{"gene":"USE1","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2010,"finding":"USE1 (UBA6-specific E2 enzyme) was identified as an interaction partner of FAT10; activated FAT10 can be transferred from UBA6 onto USE1 in vitro, establishing USE1 as an E2 enzyme in the FAT10 conjugation cascade. Additionally, USE1 is the first known substrate of FAT10 conjugation, as it auto-FAT10ylates itself in cis but not in trans.","method":"In vitro transfer assay, co-immunoprecipitation from intact cells, siRNA knockdown with FAT10 conjugate readout","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro reconstitution of FAT10 transfer, reciprocal co-IP, siRNA knockdown with defined biochemical phenotype, multiple orthogonal methods in one study","pmids":["20975683"],"is_preprint":false},{"year":2013,"finding":"Uba6 and Use1 promote proteasomal turnover of the E3 ubiquitin ligase Ube3a (E6-AP) by catalyzing Ube3a ubiquitylation in vitro and in mouse embryo fibroblasts. Loss of neuronal Uba6 leads to elevated Ube3a and Shank3 levels, reduced Arc levels, decreased dendritic spine density, and altered neuronal patterning in hippocampus and amygdala. These activities occur in parallel with but spatially distinct from the Uba1-UbcH7 pathway.","method":"Conditional knockout mouse (neuronal Uba6), in vitro ubiquitylation assay, western blot, confocal microscopy/fractionation for spatial localization, behavioral assays","journal":"Molecular Cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro ubiquitylation reconstitution, genetic knockout with defined cellular and behavioral phenotypes, multiple orthogonal methods across independent experiments","pmids":["23499007"],"is_preprint":false},{"year":2005,"finding":"The mammalian D12 protein (USE1 ortholog) was identified as a Q-SNARE that forms a tight complex with syntaxin 18, Sec22b, and binds α-SNAP. D12 localizes predominantly to the ER and ER-Golgi intermediate compartments. Knockdown of D12 caused impaired post-Golgi maturation of cathepsin D and rapid accumulation of lipofuscin granules with apoptotic cell death, implicating D12 in lysosomal degradative function. D12 also associates with VAMP7, a SNARE of the endosomal-lysosomal pathway.","method":"Co-immunoprecipitation, subcellular fractionation/immunofluorescence, siRNA knockdown with lysosomal and apoptosis phenotype readouts, co-IP with VAMP7","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP for SNARE complex, direct localization by immunofluorescence/fractionation with functional consequence (lysosomal maturation defect), knockdown with defined phenotype; single lab but multiple orthogonal methods","pmids":["16354670"],"is_preprint":false},{"year":2017,"finding":"USE1 protein level is regulated by the anaphase-promoting complex (APC/C) via a conserved D-box domain. Missense mutations in USE1 identified in lung cancer patients prolong the half-life of the protein. Stable overexpression of USE1 increased cell proliferation, migration, and invasion in lung cancer cells and xenograft models; knockdown reduced these properties.","method":"Proteomics/mass spectrometry to identify interacting proteins, D-box mutation analysis, xenograft tumor models, stable overexpression and knockdown in lung cancer cell lines","journal":"Journal of the National Cancer Institute","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — APC/C regulation identified by proteomics and domain analysis, functional KD/OE with defined cellular phenotypes, single lab with multiple methods","pmids":["28376205"],"is_preprint":false},{"year":2014,"finding":"Translation of the SNARE protein Use1 is controlled by the RNA-binding protein Grsf1. The 5'UTR of mouse Use1 mRNA contains G-repeats that bind Grsf1 in RNA band-shift assays; Grsf1 concentration-dependently stimulates translation of Use1 reporter constructs. Downregulation of either Grsf1 or Use1 abrogates expansion of erythroblasts, establishing Grsf1-driven Use1 translation as required for erythroid compartment expansion.","method":"RNA band-shift assay, reporter translation assay, siRNA knockdown of Grsf1 and Use1 with erythroblast expansion readout","journal":"PLoS One","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — direct RNA-protein binding