https://alphafold.ebi.ac.uk/files/AF-P61086-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P61086-F1-predicted_aligned_error_v6.png","plddt_mean":96.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=UBE2K","jax_strain_url":"https://www.jax.org/strain/search?query=UBE2K"},"sequence":{"accession":"P61086","fasta_url":"https://rest.uniprot.org/uniprotkb/P61086.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P61086/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P61086"}},"corpus_meta":[{"pmid":"2265617","id":"PMC_2265617","title":"UBC1 encodes a novel member of an essential subfamily of yeast ubiquitin-conjugating enzymes involved in protein degradation.","date":"1990","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/2265617","citation_count":150,"is_preprint":false},{"pmid":"32167578","id":"PMC_32167578","title":"E2 conjugases UBC1 and UBC2 regulate MYB42-mediated SOS pathway in response to salt stress in Arabidopsis.","date":"2020","source":"The New phytologist","url":"https://pubmed.ncbi.nlm.nih.gov/32167578","citation_count":76,"is_preprint":false},{"pmid":"26592444","id":"PMC_26592444","title":"The molecular basis of lysine 48 ubiquitin chain synthesis by Ube2K.","date":"2015","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/26592444","citation_count":50,"is_preprint":false},{"pmid":"15328341","id":"PMC_15328341","title":"Solution structure of the flexible class II ubiquitin-conjugating enzyme Ubc1 provides insights for polyubiquitin chain 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the UBA domain interacts with ubiquitin in a regioselective manner and facilitates polyubiquitin chain formation.\",\n      \"method\": \"NMR solution structure, NMR chemical shift perturbation experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structure with functional validation of UBA-ubiquitin interaction\",\n      \"pmids\": [\"15328341\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"UBE2K (Hip-2) physically interacts with RING finger protein RNF2, and this interaction requires the RING domain of RNF2; RNF2 exhibits E3 ubiquitin ligase activity in the presence of UBE2K, establishing UBE2K as the cognate E2 for a subset of RING finger E3 ligases.\",\n      \"method\": \"Yeast two-hybrid screen, in vitro and in vivo co-immunoprecipitation, RING domain mutagenesis, ubiquitin ligase activity assay\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP with in vitro activity assay and mutagenesis confirmation\",\n      \"pmids\": [\"11513855\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"UBE2K preferentially catalyses formation of Lys48-linked ubiquitin chains via a monomeric mechanism; crystal structure of the UBE2K~ubiquitin conjugate reveals key features including acceptor ubiquitin positioning required for K48-specific chain synthesis.\",\n      \"method\": \"Crystal structure determination, site-directed mutagenesis, in vitro ubiquitin chain synthesis assay, structural modeling\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus mutagenesis plus in vitro reconstitution in single study\",\n      \"pmids\": [\"26592444\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The HIP2 (UBE2K)~ubiquitin thioester conjugate remains predominantly monomeric in solution and adopts an open/backbent conformation; the UBA domain enhances di-ubiquitin formation rate approximately 5-fold compared to UBE2K lacking the UBA domain.\",\n      \"method\": \"Analytical ultracentrifugation, SAXS, stable disulfide complex analysis, in vitro activity assays comparing full-length vs. UBA-deleted UBE2K\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple orthogonal biophysical methods plus functional activity assay\",\n      \"pmids\": [\"25799589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Crystal structure of UBE2K/E2-25K in complex with K48-linked di-ubiquitin (at 2.47 Å) reveals that interaction between acceptor di-ubiquitin and auxiliary UBE2K promotes discharge reaction and K48-linked tri-ubiquitin synthesis without an E3 ligase.\",\n      \"method\": \"Crystal structure determination, biochemical ubiquitin chain synthesis assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with biochemical functional validation\",\n      \"pmids\": [\"30336976\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The UBA domain of UBE2K (and yeast Ubc1) preferentially binds K63-linked ubiquitin chains and facilitates assembly of K48/K63-branched ubiquitin conjugates; this branched chain topology is linked to maintenance of cellular proteostasis.\",\n      \"method\": \"NMR, structural modeling, in vitro ubiquitination assays, genetic epistasis in yeast, mass spectrometry proteomics\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods (NMR, in vitro reconstitution, genetics, proteomics) across two organisms\",\n      \"pmids\": [\"33576509\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Ubiquitin variants (UbVs) bind UBE2K at a hydrophobic cleft distinct from the active site and inhibit both ubiquitin charging of UBE2K and E3-catalyzed ubiquitin transfer, likely by steric clash with E1 and allosteric disruption of E3 interactions.