{"gene":"UBA3","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2003,"finding":"Crystal structure of the 120 kDa quaternary complex of human APPBP1-UBA3 (heterodimeric E1), NEDD8, and ATP was solved, revealing a bipartite interface for selective NEDD8 recruitment involving a domain common to all ubl-activating enzymes plus eukaryotic E1-specific sequences. A single conserved arginine in APPBP1-UBA3 acts as a selectivity gate preventing misactivation of ubiquitin by NEDD8's E1.","method":"X-ray crystallography of quaternary complex + mutational analysis modeling ubiquitin into NEDD8 binding site","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure resolved with functional mutagenesis validation, foundational mechanistic study","pmids":["14690597"],"is_preprint":false},{"year":2003,"finding":"Human APPBP1-UBA3 catalyzes NEDD8 activation via a pseudo-ordered mechanism (ATP leading, NEDD8 trailing substrate), forming a stable ternary complex of NEDD8-adenylate and Uba3-NEDD8 thioester intermediate, and then transferring NEDD8 to HsUbc12 (kcat = 3.5 s⁻¹). Alanine 72 of NEDD8 is a critical specificity determinant for APPBP1-UBA3 binding, since the Ub(R72L) mutant can serve as substrate. Wild-type ubiquitin fails to support activation or HsUbc12 transthiolation.","method":"In vitro enzyme kinetics with radiolabeled ATP and 125I-NEDD8, isotope exchange, transthiolation assays, mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with kinetic characterization and mutagenesis, rigorous biochemical study","pmids":["12740388"],"is_preprint":false},{"year":2003,"finding":"In fission yeast, But1 and But2 proteins physically bind to Uba3 (the catalytic subunit of NEDD8 E1). But1 is a nuclear protein and its overexpression causes cell elongation (a phenotype of NEDD8 pathway defective mutants); overexpression of but1+ in a ned8-ts mutant was deleterious at permissive temperatures, suggesting But1 has an inhibitory role in the NEDD8 pathway.","method":"Two-hybrid screen, overexpression phenotype analysis, genetic interaction with ned8-ts mutant","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — two-hybrid binding plus genetic epistasis in fission yeast, two orthogonal approaches but indirect inhibitory mechanism","pmids":["14623327"],"is_preprint":false},{"year":2012,"finding":"The C-terminal ubiquitin-like β-grasp domain of human Uba3 (Uba3-βGD) is independently folded in solution and its E2-binding surface undergoes conformational exchange between multiple substates (residues absent from NMR spectrum in free form, adopting a kinked α-helix when complexed with E2), analogous to the E2-binding surface of SUMO E1.","method":"NMR spectroscopy of isolated Uba3 β-grasp domain, comparison with crystal structures","journal":"Proteins","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — NMR structural characterization of isolated domain, single lab, no functional mutagenesis follow-up described","pmids":["22821745"],"is_preprint":false},{"year":2014,"finding":"UBA3 point mutations I310N (in K562 cells) and Y352H (in U937 cells) confer resistance to the NAE inhibitor MLN4924 by increasing the enzyme's affinity for ATP while decreasing its affinity for NEDD8, thereby reducing MLN4924 potency while preserving sufficient NAE activity for cell survival.","method":"Selection of resistant cell lines by chronic MLN4924 exposure, sequencing, biochemical analyses of mutant enzyme kinetics","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-based resistance selection combined with biochemical characterization of mutant enzyme, single lab, two orthogonal methods","pmids":["24691136"],"is_preprint":false},{"year":2018,"finding":"Using quantitative FRET, UBA3 alone (without APPBP1) is sufficient for NEDD8 activation, whereas APPBP1 functions mainly as a scaffold to accelerate the reaction rate. This contrasts with SUMO E1, where both heterodimer subunits are required for activation.","method":"Quantitative FRET assays with individual subunits, aided by AESOP computational electrostatic analysis","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — in vitro FRET-based functional dissection, single lab, supported by computation but single method for the key claim","pmids":["29973603"],"is_preprint":false},{"year":2023,"finding":"UBE1C (UBA3)-mediated neddylation of p53 inhibits p53 acetylation at K373, suppresses p53 transcriptional activity, and reduces expression of p53 downstream targets p21 and PTEN. Overexpression of UBE1C/UBA3 and NEDD8 promotes lung cancer cell migration, invasion, and proliferation.","method":"Overexpression of UBE1C and NEDD8 in lung cancer cells, co-IP/Western blot for p53 neddylation and acetylation, promoter activity assays, functional cell assays","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — multiple