assay, functional reporter assay, knockdown with defined proliferation phenotype; single lab, multiple orthogonal methods","pmids":["25184340"],"is_preprint":false},{"year":2016,"finding":"LMO2 interacts with the C-terminal ubiquitin fold domain (UFD) of UBA6, the E1 enzyme upstream of USE1. This LMO2-UBA6 interaction competitively blocks the UBA6-USE1 interaction, reducing overall cellular FAT10ylation and the FAT10ylation-dependent degradation of the FAT10 substrate p62.","method":"Co-immunoprecipitation, FAT10ylation assays in cells with LMO2 overexpression, domain-mapping experiments","journal":"Biochemical and Biophysical Research Communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP and functional FAT10ylation assay, single lab, multiple methods confirming pathway placement of USE1 downstream of LMO2-regulated UBA6","pmids":["27569286"],"is_preprint":false},{"year":2023,"finding":"Under inflammatory conditions induced by TNF, FAT10 conjugation becomes independent of USE1. USE1 knockout strongly diminishes FAT10 conjugation under basal conditions, but TNF-stimulated FAT10 conjugation persists. Additional E2 conjugating enzymes capable of being charged with FAT10 at their active-site cysteine were identified and shown to rescue FAT10 conjugation in the absence of USE1.","method":"USE1 knockout cells, FAT10 conjugation assays under TNF stimulation, active-site cysteine charging assays for alternative E2 enzymes","journal":"Life Science Alliance","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with defined biochemical phenotype, active-site charging assays for alternative E2s; single lab, two orthogonal methods","pmids":["37604583"],"is_preprint":false},{"year":2022,"finding":"The SNARE protein USE1 physically interacts with the mumps virus fusion (F) protein and is required for complete N-linked glycosylation and expression of the MuV F protein, as well as for efficient viral propagation.","method":"Proximity labeling, co-immunoprecipitation, siRNA knockdown with viral propagation and F protein glycosylation readouts","journal":"PLoS Pathogens","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — proximity labeling plus reciprocal Co-IP plus siRNA knockdown with defined biochemical phenotype; single lab, multiple orthogonal methods","pmids":["36480520"],"is_preprint":false},{"year":2019,"finding":"RNAi-mediated knockdown of USE1 suppresses tumor cell growth via cell cycle arrest and apoptosis induction in lung cancer cells and A549 xenograft models in vivo.","method":"BRC-siRNA knockdown, cell viability assays, cell cycle analysis, apoptosis assays, xenograft tumor models","journal":"Biomaterials","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — knockdown with defined cell cycle and apoptosis phenotypes in vitro and in vivo; single lab, multiple readouts","pmids":["31791842"],"is_preprint":false}],"current_model":"USE1 is a UBA6-specific E2 ubiquitin-conjugating enzyme that functions as the primary E2 in the FAT10ylation cascade (transferring activated FAT10 from UBA6 to substrates) and also participates in the Uba6-dependent ubiquitylation of substrates such as Ube3a for proteasomal degradation; it acts as a Q-SNARE localizing to the ER/ERGIC where it forms a complex with syntaxin 18, Sec22b, and VAMP7 to support lysosomal maturation; its protein stability is regulated by the APC/C via a D-box domain; and its translation is controlled by the RNA-binding protein Grsf1, with the entire Uba6-Use1 axis being required for neuronal development, dendritic spine architecture, and erythroid expansion."},"narrative":{"mechanistic_narrative":"USE1 is a bifunctional protein that operates both as the dedicated E2 enzyme of the UBA6-driven ubiquitin-like conjugation system and as a Q-SNARE in early secretory-pathway membrane trafficking [PMID:20975683, PMID:16354670]. In its conjugation role, USE1 receives activated FAT10 from the E1 enzyme UBA6 and serves as the primary E2 of the FAT10ylation cascade, with USE1 itself being the first identified FAT10 substrate via cis-autoFAT10ylation [PMID:20975683]; the same UBA6–USE1 axis also catalyzes ubiquitylation of substrates including the E3 ligase Ube3a (E6-AP), targeting them for proteasomal turnover [PMID:23499007]. This pathway is gated