{"gene":"AUP1","run_date":"2026-06-09T22:02:44","timeline":{"discoveries":[{"year":2010,"finding":"AUP1 localizes to lipid droplets (LDs) by integrating into the LD surface in a monotopic fashion with both termini facing the cytosol, and binds the E2 ubiquitin conjugase Ube2g2 via its C-terminal G2BR (G2 binding region) domain; deletion or mutation of G2BR abolishes Ube2g2 binding without affecting LD localization.","method":"Confocal microscopy/cell fractionation (LD localization), Co-immunoprecipitation and domain deletion/mutagenesis (G2BR–Ube2g2 interaction)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal binding confirmed by domain deletion and point mutation, LD topology independently established, replicated in subsequent studies","pmids":["21127063"],"is_preprint":false},{"year":2011,"finding":"AUP1 physically associates with the mammalian HRD1-SEL1L ERAD complex; its CUE domain regulates polyubiquitylation and facilitates interaction with the HRD1 complex and dislocation substrates; AUP1 recruits UBE2G2 to the ER membrane for ERAD; AUP1 depletion impairs degradation of misfolded ER proteins; AUP1 expression level controls cellular lipid droplet abundance, representing the first protein linking LD regulation to ER quality control.","method":"Co-immunoprecipitation (HRD1-SEL1L complex association), siRNA knockdown with ERAD substrate degradation assay, domain mutagenesis (CUE domain), LD quantification upon AUP1 overexpression/depletion","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, KD, domain mutagenesis, substrate assay) in single study, later independently replicated","pmids":["21857022"],"is_preprint":false},{"year":2021,"finding":"The 27-amino acid G2BR of AUP1 binds UBE2G2 with low nanomolar affinity at the backside of the E2 enzyme; the crystal structure of the G2BR–UBE2G2 complex reveals a network of salt bridges, hydrogen bonds, and hydrophobic interactions; G2BR–UBE2G2 binding allosterically activates ubiquitination in vitro in conjunction with ERAD E3s; AUP1 G2BR is required for ER membrane recruitment of UBE2G2, prevents its rapid degradation, and is essential for multiple ERAD pathways in cells.","method":"X-ray crystallography (structure of G2BR–UBE2G2 complex), biophysical binding assays (nanomolar affinity), in vitro ubiquitination reconstitution with mutagenesis, cell-based ERAD assays with G2BR mutants","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure + in vitro reconstitution + mutagenesis + cell-based functional validation in one study","pmids":["34879065"],"is_preprint":false},{"year":2018,"finding":"AUP1 associates with dengue virus NS4A at lipid droplets; AUP1's acyltransferase domain activity is required for DENV-triggered lipophagy and virus production; mono-ubiquitylation of AUP1 disrupts the AUP1–NS4A interaction and inhibits acyltransferase activity, thereby attenuating lipophagy and virus production; upon infection AUP1 relocalizes from lipid droplets to autophagosomes.","method":"Functional proteomics ubiquitylation screen, Co-immunoprecipitation (AUP1–NS4A), acyltransferase domain point mutant rescue, CRISPR/siRNA KO with virus titer readout, fluorescence microscopy (LD-to-autophagosome relocalization)","journal":"Cell host & microbe","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (proteomics, Co-IP, domain mutant, KO, imaging) with mechanistic pathway placement","pmids":["29902443"],"is_preprint":false},{"year":2009,"finding":"Yeast Aup1 (mitochondrial protein phosphatase homolog) is required for efficient stationary-phase mitophagy; Aup1 regulates the retrograde (RTG) signaling pathway, controls phosphorylation of the transcription factor Rtg3, and is required for induction of RTG target genes under mitophagic conditions; deletion of RTG3 itself causes a defect in stationary-phase mitophagy.","method":"Genetic deletion (aup1Δ, rtg3Δ) with mitophagy assay, phosphorylation analysis of Rtg3 by gel shift, RT-qPCR of RTG target genes","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean yeast KO with multiple readouts (mitophagy, RTG target genes, Rtg3 phosphorylation), single lab study","pmids":["19840933"],"is_preprint":false},{"year":2017,"finding":"AUP1 directly interacts with apoB100 (apolipoprotein B-100) in HepG2 cells; this interaction is enhanced by proteasomal inhibition; AUP1 knockdown reduces apoB100 ubiquitination and intracellular degradation, enhancing apoB100 secretion; AUP1 knockdown increases LD size and stimulates VLDL-sized particle secretion with higher triglyceride content, independent of MEK-ERK signaling.","method":"siRNA knockdown, Co-immunoprecipitation (AUP1–apoB100), metabolic labeling/secretion assay, ubiquitination assay, LD size measurement by imaging","journal":"Arteriosclerosis, thrombosis, and