{"gene":"UBA6","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2007,"finding":"UBE1L2 (UBA6) is a second human ubiquitin-activating E1 enzyme that forms a covalent thioester with ubiquitin in vitro and in vivo in an ATP-dependent manner, and can transfer ubiquitin to the E2 enzyme UbcH5b, supporting subsequent ubiquitylation of p53 by MDM2 and autoubiquitylation of E3 ligases HectH9 and E6-AP.","method":"In vitro ubiquitylation assay, in vivo thioester formation, in vitro polyubiquitylation with recombinant proteins","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with recombinant proteins demonstrating direct enzymatic activity; multiple orthogonal assays (thioester, ATP exchange, E2 transfer, E3-dependent ubiquitylation)","pmids":["17580310"],"is_preprint":false},{"year":2008,"finding":"UBA6 activates not only ubiquitin but also the ubiquitin-like modifier FAT10, and uses a different spectrum of E2 conjugating enzymes compared to UBE1, establishing UBA6 as a bispecific E1 enzyme with its own dedicated E2 partner USE1.","method":"Biochemical characterization, E1-E2 specificity assays","journal":"Trends in biochemical sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — review summarizing experimental findings from multiple labs; bispecificity and E2 spectrum established by biochemical assays cited therein","pmids":["18353650"],"is_preprint":false},{"year":2012,"finding":"Uba6 undergoes a three-step activation process (adenylation, thioester formation, transthiolation) for both ubiquitin and FAT10, forming a ternary complex with both substrates. FAT10 binds Uba6 with higher affinity than ubiquitin but demonstrates lower catalytic activity in ATP-PPi exchange and E1-E2 transthiolation assays. A mechanism-based inhibitor (Compound 1) forms covalent adducts with both ubiquitin and FAT10 on Uba6.","method":"Biochemical assays, pre-steady state kinetics, mechanism-based inhibitor, biophysical binding measurements","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution, kinetic analysis, multiple orthogonal methods (kinetics, inhibitor trapping, binding measurements) 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 independently of the canonical Uba1 pathway. In neuronal Uba6-knockout mice, loss of Uba6 results in abnormal hippocampal and amygdala neuron patterning, decreased dendritic spine density, and elevated levels of Ube3a (E6-AP) and Shank3 in the amygdala. Uba6 and Use1 promote proteasomal turnover of Ube3a in MEFs and catalyze Ube3a ubiquitylation in vitro, acting in a spatially distinct manner from the Uba1–UbcH7 pathway.","method":"Neuronal-specific Uba6 knockout mouse, in vitro ubiquitylation assay, MEF proteasomal turnover assay, immunofluorescence, behavioral studies","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with defined cellular and molecular phenotype plus in vitro reconstitution of Ube3a ubiquitylation; multiple orthogonal methods replicated across in vivo and in vitro systems","pmids":["23499007"],"is_preprint":false},{"year":2014,"finding":"USE1 (the dedicated E2 of UBA6) undergoes self-FAT10ylation in cis, primarily at Lys323, which accelerates its proteasomal degradation and thereby limits overall FAT10ylation levels as a negative feedback mechanism. The USE1-FAT10 conjugate retains catalytic activity and can still form thioesters with both FAT10 and ubiquitin.","method":"Mass spectrometry, site-directed mutagenesis, co-immunoprecipitation, proteasome inhibitor assays, in vitro thioester assay","journal":"The FEBS journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS identification of modification site, mutagenesis, functional thioester assay; single lab but multiple orthogonal methods","pmids":["24528925"],"is_preprint":false},{"year":2016,"finding":"LMO2 interacts with the C-terminal ubiquitin fold domain (UFD) of UBA6, which is the domain that recruits the E2 USE1. This interaction blocks UBA6–USE1 binding and reduces overall cellular FAT10ylation levels, including FAT10ylation and degradation of the substrate p62.","method":"Co-immunoprecipitation, domain mapping, FAT10ylation assays, pulldown","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP and functional FAT10ylation assays showing mechanistic outcome; single lab, multiple methods","pmids":["27569286"],"is_preprint":false},{"year":2017,"finding":"UBA6 deficiency in MCF-10A mammary epithelial cells leads to failure in cell cycle arrest upon matrix detachment and spontaneous epithelial-mesenchymal transition (EMT). The Rho-GTPase CDC42 is identified as a specific target of UBA6-initiated ubiquitination, and a CDC42 inhibitor rescues UBA6-deficient cells from the EMT phenotype.","method":"shRNA knockdown, EMT assays, pharmacological inhibition of CDC42, cell biology assays","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined cellular phenotype and pathway placement via rescue experiment; single lab","pmids":["29152096"],"is_preprint":false},{"year":2019,"finding":"UBA6 and BIRC6 cooperate to monoubiquitinate LC3B, targeting it for proteasomal degradation. Knockout of UBA6 or BIRC6 increases autophagic flux under nutrient deprivation or protein synthesis inhibition, and decreases aggresome-like induced structures and α-synuclein aggregates, demonstrating that the UBA6–BIRC6 axis negatively regulates autophagy by limiting LC3B availability.","method":"Whole-genome CRISPR/Cas9 knockout screen, fluorescent LC3B reporter, KO cell lines, autophagic flux assays, neuronal aggregate assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide unbiased screen plus mechanistic validation with KO cells, defined molecular substrate (LC3B monoubiquitination), and multiple cellular readouts including neuronal models","pmids":["31692446"],"is_preprint":false},{"year":2020,"finding":"UBA6 (together with UBE1) regulates ubiquitination and expression of the cardiac sodium channel Nav1.5 at lysine residues K590 and K591, acting through the E2 enzyme UBC9. Overexpression of UBA6 increases Nav1.5 ubiquitination and decreases Nav1.5 expression