upstream: LMO2 binds the UBA6 ubiquitin-fold domain and competitively displaces USE1, dampening cellular FAT10ylation [PMID:27569286], and under TNF-driven inflammation FAT10 conjugation becomes USE1-independent through alternative FAT10-charged E2 enzymes [PMID:37604583]. The UBA6–USE1 axis is required in vivo for neuronal patterning and dendritic spine density, where loss of upstream Uba6 elevates Ube3a and Shank3 [PMID:23499007], and for erythroblast expansion, where USE1 translation is driven by the RNA-binding protein Grsf1 acting on G-repeats in the Use1 5'UTR [PMID:25184340]. Independently, USE1 forms a tight ER/ERGIC-localized SNARE complex with syntaxin 18 and Sec22b, associates with VAMP7, and is needed for post-Golgi cathepsin D maturation and lysosomal degradative function [PMID:16354670], and supports maturation, glycosylation, and propagation of mumps virus fusion protein [PMID:36480520]. USE1 protein level is controlled by APC/C through a conserved D-box, and stabilizing missense mutations or overexpression promote lung cancer cell proliferation, migration, and invasion [PMID:28376205, PMID:31791842].","teleology":[{"year":2005,"claim":"Before its role in ubiquitin-like conjugation was known, the question was what membrane-trafficking machinery USE1 belongs to; this established USE1 (D12) as a bona fide Q-SNARE of the early secretory pathway with a functional consequence for lysosomal maturation.","evidence":"Reciprocal co-IP defining a syntaxin 18/Sec22b/α-SNAP complex, immunofluorescence/fractionation localization, and siRNA knockdown with cathepsin D maturation and lipofuscin/apoptosis readouts","pmids":["16354670"],"confidence":"High","gaps":["Does not resolve the structural arrangement of the SNARE bundle","Does not connect the SNARE function to the later-discovered E2 conjugation role"]},{"year":2010,"claim":"The question of how FAT10 is transferred from its E1 to substrates was answered by placing USE1 as the dedicated E2 of the cascade and revealing it as the first FAT10 substrate.","evidence":"In vitro FAT10 transfer assay from UBA6 to USE1, co-IP from cells, and siRNA knockdown with FAT10 conjugate readout; cis-versus-trans auto-FAT10ylation tested","pmids":["20975683"],"confidence":"High","gaps":["Does not identify the broader set of FAT10 substrates downstream of USE1","Functional consequence of USE1 auto-FAT10ylation unresolved"]},{"year":2013,"claim":"It was unclear whether the UBA6–USE1 axis had physiological substrates and in vivo importance; this showed it ubiquitylates Ube3a for degradation and is required for neuronal architecture.","evidence":"In vitro ubiquitylation reconstitution, neuronal conditional Uba6 knockout mouse with Ube3a/Shank3/Arc western blots, confocal spine-density analysis, and behavioral assays","pmids":["23499007"],"confidence":"High","gaps":["Establishes Uba6 loss-of-function phenotypes but does not isolate USE1's specific contribution genetically","Does not distinguish ubiquitin versus FAT10 conjugation in the neuronal phenotypes"]},{"year":2014,"claim":"How USE1 abundance is set translationally and what tissue depends on it was addressed by identifying Grsf1-driven translation required for erythroid expansion.","evidence":"RNA band-shift mapping of G-repeats in the Use1 5'UTR, Grsf1 dose-dependent reporter translation assay, and siRNA knockdown of Grsf1/Use1 with erythroblast expansion readout","pmids":["25184340"],"confidence":"Medium","gaps":["Does not determine whether the erythroid requirement reflects USE1's SNARE or E2 function","Mechanism of Grsf1-stimulated translation initiation not defined"]},{"year":2016,"claim":"The question of how the FAT10ylation cascade is regulated upstream of USE1 was addressed by showing LMO2 competitively blocks the UBA6–USE1 interaction.","evidence":"Co-IP, domain mapping to the UBA6 UFD, and cellular FAT10ylation assays with LMO2 overexpression measuring p62 degradation","pmids":["27569286"],"confidence":"Medium","gaps":["Single lab without reciprocal in vivo validation","Does not establish physiological contexts where LMO2 levels gate USE1 charging"]},{"year":2017,"claim":"How USE1 protein stability is controlled and whether it contributes to cancer was answered by identifying APC/C-mediated