vascular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with functional validation by KD and metabolic labeling, single lab, multiple orthogonal methods","pmids":["28183703"],"is_preprint":false},{"year":2024,"finding":"AUP1 interacts with the ER-resident form of the kidney cotransporter NKCC2 and with the ER lectin OS9; AUP1 co-expression increases ER retention and proteasome-dependent degradation of NKCC2; AUP1 knockdown or dominant-negative AUP1 expression reduces NKCC2 polyubiquitination and increases NKCC2 protein levels; AUP1 also interacts with and downregulates the related cotransporter NCC.","method":"Co-immunoprecipitation (AUP1–NKCC2, AUP1–OS9, AUP1–NCC), overexpression/knockdown with Western blot and proteasome inhibitor (MG132) rescue, dominant-negative AUP1 ubiquitination assay","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IPs, dominant-negative approach, pharmacological rescue, single lab","pmids":["38474353"],"is_preprint":false},{"year":2025,"finding":"AUP1 together with UBE2G2 interacts with STING and retains it in the ER membrane, preventing STING translocation to the Golgi and limiting STING signaling at rest; AUP1 deficiency causes spontaneous STING activation and enhanced type I IFN expression; AUP1 deficiency increases resistance to DNA virus infection in vitro and in vivo; for RNA virus VSV, AUP1 deficiency reduces lipid droplet accumulation and restricts VSV replication.","method":"Co-immunoprecipitation (AUP1–STING, UBE2G2–STING), AUP1/UBE2G2 KO with STING translocation assay (ER-to-Golgi), type I IFN reporter assay, in vitro and in vivo virus infection with KO cells, lipid droplet quantification","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, KO phenotype with multiple functional readouts (STING localization, IFN induction, virus replication), single lab","pmids":["40237449"],"is_preprint":false},{"year":2002,"finding":"AUP1 binds adenovirus E4ORF3 (from Ad5, Ad9, Ad40 but not Ad12) via the central part of E4ORF3 and the C-terminal region of AUP1; AUP1 also binds Ad5 E1A via AUP1's N-terminal segment; these interactions were detected in vitro.","method":"Yeast two-hybrid screen (initial identification), GST pulldown with in vitro translated E4ORF3 and E1A proteins, domain mapping","journal":"Tsitologiia","confidence":"Low","confidence_rationale":"Tier 3 / Weak — in vitro GST pulldown only, no in-cell validation reported, single lab","pmids":["12534236"],"is_preprint":false}],"current_model":"AUP1 is a lipid droplet- and ER-resident monotopic membrane protein that functions as an adaptor linking lipid droplets to the ubiquitin-proteasome system: its C-terminal G2BR domain recruits and allosterically activates the E2 ubiquitin-conjugating enzyme UBE2G2 at the ER membrane, facilitating polyubiquitination of ERAD substrates (including NKCC2 and apoB100) via the HRD1-SEL1L complex; its acyltransferase domain drives lipophagy (exploited by flaviviruses via NS4A); and together with UBE2G2 it retains STING in the ER to suppress basal innate immune signaling, while its expression level also controls cellular lipid droplet abundance and hepatic VLDL assembly."},"narrative":{"mechanistic_narrative":"AUP1 is a monotopic membrane protein of lipid droplets and the ER that acts as an adaptor coupling lipid droplet biology to the ubiquitin-proteasome system and ER-associated degradation (ERAD) [PMID:21127063, PMID:21857022]. Its C-terminal G2BR domain binds the E2 ubiquitin-conjugating enzyme UBE2G2 with low-nanomolar affinity at the backside of the enzyme, recruiting it to the ER membrane, protecting it from rapid degradation, and allosterically activating ubiquitination in conjunction with ERAD E3 ligases [PMID:21127063, PMID:34879065]. Through its CUE domain AUP1 physically associates with the HRD1-SEL1L dislocation complex, and its depletion impairs degradation of misfolded ER proteins while altering cellular lipid droplet abundance [PMID:21857022]. AUP1-driven ubiquitination governs the turnover of specific ERAD substrates, including the ER-retained form of the cotransporter NKCC2 (acting with the ER lectin OS9) and apolipoprotein B-100, where AUP1 loss reduces substrate ubiquitination, increases lipid droplet size, and enhances apoB100/VLDL secretion [PMID:28183703, PMID:38474353]. Beyond degradation, AUP1's acyltransferase domain drives lipophagy that is co-opted by dengue virus through its NS4A protein, with AUP1 mono-ubiquitylation disrupting the NS4A interaction and inhibiting acyltransferase activity [PMID:29902443]. Together with UBE2G2, AUP1 also retains STING in the ER to restrain basal type I interferon signaling, and its loss promotes spontaneous STING activation and altered antiviral resistance [PMID:40237449].","teleology":[{"year":2009,"claim":"Established a conserved autophagy-related role for the