and sodium current density, while knockdown has the opposite effect.","method":"Western blot, patch-clamp electrophysiology, overexpression/knockdown, site-directed mutagenesis (K590A/K591A), neonatal cardiomyocytes","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function and gain-of-function with electrophysiological readout, mutagenesis confirming specific ubiquitination sites; single lab","pmids":["32315024"],"is_preprint":false},{"year":2021,"finding":"UBA6 regulates IFN-γ production in T cells by promoting IκBα degradation, thereby increasing NF-κB p65 activation. T cell-specific UBA6-deficient mice show elevated IFN-γ production from CD4 and CD8 T cells, and transfer of UBA6-deficient T cells exacerbates multi-organ inflammation.","method":"T cell-specific conditional knockout mice, cytokine production assays, NF-κB pathway analysis, adoptive transfer experiments","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with defined cellular and molecular phenotype; pathway placement via IκBα/NF-κB analysis; single lab","pmids":["35011668"],"is_preprint":false},{"year":2022,"finding":"UBA6 and the adaptor protein NDFIP1 are required for hepcidin-induced ubiquitination and degradation of ferroportin. siRNA-mediated depletion of UBA6 prevents BMP6- and hepcidin-induced ferroportin degradation in vitro, and AAV-mediated silencing of Ndfip1 in mouse liver increases hepatic ferroportin and circulating iron.","method":"siRNA screen (88 ubiquitin pathway components), FPN-GFP reporter cell line, in vivo AAV silencing in mice, iron measurements","journal":"Haematologica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic siRNA screen with in vivo validation; single lab, orthogonal in vitro and in vivo methods","pmids":["34320783"],"is_preprint":false},{"year":2022,"finding":"Crystal structures of human Uba6/ubiquitin complex reveal two conformational states: an open conformation configured for adenylation and a closed conformation for thioester bond formation. An inositol hexakisphosphate (InsP6) molecule binds to a previously unidentified allosteric site on Uba6 and inhibits Uba6 activity by altering interconversion between the open and closed conformations while enhancing enzyme stability.","method":"X-ray crystallography, biochemical activity assays, biophysical binding measurements","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures with functional validation by biochemical and biophysical assays; multiple orthogonal methods in a single rigorous study revealing conformational mechanism and allosteric regulation","pmids":["35986001"],"is_preprint":false},{"year":2024,"finding":"UBA6 recognizes a polyalanine stretch within its cognate E2 USE1. Polyalanine expansion mutations in USE1 reduce ubiquitin transfer from UBA6 to USE1 and decrease polyubiquitination and degradation of Ube3a (E6-AP). Proteins with polyalanine expansion mutations in disease states compete for UBA6–USE1 interaction, altering E6AP levels and Arc synaptic protein levels in neurons.","method":"In vitro ubiquitin transfer assays, iPSC-derived neurons from patients, mouse primary neurons, UBA6 overexpression rescue experiments","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro mechanistic assays combined with patient iPSC-derived neurons and mouse primary neurons; multiple orthogonal methods including rescue by UBA6 overexpression","pmids":["38177505"],"is_preprint":false},{"year":2025,"finding":"BIRC6 is a UBA6-exclusive E2 enzyme that gains priority over all other UBA6-competent E2s by engaging the UBA6 ubiquitin fold domain (UFD) with exceptionally high affinity, modulated by the UBA6 Cys-Cap loop. A bespoke thioester switch mechanism disengages BIRC6 upon receiving ubiquitin, preventing inhibition of UBA6 despite BIRC6's priority. UBA6 achieves broader E2 specificity through coordinated contributions of both UFD and SCCH domains.","method":"Structural biology (cryo-EM capturing UBA6-BIRC6 complexes), biochemical E1-E2 specificity assays, mutagenesis","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structures of trapped complexes combined with biochemical specificity assays and mutagenesis; rigorous mechanistic study","pmids":["41350950"],"is_preprint":false},{"year":2026,"finding":"Cryo-EM structures of UBA6-E2 complexes at the thioester-transfer step with either FAT10 or ubiquitin reveal that UBA6 achieves E2 specificity through coordinated contributions of both the UFD and SCCH domains (contrasting with UFD-dominated selectivity of UBA1). An InsP6-binding site unique to UBA6 stabilizes an expanded SCCH cleft that pre-organizes the enzyme for selective engagement of UBA6-specific E2s, identifying InsP6 as a cofactor shaping specificity.","method":"Chemical trapping, cryo-EM (four UBA6-E2 complex structures), biochemical assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM structures of multiple trapped complexes with biochemical validation; multiple orthogonal methods revealing dual-domain E2 selectivity mechanism","pmids":["41764162"],"is_preprint":false},{"year":2025,"finding":"In UBA1M41V (VEXAS syndrome) human cells, UBA6 compensates for UBA1 dysfunction, creating an acquired dependency on UBA6. Genetic (shRNA) or pharmacological (phytic acid/InsP6) inhibition of UBA6 preferentially impairs growth and colony formation of UBA1M41V cells while sparing wild-type cells.","method":"Engineered human cell model (THP1 UBA1M41V), shRNA knockdown, pharmacological inhibition with phytic acid, competition assays, proteomic analysis","journal":"Leukemia","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional studies in engineered human cell model with genetic and pharmacological orthogonal approaches; single lab","pmids":["40588566"],"is_preprint":false}],"current_model":"UBA6 is a bispecific E1 ubiquitin-activating enzyme that ATP-dependently activates both ubiquitin and the ubiquitin-like modifier FAT10 through a three-step catalytic cycle (adenylation → thioester formation → transthiolation), working through its dedicated E2 enzyme USE1 and also the high-priority E2 BIRC6; structurally, UBA6 undergoes open/closed conformational transitions to catalyze these steps, is allosterically regulated by inositol hexakisphosphate (InsP6) at a unique allosteric site, achieves E2 selectivity through coordinated UFD and SCCH domain interactions, and physiologically controls diverse processes including autophagy (via LC3B monoubiquitination with BIRC6), neuronal development and dendritic spine maintenance (via Ube3a ubiquitylation), T cell IFN-γ production (via IκBα/NF-κB), iron homeostasis (via ferroportin degradation with NDFIP1), and epithelial homeostasis (via CDC42 ubiquitylation)."