turnover via a D-box and oncogenic stabilizing mutations.","evidence":"Proteomics of interactors, D-box mutation half-life analysis, and stable overexpression/knockdown in lung cancer lines plus xenografts measuring proliferation, migration, invasion","pmids":["28376205"],"confidence":"Medium","gaps":["Does not define which USE1 enzymatic activity drives the oncogenic phenotype","Single lab; D-box ubiquitylation not reconstituted with purified APC/C"]},{"year":2019,"claim":"Whether USE1 is required for tumor cell survival was tested, showing its knockdown triggers cell cycle arrest and apoptosis in lung cancer models.","evidence":"siRNA knockdown with viability, cell cycle, and apoptosis assays in lung cancer cells and A549 xenografts","pmids":["31791842"],"confidence":"Medium","gaps":["Does not identify the molecular mechanism linking USE1 loss to arrest/apoptosis","Off-target effects of knockdown not fully excluded"]},{"year":2022,"claim":"A pathogen-relevant role was established by showing USE1 supports mumps virus fusion protein maturation and propagation.","evidence":"Proximity labeling, reciprocal co-IP with MuV F protein, and siRNA knockdown with F glycosylation and viral propagation readouts","pmids":["36480520"],"confidence":"Medium","gaps":["Does not determine whether the SNARE complex or USE1's E2 activity mediates F protein support","Mechanism of glycosylation defect upon USE1 loss unresolved"]},{"year":2023,"claim":"Whether USE1 is the obligate E2 for FAT10 under all conditions was answered: USE1 is required basally but dispensable under TNF, where alternative FAT10-charged E2s operate.","evidence":"USE1 knockout cells with FAT10 conjugation assays under basal versus TNF stimulation and active-site cysteine charging assays for alternative E2 enzymes","pmids":["37604583"],"confidence":"Medium","gaps":["Identities and substrate specificities of the alternative E2 enzymes not fully resolved","Mechanism by which TNF redirects FAT10 conjugation away from USE1 unknown"]},{"year":null,"claim":"It remains unresolved how USE1's two distinct functions—Q-SNARE in ER/ERGIC trafficking and E2 in FAT10/ubiquitin conjugation—are coordinated, regulated, and partitioned across the tissue-specific phenotypes attributed to it.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No study dissects which activity drives the erythroid, neuronal, viral, or oncogenic phenotypes","No structural basis for dual functionality reported"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[2]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[2]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[2]}],"complexes":["syntaxin 18–Sec22b–USE1 Q-SNARE complex","FAT10 conjugation cascade (UBA6–USE1)"],"partners":["UBA6","FAT10","STX18","SEC22B","VAMP7","LMO2","GRSF1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9NZ43","full_name":"Vesicle transport protein USE1","aliases":["Putative MAPK-activating protein PM26","USE1-like protein","p31"],"length_aa":259,"mass_kda":29.4,"function":"SNARE that may be involved in targeting and fusion of Golgi-derived retrograde transport vesicles with the ER","subcellular_location":"Endoplasmic reticulum membrane","url":"https://www.uniprot.org/uniprotkb/Q9NZ43/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/USE1","classification":"Not Classified","n_dependent_lines":582,"n_total_lines":1208,"dependency_fraction":0.4817880794701987},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"BNIP1","stoichiometry":10.0},{"gene":"STX18","stoichiometry":10.0},{"gene":"SCFD1","stoichiometry":4.0},{"gene":"GOSR1","stoichiometry":0.2},{"gene":"NAPA","stoichiometry":0.2},{"gene":"STX3","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/USE1","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"},{"mim_id":"610675","title":"UNCONVENTIONAL SNARE IN THE ER 1; USE1","url":"https://www.omim.org/entry/610675"},{"mim_id":"608025","title":"NBAS SUBUNIT OF NRZ TETHERING COMPLEX; NBAS","url":"https://www.omim.org/entry/608025"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/USE1"},"hgnc":{"alias_symbol":["p31","SLT1","MDS032","D12"],"prev_symbol":[]},"alphafold":{"accession":"Q9NZ43","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NZ43","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NZ43-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NZ43-F1-predicted_aligned_error_v6.png","plddt_mean":72.