{"gene":"UBA6","run_date":"2026-04-28T21:43:00","timeline":{"discoveries":[{"year":2007,"finding":"UBA6 (UBE1L2) is a novel ubiquitin-activating E1 enzyme that forms a covalent thioester bond with ubiquitin in an ATP-dependent manner, transfers ubiquitin to the E2 enzyme UbcH5b, and supports ubiquitylation of p53 by MDM2 and autoubiquitylation of E3 ligases HectH9 and E6-AP in vitro.","method":"In vitro polyubiquitylation assay, covalent thioester bond formation assay (reducing-condition sensitivity), recombinant protein reconstitution","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro with multiple substrates and E2/E3 partners; foundational discovery paper with 133 citations","pmids":["17580310"],"is_preprint":false},{"year":2008,"finding":"UBA6 activates not only ubiquitin but also the ubiquitin-like modifier FAT10, and uses a distinct spectrum of E2 enzymes compared to UBE1/UBA1, establishing it as an orthogonal E1 enzyme in the ubiquitin system.","method":"Biochemical characterization, review of experimental literature","journal":"Trends in biochemical sciences","confidence":"High","confidence_rationale":"Tier 2 — synthesis of multiple experimental findings across labs, 99 citations","pmids":["18353650"],"is_preprint":false},{"year":2012,"finding":"UBA6 undergoes a three-step activation process (adenylation, thioester formation, transfer) for both ubiquitin and FAT10; FAT10 binds UBA6 with higher affinity than ubiquitin but shows lower catalytic activity; a mechanism-based inhibitor (Compound 1) forms covalent adducts with both ubiquitin and FAT10 on UBA6.","method":"Biochemical in vitro assays (ATP-PPi exchange, E1-E2 transthiolation), pre-steady state kinetics, inhibitor mechanism studies, biophysical binding measurements","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — multiple orthogonal biochemical and biophysical methods in a single rigorous study","pmids":["22427669"],"is_preprint":false},{"year":2013,"finding":"The UBA6-USE1 ubiquitin transfer cascade is required for mouse embryonic development independent of the canonical UBA1 pathway; neuronal loss of UBA6 causes altered hippocampal and amygdala neuron patterning, decreased dendritic spine density, and elevated levels of E3 ligase Ube3a (E6-AP) and Shank3; UBA6 and USE1 promote proteasomal turnover of Ube3a and catalyze Ube3a ubiquitylation in vitro.","method":"Conditional knockout mice, in vitro ubiquitylation assay (Ube3a ubiquitylation), immunohistochemistry, dendritic spine analysis, behavioral testing","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 — genetic KO with defined cellular phenotype plus in vitro reconstitution; 47 citations","pmids":["23499007"],"is_preprint":false},{"year":2014,"finding":"USE1 undergoes auto-FAT10ylation in cis (primarily at Lys323) via the UBA6-USE1 cascade, which accelerates its proteasomal degradation; the USE1-FAT10 conjugate retains E2 activity; auto-FAT10ylation of USE1 serves as a negative feedback mechanism limiting FAT10 conjugation.","method":"MS analysis of FAT10ylation sites, site-directed mutagenesis, thioester-linkage assays, Co-immunoprecipitation","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 1-2 — MS identification of modification site, mutagenesis, and functional readout with multiple methods","pmids":["24528925"],"is_preprint":false},{"year":2016,"finding":"LMO2 protein interacts with the C-terminal ubiquitin fold domain (UFD) of UBA6, blocking the UBA6-USE1 interaction and reducing overall cellular FAT10ylation levels, including FAT10ylation and degradation of the substrate p62.","method":"Co-immunoprecipitation, FAT10ylation assays, domain mapping","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 — single lab, Co-IP plus functional FAT10ylation assay but limited orthogonal validation","pmids":["27569286"],"is_preprint":false},{"year":2019,"finding":"UBA6 and the hybrid E2/E3 enzyme BIRC6 cooperate to monoubiquitinate LC3B, targeting it for proteasomal degradation; loss of UBA6 or BIRC6 increases autophagic flux and decreases aggresome-like induced structures and α-synuclein aggregates, demonstrating that UBA6-BIRC6 negatively regulates autophagy by limiting LC3B availability.","method":"Whole-genome CRISPR/Cas9 knockout screen, genetic epistasis (double KO), autophagic flux assays, proteasomal degradation assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 — genome-wide screen followed by mechanistic validation with multiple orthogonal methods; 82 citations","pmids":["31692446"],"is_preprint":false},{"year":2020,"finding":"UBA6 and UBE1 are E1 enzymes that ubiquitinate the cardiac sodium channel Nav1.5 at K590 and K591, reducing Nav1.5 expression and sodium current density; this is mediated through the E2 enzyme UBC9.","method":"Overexpression/knockdown western blot, patch-clamp electrophysiology, site-directed mutagenesis of ubiquitination sites, E2 knockdown epistasis","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 — multiple functional readouts (biochemical + electrophysiology) with mutagenesis, single lab","pmids":["32315024"],"is_preprint":false},{"year":2021,"finding":"UBA6 deficiency in T cells increases IFN-γ production by destabilizing IκBα degradation and thereby increasing NF-κB p65 activation; T cell-specific UBA6 knockout mice develop exacerbated multi-organ inflammation.","method":"T cell-specific conditional knockout mice, IFN-γ ELISA, NF-κB western blot, adoptive transfer experiments","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 — clean genetic KO with defined cellular phenotype and pathway placement, but limited mechanistic biochemistry on the direct substrate","pmids":["35011668"],"is_preprint":false},{"year":2022,"finding":"UBA6 is required for hepcidin/BMP6-induced ubiquitination and lysosomal degradation of ferroportin; siRNA-mediated depletion of UBA6 prevents ferroportin degradation in HepG2 cells, and AAV-mediated silencing of Ndfip1 in mouse liver increases hepatic ferroportin and circulating iron.","method":"siRNA screen (88 ubiquitin pathway components), HepG2 ferroportin-GFP reporter cell line, AAV silencing in mice, western blot","journal":"Haematologica","confidence":"Medium","confidence_rationale":"Tier 2 — siRNA screen with functional readout plus in vivo validation, single lab","pmids":["34320783"],"is_preprint":false},{"year":2022,"finding":"Crystal structures of human UBA6 in complex with ubiquitin reveal two conformational states: an open conformation configured for adenylation and a closed conformation configured for thioester bond formation; inositol hexakisphosphate (InsP6) binds an allosteric site on UBA6 and inhibits its activity by altering interconversion between open and closed conformations while enhancing protein stability.","method":"X-ray crystallography, biochemical activity assays, biophysical binding measurements, mutagenesis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 — crystal structures with functional biochemical and biophysical validation of allosteric mechanism","pmids":["35986001"],"is_preprint":false},{"year":2022,"finding":"Inosine directly inhibits UBA6 in tumor cells, reducing UBA6 activity and increasing tumor immunogenicity, thereby sensitizing tumors to immune checkpoint