{"gene":"UBE2K","run_date":"2026-06-10T10:51:56","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; ubc1 deletion causes slow growth and impaired growth after spore germination, establishing its role in protein degradation during early growth transitions.","method":"Gene disruption (ubc1 deletion mutants), growth phenotype analysis, double mutant analysis","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic loss-of-function with defined phenotypic readouts, replicated across multiple allele combinations including double mutants","pmids":["2265617"],"is_preprint":false},{"year":2004,"finding":"Ubc1 (yeast ortholog of UBE2K) is a class II E2 enzyme with an N-terminal catalytic alpha/beta domain separated by a 22-residue flexible tether from a C-terminal three-helix bundle identified as a ubiquitin-associated (UBA) domain; NMR chemical shift perturbation experiments show the UBA domain interacts in a regioselective manner with ubiquitin, suggesting a role in polyubiquitin chain formation.","method":"NMR solution structure determination; NMR chemical shift perturbation experiments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure with functional validation by binding experiments, single lab but multiple orthogonal methods","pmids":["15328341"],"is_preprint":false},{"year":2015,"finding":"Ube2K preferentially catalyses formation of Lys48-linked ubiquitin chains; crystal structure of the Ube2K~ubiquitin conjugate reveals that the enzyme is monomeric during chain synthesis, and site-directed mutagenesis identifies key residues required for positioning the acceptor ubiquitin to enable K48-specific chain synthesis.","method":"Crystal structure of Ube2K~ubiquitin conjugate; site-directed mutagenesis; in vitro ubiquitin chain synthesis assays","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure combined with mutagenesis and in vitro activity assays in a single study with multiple orthogonal methods","pmids":["26592444"],"is_preprint":false},{"year":2001,"finding":"Hip-2 (UBE2K) interacts with RING finger protein RNF2 via RNF2's RING domain; RNF2 exhibits E3 ubiquitin ligase activity in the presence of Hip-2, identifying a subset of RING finger proteins as E3 partners for UBE2K.","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 / Moderate — reciprocal in vitro and in vivo interaction confirmation with mutagenesis and functional E3 activity assay, single lab but multiple orthogonal methods","pmids":["11513855"],"is_preprint":false},{"year":2021,"finding":"The UBA domain of Ubc1/Ube2K preferentially binds K63-linked ubiquitin chains; structural modeling, in vitro ubiquitination experiments, and NMR studies show that the UBA domain aligns the enzyme with K63-linked polyubiquitin to facilitate selective assembly of K48/K63-branched ubiquitin conjugates, linking this branched-chain topology to cellular proteostasis maintenance.","method":"NMR binding studies; in vitro ubiquitination assays; structural modeling; genetic experiments in yeast; proteomics","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal methods (NMR, in vitro reconstitution, structural modeling, genetics, proteomics) across a single rigorous study","pmids":["33576509"],"is_preprint":false},{"year":2018,"finding":"Crystal structure of Ube2K/E2-25K with K48-linked di-ubiquitin at 2.47 Å resolution reveals that a novel interaction between the acceptor di-ubiquitin and Ube2K promotes the discharging reaction and tri-ubiquitin production by guiding and positioning K48 of the distal ubiquitin in the active site; Ube2K can synthesize K48-linked chains without an E3.","method":"Crystal structure (2.47 Å resolution); in vitro ubiquitination assays; structural modeling","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with biochemical validation of the proposed mechanism, single lab with two orthogonal methods","pmids":["30336976"],"is_preprint":false},{"year":2021,"finding":"Ubiquitin variants (UbVs) bind a hydrophobic cleft on Ube2K distinct from the active site and known ubiquitin-binding sites; crystal structures show UbVs are potent inhibitors blocking both ubiquitin charging of Ube2K and E3-catalyzed ubiquitin transfer, likely by steric clash with the E1 activating enzyme and allosteric disruption of E3 interactions.","method":"Phage-display selection of ubiquitin variants; crystal structures of Ube2K-UbV complexes; in vitro ubiquitin charging and transfer assays","journal":"ACS chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structures with functional inhibition assays, single lab with multiple orthogonal methods","pmids":["34397214"],"is_preprint":false},{"year":2015,"finding":"HIP2~ubiquitin (Ube2K~Ub) thioester conjugate remains predominantly monomeric in solution as shown by analytical ultracentrifugation and small-angle X-ray scattering; the complex forms a non-compact 'backbent' conformation with UBA domain and covalently attached ubiquitin on opposite ends of the catalytic domain; full-length HIP2 shows a five-fold increase in di-ubiquitin formation rate compared to HIP2 lacking the UBA domain.","method":"Analytical ultracentrifugation; small-angle X-ray scattering (SAXS); stable disulfide complex analysis; in vitro di-ubiquitin formation activity assays","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Moderate — biophysical structure determination with functional activity assays, single lab with multiple orthogonal methods","pmids":["25799589"],"is_preprint":false},{"year":2020,"finding":"UBE2K binds histone H3 and induces its polyubiquitination and degradation by the proteasome in human embryonic stem cells; loss of UBE2K upregulates the trimethyltransferase SETDB1, resulting in H3K9 trimethylation and repression of neurogenic genes during differentiation; the C. elegans ortholog ubc-20 similarly regulates histone H3 levels and H3K9 trimethylation.","method":"Co-immunoprecipitation (binding of UBE2K to histone H3); siRNA knockdown; proteasome inhibitor treatment; Western blotting for H3K9me3 and H3 levels; C. elegans genetic experiments","journal":"Communications biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function with defined molecular phenotype, interaction confirmed by Co-IP, replicated in C. elegans ortholog, multiple orthogonal methods","pmids":["32451438"],"is_preprint":false},{"year":2016,"finding":"The nuclear E3 ligase San1 functions preferentially with Ubc1 (ortholog of UBE2K) over Cdc34 during protein quality control substrate ubiquitylation; in vitro reconstitution showed San1 activity is faster with Ubc1, and Ubc1 retains preference even when both E2s are present simultaneously.","method":"In vitro reconstitution of San1-catalyzed ubiquitylation; kinetic quantification using peptide substrate; competition assays with both E2s","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with kinetic measurements, single lab with rigorous quantitative functional assays","pmids":["27405755"],"is_preprint":false},{"year":2000,"finding":"Yeast Ubc1 is required for import of fructose-1,6-bisphosphatase (FBPase) into Vid (vacuole import and degradation) vesicles; ubc1 deletion reduces Vid vesicle-specific marker Vid24p in the vesicle fraction, implicating Ubc1 in Vid vesicle biogenesis or Vid24p binding to vesicles.","method":"Biochemical