orthogonal methods (neddylation detection, acetylation, transcription assays) in single lab, no in vitro reconstitution","pmids":["37668436"],"is_preprint":false},{"year":2023,"finding":"UBA3-dependent neddylation in lung adenocarcinoma cells promotes NF-κB signaling by preventing IκBα phosphorylation/accumulation; blocking UBA3 increases p-IκBα and reduces gene expression of tumor-derived cytokines (CCL2, CXCL1, CXCL2, CSF1, CSF2, IL-6, IL-1B), and decreases recruitment of immunosuppressive cells (TAMs, pDCs, Th2, Tregs).","method":"mRNA sequencing, functional enrichment, Western blotting, real-time PCR after UBA3 knockdown in LUAD cells","journal":"Medical oncology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, indirect pathway evidence from KD + transcriptomics, no direct biochemical reconstitution of NF-κB pathway step","pmids":["37656220"],"is_preprint":false},{"year":2024,"finding":"UBA3 promotes intrahepatic cholangiocarcinoma proliferation, invasion, and migration by affecting ANXA2 through the MAPK signaling pathway; knockdown of UBA3 inhibits these processes. Bufalin was identified as a targeting inhibitor of UBA3 that suppresses ICC development through this pathway.","method":"shRNA knockdown of UBA3 in ICC cell lines, mechanistic experiments linking UBA3 to ANXA2/MAPK, bufalin inhibitor studies","journal":"Acta biochimica et biophysica Sinica","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single mechanism study with limited orthogonal validation; pathway placement indirect","pmids":["38298057"],"is_preprint":false},{"year":2026,"finding":"NAE1/UBA3 and UBE2M function as E1 and E2 enzymes, respectively, for the urmylation (URM1 conjugation) pathway in human cells under normal and oxidative stress conditions. An activity-based URM1 probe covalently captured cysteine enzymes in the URM1 signaling pathway, identifying this new substrate specificity. Pharmacologic inhibition of NAE1/UBA3 by pevonedistat blocks protein urmylation and exhibits synergy with cisplatin in killing liver cancer cells.","method":"Activity-based URM1 probe capture, proteomic characterization, cell-based validation, pharmacologic perturbation","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — activity-based probe with proteomic ID plus cell-based validation and pharmacologic inhibition, multiple orthogonal methods in single study","pmids":["42056084"],"is_preprint":false}],"current_model":"UBA3 is the catalytic subunit of the heterodimeric NEDD8-activating enzyme (NAE/E1; together with APPBP1/NAE1): it alone is sufficient for NEDD8 activation via adenylation of NEDD8's C-terminus followed by thioester formation at UBA3's active-site cysteine and transfer to the E2 Ubc12/UBE2M, with APPBP1 acting as a scaffold to accelerate catalysis; substrate selectivity is governed by a conserved arginine selectivity gate and NEDD8 residue Ala72; the β-grasp domain of UBA3 undergoes conformational exchange to engage E2 enzymes; additionally, NAE1/UBA3-UBE2M mediate urmylation (URM1 conjugation), and UBA3-driven neddylation of substrates including p53 and cullins feeds into NF-κB and MAPK signaling pathways relevant to cancer biology."},"narrative":{"mechanistic_narrative":"UBA3 is the catalytic subunit of the heterodimeric NEDD8-activating enzyme (E1), partnering with APPBP1/NAE1 to selectively activate the ubiquitin-like protein NEDD8 and initiate the neddylation cascade [PMID:14690597, PMID:12740388]. Activation proceeds by a pseudo-ordered mechanism in which ATP binds first and NEDD8 second, generating a NEDD8-adenylate and a UBA3-NEDD8 thioester intermediate that is then transthiolated onto the E2 enzyme HsUbc12/UBE2M [PMID:12740388]. Substrate selectivity is enforced both by a single conserved arginine \"selectivity gate\" in the APPBP1-UBA3 interface that excludes ubiquitin and by NEDD8 residue Ala72, mutation of which (Ub R72L) confers E1 activity on ubiquitin [PMID:14690597, PMID:12740388]. Although the enzyme is heterodimeric, UBA3 alone is sufficient for NEDD8 activation, with APPBP1 acting principally as a scaffold that accelerates catalysis [PMID:29973603]; the C-terminal ubiquitin-like β-grasp domain of UBA3 engages the E2 through a conformationally dynamic surface that adopts a defined kinked helix upon complex formation [PMID:22821745]. Beyond NEDD8, UBA3 (with NAE1 and UBE2M) also serves as the E1 for URM1 conjugation (urmylation), a specificity captured by activity-based probes under normal and oxidative-stress conditions [PMID:42056084]. UBA3-driven neddylation modulates oncogenic signaling: it neddylates p53 to block its K373 acetylation and suppress p21/PTEN transcription [PMID:37668436], and supports NF-κB signaling by limiting IκBα phosphorylation [PMID:37656220]. UBA3 is the pharmacological target of NAE