blockade; genetic ablation of Uba6 in mouse tumor cells phenocopies inosine treatment.","method":"Metabolic profiling, genetic ablation (Uba6 knockout), tumor transplant models, T cell cytotoxicity assays, biochemical inhibition assay","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 — direct biochemical inhibition plus genetic KO with defined immune phenotype, single lab","pmids":["36109526"],"is_preprint":false},{"year":2024,"finding":"UBA6 recognizes a polyalanine stretch within its cognate E2 enzyme USE1; expansion mutations in polyalanine tract proteins compete for the UBA6-USE1 interaction, reducing ubiquitin transfer to USE1 and subsequent polyubiquitination and degradation of E6AP, which in turn affects levels of synaptic protein Arc; UBA6 overexpression increases neuronal resilience to cell death in patient-derived iPSC neurons.","method":"Binding assays, ubiquitination assays, mouse primary neuron experiments, patient-derived iPSC-autonomic neuron experiments, UBA6 overexpression rescue","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1-2 — mechanistic biochemistry plus patient-derived cell validation with multiple orthogonal approaches","pmids":["38177505"],"is_preprint":false},{"year":2025,"finding":"BIRC6 is a UBA6-exclusive E2 enzyme that gains priority over all other UBA6-competent E2s through an exceptionally high-affinity interaction with the UBA6 ubiquitin fold domain (UFD), modulated by the UBA6 Cys-Cap loop; upon receiving ubiquitin via thioester transfer, BIRC6 undergoes a thioester switch mechanism that disengages it from UBA6, preventing inhibition of UBA6's activity toward other E2s.","method":"Structural capture of UBA6-BIRC6 complexes, biochemical E2 competition assays, mutagenesis, E1-E2 specificity assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 — structural and biochemical reconstitution with mechanistic mutagenesis defining the priority and thioester switch mechanism","pmids":["41350950"],"is_preprint":false},{"year":2026,"finding":"Cryo-EM structures of UBA6 in complex with E2 enzymes (including BIRC6) during thioester transfer with either FAT10 or ubiquitin reveal that UBA6 achieves E2 specificity through coordinated contributions of both the UFD and SCCH domains (contrasting with the UFD-dominated selectivity of UBA1); InsP6 binding stabilizes an expanded SCCH cleft that pre-organizes UBA6 for selective engagement of UBA6-specific E2s.","method":"Chemical trapping, high-resolution cryo-EM of four UBA6-E2 complex structures, biochemical specificity assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 — multiple high-resolution cryo-EM structures with biochemical validation defining dual-domain E2 specificity mechanism","pmids":["41764162"],"is_preprint":false}],"current_model":"UBA6 is a dual-specificity E1 ubiquitin-activating enzyme that uses ATP-dependent adenylation and thioester bond formation to activate both ubiquitin and the ubiquitin-like modifier FAT10, transferring them to a distinct set of E2 enzymes (most prominently BIRC6 and USE1) via a mechanism regulated by conformational switching between open and closed states and allosterically modulated by inositol hexakisphosphate (InsP6); through the UBA6-BIRC6 axis it monoubiquitinates LC3B to suppress autophagy, through the UBA6-USE1 axis it promotes FAT10ylation and ubiquitin-dependent proteasomal degradation of substrates including Ube3a/E6-AP, and it also regulates ferroportin stability, Nav1.5 channel ubiquitination, NF-κB signaling in T cells, and neuronal development."},"narrative":{"teleology":[{"year":2007,"claim":"The discovery that UBA6 forms thioester bonds with ubiquitin and transfers it to E2 enzymes established the existence of a second ubiquitin-activating E1 enzyme in mammals, overturning the assumption that UBA1 was the sole E1 for ubiquitin.","evidence":"In vitro reconstitution of thioester bond formation, ubiquitin transfer to UbcH5b, and support of p53 ubiquitylation by MDM2","pmids":["17580310"],"confidence":"High","gaps":["Physiological substrates and E2 partners specific to UBA6 versus UBA1 were unknown","Whether UBA6 activates ubiquitin-like modifiers besides ubiquitin was untested"]},{"year":2008,"claim":"Demonstrating that UBA6 activates both ubiquitin and the ubiquitin-like modifier FAT10 with a distinct E2 spectrum revealed it as an orthogonal E1 pathway, raising the question of how dual specificity is achieved.","evidence":"Biochemical characterization and synthesis of experimental evidence across laboratories","pmids":["18353650"],"confidence":"High","gaps":["Kinetic parameters for ubiquitin versus FAT10 activation were not yet quantified","Structural basis for dual modifier recognition was unknown"]},{"year":2012,"claim":"Kinetic dissection of the three-step activation mechanism showed that FAT10 binds UBA6 with higher affinity than ubiquitin but is activated with lower catalytic efficiency, explaining how a single E1 balances two modifier pathways.","evidence":"Pre-steady state kinetics, ATP-PPi exchange, and mechanism-based inhibitor trapping","pmids":["22427669"],"confidence":"High","gaps":["How UBA6 structurally discriminates between ubiquitin and FAT10 was unresolved","Cellular regulation of the ubiquitin/FAT10 balance by UBA6 was not addressed"]},{"year":2013,"claim":"Genetic ablation of UBA6 in mice demonstrated that the UBA6–USE1 cascade is essential for embryonic development and neuronal patterning, identifying E3 ligase Ube3a as a physiological substrate whose proteasomal degradation depends on this pathway.","evidence":"Conditional knockout mice with hippocampal neuron analysis, dendritic spine quantification, and in vitro reconstitution of Ube3a ubiquitylation","pmids":["23499007"],"confidence":"High","gaps":["Whether Ube3a is a direct or indirect substrate of UBA6-USE1 in vivo required further clarification","Downstream synaptic consequences beyond Shank3 elevation were not mapped"]},{"year":2014,"claim":"The finding that USE1 auto-FAT10ylates at K323, accelerating its own proteasomal degradation, revealed a negative feedback loop that limits cellular FAT10 conjugation capacity.","evidence":"Mass spectrometry identification of FAT10ylation site, site-directed mutagenesis, and thioester-linkage assays","pmids":["24528925"],"confidence":"High","gaps":["Whether this feedback operates under physiological FAT10-inducing conditions (e.g., cytokine stimulation) was not tested in vivo","Other regulatory post-translational modifications of USE1 were unexplored"]},{"year":2016,"claim":"Identification of LMO2 as an interactor that blocks the UBA6 UFD–USE1 interaction and reduces cellular FAT10ylation provided the first evidence of an endogenous protein-level regulatory mechanism for UBA6 substrate selection.","evidence":"Co-immunoprecipitation, domain mapping, and FAT10ylation assays including p62 as substrate","pmids":["27569286"],"confidence":"Medium","gaps":["LMO2–UBA6 interaction was demonstrated by Co-IP without reciprocal structural or biophysical validation","Physiological contexts in which LMO2 levels regulate FAT10ylation were not established"]},{"year":2019,"claim":"A genome-wide CRISPR screen identified UBA6 and BIRC6 as negative regulators of autophagy, establishing that