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=USE1","jax_strain_url":"https://www.jax.org/strain/search?query=USE1"},"sequence":{"accession":"Q9NZ43","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NZ43.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NZ43/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NZ43"}},"corpus_meta":[{"pmid":"20975683","id":"PMC_20975683","title":"USE1 is a bispecific conjugating enzyme for ubiquitin and FAT10, which FAT10ylates itself in cis.","date":"2010","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/20975683","citation_count":85,"is_preprint":false},{"pmid":"7929111","id":"PMC_7929111","title":"Intrinsic RNA (guanine-7) methyltransferase activity of the vaccinia virus capping enzyme D1 subunit is stimulated by the D12 subunit. 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Additionally, USE1 is the first known substrate of FAT10 conjugation, as it auto-FAT10ylates itself in cis but not in trans.\",\n      \"method\": \"In vitro transfer assay, co-immunoprecipitation from intact cells, siRNA knockdown with FAT10 conjugate readout\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro reconstitution of FAT10 transfer, reciprocal co-IP, siRNA knockdown with defined biochemical phenotype, multiple orthogonal methods in one study\",\n      \"pmids\": [\"20975683\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Uba6 and Use1 promote proteasomal turnover of the E3 ubiquitin ligase Ube3a (E6-AP) by catalyzing Ube3a ubiquitylation in vitro and in mouse embryo fibroblasts. Loss of neuronal Uba6 leads to elevated Ube3a and Shank3 levels, reduced Arc levels, decreased dendritic spine density, and altered neuronal patterning in hippocampus and amygdala. These activities occur in parallel with but spatially distinct from the Uba1-UbcH7 pathway.\",\n      \"method\": \"Conditional knockout mouse (neuronal Uba6), in vitro ubiquitylation assay, western blot, confocal microscopy/fractionation for spatial localization, behavioral assays\",\n      \"journal\": \"Molecular Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro ubiquitylation reconstitution, genetic knockout with defined cellular and behavioral phenotypes, multiple orthogonal methods across independent experiments\",\n      \"pmids\": [\"23499007\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The mammalian D12 protein (USE1 ortholog) was identified as a Q-SNARE that forms a tight complex with syntaxin 18, Sec22b, and binds α-SNAP. D12 localizes predominantly to the ER and ER-Golgi intermediate compartments. Knockdown of D12 caused impaired post-Golgi maturation of cathepsin D and rapid accumulation of lipofuscin granules with apoptotic cell death, implicating D12 in lysosomal degradative function. D12 also associates with VAMP7, a SNARE of the endosomal-lysosomal pathway.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation/immunofluorescence, siRNA knockdown with lysosomal and apoptosis phenotype readouts, co-IP with VAMP7\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP for SNARE complex, direct localization by immunofluorescence/fractionation with functional consequence (lysosomal maturation defect), knockdown with defined phenotype; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"16354670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"USE1 protein level is regulated by the anaphase-promoting complex (APC/C) via a conserved D-box domain. Missense mutations in USE1 identified in lung cancer patients prolong the half-life of the protein. Stable overexpression of USE1 increased cell proliferation, migration, and invasion in lung cancer cells and xenograft models; knockdown reduced these properties.