fractionation; gene deletion (Δubc1); overexpression of ubiquitin chain mutants (K48R/K63R); FBPase import assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic loss-of-function with biochemical fractionation readout, single lab, functional consequence established for vesicle biogenesis","pmids":["11134048"],"is_preprint":false},{"year":2010,"finding":"Hip2 (UBE2K) interacts with cyclin B1 and promotes its degradation through the ubiquitin-proteasome pathway; overexpression of Hip2 blocked cyclin B1-induced cell death.","method":"Co-immunoprecipitation; ubiquitin-proteasome degradation assay; cell death assay with cyclin B1 overexpression","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus proteasomal degradation assay with functional cell death readout, single lab","pmids":["20965177"],"is_preprint":false},{"year":2010,"finding":"Hip2 (UBE2K) interacts with the proapoptotic protein Smac/DIABLO and promotes its degradation through the ubiquitin-proteasome pathway; Hip2 overexpression blocked staurosporine- and Smac-induced cell death.","method":"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 / Moderate — reciprocal in vivo/in vitro interaction with functional degradation and cell death assay, single lab","pmids":["20537984"],"is_preprint":false},{"year":2013,"finding":"Hip2 (UBE2K) interacts with p53 and targets it for degradation via the ubiquitin-proteasome system, resulting in activation of cdc2-cyclin B1 kinase and abrogation of radiation-induced G2/M arrest to promote mitotic entry; radiation-increased Hip2 stability was confirmed by live-cell fluorescence imaging.","method":"Bimolecular fluorescence complementation; co-immunoprecipitation; flow cytometry; live-cell fluorescence imaging; molecular analysis of cdc2-cyclin B1 activity","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — BiFC and co-IP confirm interaction, functional consequence (G2/M arrest abrogation) measured by flow cytometry, single lab","pmids":["23933584"],"is_preprint":false},{"year":2018,"finding":"Hip2 (UBE2K) is phosphorylated following UV radiation; phosphorylated Hip2 inhibits p53, suppressing p21 expression and stimulating cyclin D and E to promote cell cycle re-entry after UV-induced G1/S arrest.","method":"Cell synchronization; UV irradiation; Western blotting for p53, p21, cyclin D, cyclin E; pIMAGO phosphorylation assay","journal":"Genes & genomics","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — phosphorylation detection by specialized assay plus Western blot pathway analysis, single lab, no mutagenesis of phosphorylation site","pmids":["30264212"],"is_preprint":false},{"year":2021,"finding":"HIP2 (UBE2K) interacts with mutant SOD1 (but not wild-type SOD1), is upregulated in response to mutant SOD1 expression and in spinal cord of SOD1-G93A transgenic mice, modifies mutant SOD1 via K48-linked polyubiquitination, and promotes its degradation through the UPS; upregulation of HIP2 protects cells from mutant SOD1-induced toxicity.","method":"Co-immunoprecipitation; K48-linkage-specific ubiquitination assay; Western blotting in transgenic mouse spinal cord; cell viability assay","journal":"Biochimica et biophysica acta. Molecular basis of disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with linkage-specific ubiquitination assay and in vivo mouse data, single lab with multiple methods","pmids":["34856358"],"is_preprint":false},{"year":2015,"finding":"UbcD4, the Drosophila ortholog of E2-25K/Ube2K, is required for activation of the immune deficiency (IMD) pathway; RNAi knockdown of the mammalian ortholog UBE2K inhibited TNFα- and LPS-mediated NF-κB pathway activation, indicating a conserved role as a positive regulator in innate immune signaling.","method":"RNAi knockdown in Drosophila (UbcD4); RNAi knockdown of UBE2K in mammalian cells; immunoblot-based IMD/NF-κB pathway activation assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function in two species with defined pathway readout, single lab","pmids":["26707646"],"is_preprint":false},{"year":2021,"finding":"Ubc1 (yeast ortholog of UBE2K) levels are regulated by the spindle assembly checkpoint: checkpoint activation or direct APC inhibition decreases Ubc1 levels, charging, and half-life; Ubc1 overexpression increases resistance to microtubule poisons, and stabilized Ubc1 prevents checkpoint-mediated down-regulation and increases resistance to checkpoint-activating drugs.","method":"Overexpression and shut-off of Ubc1; microtubule poison sensitivity assay; APC inhibition; measurement of Ubc1 charging and half-life","journal":"G3 (Bethesda, Md.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic gain/loss-of-function with multiple pathway-level readouts, single lab","pmids":["34586382"],"is_preprint":false},{"year":2025,"finding":"The microsporidian deubiquitinase ehOTUB1 selectively binds UBE2K and inhibits its ubiquitin conjugation activity independent of DUB catalytic activity; mechanistically, ehOTUB1 obstructs docking of UBE2K onto the E1 ubiquitin-activating enzyme via steric conflict with ubiquitin in the E1 adenylation site, thereby preventing ubiquitin transfer to UBE2K.","method":"Binding assays; in vitro ubiquitin conjugation activity assays; DUB-dead mutant analysis; structural modeling of steric clash with E1","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional inhibition assay with mutagenesis and structural rationale, preprint, single lab","pmids":["40766534"],"is_preprint":true},{"year":2025,"finding":"PRRSV NSP8 hijacks host UBE2K and induces degradation of IKKα to suppress NF-κB signaling; UBE2K was upregulated during PRRSV infection, directly interacted with NSP8, and acted as a cofactor enhancing NSP8-mediated proteasomal degradation of IKKα, blocking downstream antiviral factor expression and facilitating viral replication.","method":"Co-immunoprecipitation (NSP8-IKKα, NSP8-UBE2K); proteasome inhibitor rescue; knockdown of UBE2K; NF-κB reporter assay; viral replication measurement","journal":"Viruses","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP with functional assays and knockdown confirmation, single lab","pmids":["42198770"],"is_preprint":false},{"year":2022,"finding":"Ubiquitin-conjugating enzymes Ubc1 and Ubc4 are required for proteasome-dependent turnover of Hap4, the regulatory subunit of the yeast Hap2/3/4/5 transcriptional complex for mitochondrial biogenesis; stabilization of Hap4 in the ubc1 ubc4 double mutant led to increased expression of Hap2/3/4/5-target genes.","method":"Genetic double mutant analysis (ubc1 ubc4); proteasome inhibitor treatment; Hap4 stability measurement; target gene expression analysis","journal":"Microorganisms","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean double-mutant epistasis with proteasome inhibitor control and gene expression readout, single lab","pmids":["36557625"],"is_preprint":false},{"year":2025,"finding":"UBE2K ubiquitinates and promotes degradation of STUB1 (CHIP E3 ligase) via the proteasome; this activates the PKA/CREB1 signaling pathway, and CREB1 in turn acts as a transcription factor for UBE2K, forming a positive feedback loop driving breast cancer cell proliferation and migration.","method":"Co-immunoprecipitation; ubiquitination assay; Western blotting; chromatin immunoprecipitation (ChIP); dual-luciferase reporter assay; CCK-8; Transwell assays","journal":"Biochimica et biophysica acta. Molecular basis of disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ubiquitination assay plus ChIP/luciferase validation of transcriptional feedback, single lab with multiple methods","pmids":["41435988"],"is_preprint":false},{"year":2025,"finding":"Amuc_1409 (derived from Akkermansia muciniphila) interacts with Ube2K to reduce ubiquitination-mediated degradation of Foxp3, thereby promoting Treg differentiation and IL-10 production to suppress inflammation in severe acute pancreatitis.","method":"Co-immunoprecipitation (Amuc_1409-Ube2K interaction); ubiquitination assay for Foxp3; Foxp3-DTR and IL-10-KO mouse models; Treg differentiation assay","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with functional ubiquitination assay and genetic mouse model validation, single lab","pmids":["40464424"],"is_preprint":false},{"year":2004,"finding":"NUB1 interacts with ubiquitin precursor UbC1 (the polyubiquitin precursor encoded by tandem ubiquitin repeats) through NUB1's UBA domain, which specifically recognizes alpha-peptide bond-linked polyubiquitin but not isopeptide bond-linked polyubiquitin; an unidentified ubiquitin C-terminal hydrolase co-immunoprecipitated with NUB1 cleaves UbC1 to generate ubiquitin monomers.","method":"Yeast two-hybrid; co-immunoprecipitation; UBA domain binding specificity assay; ubiquitin C-terminal hydrolase activity assay; Northern blot; in situ hybridization","journal":"European journal of biochemistry","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Note: UbC1 here is a polyubiquitin precursor gene, not the E2 UBE2K/UBC1; this paper may be about a different gene. Including as low confidence because the interaction with NUB1 UBA domain and alpha-peptide ubiquitin specificity is mechanistically characterized, but the target gene identity is ambiguous.","pmids":["15009209"],"is_preprint":false},{"year":2011,"finding":"Serine phosphorylation of yeast Ubc1 at MAP kinase sites S97 and S115 regulates stress tolerance; Ubc1S97A and Ubc1S115D expression increased thermotolerance, and Ubc1S97A expression correlated with increased reductive stress tolerance; Ubc1 phosphorylation decreases upon heat shock exposure.","method":"Site-directed mutagenesis of Ubc1 phosphorylation sites; phosphoserine detection; cell survival assays under thermal and reductive stress; hog1Δ and slt2Δ genetic backgrounds","journal":"Yeast","confidence":"Low","confidence_rationale":"Tier 3 / Moderate — mutagenesis with phenotypic readout, single lab, only in yeast ortholog and no direct mechanistic link to downstream E2 activity","pmids":["21996927"],"is_preprint":false}],"current_model":"UBE2K (HIP2/E2-25K) is a class II ubiquitin-conjugating E2 enzyme with a catalytic UBC domain and a C-terminal UBA domain connected by a flexible linker; it exclusively generates K48-linked polyubiquitin chains (established by crystal structures and mutagenesis) and can uniquely also facilitate assembly of K48/K63-branched chains by using its UBA domain to engage K63-linked polyubiquitin as a scaffold; it functions with E3 ligases such as RING-finger protein RNF2 and the nuclear quality-control ligase San1, targets substrates including histone H3, cyclin B1, Smac/DIABLO, mutant SOD1, p53, STUB1, and Foxp3 for proteasomal degradation, and acts as a positive regulator of NF-κB/IMD innate immune signaling; its activity is subject to phosphorylation-dependent regulation and can be allosterically inhibited by ubiquitin variants or pathogen-derived proteins (microsporidian ehOTUB1, PRRSV NSP8) that block E1-to-E2 ubiquitin transfer."},"narrative":{"mechanistic_narrative":"UBE2K (HIP2/E2-25K; yeast ortholog Ubc1) is a ubiquitin-conjugating (E2) enzyme that mediates selective proteasomal degradation of short-lived and abnormal proteins [PMID:2265617]. It is a class II E2 with an N-terminal catalytic UBC domain joined by a flexible tether to a C-terminal ubiquitin-associated (UBA) domain that binds ubiquitin regioselectively [PMID:15328341]. UBE2K preferentially builds Lys48-linked polyubiquitin chains, acting as a monomer during chain synthesis; structures of the Ube2K~ubiquitin conjugate and of Ube2K bound to K48-linked di-ubiquitin define how acceptor ubiquitin is positioned to direct K48 specificity and enable chain elongation even without an E3 [PMID:26592444, PMID:30336976]. Its UBA domain preferentially engages K63-linked ubiquitin and aligns the enzyme on K63 chains to assemble K48/K63-branched conjugates, accelerating di-ubiquitin formation relative to UBA-deletion enzyme and linking this branched topology to proteostasis [PMID:33576509, PMID:25799589]. UBE2K works with RING-finger E3 ligases such as RNF2 and with the nuclear quality-control ligase San1, for which it is the preferred E2 [PMID:11513855, PMID:27405755]. Through K48-linked ubiquitination it targets diverse substrates for degradation, including histone H3 to control H3K9 trimethylation and neurogenic gene expression in stem cells [PMID:32451438], cyclin B1, Smac/DIABLO, and p53 to modulate cell death and cell-cycle progression [PMID:20965177, PMID:20537984, PMID:23933584], aggregation-prone mutant SOD1 [PMID:34856358], and STUB1, where a CREB1-driven transcriptional feedback loop supports breast cancer cell proliferation [PMID:41435988]. UBE2K is a conserved positive regulator of IMD/NF-κB innate immune signaling [PMID:26707646], and its activity is regulated by phosphorylation and by allosteric inhibitors including engineered ubiquitin variants and pathogen-derived proteins that block ubiquitin charging at the E1→E2 step [PMID:34397214, PMID:30264212, PMID:40766534, PMID:42198770].","teleology":[{"year":1990,"claim":"Established that the gene encodes a functional ubiquitin-conjugating enzyme controlling degradation of short-lived and abnormal proteins, defining its core cellular role.","evidence":"ubc1 deletion and double-mutant growth-phenotype analysis in yeast","pmids":["2265617"],"confidence":"High","gaps":["Functional redundancy with UBC4/UBC5 obscures unique substrates","No molecular structure or chain-linkage specificity defined"]},{"year":2004,"claim":"Defined the two-domain architecture—a catalytic UBC domain tethered to a C-terminal UBA domain—and showed the UBA domain binds ubiquitin regioselectively, implicating it in chain assembly.","evidence":"NMR solution structure and chemical-shift perturbation in the yeast ortholog Ubc1","pmids":["15328341"],"confidence":"High","gaps":["Chain linkage specificity not directly demonstrated","Functional role of UBA binding in cells untested"]},{"year":2015,"claim":"Resolved how the enzyme achieves K48 specificity and remains monomeric during synthesis, and established the contribution of the UBA domain to catalytic rate.","evidence":"Crystal structure of Ube2K~ubiquitin conjugate plus mutagenesis; AUC/SAXS conformational analysis with di-ubiquitin formation assays","pmids":["26592444","25799589"],"confidence":"High","gaps":["E3-dependent chain assembly geometry not addressed","Physiological substrate context absent"]},{"year":2018,"claim":"Showed acceptor ubiquitin engagement promotes the discharging reaction and E3-independent chain elongation, clarifying