UBA6

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).

1. 2007 High

   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

   PMID:17580310

   Open questions at the time

   • Physiological substrates and E2 partners specific to UBA6 versus UBA1 were unknown
   • Whether UBA6 activates ubiquitin-like modifiers besides ubiquitin was untested
2. 2008 High

   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

   PMID:18353650

   Open questions at the time

   • Kinetic parameters for ubiquitin versus FAT10 activation were not yet quantified
   • Structural basis for dual modifier recognition was unknown
3. 2012 High

   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

   PMID:22427669

   Open questions at the time

   • How UBA6 structurally discriminates between ubiquitin and FAT10 was unresolved
   • Cellular regulation of the ubiquitin/FAT10 balance by UBA6 was not addressed
4. 2013 High

   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

   PMID:23499007

   Open questions at the time

   • 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
5. 2014 High

   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

   PMID:24528925

   Open questions at the time

   • 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
6. 2016 Medium

   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

   PMID:27569286

   Open questions at the time

   • 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
7. 2019 High

   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

   PMID:31692446

   Open questions at the time

   • Whether UBA6-BIRC6 directly monoubiquitinates LC3B or acts through intermediates was not structurally resolved
   • The ubiquitin attachment site(s) on LC3B were not mapped
8. 2020 Medium

   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

   PMID:32315024

   Open questions at the time

   • 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
9. 2021 Medium

   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

   PMID:35011668

   Open questions at the time

   • 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
10. 2022 High

    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

    PMID:35986001

    Open questions at the time

    • How InsP6 concentrations are regulated in cells to control UBA6 activity was unknown
    • Structures with FAT10 rather than ubiquitin were not obtained
11. 2022 Medium

    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

    PMID:36109526

    Open questions at the time

    • The precise mechanism by which inosine inhibits UBA6 (competitive, allosteric) was not structurally defined
    • Whether inosine affects FAT10 activation by UBA6 was not tested
12. 2024 High

    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

    PMID:38177505

    Open questions at the time

    • Whether therapeutic UBA6 overexpression can rescue disease phenotypes in vivo remains untested
    • The full repertoire of polyalanine-containing UBA6 interactors was not mapped
13. 2025 High

    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

    PMID:41350950

    Open questions at the time

    • 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
14. 2026 High

    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

    PMID:41764162

    Open questions at the time

    • 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

• 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.
• 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