gene by showing the yeast ortholog is required for stationary-phase mitophagy and retrograde signaling, framing AUP1 within organelle quality-control biology.","evidence":"Genetic deletion (aup1Δ, rtg3Δ) with mitophagy assays, Rtg3 phosphorylation gel-shift, and RT-qPCR of RTG target genes in yeast","pmids":["19840933"],"confidence":"Medium","gaps":["Whether the mammalian protein retains the mitophagy/RTG-signaling role is not addressed","Mechanism linking Aup1 to Rtg3 phosphorylation not defined"]},{"year":2010,"claim":"Defined AUP1 as a monotopic lipid droplet surface protein that binds the E2 enzyme UBE2G2 through a discrete C-terminal G2BR domain, separating its membrane targeting from its enzyme-recruiting function.","evidence":"Confocal microscopy/fractionation for LD topology plus Co-IP and domain deletion/point mutation for G2BR–UBE2G2 binding","pmids":["21127063"],"confidence":"High","gaps":["Catalytic consequence of UBE2G2 recruitment not yet established","E3 ligase partners unidentified at this stage"]},{"year":2011,"claim":"Placed AUP1 mechanistically in ERAD by showing it associates with the HRD1-SEL1L complex, uses its CUE domain to regulate polyubiquitylation, and links lipid droplet abundance to ER quality control.","evidence":"Co-IP with HRD1-SEL1L, siRNA knockdown ERAD substrate degradation assays, CUE domain mutagenesis, and LD quantification","pmids":["21857022"],"confidence":"High","gaps":["Specific endogenous ERAD substrates not identified here","Structural basis of UBE2G2 activation unresolved"]},{"year":2017,"claim":"Identified apoB100 as a physiologically relevant AUP1-controlled substrate, connecting AUP1-mediated ubiquitination to hepatic lipoprotein secretion and lipid droplet size.","evidence":"siRNA knockdown, Co-IP of AUP1–apoB100, metabolic labeling/secretion assays, ubiquitination assays, and LD imaging in HepG2 cells","pmids":["28183703"],"confidence":"Medium","gaps":["Direct vs. indirect nature of AUP1–apoB100 interaction not fully resolved","In vivo VLDL regulation not tested"]},{"year":2018,"claim":"Revealed a degradation-independent function: AUP1's acyltransferase domain drives lipophagy that dengue virus exploits via NS4A, with mono-ubiquitylation acting as a switch on this activity.","evidence":"Ubiquitylation proteomics screen, Co-IP of AUP1–NS4A, acyltransferase point-mutant rescue, CRISPR/siRNA KO with virus titer, and imaging of LD-to-autophagosome relocalization","pmids":["29902443"],"confidence":"High","gaps":["Endogenous (non-viral) substrates of the acyltransferase activity unknown","Enzyme(s) catalyzing AUP1 mono-ubiquitylation not identified"]},{"year":2021,"claim":"Provided atomic-resolution mechanism for E2 activation by solving the G2BR–UBE2G2 structure and showing G2BR binding allosterically stimulates ubiquitination and stabilizes UBE2G2 at the ER.","evidence":"X-ray crystallography of the G2BR–UBE2G2 complex, biophysical affinity measurement, in vitro ubiquitination reconstitution, and cell-based ERAD assays with G2BR mutants","pmids":["34879065"],"confidence":"High","gaps":["How allosteric activation integrates with specific E3 ligases at the membrane not fully mapped"]},{"year":2024,"claim":"Extended AUP1's substrate repertoire to membrane transporters by demonstrating it promotes OS9-coupled ER retention and proteasomal degradation of NKCC2 and the related NCC.","evidence":"Reciprocal Co-IPs (AUP1–NKCC2, AUP1–OS9, AUP1–NCC), overexpression/knockdown with MG132 rescue, and dominant-negative ubiquitination assays","pmids":["38474353"],"confidence":"Medium","gaps":["Physiological impact on renal ion transport not established in vivo","Which E3 ligase mediates NKCC2 ubiquitination not defined"]},{"year":2025,"claim":"Uncovered an innate-immune role: AUP1 with UBE2G2 retains STING in the ER to suppress basal type I IFN, linking AUP1's ER residency to antiviral signaling control.","evidence":"Co-IP (AUP1–STING, UBE2G2–STING), AUP1/UBE2G2 KO STING ER-to-Golgi translocation assays, IFN reporter assays, and in vitro/in vivo virus infection","pmids":["40237449"],"confidence":"Medium","gaps":["Whether STING retention requires ubiquitination or only physical sequestration unclear","Reciprocal validation across cell types limited to single lab"]},{"year":null,"claim":"How AUP1's two biochemical activities — UBE2G2-dependent ubiquitination and lipid-droplet acyltransferase activity — are coordinated within a single protein, and what determines substrate and pathway selection, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model coupling acyltransferase and ubiquitin-adaptor functions","Regulatory inputs (e.g. AUP1 ubiquitylation) governing pathway choice not characterized","Disease relevance in mammals not established by direct genetic evidence in the