\",\n      \"method\": \"Crystal structure of UbV-UBE2K complex, in vitro charging and transfer assays, mutagenesis\",\n      \"journal\": \"ACS chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus in vitro functional assays demonstrating inhibitory mechanism\",\n      \"pmids\": [\"34397214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"UBE2K in human embryonic stem cells binds histone H3 and induces its K48-linked polyubiquitination and proteasomal degradation; loss of UBE2K upregulates SETDB1, causing H3K9 trimethylation and repression of neurogenic genes during differentiation. The worm ortholog ubc-20 similarly regulates H3 levels and H3K9me3 in C. elegans germ cells.\",\n      \"method\": \"RNAi/shRNA knockdown, co-immunoprecipitation, ubiquitination assays, Western blot, differentiation assays in hESCs, C. elegans genetics\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (Co-IP, ubiquitination assay, KD phenotype) replicated in two organisms\",\n      \"pmids\": [\"32451438\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Hip2 (UBE2K) overcomes radiation-induced G2/M arrest by interacting with p53 and targeting it for ubiquitin-proteasome-mediated degradation, thereby activating cdc2-cyclin B1 kinase to promote mitotic entry.\",\n      \"method\": \"Bimolecular fluorescence complementation, co-immunoprecipitation, flow cytometry, live cell fluorescence imaging, overexpression studies\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — BiFC plus Co-IP with defined cellular phenotype, single lab\",\n      \"pmids\": [\"23933584\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Hip2 (UBE2K) interacts with cyclin B1 and promotes its degradation through the ubiquitin proteasome pathway, thereby blocking cyclin B1-mediated cell death.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitin proteasome degradation assay, cell death assay\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP plus degradation assay, single lab\",\n      \"pmids\": [\"20965177\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Hip2 (UBE2K) interacts with and promotes proteasomal degradation of the pro-apoptotic protein Smac/DIABLO, blocking Smac-mediated cell death.\",\n      \"method\": \"Co-immunoprecipitation (in vivo and in vitro), ubiquitin proteasome degradation assay, cell death assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — reciprocal in vivo/in vitro Co-IP plus degradation assay, single lab\",\n      \"pmids\": [\"20537984\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Hip2 (UBE2K) is phosphorylated after UV radiation, and this phosphorylation inhibits p53 to suppress p21 expression and allow cell cycle re-entry from G1/S arrest.\",\n      \"method\": \"pIMAGO phosphorylation assay, thymidine synchronization, Western blot, flow cytometry\",\n      \"journal\": \"Genes & genomics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, phosphorylation detection without identified kinase\",\n      \"pmids\": [\"30264212\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HIP2 (UBE2K) interacts specifically with mutant (not wild-type) SOD1, is upregulated in response to mutant SOD1 expression, and modifies mutant SOD1 via K48-linked polyubiquitination leading to UPS-mediated degradation, protecting cells from mutant SOD1 proteotoxicity.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay with linkage-specific antibodies, Western blot in transgenic mouse spinal cord, cell viability assay\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular basis of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP plus in cellulo ubiquitination assay plus in vivo mouse model, single lab\",\n      \"pmids\": [\"34856358\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"UbcD4, the Drosophila ortholog of UBE2K/E2-25K, is required for activation of the IMD (NF-κB) immune deficiency pathway; RNAi knockdown of UBE2K in human cells similarly inhibited TNFα- and LPS-mediated NF-κB pathway activation.\",\n      \"method\": \"RNAi knockdown in Drosophila, immunoblot-based pathway activity assay, RNAi knockdown in human cells\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — loss-of-function with defined pathway phenotype confirmed in two organisms\",\n      \"pmids\": [\"26707646\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The E3 ligase San1 functions preferentially with yeast Ubc1 (ortholog of UBE2K) over Cdc34 for ubiquitylation of misfolded protein substrates during nuclear protein quality control, as demonstrated by reconstituted in vitro ubiquitylation kinetics.