UBA6-BIRC6-mediated monoubiquitination targets LC3B for proteasomal degradation and limits autophagic flux.","evidence":"Genome-wide CRISPR/Cas9 knockout screen, genetic epistasis with double knockouts, autophagic flux and α-synuclein aggregation assays","pmids":["31692446"],"confidence":"High","gaps":["Whether UBA6-BIRC6 directly monoubiquitinates LC3B or acts through intermediates was not structurally resolved","The ubiquitin attachment site(s) on LC3B were not mapped"]},{"year":2020,"claim":"Demonstrating that UBA6 ubiquitinates Nav1.5 at K590/K591 to reduce sodium channel expression and current density extended UBA6 function to cardiac electrophysiology and ion channel regulation.","evidence":"Overexpression/knockdown, patch-clamp electrophysiology, and site-directed mutagenesis of ubiquitination sites","pmids":["32315024"],"confidence":"Medium","gaps":["The contribution of UBA6 versus UBA1 to Nav1.5 regulation in cardiomyocytes was not cleanly separated","The claimed E2 UBC9 is canonically a SUMO E2, raising questions about the conjugation specificity"]},{"year":2021,"claim":"T cell-specific UBA6 knockout revealed that UBA6 restrains NF-κB activation by stabilizing IκBα, placing UBA6 in immune homeostasis and explaining the multi-organ inflammation observed upon its loss.","evidence":"T cell-conditional knockout mice, IκBα and NF-κB western blots, IFN-γ ELISA, adoptive transfer experiments","pmids":["35011668"],"confidence":"Medium","gaps":["Whether UBA6 directly ubiquitinates IκBα or acts through an intermediate E3 was not determined","The specific E2 mediating this UBA6-dependent pathway in T cells was not identified"]},{"year":2022,"claim":"Crystal structures of UBA6–ubiquitin complexes in open and closed states, combined with identification of InsP6 as an allosteric inhibitor, provided the first structural framework for the conformational switch governing UBA6 catalysis.","evidence":"X-ray crystallography, mutagenesis, biochemical activity and biophysical binding assays","pmids":["35986001"],"confidence":"High","gaps":["How InsP6 concentrations are regulated in cells to control UBA6 activity was unknown","Structures with FAT10 rather than ubiquitin were not obtained"]},{"year":2022,"claim":"Identification of UBA6 as a target of the metabolite inosine in tumor cells linked UBA6 activity to tumor immunogenicity and immune checkpoint sensitivity, providing a metabolic axis of UBA6 regulation.","evidence":"Metabolic profiling, Uba6 genetic ablation in mouse tumor models, T cell cytotoxicity assays, biochemical inhibition","pmids":["36109526"],"confidence":"Medium","gaps":["The precise mechanism by which inosine inhibits UBA6 (competitive, allosteric) was not structurally defined","Whether inosine affects FAT10 activation by UBA6 was not tested"]},{"year":2024,"claim":"Discovery that UBA6 recognizes USE1 via a polyalanine stretch, and that polyalanine expansion mutations in disease proteins competitively disrupt this interaction, mechanistically linked UBA6-USE1 dysfunction to polyalanine expansion diseases by showing impaired Ube3a degradation and reduced neuronal viability.","evidence":"Binding and ubiquitination assays, mouse primary neurons, patient-derived iPSC-autonomic neurons, UBA6 overexpression rescue","pmids":["38177505"],"confidence":"High","gaps":["Whether therapeutic UBA6 overexpression can rescue disease phenotypes in vivo remains untested","The full repertoire of polyalanine-containing UBA6 interactors was not mapped"]},{"year":2025,"claim":"Structural and biochemical characterization of the UBA6–BIRC6 complex revealed that BIRC6 achieves priority over other UBA6 E2s through exceptionally high UFD affinity modulated by the Cys-Cap loop, and disengages via a thioester switch upon ubiquitin loading, preventing inhibition of UBA6 activity toward other E2s.","evidence":"Structural capture of UBA6–BIRC6 complexes, E2 competition assays, mutagenesis of Cys-Cap loop","pmids":["41350950"],"confidence":"High","gaps":["How the thioester switch is regulated in cells and whether it applies to FAT10 transfer was not addressed","Structures of UBA6 with E2s other than BIRC6 in this study were limited"]},{"year":2026,"claim":"Cryo-EM structures of UBA6 with multiple E2 partners during thioester transfer resolved that UBA6 achieves E2 selectivity through coordinated contributions of both UFD and SCCH domains — a dual-domain mechanism distinct from UBA1's UFD-dominated selectivity — and that InsP6 binding stabilizes an expanded SCCH cleft to pre-organize this selectivity.","evidence":"Chemical trapping, four high-resolution cryo-EM structures (including BIRC6 and other E2s), biochemical specificity assays","pmids":["41764162"],"confidence":"High","gaps":["How InsP6-mediated allosteric regulation integrates with cellular signaling pathways is not established","Structural basis for FAT10 versus ubiquitin discrimination during E2 transfer remains unresolved"]},{"year":null,"claim":"Key unresolved questions include the structural basis for UBA6 discrimination between ubiquitin and FAT10, the complete cellular substrate landscape of UBA6-specific ubiquitination, and whether the InsP6 allosteric mechanism is physiologically regulated to tune UBA6 pathway output.","evidence":"","pmids":[],"confidence":"High","gaps":["No structure of UBA6 bound to FAT10 exists","Comprehensive proteomics of UBA6-specific versus UBA1-specific substrates has not been performed","In vivo regulation of UBA6 by InsP6 or inosine has not been validated in physiological models"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0,2,10]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,3,6,12]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,2,10]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,3]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,2,3,4,6,12]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[6]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[8,11]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[12]}],"complexes":[],"partners":["USE1","BIRC6","FAT10","UBE3A","LMO2","LC3B","UBCH5B"],"other_free_text":[]},"mechanistic_narrative":"UBA6 is a dual-specificity E1 ubiquitin-activating enzyme that activates both ubiquitin and the ubiquitin-like modifier FAT10 through ATP-dependent adenylation and thioester bond formation, operating as an orthogonal arm of the ubiquitin system with a distinct E2 enzyme repertoire [PMID:17580310, PMID:18353650, PMID:22427669]. UBA6 transfers ubiquitin to cognate E2s including USE1 and BIRC6, where BIRC6 gains priority access through an exceptionally high-affinity interaction with the UBA6 ubiquitin fold domain (UFD) and is released upon thioester loading via a thioester switch mechanism, while USE1 is recognized via a polyalanine stretch and mediates ubiquitin-dependent proteasomal degradation of substrates such as Ube3a/E6-AP [PMID:41350950, PMID:38177505, PMID:23499007]. Crystal and cryo-EM structures reveal that UBA6 cycles between open (adenylation-competent) and closed (thioester-competent) conformations, with inositol hexakisphosphate (InsP6) binding an allosteric site that stabilizes an expanded SCCH cleft to pre-organize E2 selectivity through coordinated UFD and SCCH domain contributions [PMID:35986001, PMID:41764162]. Through the UBA6–BIRC6 axis, UBA6 monoubiquitinates LC3B to suppress autophagy, and through the UBA6–USE1 axis it controls neuronal Ube3a levels, dendritic spine density, and hippocampal neuron patterning; UBA6 also regulates ferroportin degradation, NF-κB signaling in T cells, and tumor immunogenicity [PMID:31692446, PMID:23499007, PMID:34320783, PMID:35011668, PMID:36109526]."