\",\n      \"method\": \"Proteomics/mass spectrometry to identify interacting proteins, D-box mutation analysis, xenograft tumor models, stable overexpression and knockdown in lung cancer cell lines\",\n      \"journal\": \"Journal of the National Cancer Institute\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — APC/C regulation identified by proteomics and domain analysis, functional KD/OE with defined cellular phenotypes, single lab with multiple methods\",\n      \"pmids\": [\"28376205\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Translation of the SNARE protein Use1 is controlled by the RNA-binding protein Grsf1. The 5'UTR of mouse Use1 mRNA contains G-repeats that bind Grsf1 in RNA band-shift assays; Grsf1 concentration-dependently stimulates translation of Use1 reporter constructs. Downregulation of either Grsf1 or Use1 abrogates expansion of erythroblasts, establishing Grsf1-driven Use1 translation as required for erythroid compartment expansion.\",\n      \"method\": \"RNA band-shift assay, reporter translation assay, siRNA knockdown of Grsf1 and Use1 with erythroblast expansion readout\",\n      \"journal\": \"PLoS One\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — direct RNA-protein binding assay, functional reporter assay, knockdown with defined proliferation phenotype; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"25184340\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"LMO2 interacts with the C-terminal ubiquitin fold domain (UFD) of UBA6, the E1 enzyme upstream of USE1. This LMO2-UBA6 interaction competitively blocks the UBA6-USE1 interaction, reducing overall cellular FAT10ylation and the FAT10ylation-dependent degradation of the FAT10 substrate p62.\",\n      \"method\": \"Co-immunoprecipitation, FAT10ylation assays in cells with LMO2 overexpression, domain-mapping experiments\",\n      \"journal\": \"Biochemical and Biophysical Research Communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP and functional FAT10ylation assay, single lab, multiple methods confirming pathway placement of USE1 downstream of LMO2-regulated UBA6\",\n      \"pmids\": [\"27569286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Under inflammatory conditions induced by TNF, FAT10 conjugation becomes independent of USE1. USE1 knockout strongly diminishes FAT10 conjugation under basal conditions, but TNF-stimulated FAT10 conjugation persists. Additional E2 conjugating enzymes capable of being charged with FAT10 at their active-site cysteine were identified and shown to rescue FAT10 conjugation in the absence of USE1.\",\n      \"method\": \"USE1 knockout cells, FAT10 conjugation assays under TNF stimulation, active-site cysteine charging assays for alternative E2 enzymes\",\n      \"journal\": \"Life Science Alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with defined biochemical phenotype, active-site charging assays for alternative E2s; single lab, two orthogonal methods\",\n      \"pmids\": [\"37604583\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The SNARE protein USE1 physically interacts with the mumps virus fusion (F) protein and is required for complete N-linked glycosylation and expression of the MuV F protein, as well as for efficient viral propagation.\",\n      \"method\": \"Proximity labeling, co-immunoprecipitation, siRNA knockdown with viral propagation and F protein glycosylation readouts\",\n      \"journal\": \"PLoS Pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — proximity labeling plus reciprocal Co-IP plus siRNA knockdown with defined biochemical phenotype; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"36480520\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RNAi-mediated knockdown of USE1 suppresses tumor cell growth via cell cycle arrest and apoptosis induction in lung cancer cells and A549 xenograft models in vivo.\",\n      \"method\": \"BRC-siRNA knockdown, cell viability assays, cell cycle analysis, apoptosis assays, xenograft tumor models\",\n      \"journal\": \"Biomaterials\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — knockdown with defined cell cycle and apoptosis phenotypes in vitro and in vivo; single lab, multiple readouts\",\n      \"pmids\": [\"31791842\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"USE1 is a UBA6-specific E2 ubiquitin-conjugating enzyme that functions as the primary E2 in the FAT10ylation cascade (transferring activated FAT10 from UBA6 to substrates) and also participates in the Uba6-dependent ubiquitylation of substrates such as Ube3a for proteasomal degradation; it acts as a Q-SNARE localizing to the ER/ERGIC where it forms a complex with syntaxin 18, Sec22b, and VAMP7 to support lysosomal maturation; its protein stability is regulated by the APC/C via a D-box domain; and its translation is controlled by the RNA-binding protein Grsf1, with the entire Uba6-Use1 axis being required for neuronal development, dendritic spine architecture, and erythroid expansion.