inhibitors, and point mutations (I310N, Y352H) that alter ATP and NEDD8 affinity confer resistance to MLN4924 [PMID:24691136].","teleology":[{"year":2003,"claim":"Established the structural and kinetic basis for how UBA3-APPBP1 activates NEDD8 and discriminates it from ubiquitin, defining the first mechanistic picture of the NEDD8 E1.","evidence":"X-ray crystallography of the APPBP1-UBA3-NEDD8-ATP quaternary complex with mutational analysis, plus in vitro enzyme kinetics, isotope exchange, and transthiolation assays","pmids":["14690597","12740388"],"confidence":"High","gaps":["Did not resolve how the E2 (Ubc12) is recruited at atomic resolution","Selectivity gate defined biochemically; in-cell consequences of gate mutation not tested","No structure of the thioester intermediate captured"]},{"year":2003,"claim":"Identified physical and genetic regulators of Uba3 in fission yeast, raising the possibility of negative regulation of the NEDD8 pathway by binding partners.","evidence":"Two-hybrid screen and overexpression/genetic epistasis with a ned8-ts mutant in fission yeast","pmids":["14623327"],"confidence":"Medium","gaps":["Mechanism of inhibition by But1/But2 not biochemically defined","Human orthologs and relevance not established","Indirect inhibitory role inferred from overexpression phenotypes"]},{"year":2012,"claim":"Resolved that the E2-binding β-grasp domain of UBA3 is conformationally dynamic, explaining how the static crystal interface accommodates E2 engagement.","evidence":"NMR spectroscopy of the isolated Uba3 β-grasp domain compared with crystal structures","pmids":["22821745"],"confidence":"Medium","gaps":["Isolated domain studied out of full-length enzyme context","No functional mutagenesis linking dynamics to catalysis","Single lab, single method"]},{"year":2014,"claim":"Demonstrated that UBA3 is the direct target of NAE inhibitors and that defined active-site mutations rewire ATP/NEDD8 affinity to confer drug resistance, clarifying the enzyme's inhibitor pharmacology.","evidence":"Selection of MLN4924-resistant cell lines, sequencing, and kinetic characterization of mutant enzymes","pmids":["24691136"],"confidence":"Medium","gaps":["Resistance mutations not validated structurally","Clinical relevance of these specific mutations not addressed","Single lab"]},{"year":2018,"claim":"Dissected the contribution of each E1 subunit, showing UBA3 alone catalyzes NEDD8 activation while APPBP1 acts as a rate-accelerating scaffold, distinguishing NEDD8 E1 from SUMO E1.","evidence":"Quantitative FRET assays with individual subunits supported by computational electrostatic analysis","pmids":["29973603"],"confidence":"Medium","gaps":["Key claim rests on a single FRET-based method","Physiological role of UBA3-only activation in cells not shown","Rate enhancement by APPBP1 not quantified at all reaction steps"]},{"year":2023,"claim":"Connected UBA3 neddylation activity to oncogenic signaling, showing neddylation suppresses p53 acetylation/activity and promotes NF-κB-driven cytokine production in lung cancer.","evidence":"Overexpression and knockdown in lung cancer/LUAD cells with co-IP, acetylation and promoter assays, mRNA-seq, and functional migration/invasion assays","pmids":["37668436","37656220"],"confidence":"Medium","gaps":["p53 and IκBα effects not reconstituted in vitro","Whether p53 is a direct UBA3 neddylation substrate vs. indirect not fully resolved","NF-κB pathway link is transcriptomic/correlative without direct biochemical step"]},{"year":2024,"claim":"Extended UBA3's tumor-promoting role to cholangiocarcinoma via an ANXA2/MAPK axis and identified bufalin as a UBA3-targeting inhibitor.","evidence":"shRNA knockdown in ICC cell lines with mechanistic and inhibitor studies","pmids":["38298057"],"confidence":"Low","gaps":["Pathway placement is indirect with limited orthogonal validation","Direct UBA3-ANXA2 biochemical relationship not established","Single lab"]},{"year":2026,"claim":"Revealed an expanded substrate specificity in which NAE1/UBA3 and UBE2M also act as the E1/E2 for URM1 conjugation (urmylation), linking the enzyme to oxidative-stress signaling and chemosensitization.","evidence":"Activity-based URM1 probe capture with proteomics, cell-based validation, and pevonedistat/cisplatin synergy assays in liver cancer cells","pmids":["42056084"],"confidence":"Medium","gaps":["Kinetics of URM1 activation by UBA3 not characterized","Structural basis for dual NEDD8/URM1 selectivity unknown","Physiological URM1 substrates not fully enumerated"]},{"year":null,"claim":"How UBA3 partitions catalytic capacity between NEDD8 and URM1 conjugation, and how this dual activity is regulated in vivo, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural or kinetic comparison of NEDD8 vs URM1 activation by UBA3","Regulatory