},"narrative":{"mechanistic_narrative":"UBA6 is a bispecific E1 activating enzyme that initiates protein modification by ATP-dependently charging both ubiquitin and the ubiquitin-like modifier FAT10, and channels these modifiers into downstream conjugation distinct from the canonical UBA1 pathway [PMID:17580310, PMID:18353650]. It activates each substrate through a three-step cycle of adenylation, thioester formation, and transthiolation, forming a ternary complex in which FAT10 binds more tightly than ubiquitin yet is transferred less efficiently [PMID:22427669]; crystal and cryo-EM structures resolve open (adenylation) and closed (thioester) conformational states and reveal that UBA6 attains its broader E2 selectivity through coordinated contributions of both its UFD and SCCH domains, in contrast to the UFD-dominated selectivity of UBA1 [PMID:35986001, PMID:41764162]. A distinctive allosteric site binds inositol hexakisphosphate (InsP6), which shapes E2 specificity and modulates open/closed interconversion and enzyme stability [PMID:35986001, PMID:41764162]. UBA6 works through its dedicated E2 USE1—whose recognition depends on a polyalanine stretch—and through the UBA6-exclusive, high-priority E2 BIRC6, which engages the UFD with exceptional affinity governed by a Cys-Cap loop and a thioester switch that disengages BIRC6 upon ubiquitin loading [PMID:38177505, PMID:41350950]. Physiologically, the UBA6–USE1 cascade is required for embryonic development and neuronal patterning, controlling dendritic spine density via Ube3a (E6-AP) turnover [PMID:23499007, PMID:38177505], while UBA6–BIRC6 monoubiquitinates LC3B to negatively regulate autophagy [PMID:31692446]. Additional UBA6-initiated ubiquitination events govern epithelial homeostasis through CDC42 [PMID:29152096], T cell IFN-γ production via IκBα/NF-κB [PMID:35011668], hepcidin-driven ferroportin degradation with the adaptor NDFIP1 [PMID:34320783], and cardiac Nav1.5 channel abundance [PMID:32315024]. In VEXAS-associated UBA1-mutant cells, UBA6 compensates for UBA1 dysfunction, creating an acquired, InsP6-targetable dependency [PMID:40588566].","teleology":[{"year":2007,"claim":"Established that ubiquitin activation is not the exclusive province of UBA1 by identifying UBA6 as a second human E1 that charges ubiquitin and feeds E3-dependent ubiquitylation.","evidence":"In vitro reconstitution with recombinant proteins, thioester and ATP-exchange assays, E2 transfer to UbcH5b supporting MDM2/HectH9/E6-AP activity","pmids":["17580310"],"confidence":"High","gaps":["Did not establish FAT10 as a substrate","Did not define a dedicated E2 partner distinct from UbcH5b","No in vivo physiological role"]},{"year":2008,"claim":"Defined UBA6 as bispecific by showing it activates FAT10 in addition to ubiquitin and uses a different E2 spectrum, including the dedicated E2 USE1.","evidence":"Biochemical characterization and E1-E2 specificity assays (review synthesizing multiple labs)","pmids":["18353650"],"confidence":"Medium","gaps":["Structural basis of bispecificity unresolved","Kinetic discrimination between ubiquitin and FAT10 not quantified"]},{"year":2012,"claim":"Resolved the catalytic logic by demonstrating a three-step activation cycle for both modifiers and quantifying that FAT10 binds tighter but turns over slower than ubiquitin.","evidence":"Pre-steady-state kinetics, biophysical binding measurements, mechanism-based covalent inhibitor trapping","pmids":["22427669"],"confidence":"High","gaps":["No structural view of the conformational steps","Allosteric regulation not yet identified"]},{"year":2013,"claim":"Demonstrated a non-redundant in vivo role: the UBA6–USE1 cascade drives embryonic and neuronal development and controls Ube3a turnover, spatially distinct from the UBA1–UbcH7 pathway.","evidence":"Neuronal-specific Uba6 knockout mice, in vitro Ube3a ubiquitylation, MEF turnover assays, immunofluorescence and behavior","pmids":["23499007"],"confidence":"High","gaps":["Whether ubiquitin or FAT10 mediates the neuronal phenotype not separated","Direct E3 ligases for Ube3a turnover not defined"]},{"year":2014,"claim":"Identified a negative-feedback mechanism in which the dedicated E2 USE1 self-FAT10ylates to drive its own degradation and limit global FAT10ylation.","evidence":"Mass spectrometry of Lys323 site, mutagenesis, co-IP, proteasome inhibition, in vitro thioester assays","pmids":["24528925"],"confidence":"Medium","gaps":["Physiological consequences of feedback in vivo unknown","Single lab"]},{"year":2016,"claim":"Showed that the UBA6 UFD is the E2-recruitment hub and a competitive target, since LMO2 binding to the UFD blocks USE1 engagement and dampens FAT10ylation.","evidence":"Co-IP, domain mapping, pulldown, FAT10ylation assays on substrate p62","pmids":["27569286"],"confidence":"Medium","gaps":["Structural details of LMO2–UFD contact unresolved","Physiological context of LMO2 regulation unknown"]},{"year":2017,"claim":"Linked UBA6 to epithelial homeostasis by identifying CDC42 as a UBA6-initiated ubiquitination target whose dysregulation drives EMT.","evidence":"shRNA knockdown in MCF-10A cells, EMT assays, CDC42 inhibitor rescue","pmids":["29152096"],"confidence":"Medium","gaps":["E2/E3 partners for CDC42 ubiquitination not defined","Whether direct or indirect ubiquitination unclear"]},{"year":2019,"claim":"Defined the UBA6–BIRC6 axis as a negative