corpus"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[3]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2]}],"localization":[{"term_id":"GO:0005811","term_label":"lipid droplet","supporting_discovery_ids":[0,3]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[1,2,6,7]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,2,5,6]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[3]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[7]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[5]}],"complexes":["HRD1-SEL1L ERAD complex"],"partners":["UBE2G2","HRD1","SEL1L","OS9","NKCC2","APOB","STING1","NCC"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9Y679","full_name":"Lipid droplet-regulating VLDL assembly factor AUP1","aliases":["Ancient ubiquitous protein 1"],"length_aa":410,"mass_kda":45.8,"function":"Plays a role in the translocation of terminally misfolded proteins from the endoplasmic reticulum lumen to the cytoplasm and their degradation by the proteasome (PubMed:18711132, PubMed:21857022). Plays a role in lipid droplet formation (PubMed:21857022). Induces lipid droplet clustering (PubMed:24039768). Recruits ubiquitin-conjugating enzyme UBE2G2 to lipid droplets which facilitates its interaction with ubiquitin ligases AMFR/gp78 and RNF139/TRC8, leading to sterol-induced ubiquitination of HMGCR and its subsequent proteasomal degradation (PubMed:21127063, PubMed:23223569). Also required for the degradation of INSIG1, SREBF1 and SREBF2 (PubMed:23223569). Plays a role in regulating assembly and secretion of very low density lipoprotein particles and stability of apolipoprotein APOB (PubMed:28183703) (Microbial infection) Following Dengue virus infection, required for induction of lipophagy which facilitates production of virus progeny particles","subcellular_location":"Cytoplasmic vesicle, autophagosome","url":"https://www.uniprot.org/uniprotkb/Q9Y679/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/AUP1","classification":"Not Classified","n_dependent_lines":60,"n_total_lines":1208,"dependency_fraction":0.04966887417218543},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CANX","stoichiometry":0.2},{"gene":"VCP","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/AUP1","total_profiled":1310},"omim":[{"mim_id":"616175","title":"UBIQUITIN-CONJUGATING ENZYME E2 J1; UBE2J1","url":"https://www.omim.org/entry/616175"},{"mim_id":"610304","title":"DER1-LIKE DOMAIN FAMILY, MEMBER 2; DERL2","url":"https://www.omim.org/entry/610304"},{"mim_id":"609677","title":"OS9 ENDOPLASMIC RETICULUM LECTIN; OS9","url":"https://www.omim.org/entry/609677"},{"mim_id":"608046","title":"SYNOVIAL APOPTOSIS INHIBITOR 1; SYVN1","url":"https://www.omim.org/entry/608046"},{"mim_id":"602434","title":"ANCIENT UBIQUITOUS PROTEIN 1; AUP1","url":"https://www.omim.org/entry/602434"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Vesicles","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/AUP1"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"Q9Y679","domains":[{"cath_id":"-","chopping":"10-260","consensus_level":"high","plddt":87.4403,"start":10,"end":260},{"cath_id":"1.10.8.10","chopping":"294-336","consensus_level":"high","plddt":77.4967,"start":294,"end":336}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y679","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y679-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y679-F1-predicted_aligned_error_v6.png","plddt_mean":79.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=AUP1","jax_strain_url":"https://www.jax.org/strain/search?query=AUP1"},"sequence":{"accession":"Q9Y679","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y679.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y679/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y679"}},"corpus_meta":[{"pmid":"29902443","id":"PMC_29902443","title":"Flaviviruses Exploit the Lipid Droplet Protein AUP1 to Trigger Lipophagy and Drive Virus Production.","date":"2018","source":"Cell host & microbe","url":"https://pubmed.ncbi.nlm.nih.gov/29902443","citation_count":158,"is_preprint":false},{"pmid":"21127063","id":"PMC_21127063","title":"Ancient ubiquitous protein 1 (AUP1) localizes to lipid droplets and binds the E2 ubiquitin conjugase G2 (Ube2g2) via its G2 binding region.","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21127063","citation_count":105,"is_preprint":false},{"pmid":"21857022","id":"PMC_21857022","title":"Dual role of ancient ubiquitous protein 1 (AUP1) in lipid droplet accumulation and endoplasmic reticulum (ER) protein quality control.