},"prefetch_data":{"uniprot":{"accession":"A0AVT1","full_name":"Ubiquitin-like modifier-activating enzyme 6","aliases":["Monocyte protein 4","MOP-4","Ubiquitin-activating enzyme E1-like protein 2","E1-L2"],"length_aa":1052,"mass_kda":118.0,"function":"Activates ubiquitin by first adenylating its C-terminal glycine residue with ATP, and thereafter linking this residue to the side chain of a cysteine residue in E1, yielding a ubiquitin-E1 thioester and free AMP (PubMed:35970836, PubMed:35986001). Specific for ubiquitin, does not activate ubiquitin-like peptides. Also activates UBD/FAT10 conjugation via adenylation of its C-terminal glycine (PubMed:17889673, PubMed:35970836, PubMed:35986001). Differs from UBE1 in its specificity for substrate E2 charging. Does not charge cell cycle E2s, such as CDC34. Essential for embryonic development. Isoform 2 may play a key role in ubiquitin system and may influence spermatogenesis and male fertility","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/A0AVT1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/UBA6","classification":"Not Classified","n_dependent_lines":419,"n_total_lines":1208,"dependency_fraction":0.3468543046357616},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"SAR1B","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/UBA6","total_profiled":1310},"omim":[{"mim_id":"611362","title":"UBIQUITIN-CONJUGATING ENZYME E2 Z; UBE2Z","url":"https://www.omim.org/entry/611362"},{"mim_id":"611361","title":"UBIQUITIN-LIKE MODIFIER-ACTIVATING ENZYME 6; UBA6","url":"https://www.omim.org/entry/611361"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"},{"location":"Cytosol","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/UBA6"},"hgnc":{"alias_symbol":["FLJ10808"],"prev_symbol":["UBE1L2"]},"alphafold":{"accession":"A0AVT1","domains":[{"cath_id":"3.40.50.720","chopping":"42-206","consensus_level":"medium","plddt":96.7017,"start":42,"end":206},{"cath_id":"2.40.30.180","chopping":"216-292","consensus_level":"high","plddt":93.9173,"start":216,"end":292},{"cath_id":"3.40.50.720","chopping":"453-622_893-924","consensus_level":"medium","plddt":95.5656,"start":453,"end":924},{"cath_id":"3.10.290.60","chopping":"944-1047","consensus_level":"high","plddt":89.5486,"start":944,"end":1047}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/A0AVT1","model_url":"https://alphafold.ebi.ac.uk/files/AF-A0AVT1-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-A0AVT1-F1-predicted_aligned_error_v6.png","plddt_mean":91.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=UBA6","jax_strain_url":"https://www.jax.org/strain/search?query=UBA6"},"sequence":{"accession":"A0AVT1","fasta_url":"https://rest.uniprot.org/uniprotkb/A0AVT1.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/A0AVT1/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/A0AVT1"}},"corpus_meta":[{"pmid":"17580310","id":"PMC_17580310","title":"UBE1L2, a novel E1 enzyme specific for ubiquitin.","date":"2007","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17580310","citation_count":133,"is_preprint":false},{"pmid":"18353650","id":"PMC_18353650","title":"Activating the ubiquitin family: UBA6 challenges the field.","date":"2008","source":"Trends in biochemical sciences","url":"https://pubmed.ncbi.nlm.nih.gov/18353650","citation_count":99,"is_preprint":false},{"pmid":"31692446","id":"PMC_31692446","title":"Negative regulation of autophagy by UBA6-BIRC6-mediated ubiquitination of LC3.","date":"2019","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/31692446","citation_count":82,"is_preprint":false},{"pmid":"36109526","id":"PMC_36109526","title":"Inhibition of UBA6 by inosine augments tumour immunogenicity and responses.","date":"2022","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/36109526","citation_count":63,"is_preprint":false},{"pmid":"23499007","id":"PMC_23499007","title":"Altered social behavior and neuronal development in mice lacking the Uba6-Use1 ubiquitin transfer system.","date":"2013","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/23499007","citation_count":47,"is_preprint":false},{"pmid":"22427669","id":"PMC_22427669","title":"Mechanistic studies on activation of ubiquitin and di-ubiquitin-like protein, FAT10, by ubiquitin-like modifier activating enzyme 6, Uba6.","date":"2012","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22427669","citation_count":31,"is_preprint":false},{"pmid":"24528925","id":"PMC_24528925","title":"Investigations into the auto-FAT10ylation of the bispecific E2 conjugating enzyme UBA6-specific E2 enzyme 1.","date":"2014","source":"The FEBS journal","url":"https://pubmed.ncbi.nlm.nih.gov/24528925","citation_count":29,"is_preprint":false},{"pmid":"31067743","id":"PMC_31067743","title":"UBA6 and Its Bispecific Pathways for Ubiquitin and FAT10.","date":"2019","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/31067743","citation_count":27,"is_preprint":false},{"pmid":"35011668","id":"PMC_35011668","title":"Ubiquitin Activating Enzyme UBA6 Regulates Th1 and Tc1 Cell Differentiation.","date":"2021","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/35011668","citation_count":27,"is_preprint":false},{"pmid":"34320783","id":"PMC_34320783","title":"UBA6 and NDFIP1 regulate the degradation of ferroportin.","date":"2022","source":"Haematologica","url":"https://pubmed.ncbi.nlm.nih.gov/34320783","citation_count":22,"is_preprint":false},{"pmid":"28376205","id":"PMC_28376205","title":"Association of Uba6-Specific-E2 (USE1) With Lung Tumorigenesis.","date":"2017","source":"Journal of the National Cancer Institute","url":"https://pubmed.ncbi.nlm.nih.gov/28376205","citation_count":21,"is_preprint":false},{"pmid":"34951345","id":"PMC_34951345","title":"Long noncoding RNA UBA6-AS1 inhibits the malignancy of ovarian cancer cells via suppressing the decay of UBA6 mRNA.","date":"2022","source":"Bioengineered","url":"https://pubmed.ncbi.nlm.nih.gov/34951345","citation_count":19,"is_preprint":false},{"pmid":"21222287","id":"PMC_21222287","title":"FAT10 : Activated by UBA6 and Functioning in Protein Degradation.","date":"2010","source":"Sub-cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21222287","citation_count":19,"is_preprint":false},{"pmid":"25523030","id":"PMC_25523030","title":"Impairment of social behavior and communication in mice lacking the Uba6-dependent ubiquitin activation system.","date":"2014","source":"Behavioural brain research","url":"https://pubmed.ncbi.nlm.nih.gov/25523030","citation_count":19,"is_preprint":false},{"pmid":"29152096","id":"PMC_29152096","title":"The non-canonical ubiquitin activating enzyme UBA6 suppresses epithelial-mesenchymal transition of mammary epithelial cells.","date":"2017","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/29152096","citation_count":17,"is_preprint":false},{"pmid":"35137449","id":"PMC_35137449","title":"Amino