regulator of autophagy by monoubiquitinating LC3B to limit its availability.","evidence":"Genome-wide CRISPR screen, LC3B fluorescent reporter, KO cell lines, autophagic flux and neuronal aggregate assays","pmids":["31692446"],"confidence":"High","gaps":["Structural basis of BIRC6 partnership not yet resolved","Whether FAT10 contributes unclear"]},{"year":2020,"claim":"Extended UBA6 substrate scope to ion-channel control, regulating cardiac Nav1.5 abundance and sodium current via ubiquitination at K590/K591.","evidence":"Overexpression/knockdown, patch-clamp, K590A/K591A mutagenesis in neonatal cardiomyocytes","pmids":["32315024"],"confidence":"Medium","gaps":["Direct vs UBC9-bridged mechanism not fully separated from UBE1 contribution","In vivo cardiac relevance untested"]},{"year":2021,"claim":"Placed UBA6 in immune regulation, showing it promotes IκBα degradation to enable NF-κB activation and restrain T cell IFN-γ production.","evidence":"T cell-specific conditional knockout mice, cytokine and NF-κB analysis, adoptive transfer","pmids":["35011668"],"confidence":"Medium","gaps":["Direct ubiquitination of IκBα by UBA6 cascade not biochemically reconstituted","E2/E3 partners unspecified"]},{"year":2022,"claim":"Connected UBA6 to systemic iron control by showing UBA6 and the adaptor NDFIP1 are required for hepcidin-induced ferroportin degradation.","evidence":"siRNA screen of ubiquitin pathway, FPN-GFP reporter cells, in vivo AAV Ndfip1 silencing with iron measurements","pmids":["34320783"],"confidence":"Medium","gaps":["Direct ferroportin ubiquitination by UBA6 cascade not shown in vitro","E3 ligase identity unconfirmed"]},{"year":2022,"claim":"Provided the first structural mechanism, capturing open (adenylation) and closed (thioester) states and discovering an InsP6 allosteric site that inhibits UBA6 and stabilizes it.","evidence":"X-ray crystallography of Uba6/ubiquitin complex with biochemical and biophysical validation","pmids":["35986001"],"confidence":"High","gaps":["E2-bound transthiolation state not yet captured","Physiological source/regulation of InsP6 occupancy unknown"]},{"year":2024,"claim":"Defined the molecular code for USE1 recognition—a polyalanine stretch—and showed that polyalanine-expansion proteins competitively disrupt UBA6–USE1, altering E6-AP and Arc levels in neurons.","evidence":"In vitro ubiquitin transfer, patient iPSC-derived and mouse primary neurons, UBA6 overexpression rescue","pmids":["38177505"],"confidence":"High","gaps":["Structural basis of polyalanine recognition not resolved here","Breadth of competing polyalanine proteins not enumerated"]},{"year":2025,"claim":"Explained E2 prioritization by showing BIRC6 is UBA6-exclusive, binds the UFD with exceptional affinity gated by a Cys-Cap loop, and uses a thioester switch to disengage and avoid inhibiting UBA6.","evidence":"Cryo-EM of trapped UBA6–BIRC6 complexes, E1-E2 specificity assays, mutagenesis","pmids":["41350950"],"confidence":"High","gaps":["Dynamics of switching under physiological E2 competition not quantified","Cellular consequences of altering the switch untested"]},{"year":2026,"claim":"Completed the specificity model by capturing thioester-transfer complexes with FAT10 or ubiquitin, showing dual UFD+SCCH domain control and that InsP6 pre-organizes an expanded SCCH cleft as a specificity cofactor.","evidence":"Chemical trapping and cryo-EM of four UBA6-E2 complexes with biochemical validation","pmids":["41764162"],"confidence":"High","gaps":["Whether InsP6 occupancy is dynamically regulated in cells unresolved","Quantitative discrimination of all E2s not fully mapped"]},{"year":2025,"claim":"Revealed a disease-relevant compensation: in UBA1-mutant VEXAS cells UBA6 substitutes for UBA1, creating an acquired, InsP6-targetable dependency.","evidence":"Engineered THP1 UBA1M41V cells, shRNA, phytic acid (InsP6) inhibition, competition and proteomic assays","pmids":["40588566"],"confidence":"Medium","gaps":["Substrate set rerouted to UBA6 not defined","In vivo therapeutic window unestablished"]},{"year":null,"claim":"How UBA6 partitions its many physiological outputs between ubiquitin and FAT10 conjugation, and how InsP6 occupancy is regulated in vivo to tune E2 selectivity, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No systematic mapping of which substrates require FAT10 vs ubiquitin","Endogenous control of InsP6-dependent specificity unknown","E3 ligases for most UBA6-dependent substrates unidentified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0,2,11]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,3,7]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,1]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[11,14]}],"localization":[],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,2,3]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[7]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[9]}],"complexes":[],"partners":["USE1","BIRC6","FAT10","NDFIP1","LMO2"],"other_free_text":[]}},"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":134,"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":101,"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":85,"is_preprint":false},{"pmid":"36109526","id":"PMC_36109526","title":"Inhibition of UBA6 by inosine augments tumour immunogenicity and responses.","date":"2022","source":"Nature 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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 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[Retraction].","date":"2021","source":"Cancer management and research","url":"https://pubmed.ncbi.nlm.nih.gov/34616182","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}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16543,"output_tokens":4393,"usd":0.057762,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12444,"output_tokens":4901,"usd":0.092372,"stage2_stop_reason":"end_turn"},"total_usd":0.150134,"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\": 2007,\n      \"finding\": \"UBE1L2 (UBA6) is a second human ubiquitin-activating E1 enzyme that forms a covalent thioester with ubiquitin in vitro and in vivo in an ATP-dependent manner, and can transfer ubiquitin to the E2 enzyme UbcH5b, supporting subsequent ubiquitylation of p53 by MDM2 and autoubiquitylation of E3 ligases HectH9 and E6-AP.