processivity.","evidence":"Crystal structure of Ube2K with K48-linked di-ubiquitin at 2.47 Å plus in vitro ubiquitination assays","pmids":["30336976"],"confidence":"High","gaps":["Relevance of E3-free synthesis in cells unknown"]},{"year":2021,"claim":"Defined a unique capacity to build K48/K63-branched chains by using the UBA domain to read K63 chains as a scaffold, connecting chain topology to proteostasis.","evidence":"NMR binding, in vitro ubiquitination, structural modeling, yeast genetics and proteomics","pmids":["33576509"],"confidence":"High","gaps":["In vivo substrates marked by branched chains not identified","Proteasomal decoding of branched signal not directly tested here"]},{"year":2021,"claim":"Identified a druggable allosteric site by which ubiquitin variants block both charging and E3-catalyzed transfer, revealing regulatory surfaces beyond the active site.","evidence":"Phage-display ubiquitin variants, Ube2K-UbV crystal structures, charging/transfer assays","pmids":["34397214"],"confidence":"High","gaps":["No endogenous ligand of this cleft identified","Cellular efficacy of UbV inhibitors untested"]},{"year":2001,"claim":"Identified RING-finger E3 partners (RNF2) that confer ligase activity with UBE2K, placing the E2 in defined E2–E3 pairs.","evidence":"Yeast two-hybrid, reciprocal co-IP, RING mutagenesis, ligase activity assay","pmids":["11513855"],"confidence":"High","gaps":["RNF2 substrates ubiquitinated by UBE2K not defined"]},{"year":2016,"claim":"Established that the nuclear quality-control ligase San1 preferentially uses Ubc1 over Cdc34, assigning a specific E3 partner for protein quality control.","evidence":"In vitro reconstitution with kinetic quantification and E2 competition assays","pmids":["27405755"],"confidence":"High","gaps":["Structural basis of San1–Ubc1 preference unknown","Human San1 ortholog context not addressed"]},{"year":2020,"claim":"Linked UBE2K-driven degradation of histone H3 to epigenetic control of differentiation, expanding its role beyond generic quality control.","evidence":"Co-IP, siRNA knockdown, proteasome inhibition, H3K9me3 Western blots, C. elegans genetics","pmids":["32451438"],"confidence":"High","gaps":["E3 ligase mediating H3 degradation not identified","Mechanism of SETDB1 upregulation indirect"]},{"year":2013,"claim":"Connected UBE2K to cell-cycle and apoptotic control by targeting cyclin B1, Smac/DIABLO, and p53 for degradation, modulating cell death and G2/M transition.","evidence":"Co-IP/BiFC, proteasomal degradation assays, flow cytometry and cell-death assays (multiple studies)","pmids":["20965177","20537984","23933584"],"confidence":"Medium","gaps":["E3 ligases for these substrates not defined","Direct vs indirect degradation not fully resolved","Single-lab findings"]},{"year":2018,"claim":"Showed phosphorylation regulates UBE2K-dependent p53 suppression and stability following radiation, indicating signal-controlled E2 activity.","evidence":"UV irradiation, phosphorylation detection (pIMAGO), live-cell imaging, pathway Western blots","pmids":["23933584","30264212"],"confidence":"Medium","gaps":["Phosphorylation sites not mutated to test causality","Responsible kinase unidentified"]},{"year":2021,"claim":"Demonstrated cytoprotection against aggregation-prone protein toxicity via K48-linked ubiquitination of mutant SOD1, implicating UBE2K in neurodegeneration-relevant clearance.","evidence":"Co-IP, K48-linkage-specific ubiquitination, SOD1-G93A mouse spinal cord, viability assays","pmids":["34856358"],"confidence":"Medium","gaps":["E3 partner for mutant SOD1 not identified","In vivo therapeutic relevance untested"]},{"year":2021,"claim":"Showed Ubc1 levels are coupled to the spindle assembly checkpoint and APC activity, with consequences for microtubule-poison resistance.","evidence":"Overexpression/shut-off, charging and half-life measurement, APC inhibition, drug-sensitivity assays in yeast","pmids":["34586382"],"confidence":"Medium","gaps":["Mechanism of checkpoint-dependent Ubc1 turnover unclear","Human relevance not established"]},{"year":2016,"claim":"Established a conserved role as a positive regulator of IMD/NF-κB innate immune signaling across Drosophila and mammalian cells.","evidence":"RNAi knockdown of UbcD4 and UBE2K with pathway-activation immunoblot readouts","pmids":["26707646"],"confidence":"Medium","gaps":["Direct substrate within the NF-κB pathway not identified","E3 partner unknown"]},{"year":2025,"claim":"Revealed that pathogens hijack or inhibit UBE2K to subvert immune signaling, including PRRSV NSP8-driven IKKα degradation and microsporidian ehOTUB1 blockade of E1-to-E2 transfer.","evidence":"Co-IP, proteasome rescue, knockdown, NF-κB reporters, charging assays and structural modeling of E1 steric clash (one preprint)","pmids":["42198770","40766534"],"confidence":"Medium","gaps":["ehOTUB1 finding is a single-lab preprint","Structural details of NSP8–UBE2K–IKKα complex unresolved"]},{"year":2025,"claim":"Connected UBE2K to disease via degradation of STUB1 with CREB1 transcriptional feedback in cancer, and Foxp3 stabilization controlling Treg-mediated inflammation.","evidence":"Co-IP, ubiquitination assays, ChIP, luciferase, proliferation/migration assays; mouse Foxp3-DTR/IL-10-KO models","pmids":["41435988","40464424"],"confidence":"Medium","gaps":["Direct vs indirect ubiquitination of substrates not fully separated","Single-lab disease models"]},{"year":null,"claim":"The complete set of physiological E3 partners and the in vivo substrate landscape decoded by UBE2K-built K48 and K48/K63-branched chains remain to be mapped.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No comprehensive E3 partner census","Branched-chain substrate repertoire in cells undefined","Phosphorylation-site causality untested by mutagenesis"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,3,5,8,15]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[2,5,9]},{"term_id":"GO:0031386","term_label":"protein tag activity","supporting_discovery_ids":[2,4,5]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[8,9]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,2,5]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[16,19]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[11,13,17]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[12,13]}],"complexes":[],"partners":["RNF2","SAN1","STUB1","TP53","CCNB1","DIABLO","SOD1","FOXP3"],"other_free_text":[]}},"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 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ubc1 deletion causes slow growth and impaired growth after spore germination, establishing its role in protein degradation during early growth transitions.\",\n      \"method\": \"Gene disruption (ubc1 deletion mutants), growth phenotype analysis, double mutant analysis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic loss-of-function with defined phenotypic readouts, replicated across multiple allele combinations including double mutants\",\n      \"pmids\": [\"2265617\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Ubc1 (yeast ortholog of UBE2K) is a class II E2 enzyme with an N-terminal catalytic alpha/beta domain separated by a 22-residue flexible tether from a C-terminal three-helix bundle identified as a ubiquitin-associated (UBA) domain; NMR chemical shift perturbation experiments show the UBA domain interacts in a regioselective manner with ubiquitin, suggesting a role in polyubiquitin chain formation.