acid restriction induces a long non-coding RNA UBA6-AS1 to regulate GCN2-mediated integrated stress response in breast cancer.","date":"2022","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/35137449","citation_count":16,"is_preprint":false},{"pmid":"32315024","id":"PMC_32315024","title":"Ubiquitination-activating enzymes UBE1 and UBA6 regulate ubiquitination and expression of cardiac sodium channel Nav1.5.","date":"2020","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/32315024","citation_count":15,"is_preprint":false},{"pmid":"35986001","id":"PMC_35986001","title":"Crystal structures reveal catalytic and regulatory mechanisms of the dual-specificity ubiquitin/FAT10 E1 enzyme Uba6.","date":"2022","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/35986001","citation_count":14,"is_preprint":false},{"pmid":"26284580","id":"PMC_26284580","title":"Interstitial microdeletions including the chromosome band 4q13.2 and the UBA6 gene as possible causes of intellectual disability and behavior disorder.","date":"2015","source":"American journal of medical genetics. Part A","url":"https://pubmed.ncbi.nlm.nih.gov/26284580","citation_count":11,"is_preprint":false},{"pmid":"27569286","id":"PMC_27569286","title":"LMO2 blocks the UBA6-USE1 interaction and downstream FAT10ylation by targeting the ubiquitin fold domain of UBA6.","date":"2016","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/27569286","citation_count":9,"is_preprint":false},{"pmid":"38177505","id":"PMC_38177505","title":"Disease-associated polyalanine expansion mutations impair UBA6-dependent ubiquitination.","date":"2024","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/38177505","citation_count":7,"is_preprint":false},{"pmid":"32497710","id":"PMC_32497710","title":"Down-regulation of UBA6 exacerbates brain injury by inhibiting the activation of Notch signaling pathway to promote cerebral cell apoptosis in rat acute cerebral infarction model.","date":"2020","source":"Molecular and cellular probes","url":"https://pubmed.ncbi.nlm.nih.gov/32497710","citation_count":6,"is_preprint":false},{"pmid":"33469379","id":"PMC_33469379","title":"Long Non-Coding RNA UBA6-AS1 Promotes the Malignant Properties of Glioblastoma by Competitively Binding to microRNA-760 and Enhancing Homeobox A2 Expression.","date":"2021","source":"Cancer management and research","url":"https://pubmed.ncbi.nlm.nih.gov/33469379","citation_count":6,"is_preprint":false},{"pmid":"31060776","id":"PMC_31060776","title":"Early-stage paired housing improves social interaction in neuronal Uba6-deficient mice.","date":"2019","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/31060776","citation_count":5,"is_preprint":false},{"pmid":"41350950","id":"PMC_41350950","title":"UBA6 specificity for ubiquitin E2 conjugating enzymes reveals a priority mechanism of BIRC6.","date":"2025","source":"Nature structural & molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/41350950","citation_count":3,"is_preprint":false},{"pmid":"40588566","id":"PMC_40588566","title":"Characterization of E1 enzyme dependencies in mutant-UBA1 human cells reveals UBA6 as a novel therapeutic target in VEXAS syndrome.","date":"2025","source":"Leukemia","url":"https://pubmed.ncbi.nlm.nih.gov/40588566","citation_count":1,"is_preprint":false},{"pmid":"38474091","id":"PMC_38474091","title":"UBA6 Inhibition Accelerates Lysosomal TRPML1 Depletion and Exosomal Secretion in Lung Cancer Cells.","date":"2024","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/38474091","citation_count":1,"is_preprint":false},{"pmid":"40766763","id":"PMC_40766763","title":"Ethyl acetate extract of Knoxia roxburghii (Rubiaceae) down-regulates ECHDC1, CAMK2D, DDB1, UBA6, BIRC6, and HK1 proteins and ameliorates the symptoms of diabetes mellitus.","date":"2025","source":"Frontiers in pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/40766763","citation_count":0,"is_preprint":false},{"pmid":"41764162","id":"PMC_41764162","title":"Cryo-EM structures of UBA6 reveal mechanisms of E1-E2 specificity and dual FAT10/ubiquitin thioester transfer.","date":"2026","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/41764162","citation_count":0,"is_preprint":false},{"pmid":"41720079","id":"PMC_41720079","title":"Decoding E1-E2 specificity: How UBA6 prioritizes BIRC6 for ubiquitin conjugation.","date":"2026","source":"Cell chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/41720079","citation_count":0,"is_preprint":false},{"pmid":"37445840","id":"PMC_37445840","title":"Involvement in Fertilization and Expression of Gamete Ubiquitin-Activating Enzymes UBA1 and UBA6 in the Ascidian Halocynthia roretzi.","date":"2023","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/37445840","citation_count":0,"is_preprint":false},{"pmid":"34616182","id":"PMC_34616182","title":"Long Non-Coding RNA UBA6-AS1 Promotes the Malignant Properties of Glioblastoma by Competitively Binding to microRNA-760 and Enhancing Homeobox A2 Expression [Retraction].","date":"2021","source":"Cancer management and research","url":"https://pubmed.ncbi.nlm.nih.gov/34616182","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":15779,"output_tokens":3671,"usd":0.051201},"stage2":{"model":"claude-opus-4-6","input_tokens":7096,"output_tokens":3780,"usd":0.19497},"total_usd":0.246171,"stage1_batch_id":"msgbatch_01RPbVc9PDH9zQmYK5Hs6srm","stage2_batch_id":"msgbatch_01JVbVLzeKN2yVrsGp3zC8aM","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2007,\n      \"finding\": \"UBA6 (UBE1L2) is a novel ubiquitin-activating E1 enzyme that forms a covalent thioester bond with ubiquitin in an ATP-dependent manner, transfers ubiquitin to the E2 enzyme UbcH5b, and supports ubiquitylation of p53 by MDM2 and autoubiquitylation of E3 ligases HectH9 and E6-AP in vitro.\",\n      \"method\": \"In vitro polyubiquitylation assay, covalent thioester bond formation assay (reducing-condition sensitivity), recombinant protein reconstitution\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro with multiple substrates and E2/E3 partners; foundational discovery paper with 133 citations\",\n      \"pmids\": [\"17580310\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"UBA6 activates not only ubiquitin but also the ubiquitin-like modifier FAT10, and uses a distinct spectrum of E2 enzymes compared to UBE1/UBA1, establishing it as an orthogonal E1 enzyme in the ubiquitin system.\",\n      \"method\": \"Biochemical characterization, review of experimental literature\",\n      \"journal\": \"Trends in biochemical sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — synthesis of multiple experimental findings across labs, 99 citations\",\n      \"pmids\": [\"18353650\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"UBA6 undergoes a three-step activation process (adenylation, thioester formation, transfer) for both ubiquitin and FAT10; FAT10 binds UBA6 with higher affinity than ubiquitin but shows lower catalytic activity; a mechanism-based inhibitor (Compound 1) forms covalent adducts with both ubiquitin and FAT10 on UBA6.