\",\n      \"method\": \"In vitro ubiquitylation assay, in vivo thioester formation, in vitro polyubiquitylation with recombinant proteins\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with recombinant proteins demonstrating direct enzymatic activity; multiple orthogonal assays (thioester, ATP exchange, E2 transfer, E3-dependent ubiquitylation)\",\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 different spectrum of E2 conjugating enzymes compared to UBE1, establishing UBA6 as a bispecific E1 enzyme with its own dedicated E2 partner USE1.\",\n      \"method\": \"Biochemical characterization, E1-E2 specificity assays\",\n      \"journal\": \"Trends in biochemical sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — review summarizing experimental findings from multiple labs; bispecificity and E2 spectrum established by biochemical assays cited therein\",\n      \"pmids\": [\"18353650\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Uba6 undergoes a three-step activation process (adenylation, thioester formation, transthiolation) for both ubiquitin and FAT10, forming a ternary complex with both substrates. FAT10 binds Uba6 with higher affinity than ubiquitin but demonstrates lower catalytic activity in ATP-PPi exchange and E1-E2 transthiolation assays. A mechanism-based inhibitor (Compound 1) forms covalent adducts with both ubiquitin and FAT10 on Uba6.\",\n      \"method\": \"Biochemical assays, pre-steady state kinetics, mechanism-based inhibitor, biophysical binding measurements\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution, kinetic analysis, multiple orthogonal methods (kinetics, inhibitor trapping, binding measurements) 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 independently of the canonical Uba1 pathway. In neuronal Uba6-knockout mice, loss of Uba6 results in abnormal hippocampal and amygdala neuron patterning, decreased dendritic spine density, and elevated levels of Ube3a (E6-AP) and Shank3 in the amygdala. Uba6 and Use1 promote proteasomal turnover of Ube3a in MEFs and catalyze Ube3a ubiquitylation in vitro, acting in a spatially distinct manner from the Uba1–UbcH7 pathway.\",\n      \"method\": \"Neuronal-specific Uba6 knockout mouse, in vitro ubiquitylation assay, MEF proteasomal turnover assay, immunofluorescence, behavioral studies\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with defined cellular and molecular phenotype plus in vitro reconstitution of Ube3a ubiquitylation; multiple orthogonal methods replicated across in vivo and in vitro systems\",\n      \"pmids\": [\"23499007\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"USE1 (the dedicated E2 of UBA6) undergoes self-FAT10ylation in cis, primarily at Lys323, which accelerates its proteasomal degradation and thereby limits overall FAT10ylation levels as a negative feedback mechanism. The USE1-FAT10 conjugate retains catalytic activity and can still form thioesters with both FAT10 and ubiquitin.\",\n      \"method\": \"Mass spectrometry, site-directed mutagenesis, co-immunoprecipitation, proteasome inhibitor assays, in vitro thioester assay\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS identification of modification site, mutagenesis, functional thioester assay; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"24528925\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"LMO2 interacts with the C-terminal ubiquitin fold domain (UFD) of UBA6, which is the domain that recruits the E2 USE1. This interaction blocks UBA6–USE1 binding and reduces overall cellular FAT10ylation levels, including FAT10ylation and degradation of the substrate p62.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping, FAT10ylation assays, pulldown\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP and functional FAT10ylation assays showing mechanistic outcome; single lab, multiple methods\",\n      \"pmids\": [\"27569286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"UBA6 deficiency in MCF-10A mammary epithelial cells leads to failure in cell cycle arrest upon matrix detachment and spontaneous epithelial-mesenchymal transition (EMT). The Rho-GTPase CDC42 is identified as a specific target of UBA6-initiated ubiquitination, and a CDC42 inhibitor rescues UBA6-deficient cells from the EMT phenotype.\",\n      \"method\": \"shRNA knockdown, EMT assays, pharmacological inhibition of CDC42, cell biology assays\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined cellular phenotype and pathway placement via rescue experiment; single lab\",\n      \"pmids\": [\"29152096\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"UBA6 and BIRC6 cooperate to monoubiquitinate LC3B, targeting it for proteasomal degradation. Knockout of UBA6 or BIRC6 increases autophagic flux under nutrient deprivation or protein synthesis inhibition, and decreases aggresome-like induced structures and α-synuclein aggregates, demonstrating that the UBA6–BIRC6 axis negatively regulates autophagy by limiting LC3B availability.\",\n      \"method\": \"Whole-genome CRISPR/Cas9 knockout screen, fluorescent LC3B reporter, KO cell lines, autophagic flux assays, neuronal aggregate assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide unbiased screen plus mechanistic validation with KO cells, defined molecular substrate (LC3B monoubiquitination), and multiple cellular readouts including neuronal models\",\n      \"pmids\": [\"31692446\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"UBA6 (together with UBE1) regulates ubiquitination and expression of the cardiac sodium channel Nav1.5 at lysine residues K590 and K591, acting through the E2 enzyme UBC9. Overexpression of UBA6 increases Nav1.5 ubiquitination and decreases Nav1.5 expression and sodium current density, while knockdown has the opposite effect.