\",\n      \"method\": \"NMR solution structure determination; NMR chemical shift perturbation experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure with functional validation by binding experiments, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"15328341\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Ube2K preferentially catalyses formation of Lys48-linked ubiquitin chains; crystal structure of the Ube2K~ubiquitin conjugate reveals that the enzyme is monomeric during chain synthesis, and site-directed mutagenesis identifies key residues required for positioning the acceptor ubiquitin to enable K48-specific chain synthesis.\",\n      \"method\": \"Crystal structure of Ube2K~ubiquitin conjugate; site-directed mutagenesis; in vitro ubiquitin chain synthesis assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure combined with mutagenesis and in vitro activity assays in a single study with multiple orthogonal methods\",\n      \"pmids\": [\"26592444\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Hip-2 (UBE2K) interacts with RING finger protein RNF2 via RNF2's RING domain; RNF2 exhibits E3 ubiquitin ligase activity in the presence of Hip-2, identifying a subset of RING finger proteins as E3 partners for UBE2K.\",\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 / Moderate — reciprocal in vitro and in vivo interaction confirmation with mutagenesis and functional E3 activity assay, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"11513855\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The UBA domain of Ubc1/Ube2K preferentially binds K63-linked ubiquitin chains; structural modeling, in vitro ubiquitination experiments, and NMR studies show that the UBA domain aligns the enzyme with K63-linked polyubiquitin to facilitate selective assembly of K48/K63-branched ubiquitin conjugates, linking this branched-chain topology to cellular proteostasis maintenance.\",\n      \"method\": \"NMR binding studies; in vitro ubiquitination assays; structural modeling; genetic experiments in yeast; proteomics\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal methods (NMR, in vitro reconstitution, structural modeling, genetics, proteomics) across a single rigorous study\",\n      \"pmids\": [\"33576509\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Crystal structure of Ube2K/E2-25K with K48-linked di-ubiquitin at 2.47 Å resolution reveals that a novel interaction between the acceptor di-ubiquitin and Ube2K promotes the discharging reaction and tri-ubiquitin production by guiding and positioning K48 of the distal ubiquitin in the active site; Ube2K can synthesize K48-linked chains without an E3.\",\n      \"method\": \"Crystal structure (2.47 Å resolution); in vitro ubiquitination assays; structural modeling\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with biochemical validation of the proposed mechanism, single lab with two orthogonal methods\",\n      \"pmids\": [\"30336976\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Ubiquitin variants (UbVs) bind a hydrophobic cleft on Ube2K distinct from the active site and known ubiquitin-binding sites; crystal structures show UbVs are potent inhibitors blocking both ubiquitin charging of Ube2K and E3-catalyzed ubiquitin transfer, likely by steric clash with the E1 activating enzyme and allosteric disruption of E3 interactions.\",\n      \"method\": \"Phage-display selection of ubiquitin variants; crystal structures of Ube2K-UbV complexes; in vitro ubiquitin charging and transfer assays\",\n      \"journal\": \"ACS chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structures with functional inhibition assays, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"34397214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"HIP2~ubiquitin (Ube2K~Ub) thioester conjugate remains predominantly monomeric in solution as shown by analytical ultracentrifugation and small-angle X-ray scattering; the complex forms a non-compact 'backbent' conformation with UBA domain and covalently attached ubiquitin on opposite ends of the catalytic domain; full-length HIP2 shows a five-fold increase in di-ubiquitin formation rate compared to HIP2 lacking the UBA domain.\",\n      \"method\": \"Analytical ultracentrifugation; small-angle X-ray scattering (SAXS); stable disulfide complex analysis; in vitro di-ubiquitin formation activity assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — biophysical structure determination with functional activity assays, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"25799589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"UBE2K binds histone H3 and induces its polyubiquitination and degradation by the proteasome in human embryonic stem cells; loss of UBE2K upregulates the trimethyltransferase SETDB1, resulting in H3K9 trimethylation and repression of neurogenic genes during differentiation; the C. elegans ortholog ubc-20 similarly regulates histone H3 levels and H3K9 trimethylation.\",\n      \"method\": \"Co-immunoprecipitation (binding of UBE2K to histone H3); siRNA knockdown; proteasome inhibitor treatment; Western blotting for H3K9me3 and H3 levels; C. elegans genetic experiments\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function with defined molecular phenotype, interaction confirmed by Co-IP, replicated in C. elegans ortholog, multiple orthogonal methods\",\n      \"pmids\": [\"32451438\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The nuclear E3 ligase San1 functions preferentially with Ubc1 (ortholog of UBE2K) over Cdc34 during protein quality control substrate ubiquitylation; in vitro reconstitution showed San1 activity is faster with Ubc1, and Ubc1 retains preference even when both E2s are present simultaneously.\",\n      \"method\": \"In vitro reconstitution of San1-catalyzed ubiquitylation; kinetic quantification using peptide substrate; competition assays with both E2s\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with kinetic measurements, single lab with rigorous quantitative functional assays\",\n      \"pmids\": [\"27405755\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Yeast Ubc1 is required for import of fructose-1,6-bisphosphatase (FBPase) into Vid (vacuole import and degradation) vesicles; ubc1 deletion reduces Vid vesicle-specific marker Vid24p in the vesicle fraction, implicating Ubc1 in Vid vesicle biogenesis or Vid24p binding to vesicles.\",\n      \"method\": \"Biochemical fractionation; gene deletion (Δubc1); overexpression of ubiquitin chain mutants (K48R/K63R); FBPase import assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic loss-of-function with biochemical fractionation readout, single lab, functional consequence established for vesicle biogenesis\",\n      \"pmids\": [\"11134048\"],\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; overexpression of Hip2 blocked cyclin B1-induced cell death.