\",\n      \"method\": \"Biochemical in vitro assays (ATP-PPi exchange, E1-E2 transthiolation), pre-steady state kinetics, inhibitor mechanism studies, biophysical binding measurements\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple orthogonal biochemical and biophysical methods in a single rigorous study\",\n      \"pmids\": [\"22427669\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The UBA6-USE1 ubiquitin transfer cascade is required for mouse embryonic development independent of the canonical UBA1 pathway; neuronal loss of UBA6 causes altered hippocampal and amygdala neuron patterning, decreased dendritic spine density, and elevated levels of E3 ligase Ube3a (E6-AP) and Shank3; UBA6 and USE1 promote proteasomal turnover of Ube3a and catalyze Ube3a ubiquitylation in vitro.\",\n      \"method\": \"Conditional knockout mice, in vitro ubiquitylation assay (Ube3a ubiquitylation), immunohistochemistry, dendritic spine analysis, behavioral testing\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — genetic KO with defined cellular phenotype plus in vitro reconstitution; 47 citations\",\n      \"pmids\": [\"23499007\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"USE1 undergoes auto-FAT10ylation in cis (primarily at Lys323) via the UBA6-USE1 cascade, which accelerates its proteasomal degradation; the USE1-FAT10 conjugate retains E2 activity; auto-FAT10ylation of USE1 serves as a negative feedback mechanism limiting FAT10 conjugation.\",\n      \"method\": \"MS analysis of FAT10ylation sites, site-directed mutagenesis, thioester-linkage assays, Co-immunoprecipitation\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — MS identification of modification site, mutagenesis, and functional readout with multiple methods\",\n      \"pmids\": [\"24528925\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"LMO2 protein interacts with the C-terminal ubiquitin fold domain (UFD) of UBA6, blocking the UBA6-USE1 interaction and reducing overall cellular FAT10ylation levels, including FAT10ylation and degradation of the substrate p62.\",\n      \"method\": \"Co-immunoprecipitation, FAT10ylation assays, domain mapping\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab, Co-IP plus functional FAT10ylation assay but limited orthogonal validation\",\n      \"pmids\": [\"27569286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"UBA6 and the hybrid E2/E3 enzyme BIRC6 cooperate to monoubiquitinate LC3B, targeting it for proteasomal degradation; loss of UBA6 or BIRC6 increases autophagic flux and decreases aggresome-like induced structures and α-synuclein aggregates, demonstrating that UBA6-BIRC6 negatively regulates autophagy by limiting LC3B availability.\",\n      \"method\": \"Whole-genome CRISPR/Cas9 knockout screen, genetic epistasis (double KO), autophagic flux assays, proteasomal degradation assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genome-wide screen followed by mechanistic validation with multiple orthogonal methods; 82 citations\",\n      \"pmids\": [\"31692446\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"UBA6 and UBE1 are E1 enzymes that ubiquitinate the cardiac sodium channel Nav1.5 at K590 and K591, reducing Nav1.5 expression and sodium current density; this is mediated through the E2 enzyme UBC9.\",\n      \"method\": \"Overexpression/knockdown western blot, patch-clamp electrophysiology, site-directed mutagenesis of ubiquitination sites, E2 knockdown epistasis\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple functional readouts (biochemical + electrophysiology) with mutagenesis, single lab\",\n      \"pmids\": [\"32315024\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"UBA6 deficiency in T cells increases IFN-γ production by destabilizing IκBα degradation and thereby increasing NF-κB p65 activation; T cell-specific UBA6 knockout mice develop exacerbated multi-organ inflammation.\",\n      \"method\": \"T cell-specific conditional knockout mice, IFN-γ ELISA, NF-κB western blot, adoptive transfer experiments\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic KO with defined cellular phenotype and pathway placement, but limited mechanistic biochemistry on the direct substrate\",\n      \"pmids\": [\"35011668\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"UBA6 is required for hepcidin/BMP6-induced ubiquitination and lysosomal degradation of ferroportin; siRNA-mediated depletion of UBA6 prevents ferroportin degradation in HepG2 cells, and AAV-mediated silencing of Ndfip1 in mouse liver increases hepatic ferroportin and circulating iron.\",\n      \"method\": \"siRNA screen (88 ubiquitin pathway components), HepG2 ferroportin-GFP reporter cell line, AAV silencing in mice, western blot\",\n      \"journal\": \"Haematologica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — siRNA screen with functional readout plus in vivo validation, single lab\",\n      \"pmids\": [\"34320783\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Crystal structures of human UBA6 in complex with ubiquitin reveal two conformational states: an open conformation configured for adenylation and a closed conformation configured for thioester bond formation; inositol hexakisphosphate (InsP6) binds an allosteric site on UBA6 and inhibits its activity by altering interconversion between open and closed conformations while enhancing protein stability.\",\n      \"method\": \"X-ray crystallography, biochemical activity assays, biophysical binding measurements, mutagenesis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structures with functional biochemical and biophysical validation of allosteric mechanism\",\n      \"pmids\": [\"35986001\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Inosine directly inhibits UBA6 in tumor cells, reducing UBA6 activity and increasing tumor immunogenicity, thereby sensitizing tumors to immune checkpoint blockade; genetic ablation of Uba6 in mouse tumor cells phenocopies inosine treatment.\",\n      \"method\": \"Metabolic profiling, genetic ablation (Uba6 knockout), tumor transplant models, T cell cytotoxicity assays, biochemical inhibition assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct biochemical inhibition plus genetic KO with defined immune phenotype, single lab\",\n      \"pmids\": [\"36109526\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"UBA6 recognizes a polyalanine stretch within its cognate E2 enzyme USE1; expansion mutations in polyalanine tract proteins compete for the UBA6-USE1 interaction, reducing ubiquitin transfer to USE1 and subsequent polyubiquitination and degradation of E6AP, which in turn affects levels of synaptic protein Arc; UBA6 overexpression increases neuronal resilience to cell death in patient-derived iPSC neurons.\",\n      \"method\": \"Binding assays, ubiquitination assays, mouse primary neuron experiments, patient-derived iPSC-autonomic neuron experiments, UBA6 overexpression rescue\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mechanistic biochemistry plus patient-derived cell validation with multiple orthogonal approaches\",\n      \"pmids\": [\"38177505\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"BIRC6 is a UBA6-exclusive E2 enzyme that gains priority over all other UBA6-competent E2s through an exceptionally high-affinity interaction with the UBA6 ubiquitin fold domain (UFD), modulated by the UBA6 Cys-Cap loop; upon receiving ubiquitin via thioester transfer, BIRC6 undergoes a thioester switch mechanism that disengages it from UBA6, preventing inhibition of UBA6's activity toward other E2s.