\",\n      \"method\": \"Western blot, patch-clamp electrophysiology, overexpression/knockdown, site-directed mutagenesis (K590A/K591A), neonatal cardiomyocytes\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function and gain-of-function with electrophysiological readout, mutagenesis confirming specific ubiquitination sites; single lab\",\n      \"pmids\": [\"32315024\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"UBA6 regulates IFN-γ production in T cells by promoting IκBα degradation, thereby increasing NF-κB p65 activation. T cell-specific UBA6-deficient mice show elevated IFN-γ production from CD4 and CD8 T cells, and transfer of UBA6-deficient T cells exacerbates multi-organ inflammation.\",\n      \"method\": \"T cell-specific conditional knockout mice, cytokine production assays, NF-κB pathway analysis, adoptive transfer experiments\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with defined cellular and molecular phenotype; pathway placement via IκBα/NF-κB analysis; single lab\",\n      \"pmids\": [\"35011668\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"UBA6 and the adaptor protein NDFIP1 are required for hepcidin-induced ubiquitination and degradation of ferroportin. siRNA-mediated depletion of UBA6 prevents BMP6- and hepcidin-induced ferroportin degradation in vitro, and AAV-mediated silencing of Ndfip1 in mouse liver increases hepatic ferroportin and circulating iron.\",\n      \"method\": \"siRNA screen (88 ubiquitin pathway components), FPN-GFP reporter cell line, in vivo AAV silencing in mice, iron measurements\",\n      \"journal\": \"Haematologica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic siRNA screen with in vivo validation; single lab, orthogonal in vitro and in vivo methods\",\n      \"pmids\": [\"34320783\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Crystal structures of human Uba6/ubiquitin complex reveal two conformational states: an open conformation configured for adenylation and a closed conformation for thioester bond formation. An inositol hexakisphosphate (InsP6) molecule binds to a previously unidentified allosteric site on Uba6 and inhibits Uba6 activity by altering interconversion between the open and closed conformations while enhancing enzyme stability.\",\n      \"method\": \"X-ray crystallography, biochemical activity assays, biophysical binding measurements\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures with functional validation by biochemical and biophysical assays; multiple orthogonal methods in a single rigorous study revealing conformational mechanism and allosteric regulation\",\n      \"pmids\": [\"35986001\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"UBA6 recognizes a polyalanine stretch within its cognate E2 USE1. Polyalanine expansion mutations in USE1 reduce ubiquitin transfer from UBA6 to USE1 and decrease polyubiquitination and degradation of Ube3a (E6-AP). Proteins with polyalanine expansion mutations in disease states compete for UBA6–USE1 interaction, altering E6AP levels and Arc synaptic protein levels in neurons.\",\n      \"method\": \"In vitro ubiquitin transfer assays, iPSC-derived neurons from patients, mouse primary neurons, UBA6 overexpression rescue experiments\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro mechanistic assays combined with patient iPSC-derived neurons and mouse primary neurons; multiple orthogonal methods including rescue by UBA6 overexpression\",\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 by engaging the UBA6 ubiquitin fold domain (UFD) with exceptionally high affinity, modulated by the UBA6 Cys-Cap loop. A bespoke thioester switch mechanism disengages BIRC6 upon receiving ubiquitin, preventing inhibition of UBA6 despite BIRC6's priority. UBA6 achieves broader E2 specificity through coordinated contributions of both UFD and SCCH domains.\",\n      \"method\": \"Structural biology (cryo-EM capturing UBA6-BIRC6 complexes), biochemical E1-E2 specificity assays, mutagenesis\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structures of trapped complexes combined with biochemical specificity assays and mutagenesis; rigorous mechanistic study\",\n      \"pmids\": [\"41350950\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Cryo-EM structures of UBA6-E2 complexes at the thioester-transfer step with either FAT10 or ubiquitin reveal that UBA6 achieves E2 specificity through coordinated contributions of both the UFD and SCCH domains (contrasting with UFD-dominated selectivity of UBA1). An InsP6-binding site unique to UBA6 stabilizes an expanded SCCH cleft that pre-organizes the enzyme for selective engagement of UBA6-specific E2s, identifying InsP6 as a cofactor shaping specificity.\",\n      \"method\": \"Chemical trapping, cryo-EM (four UBA6-E2 complex structures), biochemical assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM structures of multiple trapped complexes with biochemical validation; multiple orthogonal methods revealing dual-domain E2 selectivity mechanism\",\n      \"pmids\": [\"41764162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In UBA1M41V (VEXAS syndrome) human cells, UBA6 compensates for UBA1 dysfunction, creating an acquired dependency on UBA6. Genetic (shRNA) or pharmacological (phytic acid/InsP6) inhibition of UBA6 preferentially impairs growth and colony formation of UBA1M41V cells while sparing wild-type cells.