inputs controlling substrate choice unknown","In vivo physiological significance of urmylation by UBA3 not defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0,1]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,1,9]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[6]}],"localization":[],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,9]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[6,7]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[6,8]}],"complexes":["NEDD8-activating enzyme (NAE/E1) heterodimer (UBA3-APPBP1/NAE1)"],"partners":["NAE1","UBE2M","NEDD8","URM1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8TBC4","full_name":"NEDD8-activating enzyme E1 catalytic subunit","aliases":["NEDD8-activating enzyme E1C","Ubiquitin-activating enzyme E1C","Ubiquitin-like modifier-activating enzyme 3","Ubiquitin-activating enzyme 3"],"length_aa":463,"mass_kda":51.9,"function":"Catalytic subunit of the dimeric UBA3-NAE1 E1 enzyme. E1 activates NEDD8 by first adenylating its C-terminal glycine residue with ATP, thereafter linking this residue to the side chain of the catalytic cysteine, yielding a NEDD8-UBA3 thioester and free AMP. E1 finally transfers NEDD8 to the catalytic cysteine of UBE2M. Down-regulates steroid receptor activity. Necessary for cell cycle progression","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q8TBC4/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/UBA3","classification":"Common Essential","n_dependent_lines":1046,"n_total_lines":1208,"dependency_fraction":0.8658940397350994},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"SAR1B","stoichiometry":0.2},{"gene":"UBE2M","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/UBA3","total_profiled":1310},"omim":[{"mim_id":"617700","title":"UBIQUITIN-CONJUGATING ENZYME E2 F; UBE2F","url":"https://www.omim.org/entry/617700"},{"mim_id":"616522","title":"DCN1 DOMAIN-CONTAINING PROTEIN 5; DCUN1D5","url":"https://www.omim.org/entry/616522"},{"mim_id":"608760","title":"AUTOPHAGY-RELATED 7; ATG7","url":"https://www.omim.org/entry/608760"},{"mim_id":"603385","title":"NEDD8-ACTIVATING ENZYME E1, SUBUNIT 1; NAE1","url":"https://www.omim.org/entry/603385"},{"mim_id":"603173","title":"UBIQUITIN-CONJUGATING ENZYME E2 M; UBE2M","url":"https://www.omim.org/entry/603173"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"},{"location":"Nucleoplasm","reliability":"Additional"},{"location":"Centrosome","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/UBA3"},"hgnc":{"alias_symbol":["hUba3","NAE2"],"prev_symbol":["UBE1C"]},"alphafold":{"accession":"Q8TBC4","domains":[{"cath_id":"3.40.50.720","chopping":"65-205_312-339","consensus_level":"high","plddt":97.6058,"start":65,"end":339},{"cath_id":"1.10.10.520","chopping":"235-306","consensus_level":"medium","plddt":96.3519,"start":235,"end":306},{"cath_id":"3.10.290.20","chopping":"371-460","consensus_level":"high","plddt":94.3871,"start":371,"end":460}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TBC4","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TBC4-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TBC4-F1-predicted_aligned_error_v6.png","plddt_mean":92.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=UBA3","jax_strain_url":"https://www.jax.org/strain/search?query=UBA3"},"sequence":{"accession":"Q8TBC4","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8TBC4.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8TBC4/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TBC4"}},"corpus_meta":[{"pmid":"14690597","id":"PMC_14690597","title":"The structure of the APPBP1-UBA3-NEDD8-ATP complex reveals the basis for selective ubiquitin-like protein activation by an E1.","date":"2003","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/14690597","citation_count":242,"is_preprint":false},{"pmid":"12740388","id":"PMC_12740388","title":"Conservation in the mechanism of Nedd8 activation by the human AppBp1-Uba3 heterodimer.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12740388","citation_count":110,"is_preprint":false},{"pmid":"24691136","id":"PMC_24691136","title":"Mutations in UBA3 confer resistance to the NEDD8-activating enzyme inhibitor MLN4924 in human leukemic cells.