\",\n      \"method\": \"In vitro reconstitution of San1-mediated ubiquitylation, kinetic measurements with synthetic misfolded substrate peptide, competition assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with quantitative kinetics, rigorous controls\",\n      \"pmids\": [\"27405755\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"A microsporidial otubain deubiquitinase (ehOTUB1) selectively binds UBE2K and inhibits its ubiquitin conjugation by sterically blocking UBE2K docking onto the E1 adenylation site, preventing ubiquitin transfer to UBE2K independent of DUB catalytic activity.\",\n      \"method\": \"In vitro binding assays, ubiquitin conjugation/charging assays, structural modeling, mutagenesis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple in vitro functional assays revealing novel mechanism, preprint not yet peer-reviewed\",\n      \"pmids\": [\"40766534\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"UBE2K promotes ubiquitination and proteasomal degradation of STUB1 (CHIP) in breast cancer cells, activating the PKA/CREB1 signaling pathway; CREB1 in turn transcriptionally activates UBE2K, forming a positive feedback loop.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, ChIP assay, dual-luciferase reporter assay, Western blot, functional rescue experiments\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular basis of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — multiple orthogonal methods (Co-IP, ChIP, reporter assay, ubiquitination assay), single lab\",\n      \"pmids\": [\"41435988\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Amuc_1409 (an Akkermansia muciniphila protein) interacts with UBE2K to reduce ubiquitination-mediated degradation of Foxp3 in regulatory T cells, promoting Treg differentiation and IL-10 production to suppress inflammatory responses.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, Foxp3-DTR and IL-10-KO mouse models, cell differentiation assays\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP plus ubiquitination assay plus in vivo mouse models, identifies UBE2K as ubiquitin writer for Foxp3\",\n      \"pmids\": [\"40464424\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Circular RNA circ-UBE2K binds HNRNPU to form a complex that upregulates expression of the parental gene UBE2K, leading to abnormal microglial activation and neuroinflammation promoting major depressive disorder.\",\n      \"method\": \"Pull-down mass spectrometry, RNA immunoprecipitation (RIP), FISH, lentiviral/AAV overexpression and knockdown in mouse hippocampus, behavioral tests\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — RIP and pulldown-MS identify HNRNPU as circ-UBE2K binding partner; in vivo functional validation links to UBE2K upregulation\",\n      \"pmids\": [\"38994030\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"UBE2K is a class II E2 ubiquitin-conjugating enzyme that autonomously synthesizes K48-linked polyubiquitin chains (and K48/K63-branched chains facilitated by its UBA domain's preferential binding to K63-linked ubiquitin) to target substrates—including histone H3, p53, cyclin B1, Smac/DIABLO, mutant SOD1, Foxp3, and STUB1—for proteasomal degradation, acting with diverse RING-finger E3 ligases (e.g., RNF2, San1) to regulate protein quality control, cell cycle progression, apoptosis suppression, neurogenic potential, and immune signaling.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"UBE2K is a class II E2 ubiquitin-conjugating enzyme that autonomously synthesizes K48-linked polyubiquitin chains through a monomeric mechanism, with its C-terminal UBA domain enhancing chain formation rate and conferring preferential binding to K63-linked ubiquitin to facilitate assembly of K48/K63-branched chains important for proteostasis [PMID:26592444, PMID:33576509, PMID:25799589]. UBE2K partners with diverse RING-finger E3 ligases, including RNF2 and yeast San1, to ubiquitinate substrates such as histone H3, p53, cyclin B1, Smac/DIABLO, mutant SOD1, Foxp3, and STUB1, targeting them for proteasomal degradation [PMID:11513855, PMID:27405755, PMID:32451438, PMID:34856358, PMID:40464424, PMID:41435988]. Through these substrate-directed activities, UBE2K regulates nuclear protein quality control, neurogenic differentiation via histone H3 turnover and H3K9me3 modulation, cell cycle checkpoint recovery, apoptosis suppression, NF-κB immune signaling, and regulatory T cell homeostasis [PMID:32451438, PMID:23933584, PMID:20965177, PMID:26707646, PMID:40464424]. In the original yeast system, the ortholog UBC1 is essential redundantly with UBC4/UBC5 for degradation of short-lived and abnormal proteins and for cell growth [PMID:2265617].\",\n  \"teleology\": [\n    {\n      \"year\": 1990,\n      \"claim\": \"Before characterization, it was unknown whether UBC1 contributed a specific degradation function; gene disruption in yeast established that UBC1 (the UBE2K ortholog) mediates selective turnover of short-lived and abnormal proteins and is essential for viability redundantly with UBC4/UBC5.