\",\n      \"method\": \"Engineered human cell model (THP1 UBA1M41V), shRNA knockdown, pharmacological inhibition with phytic acid, competition assays, proteomic analysis\",\n      \"journal\": \"Leukemia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional studies in engineered human cell model with genetic and pharmacological orthogonal approaches; single lab\",\n      \"pmids\": [\"40588566\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"UBA6 is a bispecific E1 ubiquitin-activating enzyme that ATP-dependently activates both ubiquitin and the ubiquitin-like modifier FAT10 through a three-step catalytic cycle (adenylation → thioester formation → transthiolation), working through its dedicated E2 enzyme USE1 and also the high-priority E2 BIRC6; structurally, UBA6 undergoes open/closed conformational transitions to catalyze these steps, is allosterically regulated by inositol hexakisphosphate (InsP6) at a unique allosteric site, achieves E2 selectivity through coordinated UFD and SCCH domain interactions, and physiologically controls diverse processes including autophagy (via LC3B monoubiquitination with BIRC6), neuronal development and dendritic spine maintenance (via Ube3a ubiquitylation), T cell IFN-γ production (via IκBα/NF-κB), iron homeostasis (via ferroportin degradation with NDFIP1), and epithelial homeostasis (via CDC42 ubiquitylation).\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"UBA6 is a bispecific E1 activating enzyme that initiates protein modification by ATP-dependently charging both ubiquitin and the ubiquitin-like modifier FAT10, and channels these modifiers into downstream conjugation distinct from the canonical UBA1 pathway [#0, #1]. It activates each substrate through a three-step cycle of adenylation, thioester formation, and transthiolation, forming a ternary complex in which FAT10 binds more tightly than ubiquitin yet is transferred less efficiently [#2]; crystal and cryo-EM structures resolve open (adenylation) and closed (thioester) conformational states and reveal that UBA6 attains its broader E2 selectivity through coordinated contributions of both its UFD and SCCH domains, in contrast to the UFD-dominated selectivity of UBA1 [#11, #14]. A distinctive allosteric site binds inositol hexakisphosphate (InsP6), which shapes E2 specificity and modulates open/closed interconversion and enzyme stability [#11, #14]. UBA6 works through its dedicated E2 USE1—whose recognition depends on a polyalanine stretch—and through the UBA6-exclusive, high-priority E2 BIRC6, which engages the UFD with exceptional affinity governed by a Cys-Cap loop and a thioester switch that disengages BIRC6 upon ubiquitin loading [#12, #13]. Physiologically, the UBA6–USE1 cascade is required for embryonic development and neuronal patterning, controlling dendritic spine density via Ube3a (E6-AP) turnover [#3, #12], while UBA6–BIRC6 monoubiquitinates LC3B to negatively regulate autophagy [#7]. Additional UBA6-initiated ubiquitination events govern epithelial homeostasis through CDC42 [#6], T cell IFN-\\u03b3 production via I\\u03baB\\u03b1/NF-\\u03baB [#9], hepcidin-driven ferroportin degradation with the adaptor NDFIP1 [#10], and cardiac Nav1.5 channel abundance [#8]. In VEXAS-associated UBA1-mutant cells, UBA6 compensates for UBA1 dysfunction, creating an acquired, InsP6-targetable dependency [#15].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Established that ubiquitin activation is not the exclusive province of UBA1 by identifying UBA6 as a second human E1 that charges ubiquitin and feeds E3-dependent ubiquitylation.\",\n      \"evidence\": \"In vitro reconstitution with recombinant proteins, thioester and ATP-exchange assays, E2 transfer to UbcH5b supporting MDM2/HectH9/E6-AP activity\",\n      \"pmids\": [\"17580310\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish FAT10 as a substrate\", \"Did not define a dedicated E2 partner distinct from UbcH5b\", \"No in vivo physiological role\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined UBA6 as bispecific by showing it activates FAT10 in addition to ubiquitin and uses a different E2 spectrum, including the dedicated E2 USE1.\",\n      \"evidence\": \"Biochemical characterization and E1-E2 specificity assays (review synthesizing multiple labs)\",\n      \"pmids\": [\"18353650\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of bispecificity unresolved\", \"Kinetic discrimination between ubiquitin and FAT10 not quantified\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Resolved the catalytic logic by demonstrating a three-step activation cycle for both modifiers and quantifying that FAT10 binds tighter but turns over slower than ubiquitin.\",\n      \"evidence\": \"Pre-steady-state kinetics, biophysical binding measurements, mechanism-based covalent inhibitor trapping\",\n      \"pmids\": [\"22427669\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural view of the conformational steps\", \"Allosteric regulation not yet identified\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstrated a non-redundant in vivo role: the UBA6–USE1 cascade drives embryonic and neuronal development and controls Ube3a turnover, spatially distinct from the UBA1–UbcH7 pathway.\",\n      \"evidence\": \"Neuronal-specific Uba6 knockout mice, in vitro Ube3a ubiquitylation, MEF turnover assays, immunofluorescence and behavior\",\n      \"pmids\": [\"23499007\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ubiquitin or FAT10 mediates the neuronal phenotype not separated\", \"Direct E3 ligases for Ube3a turnover not defined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identified a negative-feedback mechanism in which the dedicated E2 USE1 self-FAT10ylates to drive its own degradation and limit global FAT10ylation.\",\n      \"evidence\": \"Mass spectrometry of Lys323 site, mutagenesis, co-IP, proteasome inhibition, in vitro thioester assays\",\n      \"pmids\": [\"24528925\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological consequences of feedback in vivo unknown\", \"Single lab\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showed that the UBA6 UFD is the E2-recruitment hub and a competitive target, since LMO2 binding to the UFD blocks USE1 engagement and dampens FAT10ylation.\",\n      \"evidence\": \"Co-IP, domain mapping, pulldown, FAT10ylation assays on substrate p62\",\n      \"pmids\": [\"27569286\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural details of LMO2–UFD contact unresolved\", \"Physiological context of LMO2 regulation unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Linked UBA6 to epithelial homeostasis by identifying CDC42 as a UBA6-initiated ubiquitination target whose dysregulation drives EMT.