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/24691136","citation_count":36,"is_preprint":false},{"pmid":"16275315","id":"PMC_16275315","title":"Expression, purification, and characterization of the E1 for human NEDD8, the heterodimeric APPBP1-UBA3 complex.","date":"2005","source":"Methods in enzymology","url":"https://pubmed.ncbi.nlm.nih.gov/16275315","citation_count":29,"is_preprint":false},{"pmid":"38298057","id":"PMC_38298057","title":"UBA3 promotes the occurrence and metastasis of intrahepatic cholangiocarcinoma through MAPK signaling pathway.","date":"2024","source":"Acta biochimica et biophysica Sinica","url":"https://pubmed.ncbi.nlm.nih.gov/38298057","citation_count":18,"is_preprint":false},{"pmid":"29973603","id":"PMC_29973603","title":"Dissecting Distinct Roles of NEDDylation E1 Ligase Heterodimer APPBP1 and UBA3 Reveals Potential Evolution Process for Activation of Ubiquitin-related Pathways.","date":"2018","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/29973603","citation_count":16,"is_preprint":false},{"pmid":"14623327","id":"PMC_14623327","title":"But1 and But2 proteins bind to Uba3, a catalytic subunit of E1 for neddylation, in fission yeast.","date":"2003","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/14623327","citation_count":9,"is_preprint":false},{"pmid":"37668436","id":"PMC_37668436","title":"UBE1C is upregulated and promotes neddylation of p53 in lung cancer.","date":"2023","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/37668436","citation_count":6,"is_preprint":false},{"pmid":"22502714","id":"PMC_22502714","title":"Inhibition of the NEDD8 conjugation pathway by shRNA to UBA3, the subunit of the NEDD8-activating enzyme, suppresses the growth of melanoma cells.","date":"2012","source":"Asian Pacific journal of cancer prevention : APJCP","url":"https://pubmed.ncbi.nlm.nih.gov/22502714","citation_count":6,"is_preprint":false},{"pmid":"37656220","id":"PMC_37656220","title":"The NEDD8-activating enzyme E1 UBA3 orchestrates the immunosuppressive microenvironment in lung adenocarcinoma via the NF-кB pathway.","date":"2023","source":"Medical oncology (Northwood, London, England)","url":"https://pubmed.ncbi.nlm.nih.gov/37656220","citation_count":5,"is_preprint":false},{"pmid":"22821745","id":"PMC_22821745","title":"E2-binding surface on Uba3 β-grasp domain undergoes a conformational transition.","date":"2012","source":"Proteins","url":"https://pubmed.ncbi.nlm.nih.gov/22821745","citation_count":5,"is_preprint":false},{"pmid":"35622528","id":"PMC_35622528","title":"Genetic mapping of Uba3  , a pupal lethal mutation in Drosophila melanogaster.","date":"2022","source":"microPublication biology","url":"https://pubmed.ncbi.nlm.nih.gov/35622528","citation_count":5,"is_preprint":false},{"pmid":"42055937","id":"PMC_42055937","title":"UBA3 reduction sensitizes cancer cells to NAE inhibitors.","date":"2026","source":"Life science alliance","url":"https://pubmed.ncbi.nlm.nih.gov/42055937","citation_count":0,"is_preprint":false},{"pmid":"42056084","id":"PMC_42056084","title":"NAE1/UBA3-UBE2M are E1 and E2 enzymes for the URM1 modification.","date":"2026","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/42056084","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8149,"output_tokens":2755,"usd":0.032886,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10049,"output_tokens":3432,"usd":0.068022,"stage2_stop_reason":"end_turn"},"total_usd":0.100908,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2003,\n      \"finding\": \"Crystal structure of the 120 kDa quaternary complex of human APPBP1-UBA3 (heterodimeric E1), NEDD8, and ATP was solved, revealing a bipartite interface for selective NEDD8 recruitment involving a domain common to all ubl-activating enzymes plus eukaryotic E1-specific sequences. A single conserved arginine in APPBP1-UBA3 acts as a selectivity gate preventing misactivation of ubiquitin by NEDD8's E1.\",\n      \"method\": \"X-ray crystallography of quaternary complex + mutational analysis modeling ubiquitin into NEDD8 binding site\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure resolved with functional mutagenesis validation, foundational mechanistic study\",\n      \"pmids\": [\"14690597\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Human APPBP1-UBA3 catalyzes NEDD8 activation via a pseudo-ordered mechanism (ATP leading, NEDD8 trailing substrate), forming a stable ternary complex of NEDD8-adenylate and Uba3-NEDD8 thioester intermediate, and then transferring NEDD8 to HsUbc12 (kcat = 3.5 s⁻¹). Alanine 72 of NEDD8 is a critical specificity determinant for APPBP1-UBA3 binding, since the Ub(R72L) mutant can serve as substrate. Wild-type ubiquitin fails to support activation or HsUbc12 transthiolation.\",\n      \"method\": \"In vitro enzyme kinetics with radiolabeled ATP and 125I-NEDD8, isotope exchange, transthiolation assays, mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with kinetic characterization and mutagenesis, rigorous biochemical study\",\n      \"pmids\": [\"12740388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"In fission yeast, But1 and But2 proteins physically bind to Uba3 (the catalytic subunit of NEDD8 E1). But1 is a nuclear protein and its overexpression causes cell elongation (a phenotype of NEDD8 pathway defective mutants); overexpression of but1+ in a ned8-ts mutant was deleterious at permissive temperatures, suggesting But1 has an inhibitory role in the NEDD8 pathway.