\",\n      \"evidence\": \"Gene disruption and genetic epistasis in S. cerevisiae\",\n      \"pmids\": [\"2265617\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mammalian substrate specificity unknown\", \"Mechanism of chain type selectivity not addressed\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"The identity of E3 partners for UBE2K was unknown; yeast two-hybrid and Co-IP experiments demonstrated that UBE2K physically interacts with the RING domain of RNF2, establishing it as a cognate E2 for RING-finger E3 ligases.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal Co-IP, RING mutagenesis, in vitro ubiquitin ligase assay\",\n      \"pmids\": [\"11513855\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Breadth of E3 partners beyond RNF2 not known\", \"In vivo substrates of the UBE2K-RNF2 pair unidentified\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"It was unclear how the unusual UBA domain of UBE2K contributed to chain assembly; NMR structural analysis revealed the UBA domain connected by a flexible tether interacts with ubiquitin in a regioselective manner and facilitates polyubiquitin chain formation.\",\n      \"evidence\": \"NMR solution structure and chemical shift perturbation experiments\",\n      \"pmids\": [\"15328341\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Chain linkage specificity not yet defined\", \"Structural basis of acceptor ubiquitin positioning unresolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Whether UBE2K had anti-apoptotic roles in mammalian cells was unknown; Co-IP and degradation assays showed UBE2K interacts with cyclin B1 and Smac/DIABLO, promoting their proteasomal degradation and blocking cell death.\",\n      \"evidence\": \"Co-immunoprecipitation and proteasome-dependent degradation assays with cell death readouts\",\n      \"pmids\": [\"20965177\", \"20537984\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E3 ligase(s) mediating cyclin B1 and Smac degradation not identified\", \"Single-lab studies without independent replication\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"The mechanism by which cells overcome radiation-induced G2/M arrest was unclear with respect to UBE2K; experiments showed UBE2K interacts with p53 and targets it for proteasomal degradation, thereby activating cdc2-cyclin B1 kinase and promoting mitotic re-entry.\",\n      \"evidence\": \"BiFC, Co-IP, flow cytometry, live-cell imaging in irradiated cells\",\n      \"pmids\": [\"23933584\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E3 partner for p53 ubiquitination by UBE2K not defined\", \"Relationship to canonical MDM2-mediated p53 degradation unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"The structural basis for K48 linkage specificity was unresolved; crystal structures of UBE2K~ubiquitin conjugates and biophysical analyses established that UBE2K operates as a monomer in an open/backbent conformation, with the UBA domain enhancing di-ubiquitin synthesis ~5-fold and positioning the acceptor ubiquitin for K48-specific chain elongation.\",\n      \"evidence\": \"Crystal structures, AUC, SAXS, mutagenesis, in vitro chain synthesis comparing full-length vs. UBA-deleted UBE2K\",\n      \"pmids\": [\"26592444\", \"25799589\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How E3 ligases modulate the monomeric chain-building mechanism not addressed\", \"No in vivo confirmation of UBA-dependent chain enhancement\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Whether UBE2K had roles in innate immunity was unknown; RNAi of the Drosophila ortholog UbcD4 and of UBE2K in human cells demonstrated requirement for NF-κB pathway activation downstream of IMD/TNFα/LPS signaling.\",\n      \"evidence\": \"RNAi knockdown in Drosophila and human cells, immunoblot pathway readouts\",\n      \"pmids\": [\"26707646\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ubiquitination target within NF-κB cascade not identified\", \"Mechanism of action in NF-κB signaling unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"It was unknown which E2 partnered with the nuclear protein quality control E3 San1; reconstituted ubiquitylation kinetics showed San1 preferentially uses Ubc1/UBE2K over Cdc34 for misfolded protein substrates.\",\n      \"evidence\": \"In vitro reconstitution with synthetic misfolded substrates, kinetic measurements\",\n      \"pmids\": [\"27405755\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this preference extends to mammalian San1 orthologs untested\", \"In vivo validation limited\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"How UBE2K elongates chains beyond di-ubiquitin without an E3 was structurally unresolved; a crystal structure of UBE2K bound to K48-linked di-ubiquitin revealed that an auxiliary UBE2K molecule promotes discharge to synthesize tri-ubiquitin.