\",\n      \"method\": \"Co-immunoprecipitation; ubiquitin-proteasome degradation assay; cell death assay with cyclin B1 overexpression\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus proteasomal degradation assay with functional cell death readout, single lab\",\n      \"pmids\": [\"20965177\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Hip2 (UBE2K) interacts with the proapoptotic protein Smac/DIABLO and promotes its degradation through the ubiquitin-proteasome pathway; Hip2 overexpression blocked staurosporine- and Smac-induced cell death.\",\n      \"method\": \"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 / Moderate — reciprocal in vivo/in vitro interaction with functional degradation and cell death assay, single lab\",\n      \"pmids\": [\"20537984\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Hip2 (UBE2K) interacts with p53 and targets it for degradation via the ubiquitin-proteasome system, resulting in activation of cdc2-cyclin B1 kinase and abrogation of radiation-induced G2/M arrest to promote mitotic entry; radiation-increased Hip2 stability was confirmed by live-cell fluorescence imaging.\",\n      \"method\": \"Bimolecular fluorescence complementation; co-immunoprecipitation; flow cytometry; live-cell fluorescence imaging; molecular analysis of cdc2-cyclin B1 activity\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — BiFC and co-IP confirm interaction, functional consequence (G2/M arrest abrogation) measured by flow cytometry, single lab\",\n      \"pmids\": [\"23933584\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Hip2 (UBE2K) is phosphorylated following UV radiation; phosphorylated Hip2 inhibits p53, suppressing p21 expression and stimulating cyclin D and E to promote cell cycle re-entry after UV-induced G1/S arrest.\",\n      \"method\": \"Cell synchronization; UV irradiation; Western blotting for p53, p21, cyclin D, cyclin E; pIMAGO phosphorylation assay\",\n      \"journal\": \"Genes & genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — phosphorylation detection by specialized assay plus Western blot pathway analysis, single lab, no mutagenesis of phosphorylation site\",\n      \"pmids\": [\"30264212\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HIP2 (UBE2K) interacts with mutant SOD1 (but not wild-type SOD1), is upregulated in response to mutant SOD1 expression and in spinal cord of SOD1-G93A transgenic mice, modifies mutant SOD1 via K48-linked polyubiquitination, and promotes its degradation through the UPS; upregulation of HIP2 protects cells from mutant SOD1-induced toxicity.\",\n      \"method\": \"Co-immunoprecipitation; K48-linkage-specific ubiquitination assay; Western blotting 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 / Moderate — Co-IP with linkage-specific ubiquitination assay and in vivo mouse data, single lab with multiple methods\",\n      \"pmids\": [\"34856358\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"UbcD4, the Drosophila ortholog of E2-25K/Ube2K, is required for activation of the immune deficiency (IMD) pathway; RNAi knockdown of the mammalian ortholog UBE2K inhibited TNFα- and LPS-mediated NF-κB pathway activation, indicating a conserved role as a positive regulator in innate immune signaling.\",\n      \"method\": \"RNAi knockdown in Drosophila (UbcD4); RNAi knockdown of UBE2K in mammalian cells; immunoblot-based IMD/NF-κB pathway activation assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function in two species with defined pathway readout, single lab\",\n      \"pmids\": [\"26707646\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Ubc1 (yeast ortholog of UBE2K) levels are regulated by the spindle assembly checkpoint: checkpoint activation or direct APC inhibition decreases Ubc1 levels, charging, and half-life; Ubc1 overexpression increases resistance to microtubule poisons, and stabilized Ubc1 prevents checkpoint-mediated down-regulation and increases resistance to checkpoint-activating drugs.\",\n      \"method\": \"Overexpression and shut-off of Ubc1; microtubule poison sensitivity assay; APC inhibition; measurement of Ubc1 charging and half-life\",\n      \"journal\": \"G3 (Bethesda, Md.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic gain/loss-of-function with multiple pathway-level readouts, single lab\",\n      \"pmids\": [\"34586382\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The microsporidian deubiquitinase ehOTUB1 selectively binds UBE2K and inhibits its ubiquitin conjugation activity independent of DUB catalytic activity; mechanistically, ehOTUB1 obstructs docking of UBE2K onto the E1 ubiquitin-activating enzyme via steric conflict with ubiquitin in the E1 adenylation site, thereby preventing ubiquitin transfer to UBE2K.\",\n      \"method\": \"Binding assays; in vitro ubiquitin conjugation activity assays; DUB-dead mutant analysis; structural modeling of steric clash with E1\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional inhibition assay with mutagenesis and structural rationale, preprint, single lab\",\n      \"pmids\": [\"40766534\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PRRSV NSP8 hijacks host UBE2K and induces degradation of IKKα to suppress NF-κB signaling; UBE2K was upregulated during PRRSV infection, directly interacted with NSP8, and acted as a cofactor enhancing NSP8-mediated proteasomal degradation of IKKα, blocking downstream antiviral factor expression and facilitating viral replication.\",\n      \"method\": \"Co-immunoprecipitation (NSP8-IKKα, NSP8-UBE2K); proteasome inhibitor rescue; knockdown of UBE2K; NF-κB reporter assay; viral replication measurement\",\n      \"journal\": \"Viruses\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP with functional assays and knockdown confirmation, single lab\",\n      \"pmids\": [\"42198770\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Ubiquitin-conjugating enzymes Ubc1 and Ubc4 are required for proteasome-dependent turnover of Hap4, the regulatory subunit of the yeast Hap2/3/4/5 transcriptional complex for mitochondrial biogenesis; stabilization of Hap4 in the ubc1 ubc4 double mutant led to increased expression of Hap2/3/4/5-target genes.\",\n      \"method\": \"Genetic double mutant analysis (ubc1 ubc4); proteasome inhibitor treatment; Hap4 stability measurement; target gene expression analysis\",\n      \"journal\": \"Microorganisms\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean double-mutant epistasis with proteasome inhibitor control and gene expression readout, single lab\",\n      \"pmids\": [\"36557625\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"UBE2K ubiquitinates and promotes degradation of STUB1 (CHIP E3 ligase) via the proteasome; this activates the PKA/CREB1 signaling pathway, and CREB1 in turn acts as a transcription factor for UBE2K, forming a positive feedback loop driving breast cancer cell proliferation and migration.\",\n      \"method\": \"Co-immunoprecipitation; ubiquitination assay; Western blotting; chromatin immunoprecipitation (ChIP); dual-luciferase reporter assay; CCK-8; Transwell assays\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular basis of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ubiquitination assay plus ChIP/luciferase validation of transcriptional feedback, single lab with multiple methods\",\n      \"pmids\": [\"41435988\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Amuc_1409 (derived from Akkermansia muciniphila) interacts with Ube2K to reduce ubiquitination-mediated degradation of Foxp3, thereby promoting Treg differentiation and IL-10 production to suppress inflammation in severe acute pancreatitis.