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21857022","citation_count":105,"is_preprint":false},{"pmid":"19840933","id":"PMC_19840933","title":"Aup1-mediated regulation of Rtg3 during mitophagy.","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19840933","citation_count":75,"is_preprint":false},{"pmid":"35633317","id":"PMC_35633317","title":"AUP1 regulates lipid metabolism and induces lipid accumulation to accelerate the progression of renal clear cell carcinoma.","date":"2022","source":"Cancer science","url":"https://pubmed.ncbi.nlm.nih.gov/35633317","citation_count":37,"is_preprint":false},{"pmid":"28183703","id":"PMC_28183703","title":"AUP1 (Ancient Ubiquitous Protein 1) Is a Key Determinant of Hepatic Very-Low-Density Lipoprotein Assembly and Secretion.","date":"2017","source":"Arteriosclerosis, thrombosis, and vascular biology","url":"https://pubmed.ncbi.nlm.nih.gov/28183703","citation_count":18,"is_preprint":false},{"pmid":"34879065","id":"PMC_34879065","title":"A structurally conserved site in AUP1 binds the E2 enzyme UBE2G2 and is essential for ER-associated degradation.","date":"2021","source":"PLoS biology","url":"https://pubmed.ncbi.nlm.nih.gov/34879065","citation_count":14,"is_preprint":false},{"pmid":"8812468","id":"PMC_8812468","title":"Aup1, a novel gene on mouse chromosome 6 and human chromosome 2p13.","date":"1996","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/8812468","citation_count":11,"is_preprint":false},{"pmid":"38474353","id":"PMC_38474353","title":"AUP1 Regulates the Endoplasmic Reticulum-Associated Degradation and Polyubiquitination of NKCC2.","date":"2024","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/38474353","citation_count":4,"is_preprint":false},{"pmid":"39329209","id":"PMC_39329209","title":"AUP1 transcriptionally activated by KDM5B reprograms lipid metabolism to promote the malignant progression of cervical cancer.","date":"2024","source":"International journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/39329209","citation_count":4,"is_preprint":false},{"pmid":"40237449","id":"PMC_40237449","title":"AUP1 and UBE2G2 complex targets STING signaling and regulates virus-induced innate immunity.","date":"2025","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/40237449","citation_count":1,"is_preprint":false},{"pmid":"12534238","id":"PMC_12534238","title":"[A new human cellular protein AUP1. III. The intracellular localization of AUP1 protein in different human and rat cell lines].","date":"2002","source":"Tsitologiia","url":"https://pubmed.ncbi.nlm.nih.gov/12534238","citation_count":1,"is_preprint":false},{"pmid":"12534237","id":"PMC_12534237","title":"[A new human cellular protein AUP1. II. cDNA cloning, genomic organization of Aup1 gene ans preliminary characterization of human AUP1 protein].","date":"2002","source":"Tsitologiia","url":"https://pubmed.ncbi.nlm.nih.gov/12534237","citation_count":1,"is_preprint":false},{"pmid":"12534236","id":"PMC_12534236","title":"[A new human cellular protein AUP1. I. In vitro interaction of AUP1 with adenoviral proteins E4ORF3 and E1A].","date":"2002","source":"Tsitologiia","url":"https://pubmed.ncbi.nlm.nih.gov/12534236","citation_count":1,"is_preprint":false},{"pmid":"40484276","id":"PMC_40484276","title":"The role of ancient ubiquitous protein 1 (Aup1) in regulating hepatic lipid droplet levels, endoplasmic reticulum stress, and inflammation in zebrafish (Danio rerio).","date":"2025","source":"Biochimica et biophysica acta. Molecular and cell biology of lipids","url":"https://pubmed.ncbi.nlm.nih.gov/40484276","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9860,"output_tokens":2839,"usd":0.036083,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9995,"output_tokens":3400,"usd":0.067488,"stage2_stop_reason":"end_turn"},"total_usd":0.103571,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2010,\n      \"finding\": \"AUP1 localizes to lipid droplets (LDs) by integrating into the LD surface in a monotopic fashion with both termini facing the cytosol, and binds the E2 ubiquitin conjugase Ube2g2 via its C-terminal G2BR (G2 binding region) domain; deletion or mutation of G2BR abolishes Ube2g2 binding without affecting LD localization.\",\n      \"method\": \"Confocal microscopy/cell fractionation (LD localization), Co-immunoprecipitation and domain deletion/mutagenesis (G2BR–Ube2g2 interaction)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal binding confirmed by domain deletion and point mutation, LD topology independently established, replicated in subsequent studies\",\n      \"pmids\": [\"21127063\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"AUP1 physically associates with the mammalian HRD1-SEL1L ERAD complex; its CUE domain regulates polyubiquitylation and facilitates interaction with the HRD1 complex and dislocation substrates; AUP1 recruits UBE2G2 to the ER membrane for ERAD; AUP1 depletion impairs degradation of misfolded ER proteins; AUP1 expression level controls cellular lipid droplet abundance, representing the first protein linking LD regulation to ER quality control.