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"USE1 is a bifunctional protein that operates both as the dedicated E2 enzyme of the UBA6-driven ubiquitin-like conjugation system and as a Q-SNARE in early secretory-pathway membrane trafficking [#0, #2]. In its conjugation role, USE1 receives activated FAT10 from the E1 enzyme UBA6 and serves as the primary E2 of the FAT10ylation cascade, with USE1 itself being the first identified FAT10 substrate via cis-autoFAT10ylation [#0]; the same UBA6–USE1 axis also catalyzes ubiquitylation of substrates including the E3 ligase Ube3a (E6-AP), targeting them for proteasomal turnover [#1]. This pathway is gated upstream: LMO2 binds the UBA6 ubiquitin-fold domain and competitively displaces USE1, dampening cellular FAT10ylation [#5], and under TNF-driven inflammation FAT10 conjugation becomes USE1-independent through alternative FAT10-charged E2 enzymes [#6]. The UBA6–USE1 axis is required in vivo for neuronal patterning and dendritic spine density, where loss of upstream Uba6 elevates Ube3a and Shank3 [#1], and for erythroblast expansion, where USE1 translation is driven by the RNA-binding protein Grsf1 acting on G-repeats in the Use1 5'UTR [#4]. Independently, USE1 forms a tight ER/ERGIC-localized SNARE complex with syntaxin 18 and Sec22b, associates with VAMP7, and is needed for post-Golgi cathepsin D maturation and lysosomal degradative function [#2], and supports maturation, glycosylation, and propagation of mumps virus fusion protein [#7]. USE1 protein level is controlled by APC/C through a conserved D-box, and stabilizing missense mutations or overexpression promote lung cancer cell proliferation, migration, and invasion [#3, #8].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Before its role in ubiquitin-like conjugation was known, the question was what membrane-trafficking machinery USE1 belongs to; this established USE1 (D12) as a bona fide Q-SNARE of the early secretory pathway with a functional consequence for lysosomal maturation.\",\n      \"evidence\": \"Reciprocal co-IP defining a syntaxin 18/Sec22b/α-SNAP complex, immunofluorescence/fractionation localization, and siRNA knockdown with cathepsin D maturation and lipofuscin/apoptosis readouts\",\n      \"pmids\": [\"16354670\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not resolve the structural arrangement of the SNARE bundle\", \"Does not connect the SNARE function to the later-discovered E2 conjugation role\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"The question of how FAT10 is transferred from its E1 to substrates was answered by placing USE1 as the dedicated E2 of the cascade and revealing it as the first FAT10 substrate.\",\n      \"evidence\": \"In vitro FAT10 transfer assay from UBA6 to USE1, co-IP from cells, and siRNA knockdown with FAT10 conjugate readout; cis-versus-trans auto-FAT10ylation tested\",\n      \"pmids\": [\"20975683\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not identify the broader set of FAT10 substrates downstream of USE1\", \"Functional consequence of USE1 auto-FAT10ylation unresolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"It was unclear whether the UBA6–USE1 axis had physiological substrates and in vivo importance; this showed it ubiquitylates Ube3a for degradation and is required for neuronal architecture.\",\n      \"evidence\": \"In vitro ubiquitylation reconstitution, neuronal conditional Uba6 knockout mouse with Ube3a/Shank3/Arc western blots, confocal spine-density analysis, and behavioral assays\",\n      \"pmids\": [\"23499007\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Establishes Uba6 loss-of-function phenotypes but does not isolate USE1's specific contribution genetically\", \"Does not distinguish ubiquitin versus FAT10 conjugation in the neuronal phenotypes\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"How USE1 abundance is set translationally and what tissue depends on it was addressed by identifying Grsf1-driven translation required for erythroid expansion.