\",\n      \"method\": \"Co-immunoprecipitation (Amuc_1409-Ube2K interaction); ubiquitination assay for Foxp3; Foxp3-DTR and IL-10-KO mouse models; Treg differentiation assay\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with functional ubiquitination assay and genetic mouse model validation, single lab\",\n      \"pmids\": [\"40464424\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"NUB1 interacts with ubiquitin precursor UbC1 (the polyubiquitin precursor encoded by tandem ubiquitin repeats) through NUB1's UBA domain, which specifically recognizes alpha-peptide bond-linked polyubiquitin but not isopeptide bond-linked polyubiquitin; an unidentified ubiquitin C-terminal hydrolase co-immunoprecipitated with NUB1 cleaves UbC1 to generate ubiquitin monomers.\",\n      \"method\": \"Yeast two-hybrid; co-immunoprecipitation; UBA domain binding specificity assay; ubiquitin C-terminal hydrolase activity assay; Northern blot; in situ hybridization\",\n      \"journal\": \"European journal of biochemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Note: UbC1 here is a polyubiquitin precursor gene, not the E2 UBE2K/UBC1; this paper may be about a different gene. Including as low confidence because the interaction with NUB1 UBA domain and alpha-peptide ubiquitin specificity is mechanistically characterized, but the target gene identity is ambiguous.\",\n      \"pmids\": [\"15009209\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Serine phosphorylation of yeast Ubc1 at MAP kinase sites S97 and S115 regulates stress tolerance; Ubc1S97A and Ubc1S115D expression increased thermotolerance, and Ubc1S97A expression correlated with increased reductive stress tolerance; Ubc1 phosphorylation decreases upon heat shock exposure.\",\n      \"method\": \"Site-directed mutagenesis of Ubc1 phosphorylation sites; phosphoserine detection; cell survival assays under thermal and reductive stress; hog1Δ and slt2Δ genetic backgrounds\",\n      \"journal\": \"Yeast\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — mutagenesis with phenotypic readout, single lab, only in yeast ortholog and no direct mechanistic link to downstream E2 activity\",\n      \"pmids\": [\"21996927\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"UBE2K (HIP2/E2-25K) is a class II ubiquitin-conjugating E2 enzyme with a catalytic UBC domain and a C-terminal UBA domain connected by a flexible linker; it exclusively generates K48-linked polyubiquitin chains (established by crystal structures and mutagenesis) and can uniquely also facilitate assembly of K48/K63-branched chains by using its UBA domain to engage K63-linked polyubiquitin as a scaffold; it functions with E3 ligases such as RING-finger protein RNF2 and the nuclear quality-control ligase San1, targets substrates including histone H3, cyclin B1, Smac/DIABLO, mutant SOD1, p53, STUB1, and Foxp3 for proteasomal degradation, and acts as a positive regulator of NF-κB/IMD innate immune signaling; its activity is subject to phosphorylation-dependent regulation and can be allosterically inhibited by ubiquitin variants or pathogen-derived proteins (microsporidian ehOTUB1, PRRSV NSP8) that block E1-to-E2 ubiquitin transfer.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"UBE2K (HIP2/E2-25K; yeast ortholog Ubc1) is a ubiquitin-conjugating (E2) enzyme that mediates selective proteasomal degradation of short-lived and abnormal proteins [#0]. It is a class II E2 with an N-terminal catalytic UBC domain joined by a flexible tether to a C-terminal ubiquitin-associated (UBA) domain that binds ubiquitin regioselectively [#1]. UBE2K preferentially builds Lys48-linked polyubiquitin chains, acting as a monomer during chain synthesis; structures of the Ube2K~ubiquitin conjugate and of Ube2K bound to K48-linked di-ubiquitin define how acceptor ubiquitin is positioned to direct K48 specificity and enable chain elongation even without an E3 [#2, #5]. Its UBA domain preferentially engages K63-linked ubiquitin and aligns the enzyme on K63 chains to assemble K48/K63-branched conjugates, accelerating di-ubiquitin formation relative to UBA-deletion enzyme and linking this branched topology to proteostasis [#4, #7]. UBE2K works with RING-finger E3 ligases such as RNF2 and with the nuclear quality-control ligase San1, for which it is the preferred E2 [#3, #9]. Through K48-linked ubiquitination it targets diverse substrates for degradation, including histone H3 to control H3K9 trimethylation and neurogenic gene expression in stem cells [#8], cyclin B1, Smac/DIABLO, and p53 to modulate cell death and cell-cycle progression [#11, #12, #13], aggregation-prone mutant SOD1 [#15], and STUB1, where a CREB1-driven transcriptional feedback loop supports breast cancer cell proliferation [#21]. UBE2K is a conserved positive regulator of IMD/NF-\\u03baB innate immune signaling [#16], and its activity is regulated by phosphorylation and by allosteric inhibitors including engineered ubiquitin variants and pathogen-derived proteins that block ubiquitin charging at the E1\\u2192E2 step [#6, #14, #18, #19].\",\n  \"teleology\": [\n    {\n      \"year\": 1990,\n      \"claim\": \"Established that the gene encodes a functional ubiquitin-conjugating enzyme controlling degradation of short-lived and abnormal proteins, defining its core cellular role.\",\n      \"evidence\": \"ubc1 deletion and double-mutant growth-phenotype analysis in yeast\",\n      \"pmids\": [\"2265617\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional redundancy with UBC4/UBC5 obscures unique substrates\", \"No molecular structure or chain-linkage specificity defined\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defined the two-domain architecture\\u2014a catalytic UBC domain tethered to a C-terminal UBA domain\\u2014and showed the UBA domain binds ubiquitin regioselectively, implicating it in chain assembly.\",\n      \"evidence\": \"NMR solution structure and chemical-shift perturbation in the yeast ortholog Ubc1\",\n      \"pmids\": [\"15328341\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Chain linkage specificity not directly demonstrated\", \"Functional role of UBA binding in cells untested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Resolved how the enzyme achieves K48 specificity and remains monomeric during synthesis, and established the contribution of the UBA domain to catalytic rate.\",\n      \"evidence\": \"Crystal structure of Ube2K~ubiquitin conjugate plus mutagenesis; AUC/SAXS conformational analysis with di-ubiquitin formation assays\",\n      \"pmids\": [\"26592444\", \"25799589\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"E3-dependent chain assembly geometry not addressed\", \"Physiological substrate context absent\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Showed acceptor ubiquitin engagement promotes the discharging reaction and E3-independent chain elongation, clarifying processivity.