\",\n      \"evidence\": \"Crystal structure at 2.47 Å, biochemical chain synthesis assays\",\n      \"pmids\": [\"30336976\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological relevance of E3-independent chain elongation in vivo not established\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Whether UBE2K regulated chromatin during differentiation was unknown; knockdown in human ESCs and C. elegans showed UBE2K ubiquitinates histone H3 for K48-linked proteasomal degradation, and its loss upregulates SETDB1, causing H3K9me3-mediated repression of neurogenic genes.\",\n      \"evidence\": \"shRNA/RNAi knockdown, Co-IP, ubiquitination assays, hESC differentiation, C. elegans genetics\",\n      \"pmids\": [\"32451438\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How UBE2K selectively targets histone H3 over other histones is unexplained\", \"E3 ligase for H3 ubiquitination not identified\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"The chain topology produced by UBE2K in the context of mixed-linkage ubiquitin was unclear; NMR, proteomics, and genetics demonstrated the UBA domain preferentially binds K63-linked chains and facilitates K48/K63-branched conjugate assembly, linking this topology to proteostasis.\",\n      \"evidence\": \"NMR, in vitro ubiquitination, yeast genetic epistasis, mass spectrometry\",\n      \"pmids\": [\"33576509\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of in vivo substrates receiving branched chains from UBE2K unknown\", \"Quantitative contribution to total branched ubiquitin pool not measured\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"No selective small-molecule or protein-based inhibitors of UBE2K existed; engineered ubiquitin variants were shown to bind a hydrophobic cleft on UBE2K and inhibit both E1 charging and E3-mediated transfer by steric and allosteric mechanisms.\",\n      \"evidence\": \"Crystal structure of UbV-UBE2K complex, mutagenesis, in vitro charging and transfer assays\",\n      \"pmids\": [\"34397214\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo efficacy and selectivity of UbV inhibitors not tested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Whether UBE2K contributed to clearance of ALS-linked mutant SOD1 was unknown; Co-IP and linkage-specific ubiquitination assays showed UBE2K selectively binds and K48-polyubiquitinates mutant (not wild-type) SOD1, promoting its proteasomal degradation and protecting against proteotoxicity.\",\n      \"evidence\": \"Co-IP, K48-specific ubiquitination assay, transgenic mouse spinal cord, cell viability assay\",\n      \"pmids\": [\"34856358\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E3 ligase mediating mutant SOD1 ubiquitination with UBE2K not identified\", \"Single-lab finding awaiting independent replication\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"New substrate-level roles and regulatory inputs for UBE2K continue to emerge: UBE2K ubiquitinates STUB1/CHIP in breast cancer cells within a CREB1-driven positive feedback loop, and UBE2K-mediated Foxp3 ubiquitination in Tregs is modulated by the gut microbial protein Amuc_1409.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, ChIP, dual-luciferase reporter, mouse Treg models\",\n      \"pmids\": [\"41435988\", \"40464424\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether UBE2K acts directly or through an E3 for Foxp3 ubiquitination is unresolved\", \"Physiological relevance of UBE2K-STUB1 axis beyond breast cancer cell lines not established\", \"Feedback loop dynamics not modeled quantitatively\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A systematic map of E3 ligase partners and substrates of UBE2K in vivo, the structural basis of E3-dependent substrate selection, and the relative contributions of linear vs. branched chain building to UBE2K's physiological functions remain to be established.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No comprehensive E3 partner catalog exists\", \"No structural model of UBE2K within an E3-substrate ternary complex\", \"In vivo phenotypes in mammalian knockout models not systematically characterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 3, 5, 6, 8, 13, 17, 18]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [3, 5, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [8, 15]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [4, 9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 3, 5, 6, 8, 13, 15, 17, 18]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [6, 15]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [14, 18]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [9, 10]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [10, 11]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"RNF2\",\n      \"STUB1\",\n      \"H3\",\n      \"TP53\",\n      \"CCNB1\",\n      \"DIABLO\",\n      \"SOD1\",\n      \"FOXP3\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}