\",\n      \"method\": \"Two-hybrid screen, overexpression phenotype analysis, genetic interaction with ned8-ts mutant\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — two-hybrid binding plus genetic epistasis in fission yeast, two orthogonal approaches but indirect inhibitory mechanism\",\n      \"pmids\": [\"14623327\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The C-terminal ubiquitin-like β-grasp domain of human Uba3 (Uba3-βGD) is independently folded in solution and its E2-binding surface undergoes conformational exchange between multiple substates (residues absent from NMR spectrum in free form, adopting a kinked α-helix when complexed with E2), analogous to the E2-binding surface of SUMO E1.\",\n      \"method\": \"NMR spectroscopy of isolated Uba3 β-grasp domain, comparison with crystal structures\",\n      \"journal\": \"Proteins\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — NMR structural characterization of isolated domain, single lab, no functional mutagenesis follow-up described\",\n      \"pmids\": [\"22821745\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"UBA3 point mutations I310N (in K562 cells) and Y352H (in U937 cells) confer resistance to the NAE inhibitor MLN4924 by increasing the enzyme's affinity for ATP while decreasing its affinity for NEDD8, thereby reducing MLN4924 potency while preserving sufficient NAE activity for cell survival.\",\n      \"method\": \"Selection of resistant cell lines by chronic MLN4924 exposure, sequencing, biochemical analyses of mutant enzyme kinetics\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-based resistance selection combined with biochemical characterization of mutant enzyme, single lab, two orthogonal methods\",\n      \"pmids\": [\"24691136\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Using quantitative FRET, UBA3 alone (without APPBP1) is sufficient for NEDD8 activation, whereas APPBP1 functions mainly as a scaffold to accelerate the reaction rate. This contrasts with SUMO E1, where both heterodimer subunits are required for activation.\",\n      \"method\": \"Quantitative FRET assays with individual subunits, aided by AESOP computational electrostatic analysis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — in vitro FRET-based functional dissection, single lab, supported by computation but single method for the key claim\",\n      \"pmids\": [\"29973603\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"UBE1C (UBA3)-mediated neddylation of p53 inhibits p53 acetylation at K373, suppresses p53 transcriptional activity, and reduces expression of p53 downstream targets p21 and PTEN. Overexpression of UBE1C/UBA3 and NEDD8 promotes lung cancer cell migration, invasion, and proliferation.\",\n      \"method\": \"Overexpression of UBE1C and NEDD8 in lung cancer cells, co-IP/Western blot for p53 neddylation and acetylation, promoter activity assays, functional cell assays\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — multiple orthogonal methods (neddylation detection, acetylation, transcription assays) in single lab, no in vitro reconstitution\",\n      \"pmids\": [\"37668436\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"UBA3-dependent neddylation in lung adenocarcinoma cells promotes NF-κB signaling by preventing IκBα phosphorylation/accumulation; blocking UBA3 increases p-IκBα and reduces gene expression of tumor-derived cytokines (CCL2, CXCL1, CXCL2, CSF1, CSF2, IL-6, IL-1B), and decreases recruitment of immunosuppressive cells (TAMs, pDCs, Th2, Tregs).\",\n      \"method\": \"mRNA sequencing, functional enrichment, Western blotting, real-time PCR after UBA3 knockdown in LUAD cells\",\n      \"journal\": \"Medical oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, indirect pathway evidence from KD + transcriptomics, no direct biochemical reconstitution of NF-κB pathway step\",\n      \"pmids\": [\"37656220\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"UBA3 promotes intrahepatic cholangiocarcinoma proliferation, invasion, and migration by affecting ANXA2 through the MAPK signaling pathway; knockdown of UBA3 inhibits these processes. Bufalin was identified as a targeting inhibitor of UBA3 that suppresses ICC development through this pathway.\",\n      \"method\": \"shRNA knockdown of UBA3 in ICC cell lines, mechanistic experiments linking UBA3 to ANXA2/MAPK, bufalin inhibitor studies\",\n      \"journal\": \"Acta biochimica et biophysica Sinica\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single mechanism study with limited orthogonal validation; pathway placement indirect\",\n      \"pmids\": [\"38298057\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"NAE1/UBA3 and UBE2M function as E1 and E2 enzymes, respectively, for the urmylation (URM1 conjugation) pathway in human cells under normal and oxidative stress conditions. An activity-based URM1 probe covalently captured cysteine enzymes in the URM1 signaling pathway, identifying this new substrate specificity. Pharmacologic inhibition of NAE1/UBA3 by pevonedistat blocks protein urmylation and exhibits synergy with cisplatin in killing liver cancer cells.