\",\n      \"evidence\": \"Crystal structure of Ube2K with K48-linked di-ubiquitin at 2.47 \\u00c5 plus in vitro ubiquitination assays\",\n      \"pmids\": [\"30336976\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relevance of E3-free synthesis in cells unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined a unique capacity to build K48/K63-branched chains by using the UBA domain to read K63 chains as a scaffold, connecting chain topology to proteostasis.\",\n      \"evidence\": \"NMR binding, in vitro ubiquitination, structural modeling, yeast genetics and proteomics\",\n      \"pmids\": [\"33576509\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo substrates marked by branched chains not identified\", \"Proteasomal decoding of branched signal not directly tested here\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified a druggable allosteric site by which ubiquitin variants block both charging and E3-catalyzed transfer, revealing regulatory surfaces beyond the active site.\",\n      \"evidence\": \"Phage-display ubiquitin variants, Ube2K-UbV crystal structures, charging/transfer assays\",\n      \"pmids\": [\"34397214\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No endogenous ligand of this cleft identified\", \"Cellular efficacy of UbV inhibitors untested\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Identified RING-finger E3 partners (RNF2) that confer ligase activity with UBE2K, placing the E2 in defined E2\\u2013E3 pairs.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal co-IP, RING mutagenesis, ligase activity assay\",\n      \"pmids\": [\"11513855\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"RNF2 substrates ubiquitinated by UBE2K not defined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Established that the nuclear quality-control ligase San1 preferentially uses Ubc1 over Cdc34, assigning a specific E3 partner for protein quality control.\",\n      \"evidence\": \"In vitro reconstitution with kinetic quantification and E2 competition assays\",\n      \"pmids\": [\"27405755\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of San1\\u2013Ubc1 preference unknown\", \"Human San1 ortholog context not addressed\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Linked UBE2K-driven degradation of histone H3 to epigenetic control of differentiation, expanding its role beyond generic quality control.\",\n      \"evidence\": \"Co-IP, siRNA knockdown, proteasome inhibition, H3K9me3 Western blots, C. elegans genetics\",\n      \"pmids\": [\"32451438\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"E3 ligase mediating H3 degradation not identified\", \"Mechanism of SETDB1 upregulation indirect\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Connected UBE2K to cell-cycle and apoptotic control by targeting cyclin B1, Smac/DIABLO, and p53 for degradation, modulating cell death and G2/M transition.\",\n      \"evidence\": \"Co-IP/BiFC, proteasomal degradation assays, flow cytometry and cell-death assays (multiple studies)\",\n      \"pmids\": [\"20965177\", \"20537984\", \"23933584\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E3 ligases for these substrates not defined\", \"Direct vs indirect degradation not fully resolved\", \"Single-lab findings\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Showed phosphorylation regulates UBE2K-dependent p53 suppression and stability following radiation, indicating signal-controlled E2 activity.\",\n      \"evidence\": \"UV irradiation, phosphorylation detection (pIMAGO), live-cell imaging, pathway Western blots\",\n      \"pmids\": [\"23933584\", \"30264212\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Phosphorylation sites not mutated to test causality\", \"Responsible kinase unidentified\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrated cytoprotection against aggregation-prone protein toxicity via K48-linked ubiquitination of mutant SOD1, implicating UBE2K in neurodegeneration-relevant clearance.\",\n      \"evidence\": \"Co-IP, K48-linkage-specific ubiquitination, SOD1-G93A mouse spinal cord, viability assays\",\n      \"pmids\": [\"34856358\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E3 partner for mutant SOD1 not identified\", \"In vivo therapeutic relevance untested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed Ubc1 levels are coupled to the spindle assembly checkpoint and APC activity, with consequences for microtubule-poison resistance.\",\n      \"evidence\": \"Overexpression/shut-off, charging and half-life measurement, APC inhibition, drug-sensitivity assays in yeast\",\n      \"pmids\": [\"34586382\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of checkpoint-dependent Ubc1 turnover unclear\", \"Human relevance not established\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Established a conserved role as a positive regulator of IMD/NF-\\u03baB innate immune signaling across Drosophila and mammalian cells.\",\n      \"evidence\": \"RNAi knockdown of UbcD4 and UBE2K with pathway-activation immunoblot readouts\",\n      \"pmids\": [\"26707646\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct substrate within the NF-\\u03baB pathway not identified\", \"E3 partner unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed that pathogens hijack or inhibit UBE2K to subvert immune signaling, including PRRSV NSP8-driven IKK\\u03b1 degradation and microsporidian ehOTUB1 blockade of E1-to-E2 transfer.\",\n      \"evidence\": \"Co-IP, proteasome rescue, knockdown, NF-\\u03baB reporters, charging assays and structural modeling of E1 steric clash (one preprint)\",\n      \"pmids\": [\"42198770\", \"40766534\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"ehOTUB1 finding is a single-lab preprint\", \"Structural details of NSP8\\u2013UBE2K\\u2013IKK\\u03b1 complex unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Connected UBE2K to disease via degradation of STUB1 with CREB1 transcriptional feedback in cancer, and Foxp3 stabilization controlling Treg-mediated inflammation.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, ChIP, luciferase, proliferation/migration assays; mouse Foxp3-DTR/IL-10-KO models\",\n      \"pmids\": [\"41435988\", \"40464424\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect ubiquitination of substrates not fully separated\", \"Single-lab disease models\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The complete set of physiological E3 partners and the in vivo substrate landscape decoded by UBE2K-built K48 and K48/K63-branched chains remain to be mapped.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No comprehensive E3 partner census\", \"Branched-chain substrate repertoire in cells undefined\", \"Phosphorylation-site causality untested by mutagenesis\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 3, 5, 8, 15]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [2, 5, 9]},\n      {\"term_id\": \"GO:0031386\", \"supporting_discovery_ids\": [2, 4, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [8, 9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 2, 5]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [16, 19]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [11, 13, 17]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [12, 13]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"RNF2\", \"San1\", \"STUB1\", \"TP53\", \"CCNB1\", \"DIABLO\", \"SOD1\", \"FOXP3\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}