\",\n      \"evidence\": \"RNA band-shift mapping of G-repeats in the Use1 5'UTR, Grsf1 dose-dependent reporter translation assay, and siRNA knockdown of Grsf1/Use1 with erythroblast expansion readout\",\n      \"pmids\": [\"25184340\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not determine whether the erythroid requirement reflects USE1's SNARE or E2 function\", \"Mechanism of Grsf1-stimulated translation initiation not defined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"The question of how the FAT10ylation cascade is regulated upstream of USE1 was addressed by showing LMO2 competitively blocks the UBA6–USE1 interaction.\",\n      \"evidence\": \"Co-IP, domain mapping to the UBA6 UFD, and cellular FAT10ylation assays with LMO2 overexpression measuring p62 degradation\",\n      \"pmids\": [\"27569286\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab without reciprocal in vivo validation\", \"Does not establish physiological contexts where LMO2 levels gate USE1 charging\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"How USE1 protein stability is controlled and whether it contributes to cancer was answered by identifying APC/C-mediated turnover via a D-box and oncogenic stabilizing mutations.\",\n      \"evidence\": \"Proteomics of interactors, D-box mutation half-life analysis, and stable overexpression/knockdown in lung cancer lines plus xenografts measuring proliferation, migration, invasion\",\n      \"pmids\": [\"28376205\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not define which USE1 enzymatic activity drives the oncogenic phenotype\", \"Single lab; D-box ubiquitylation not reconstituted with purified APC/C\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Whether USE1 is required for tumor cell survival was tested, showing its knockdown triggers cell cycle arrest and apoptosis in lung cancer models.\",\n      \"evidence\": \"siRNA knockdown with viability, cell cycle, and apoptosis assays in lung cancer cells and A549 xenografts\",\n      \"pmids\": [\"31791842\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not identify the molecular mechanism linking USE1 loss to arrest/apoptosis\", \"Off-target effects of knockdown not fully excluded\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"A pathogen-relevant role was established by showing USE1 supports mumps virus fusion protein maturation and propagation.\",\n      \"evidence\": \"Proximity labeling, reciprocal co-IP with MuV F protein, and siRNA knockdown with F glycosylation and viral propagation readouts\",\n      \"pmids\": [\"36480520\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not determine whether the SNARE complex or USE1's E2 activity mediates F protein support\", \"Mechanism of glycosylation defect upon USE1 loss unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Whether USE1 is the obligate E2 for FAT10 under all conditions was answered: USE1 is required basally but dispensable under TNF, where alternative FAT10-charged E2s operate.\",\n      \"evidence\": \"USE1 knockout cells with FAT10 conjugation assays under basal versus TNF stimulation and active-site cysteine charging assays for alternative E2 enzymes\",\n      \"pmids\": [\"37604583\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identities and substrate specificities of the alternative E2 enzymes not fully resolved\", \"Mechanism by which TNF redirects FAT10 conjugation away from USE1 unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how USE1's two distinct functions—Q-SNARE in ER/ERGIC trafficking and E2 in FAT10/ubiquitin conjugation—are coordinated, regulated, and partitioned across the tissue-specific phenotypes attributed to it.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No study dissects which activity drives the erythroid, neuronal, viral, or oncogenic phenotypes\", \"No structural basis for dual functionality reported\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"complexes\": [\"syntaxin 18–Sec22b–USE1 Q-SNARE complex\", \"FAT10 conjugation cascade (UBA6–USE1)\"],\n    \"partners\": [\"UBA6\", \"FAT10\", \"STX18\", \"SEC22B\", \"VAMP7\", \"LMO2\", \"Grsf1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}