\",\n      \"method\": \"Structural capture of UBA6-BIRC6 complexes, biochemical E2 competition assays, mutagenesis, E1-E2 specificity assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — structural and biochemical reconstitution with mechanistic mutagenesis defining the priority and thioester switch mechanism\",\n      \"pmids\": [\"41350950\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Cryo-EM structures of UBA6 in complex with E2 enzymes (including BIRC6) during thioester transfer with either FAT10 or ubiquitin reveal that UBA6 achieves E2 specificity through coordinated contributions of both the UFD and SCCH domains (contrasting with the UFD-dominated selectivity of UBA1); InsP6 binding stabilizes an expanded SCCH cleft that pre-organizes UBA6 for selective engagement of UBA6-specific E2s.\",\n      \"method\": \"Chemical trapping, high-resolution cryo-EM of four UBA6-E2 complex structures, biochemical specificity assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple high-resolution cryo-EM structures with biochemical validation defining dual-domain E2 specificity mechanism\",\n      \"pmids\": [\"41764162\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"UBA6 is a dual-specificity E1 ubiquitin-activating enzyme that uses ATP-dependent adenylation and thioester bond formation to activate both ubiquitin and the ubiquitin-like modifier FAT10, transferring them to a distinct set of E2 enzymes (most prominently BIRC6 and USE1) via a mechanism regulated by conformational switching between open and closed states and allosterically modulated by inositol hexakisphosphate (InsP6); through the UBA6-BIRC6 axis it monoubiquitinates LC3B to suppress autophagy, through the UBA6-USE1 axis it promotes FAT10ylation and ubiquitin-dependent proteasomal degradation of substrates including Ube3a/E6-AP, and it also regulates ferroportin stability, Nav1.5 channel ubiquitination, NF-κB signaling in T cells, and neuronal development.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"UBA6 is a dual-specificity E1 ubiquitin-activating enzyme that activates both ubiquitin and the ubiquitin-like modifier FAT10 through ATP-dependent adenylation and thioester bond formation, operating as an orthogonal arm of the ubiquitin system with a distinct E2 enzyme repertoire [PMID:17580310, PMID:18353650, PMID:22427669]. UBA6 transfers ubiquitin to cognate E2s including USE1 and BIRC6, where BIRC6 gains priority access through an exceptionally high-affinity interaction with the UBA6 ubiquitin fold domain (UFD) and is released upon thioester loading via a thioester switch mechanism, while USE1 is recognized via a polyalanine stretch and mediates ubiquitin-dependent proteasomal degradation of substrates such as Ube3a/E6-AP [PMID:41350950, PMID:38177505, PMID:23499007]. Crystal and cryo-EM structures reveal that UBA6 cycles between open (adenylation-competent) and closed (thioester-competent) conformations, with inositol hexakisphosphate (InsP6) binding an allosteric site that stabilizes an expanded SCCH cleft to pre-organize E2 selectivity through coordinated UFD and SCCH domain contributions [PMID:35986001, PMID:41764162]. Through the UBA6–BIRC6 axis, UBA6 monoubiquitinates LC3B to suppress autophagy, and through the UBA6–USE1 axis it controls neuronal Ube3a levels, dendritic spine density, and hippocampal neuron patterning; UBA6 also regulates ferroportin degradation, NF-κB signaling in T cells, and tumor immunogenicity [PMID:31692446, PMID:23499007, PMID:34320783, PMID:35011668, PMID:36109526].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"The discovery that UBA6 forms thioester bonds with ubiquitin and transfers it to E2 enzymes established the existence of a second ubiquitin-activating E1 enzyme in mammals, overturning the assumption that UBA1 was the sole E1 for ubiquitin.\",\n      \"evidence\": \"In vitro reconstitution of thioester bond formation, ubiquitin transfer to UbcH5b, and support of p53 ubiquitylation by MDM2\",\n      \"pmids\": [\"17580310\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological substrates and E2 partners specific to UBA6 versus UBA1 were unknown\", \"Whether UBA6 activates ubiquitin-like modifiers besides ubiquitin was untested\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrating that UBA6 activates both ubiquitin and the ubiquitin-like modifier FAT10 with a distinct E2 spectrum revealed it as an orthogonal E1 pathway, raising the question of how dual specificity is achieved.\",\n      \"evidence\": \"Biochemical characterization and synthesis of experimental evidence across laboratories\",\n      \"pmids\": [\"18353650\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinetic parameters for ubiquitin versus FAT10 activation were not yet quantified\", \"Structural basis for dual modifier recognition was unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Kinetic dissection of the three-step activation mechanism showed that FAT10 binds UBA6 with higher affinity than ubiquitin but is activated with lower catalytic efficiency, explaining how a single E1 balances two modifier pathways.\",\n      \"evidence\": \"Pre-steady state kinetics, ATP-PPi exchange, and mechanism-based inhibitor trapping\",\n      \"pmids\": [\"22427669\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How UBA6 structurally discriminates between ubiquitin and FAT10 was unresolved\", \"Cellular regulation of the ubiquitin/FAT10 balance by UBA6 was not addressed\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Genetic ablation of UBA6 in mice demonstrated that the UBA6–USE1 cascade is essential for embryonic development and neuronal patterning, identifying E3 ligase Ube3a as a physiological substrate whose proteasomal degradation depends on this pathway.\",\n      \"evidence\": \"Conditional knockout mice with hippocampal neuron analysis, dendritic spine quantification, and in vitro reconstitution of Ube3a ubiquitylation\",\n      \"pmids\": [\"23499007\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Ube3a is a direct or indirect substrate of UBA6-USE1 in vivo required further clarification\", \"Downstream synaptic consequences beyond Shank3 elevation were not mapped\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"The finding that USE1 auto-FAT10ylates at K323, accelerating its own proteasomal degradation, revealed a negative feedback loop that limits cellular FAT10 conjugation capacity.\",\n      \"evidence\": \"Mass spectrometry identification of FAT10ylation site, site-directed mutagenesis, and thioester-linkage assays\",\n      \"pmids\": [\"24528925\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this feedback operates under physiological FAT10-inducing conditions (e.g., cytokine stimulation) was not tested in vivo\", \"Other regulatory post-translational modifications of USE1 were unexplored\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identification of LMO2 as an interactor that blocks the UBA6 UFD–USE1 interaction and reduces cellular FAT10ylation provided the first evidence of an endogenous protein-level regulatory mechanism for UBA6 substrate selection.