\",\n      \"method\": \"Co-immunoprecipitation (HRD1-SEL1L complex association), siRNA knockdown with ERAD substrate degradation assay, domain mutagenesis (CUE domain), LD quantification upon AUP1 overexpression/depletion\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, KD, domain mutagenesis, substrate assay) in single study, later independently replicated\",\n      \"pmids\": [\"21857022\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The 27-amino acid G2BR of AUP1 binds UBE2G2 with low nanomolar affinity at the backside of the E2 enzyme; the crystal structure of the G2BR–UBE2G2 complex reveals a network of salt bridges, hydrogen bonds, and hydrophobic interactions; G2BR–UBE2G2 binding allosterically activates ubiquitination in vitro in conjunction with ERAD E3s; AUP1 G2BR is required for ER membrane recruitment of UBE2G2, prevents its rapid degradation, and is essential for multiple ERAD pathways in cells.\",\n      \"method\": \"X-ray crystallography (structure of G2BR–UBE2G2 complex), biophysical binding assays (nanomolar affinity), in vitro ubiquitination reconstitution with mutagenesis, cell-based ERAD assays with G2BR mutants\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure + in vitro reconstitution + mutagenesis + cell-based functional validation in one study\",\n      \"pmids\": [\"34879065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"AUP1 associates with dengue virus NS4A at lipid droplets; AUP1's acyltransferase domain activity is required for DENV-triggered lipophagy and virus production; mono-ubiquitylation of AUP1 disrupts the AUP1–NS4A interaction and inhibits acyltransferase activity, thereby attenuating lipophagy and virus production; upon infection AUP1 relocalizes from lipid droplets to autophagosomes.\",\n      \"method\": \"Functional proteomics ubiquitylation screen, Co-immunoprecipitation (AUP1–NS4A), acyltransferase domain point mutant rescue, CRISPR/siRNA KO with virus titer readout, fluorescence microscopy (LD-to-autophagosome relocalization)\",\n      \"journal\": \"Cell host & microbe\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (proteomics, Co-IP, domain mutant, KO, imaging) with mechanistic pathway placement\",\n      \"pmids\": [\"29902443\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Yeast Aup1 (mitochondrial protein phosphatase homolog) is required for efficient stationary-phase mitophagy; Aup1 regulates the retrograde (RTG) signaling pathway, controls phosphorylation of the transcription factor Rtg3, and is required for induction of RTG target genes under mitophagic conditions; deletion of RTG3 itself causes a defect in stationary-phase mitophagy.\",\n      \"method\": \"Genetic deletion (aup1Δ, rtg3Δ) with mitophagy assay, phosphorylation analysis of Rtg3 by gel shift, RT-qPCR of RTG target genes\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean yeast KO with multiple readouts (mitophagy, RTG target genes, Rtg3 phosphorylation), single lab study\",\n      \"pmids\": [\"19840933\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"AUP1 directly interacts with apoB100 (apolipoprotein B-100) in HepG2 cells; this interaction is enhanced by proteasomal inhibition; AUP1 knockdown reduces apoB100 ubiquitination and intracellular degradation, enhancing apoB100 secretion; AUP1 knockdown increases LD size and stimulates VLDL-sized particle secretion with higher triglyceride content, independent of MEK-ERK signaling.\",\n      \"method\": \"siRNA knockdown, Co-immunoprecipitation (AUP1–apoB100), metabolic labeling/secretion assay, ubiquitination assay, LD size measurement by imaging\",\n      \"journal\": \"Arteriosclerosis, thrombosis, and vascular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with functional validation by KD and metabolic labeling, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"28183703\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"AUP1 interacts with the ER-resident form of the kidney cotransporter NKCC2 and with the ER lectin OS9; AUP1 co-expression increases ER retention and proteasome-dependent degradation of NKCC2; AUP1 knockdown or dominant-negative AUP1 expression reduces NKCC2 polyubiquitination and increases NKCC2 protein levels; AUP1 also interacts with and downregulates the related cotransporter NCC.\",\n      \"method\": \"Co-immunoprecipitation (AUP1–NKCC2, AUP1–OS9, AUP1–NCC), overexpression/knockdown with Western blot and proteasome inhibitor (MG132) rescue, dominant-negative AUP1 ubiquitination assay\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IPs, dominant-negative approach, pharmacological rescue, single lab\",\n      \"pmids\": [\"38474353\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"AUP1 together with UBE2G2 interacts with STING and retains it in the ER membrane, preventing STING translocation to the Golgi and limiting STING signaling at rest; AUP1 deficiency causes spontaneous STING activation and enhanced type I IFN expression; AUP1 deficiency increases resistance to DNA virus infection in vitro and in vivo; for RNA virus VSV, AUP1 deficiency reduces lipid droplet accumulation and restricts VSV replication.