\",\n      \"method\": \"Activity-based URM1 probe capture, proteomic characterization, cell-based validation, pharmacologic perturbation\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — activity-based probe with proteomic ID plus cell-based validation and pharmacologic inhibition, multiple orthogonal methods in single study\",\n      \"pmids\": [\"42056084\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"UBA3 is the catalytic subunit of the heterodimeric NEDD8-activating enzyme (NAE/E1; together with APPBP1/NAE1): it alone is sufficient for NEDD8 activation via adenylation of NEDD8's C-terminus followed by thioester formation at UBA3's active-site cysteine and transfer to the E2 Ubc12/UBE2M, with APPBP1 acting as a scaffold to accelerate catalysis; substrate selectivity is governed by a conserved arginine selectivity gate and NEDD8 residue Ala72; the β-grasp domain of UBA3 undergoes conformational exchange to engage E2 enzymes; additionally, NAE1/UBA3-UBE2M mediate urmylation (URM1 conjugation), and UBA3-driven neddylation of substrates including p53 and cullins feeds into NF-κB and MAPK signaling pathways relevant to cancer biology.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"UBA3 is the catalytic subunit of the heterodimeric NEDD8-activating enzyme (E1), partnering with APPBP1/NAE1 to selectively activate the ubiquitin-like protein NEDD8 and initiate the neddylation cascade [#0, #1]. Activation proceeds by a pseudo-ordered mechanism in which ATP binds first and NEDD8 second, generating a NEDD8-adenylate and a UBA3-NEDD8 thioester intermediate that is then transthiolated onto the E2 enzyme HsUbc12/UBE2M [#1]. Substrate selectivity is enforced both by a single conserved arginine \\\"selectivity gate\\\" in the APPBP1-UBA3 interface that excludes ubiquitin and by NEDD8 residue Ala72, mutation of which (Ub R72L) confers E1 activity on ubiquitin [#0, #1]. Although the enzyme is heterodimeric, UBA3 alone is sufficient for NEDD8 activation, with APPBP1 acting principally as a scaffold that accelerates catalysis [#5]; the C-terminal ubiquitin-like \\u03b2-grasp domain of UBA3 engages the E2 through a conformationally dynamic surface that adopts a defined kinked helix upon complex formation [#3]. Beyond NEDD8, UBA3 (with NAE1 and UBE2M) also serves as the E1 for URM1 conjugation (urmylation), a specificity captured by activity-based probes under normal and oxidative-stress conditions [#9]. UBA3-driven neddylation modulates oncogenic signaling: it neddylates p53 to block its K373 acetylation and suppress p21/PTEN transcription [#6], and supports NF-\\u03baB signaling by limiting I\\u03baB\\u03b1 phosphorylation [#7]. UBA3 is the pharmacological target of NAE inhibitors, and point mutations (I310N, Y352H) that alter ATP and NEDD8 affinity confer resistance to MLN4924 [#4].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established the structural and kinetic basis for how UBA3-APPBP1 activates NEDD8 and discriminates it from ubiquitin, defining the first mechanistic picture of the NEDD8 E1.\",\n      \"evidence\": \"X-ray crystallography of the APPBP1-UBA3-NEDD8-ATP quaternary complex with mutational analysis, plus in vitro enzyme kinetics, isotope exchange, and transthiolation assays\",\n      \"pmids\": [\n        \"14690597\",\n        \"12740388\"\n      ],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Did not resolve how the E2 (Ubc12) is recruited at atomic resolution\",\n        \"Selectivity gate defined biochemically; in-cell consequences of gate mutation not tested\",\n        \"No structure of the thioester intermediate captured\"\n      ]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Identified physical and genetic regulators of Uba3 in fission yeast, raising the possibility of negative regulation of the NEDD8 pathway by binding partners.\",\n      \"evidence\": \"Two-hybrid screen and overexpression/genetic epistasis with a ned8-ts mutant in fission yeast\",\n      \"pmids\": [\n        \"14623327\"\n      ],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanism of inhibition by But1/But2 not biochemically defined\",\n        \"Human orthologs and relevance not established\",\n        \"Indirect inhibitory role inferred from overexpression phenotypes\"\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Resolved that the E2-binding \\u03b2-grasp domain of UBA3 is conformationally dynamic, explaining how the static crystal interface accommodates E2 engagement.