\",\n      \"evidence\": \"Co-immunoprecipitation, domain mapping, and FAT10ylation assays including p62 as substrate\",\n      \"pmids\": [\"27569286\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"LMO2–UBA6 interaction was demonstrated by Co-IP without reciprocal structural or biophysical validation\", \"Physiological contexts in which LMO2 levels regulate FAT10ylation were not established\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"A genome-wide CRISPR screen identified UBA6 and BIRC6 as negative regulators of autophagy, establishing that UBA6-BIRC6-mediated monoubiquitination targets LC3B for proteasomal degradation and limits autophagic flux.\",\n      \"evidence\": \"Genome-wide CRISPR/Cas9 knockout screen, genetic epistasis with double knockouts, autophagic flux and α-synuclein aggregation assays\",\n      \"pmids\": [\"31692446\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether UBA6-BIRC6 directly monoubiquitinates LC3B or acts through intermediates was not structurally resolved\", \"The ubiquitin attachment site(s) on LC3B were not mapped\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrating that UBA6 ubiquitinates Nav1.5 at K590/K591 to reduce sodium channel expression and current density extended UBA6 function to cardiac electrophysiology and ion channel regulation.\",\n      \"evidence\": \"Overexpression/knockdown, patch-clamp electrophysiology, and site-directed mutagenesis of ubiquitination sites\",\n      \"pmids\": [\"32315024\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The contribution of UBA6 versus UBA1 to Nav1.5 regulation in cardiomyocytes was not cleanly separated\", \"The claimed E2 UBC9 is canonically a SUMO E2, raising questions about the conjugation specificity\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"T cell-specific UBA6 knockout revealed that UBA6 restrains NF-κB activation by stabilizing IκBα, placing UBA6 in immune homeostasis and explaining the multi-organ inflammation observed upon its loss.\",\n      \"evidence\": \"T cell-conditional knockout mice, IκBα and NF-κB western blots, IFN-γ ELISA, adoptive transfer experiments\",\n      \"pmids\": [\"35011668\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether UBA6 directly ubiquitinates IκBα or acts through an intermediate E3 was not determined\", \"The specific E2 mediating this UBA6-dependent pathway in T cells was not identified\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Crystal structures of UBA6–ubiquitin complexes in open and closed states, combined with identification of InsP6 as an allosteric inhibitor, provided the first structural framework for the conformational switch governing UBA6 catalysis.\",\n      \"evidence\": \"X-ray crystallography, mutagenesis, biochemical activity and biophysical binding assays\",\n      \"pmids\": [\"35986001\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How InsP6 concentrations are regulated in cells to control UBA6 activity was unknown\", \"Structures with FAT10 rather than ubiquitin were not obtained\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identification of UBA6 as a target of the metabolite inosine in tumor cells linked UBA6 activity to tumor immunogenicity and immune checkpoint sensitivity, providing a metabolic axis of UBA6 regulation.\",\n      \"evidence\": \"Metabolic profiling, Uba6 genetic ablation in mouse tumor models, T cell cytotoxicity assays, biochemical inhibition\",\n      \"pmids\": [\"36109526\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The precise mechanism by which inosine inhibits UBA6 (competitive, allosteric) was not structurally defined\", \"Whether inosine affects FAT10 activation by UBA6 was not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Discovery that UBA6 recognizes USE1 via a polyalanine stretch, and that polyalanine expansion mutations in disease proteins competitively disrupt this interaction, mechanistically linked UBA6-USE1 dysfunction to polyalanine expansion diseases by showing impaired Ube3a degradation and reduced neuronal viability.\",\n      \"evidence\": \"Binding and ubiquitination assays, mouse primary neurons, patient-derived iPSC-autonomic neurons, UBA6 overexpression rescue\",\n      \"pmids\": [\"38177505\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether therapeutic UBA6 overexpression can rescue disease phenotypes in vivo remains untested\", \"The full repertoire of polyalanine-containing UBA6 interactors was not mapped\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Structural and biochemical characterization of the UBA6–BIRC6 complex revealed that BIRC6 achieves priority over other UBA6 E2s through exceptionally high UFD affinity modulated by the Cys-Cap loop, and disengages via a thioester switch upon ubiquitin loading, preventing inhibition of UBA6 activity toward other E2s.\",\n      \"evidence\": \"Structural capture of UBA6–BIRC6 complexes, E2 competition assays, mutagenesis of Cys-Cap loop\",\n      \"pmids\": [\"41350950\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the thioester switch is regulated in cells and whether it applies to FAT10 transfer was not addressed\", \"Structures of UBA6 with E2s other than BIRC6 in this study were limited\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Cryo-EM structures of UBA6 with multiple E2 partners during thioester transfer resolved that UBA6 achieves E2 selectivity through coordinated contributions of both UFD and SCCH domains — a dual-domain mechanism distinct from UBA1's UFD-dominated selectivity — and that InsP6 binding stabilizes an expanded SCCH cleft to pre-organize this selectivity.\",\n      \"evidence\": \"Chemical trapping, four high-resolution cryo-EM structures (including BIRC6 and other E2s), biochemical specificity assays\",\n      \"pmids\": [\"41764162\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How InsP6-mediated allosteric regulation integrates with cellular signaling pathways is not established\", \"Structural basis for FAT10 versus ubiquitin discrimination during E2 transfer remains unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis for UBA6 discrimination between ubiquitin and FAT10, the complete cellular substrate landscape of UBA6-specific ubiquitination, and whether the InsP6 allosteric mechanism is physiologically regulated to tune UBA6 pathway output.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure of UBA6 bound to FAT10 exists\", \"Comprehensive proteomics of UBA6-specific versus UBA1-specific substrates has not been performed\", \"In vivo regulation of UBA6 by InsP6 or inosine has not been validated in physiological models\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0, 2, 10]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 3, 6, 12]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 2, 10]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 2, 3, 4, 6, 12]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [8, 11]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [12]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"USE1\",\n      \"BIRC6\",\n      \"FAT10\",\n      \"UBE3A\",\n      \"LMO2\",\n      \"LC3B\",\n      \"UbcH5b\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}