\",\n      \"method\": \"Co-immunoprecipitation (AUP1–STING, UBE2G2–STING), AUP1/UBE2G2 KO with STING translocation assay (ER-to-Golgi), type I IFN reporter assay, in vitro and in vivo virus infection with KO cells, lipid droplet quantification\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, KO phenotype with multiple functional readouts (STING localization, IFN induction, virus replication), single lab\",\n      \"pmids\": [\"40237449\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"AUP1 binds adenovirus E4ORF3 (from Ad5, Ad9, Ad40 but not Ad12) via the central part of E4ORF3 and the C-terminal region of AUP1; AUP1 also binds Ad5 E1A via AUP1's N-terminal segment; these interactions were detected in vitro.\",\n      \"method\": \"Yeast two-hybrid screen (initial identification), GST pulldown with in vitro translated E4ORF3 and E1A proteins, domain mapping\",\n      \"journal\": \"Tsitologiia\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — in vitro GST pulldown only, no in-cell validation reported, single lab\",\n      \"pmids\": [\"12534236\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"AUP1 is a lipid droplet- and ER-resident monotopic membrane protein that functions as an adaptor linking lipid droplets to the ubiquitin-proteasome system: its C-terminal G2BR domain recruits and allosterically activates the E2 ubiquitin-conjugating enzyme UBE2G2 at the ER membrane, facilitating polyubiquitination of ERAD substrates (including NKCC2 and apoB100) via the HRD1-SEL1L complex; its acyltransferase domain drives lipophagy (exploited by flaviviruses via NS4A); and together with UBE2G2 it retains STING in the ER to suppress basal innate immune signaling, while its expression level also controls cellular lipid droplet abundance and hepatic VLDL assembly.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"AUP1 is a monotopic membrane protein of lipid droplets and the ER that acts as an adaptor coupling lipid droplet biology to the ubiquitin-proteasome system and ER-associated degradation (ERAD) [#0, #1]. Its C-terminal G2BR domain binds the E2 ubiquitin-conjugating enzyme UBE2G2 with low-nanomolar affinity at the backside of the enzyme, recruiting it to the ER membrane, protecting it from rapid degradation, and allosterically activating ubiquitination in conjunction with ERAD E3 ligases [#0, #2]. Through its CUE domain AUP1 physically associates with the HRD1-SEL1L dislocation complex, and its depletion impairs degradation of misfolded ER proteins while altering cellular lipid droplet abundance [#1]. AUP1-driven ubiquitination governs the turnover of specific ERAD substrates, including the ER-retained form of the cotransporter NKCC2 (acting with the ER lectin OS9) and apolipoprotein B-100, where AUP1 loss reduces substrate ubiquitination, increases lipid droplet size, and enhances apoB100/VLDL secretion [#5, #6]. Beyond degradation, AUP1's acyltransferase domain drives lipophagy that is co-opted by dengue virus through its NS4A protein, with AUP1 mono-ubiquitylation disrupting the NS4A interaction and inhibiting acyltransferase activity [#3]. Together with UBE2G2, AUP1 also retains STING in the ER to restrain basal type I interferon signaling, and its loss promotes spontaneous STING activation and altered antiviral resistance [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Established a conserved autophagy-related role for the gene by showing the yeast ortholog is required for stationary-phase mitophagy and retrograde signaling, framing AUP1 within organelle quality-control biology.\",\n      \"evidence\": \"Genetic deletion (aup1Δ, rtg3Δ) with mitophagy assays, Rtg3 phosphorylation gel-shift, and RT-qPCR of RTG target genes in yeast\",\n      \"pmids\": [\"19840933\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the mammalian protein retains the mitophagy/RTG-signaling role is not addressed\", \"Mechanism linking Aup1 to Rtg3 phosphorylation not defined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined AUP1 as a monotopic lipid droplet surface protein that binds the E2 enzyme UBE2G2 through a discrete C-terminal G2BR domain, separating its membrane targeting from its enzyme-recruiting function.