\",\n      \"evidence\": \"NMR spectroscopy of the isolated Uba3 \\u03b2-grasp domain compared with crystal structures\",\n      \"pmids\": [\n        \"22821745\"\n      ],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Isolated domain studied out of full-length enzyme context\",\n        \"No functional mutagenesis linking dynamics to catalysis\",\n        \"Single lab, single method\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrated that UBA3 is the direct target of NAE inhibitors and that defined active-site mutations rewire ATP/NEDD8 affinity to confer drug resistance, clarifying the enzyme's inhibitor pharmacology.\",\n      \"evidence\": \"Selection of MLN4924-resistant cell lines, sequencing, and kinetic characterization of mutant enzymes\",\n      \"pmids\": [\n        \"24691136\"\n      ],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Resistance mutations not validated structurally\",\n        \"Clinical relevance of these specific mutations not addressed\",\n        \"Single lab\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Dissected the contribution of each E1 subunit, showing UBA3 alone catalyzes NEDD8 activation while APPBP1 acts as a rate-accelerating scaffold, distinguishing NEDD8 E1 from SUMO E1.\",\n      \"evidence\": \"Quantitative FRET assays with individual subunits supported by computational electrostatic analysis\",\n      \"pmids\": [\n        \"29973603\"\n      ],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Key claim rests on a single FRET-based method\",\n        \"Physiological role of UBA3-only activation in cells not shown\",\n        \"Rate enhancement by APPBP1 not quantified at all reaction steps\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Connected UBA3 neddylation activity to oncogenic signaling, showing neddylation suppresses p53 acetylation/activity and promotes NF-\\u03baB-driven cytokine production in lung cancer.\",\n      \"evidence\": \"Overexpression and knockdown in lung cancer/LUAD cells with co-IP, acetylation and promoter assays, mRNA-seq, and functional migration/invasion assays\",\n      \"pmids\": [\n        \"37668436\",\n        \"37656220\"\n      ],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"p53 and I\\u03baB\\u03b1 effects not reconstituted in vitro\",\n        \"Whether p53 is a direct UBA3 neddylation substrate vs. indirect not fully resolved\",\n        \"NF-\\u03baB pathway link is transcriptomic/correlative without direct biochemical step\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended UBA3's tumor-promoting role to cholangiocarcinoma via an ANXA2/MAPK axis and identified bufalin as a UBA3-targeting inhibitor.\",\n      \"evidence\": \"shRNA knockdown in ICC cell lines with mechanistic and inhibitor studies\",\n      \"pmids\": [\n        \"38298057\"\n      ],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"Pathway placement is indirect with limited orthogonal validation\",\n        \"Direct UBA3-ANXA2 biochemical relationship not established\",\n        \"Single lab\"\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Revealed an expanded substrate specificity in which NAE1/UBA3 and UBE2M also act as the E1/E2 for URM1 conjugation (urmylation), linking the enzyme to oxidative-stress signaling and chemosensitization.\",\n      \"evidence\": \"Activity-based URM1 probe capture with proteomics, cell-based validation, and pevonedistat/cisplatin synergy assays in liver cancer cells\",\n      \"pmids\": [\n        \"42056084\"\n      ],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Kinetics of URM1 activation by UBA3 not characterized\",\n        \"Structural basis for dual NEDD8/URM1 selectivity unknown\",\n        \"Physiological URM1 substrates not fully enumerated\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How UBA3 partitions catalytic capacity between NEDD8 and URM1 conjugation, and how this dual activity is regulated in vivo, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No structural or kinetic comparison of NEDD8 vs URM1 activation by UBA3\",\n        \"Regulatory inputs controlling substrate choice unknown\",\n        \"In vivo physiological significance of urmylation by UBA3 not defined\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 1, 9]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 9]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [6, 7]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [6, 8]}\n    ],\n    \"complexes\": [\n      \"NEDD8-activating enzyme (NAE/E1) heterodimer (UBA3-APPBP1/NAE1)\"\n    ],\n    \"partners\": [\n      \"NAE1\",\n      \"UBE2M\",\n      \"NEDD8\",\n      \"URM1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}