\",\n      \"evidence\": \"shRNA knockdown in MCF-10A cells, EMT assays, CDC42 inhibitor rescue\",\n      \"pmids\": [\"29152096\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E2/E3 partners for CDC42 ubiquitination not defined\", \"Whether direct or indirect ubiquitination unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined the UBA6–BIRC6 axis as a negative regulator of autophagy by monoubiquitinating LC3B to limit its availability.\",\n      \"evidence\": \"Genome-wide CRISPR screen, LC3B fluorescent reporter, KO cell lines, autophagic flux and neuronal aggregate assays\",\n      \"pmids\": [\"31692446\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of BIRC6 partnership not yet resolved\", \"Whether FAT10 contributes unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Extended UBA6 substrate scope to ion-channel control, regulating cardiac Nav1.5 abundance and sodium current via ubiquitination at K590/K591.\",\n      \"evidence\": \"Overexpression/knockdown, patch-clamp, K590A/K591A mutagenesis in neonatal cardiomyocytes\",\n      \"pmids\": [\"32315024\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs UBC9-bridged mechanism not fully separated from UBE1 contribution\", \"In vivo cardiac relevance untested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Placed UBA6 in immune regulation, showing it promotes I\\u03baB\\u03b1 degradation to enable NF-\\u03baB activation and restrain T cell IFN-\\u03b3 production.\",\n      \"evidence\": \"T cell-specific conditional knockout mice, cytokine and NF-\\u03baB analysis, adoptive transfer\",\n      \"pmids\": [\"35011668\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ubiquitination of I\\u03baB\\u03b1 by UBA6 cascade not biochemically reconstituted\", \"E2/E3 partners unspecified\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected UBA6 to systemic iron control by showing UBA6 and the adaptor NDFIP1 are required for hepcidin-induced ferroportin degradation.\",\n      \"evidence\": \"siRNA screen of ubiquitin pathway, FPN-GFP reporter cells, in vivo AAV Ndfip1 silencing with iron measurements\",\n      \"pmids\": [\"34320783\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ferroportin ubiquitination by UBA6 cascade not shown in vitro\", \"E3 ligase identity unconfirmed\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Provided the first structural mechanism, capturing open (adenylation) and closed (thioester) states and discovering an InsP6 allosteric site that inhibits UBA6 and stabilizes it.\",\n      \"evidence\": \"X-ray crystallography of Uba6/ubiquitin complex with biochemical and biophysical validation\",\n      \"pmids\": [\"35986001\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"E2-bound transthiolation state not yet captured\", \"Physiological source/regulation of InsP6 occupancy unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined the molecular code for USE1 recognition—a polyalanine stretch—and showed that polyalanine-expansion proteins competitively disrupt UBA6–USE1, altering E6-AP and Arc levels in neurons.\",\n      \"evidence\": \"In vitro ubiquitin transfer, patient iPSC-derived and mouse primary neurons, UBA6 overexpression rescue\",\n      \"pmids\": [\"38177505\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of polyalanine recognition not resolved here\", \"Breadth of competing polyalanine proteins not enumerated\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Explained E2 prioritization by showing BIRC6 is UBA6-exclusive, binds the UFD with exceptional affinity gated by a Cys-Cap loop, and uses a thioester switch to disengage and avoid inhibiting UBA6.\",\n      \"evidence\": \"Cryo-EM of trapped UBA6–BIRC6 complexes, E1-E2 specificity assays, mutagenesis\",\n      \"pmids\": [\"41350950\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dynamics of switching under physiological E2 competition not quantified\", \"Cellular consequences of altering the switch untested\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Completed the specificity model by capturing thioester-transfer complexes with FAT10 or ubiquitin, showing dual UFD+SCCH domain control and that InsP6 pre-organizes an expanded SCCH cleft as a specificity cofactor.\",\n      \"evidence\": \"Chemical trapping and cryo-EM of four UBA6-E2 complexes with biochemical validation\",\n      \"pmids\": [\"41764162\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether InsP6 occupancy is dynamically regulated in cells unresolved\", \"Quantitative discrimination of all E2s not fully mapped\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed a disease-relevant compensation: in UBA1-mutant VEXAS cells UBA6 substitutes for UBA1, creating an acquired, InsP6-targetable dependency.\",\n      \"evidence\": \"Engineered THP1 UBA1M41V cells, shRNA, phytic acid (InsP6) inhibition, competition and proteomic assays\",\n      \"pmids\": [\"40588566\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Substrate set rerouted to UBA6 not defined\", \"In vivo therapeutic window unestablished\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How UBA6 partitions its many physiological outputs between ubiquitin and FAT10 conjugation, and how InsP6 occupancy is regulated in vivo to tune E2 selectivity, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No systematic mapping of which substrates require FAT10 vs ubiquitin\", \"Endogenous control of InsP6-dependent specificity unknown\", \"E3 ligases for most UBA6-dependent substrates unidentified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0, 2, 11]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 3, 7]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [11, 14]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 2, 3]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"USE1\", \"BIRC6\", \"FAT10\", \"NDFIP1\", \"LMO2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":7,"faith_pct":85.71428571428571}}