\",\n      \"evidence\": \"Confocal microscopy/fractionation for LD topology plus Co-IP and domain deletion/point mutation for G2BR–UBE2G2 binding\",\n      \"pmids\": [\"21127063\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Catalytic consequence of UBE2G2 recruitment not yet established\", \"E3 ligase partners unidentified at this stage\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Placed AUP1 mechanistically in ERAD by showing it associates with the HRD1-SEL1L complex, uses its CUE domain to regulate polyubiquitylation, and links lipid droplet abundance to ER quality control.\",\n      \"evidence\": \"Co-IP with HRD1-SEL1L, siRNA knockdown ERAD substrate degradation assays, CUE domain mutagenesis, and LD quantification\",\n      \"pmids\": [\"21857022\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific endogenous ERAD substrates not identified here\", \"Structural basis of UBE2G2 activation unresolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified apoB100 as a physiologically relevant AUP1-controlled substrate, connecting AUP1-mediated ubiquitination to hepatic lipoprotein secretion and lipid droplet size.\",\n      \"evidence\": \"siRNA knockdown, Co-IP of AUP1–apoB100, metabolic labeling/secretion assays, ubiquitination assays, and LD imaging in HepG2 cells\",\n      \"pmids\": [\"28183703\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs. indirect nature of AUP1–apoB100 interaction not fully resolved\", \"In vivo VLDL regulation not tested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Revealed a degradation-independent function: AUP1's acyltransferase domain drives lipophagy that dengue virus exploits via NS4A, with mono-ubiquitylation acting as a switch on this activity.\",\n      \"evidence\": \"Ubiquitylation proteomics screen, Co-IP of AUP1–NS4A, acyltransferase point-mutant rescue, CRISPR/siRNA KO with virus titer, and imaging of LD-to-autophagosome relocalization\",\n      \"pmids\": [\"29902443\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous (non-viral) substrates of the acyltransferase activity unknown\", \"Enzyme(s) catalyzing AUP1 mono-ubiquitylation not identified\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Provided atomic-resolution mechanism for E2 activation by solving the G2BR–UBE2G2 structure and showing G2BR binding allosterically stimulates ubiquitination and stabilizes UBE2G2 at the ER.\",\n      \"evidence\": \"X-ray crystallography of the G2BR–UBE2G2 complex, biophysical affinity measurement, in vitro ubiquitination reconstitution, and cell-based ERAD assays with G2BR mutants\",\n      \"pmids\": [\"34879065\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How allosteric activation integrates with specific E3 ligases at the membrane not fully mapped\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended AUP1's substrate repertoire to membrane transporters by demonstrating it promotes OS9-coupled ER retention and proteasomal degradation of NKCC2 and the related NCC.\",\n      \"evidence\": \"Reciprocal Co-IPs (AUP1–NKCC2, AUP1–OS9, AUP1–NCC), overexpression/knockdown with MG132 rescue, and dominant-negative ubiquitination assays\",\n      \"pmids\": [\"38474353\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological impact on renal ion transport not established in vivo\", \"Which E3 ligase mediates NKCC2 ubiquitination not defined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Uncovered an innate-immune role: AUP1 with UBE2G2 retains STING in the ER to suppress basal type I IFN, linking AUP1's ER residency to antiviral signaling control.\",\n      \"evidence\": \"Co-IP (AUP1–STING, UBE2G2–STING), AUP1/UBE2G2 KO STING ER-to-Golgi translocation assays, IFN reporter assays, and in vitro/in vivo virus infection\",\n      \"pmids\": [\"40237449\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether STING retention requires ubiquitination or only physical sequestration unclear\", \"Reciprocal validation across cell types limited to single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How AUP1's two biochemical activities — UBE2G2-dependent ubiquitination and lipid-droplet acyltransferase activity — are coordinated within a single protein, and what determines substrate and pathway selection, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model coupling acyltransferase and ubiquitin-adaptor functions\", \"Regulatory inputs (e.g. AUP1 ubiquitylation) governing pathway choice not characterized\", \"Disease relevance in mammals not established by direct genetic evidence in the corpus\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005811\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [1, 2, 6, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 2, 5, 6]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"complexes\": [\"HRD1-SEL1L ERAD complex\"],\n    \"partners\": [\"UBE2G2\", \"HRD1\", \"SEL1L\", \"OS9\", \"NKCC2\", \"APOB\", \"STING1\", \"NCC\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":6,"faith_total":6,"faith_pct":100.0}}