{"gene":"ATE1","run_date":"2026-04-28T17:12:37","timeline":{"discoveries":[{"year":1990,"finding":"ATE1 encodes arginyl-tRNA-protein transferase (R-transferase) that catalyzes post-translational conjugation of Arg to N-terminal residues of acceptor proteins; expression of yeast ATE1 in E. coli (which lacks R-transferases) conferred Arg-transferase activity, and null ate1 mutants lack this activity and cannot degrade N-end rule substrates requiring Arg-transferase action.","method":"Heterologous expression in E. coli, null mutant analysis, in vitro enzymatic assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — reconstituted activity in heterologous host, functional null mutant phenotype, foundational paper with 140 citations","pmids":["2185248"],"is_preprint":false},{"year":2014,"finding":"ATE1 catalyzes arginylation not only at N-terminal alpha-amino groups but also at internal side-chain carboxylates of Asp and Glu residues within intact proteins in vivo, revealing an unconventional midchain arginylation mechanism.","method":"Mass spectrometry (in vivo arginylation profiling), in vitro ATE1 arginylation assay with purified components","journal":"Chemistry & biology","confidence":"High","confidence_rationale":"Tier 1 — in vitro assay with purified enzyme plus in vivo MS identification; replicated across multiple protein targets","pmids":["24529990"],"is_preprint":false},{"year":2022,"finding":"Crystal structure of K. lactis Ate1 R-transferase at 58 kDa reveals two domains that together recognize an acidic N-terminal residue of the acceptor substrate, the Arg residue of Arg-tRNAArg, and the 3'-proximal tRNAArg segment; the active site is located between the two domains. Hemin (Fe3+-heme) inhibits Nt-arginylation activity and induces disulfide-mediated oligomerization of Ate1. Site-directed mutagenesis guided by the structure identified specific binding residues.","method":"X-ray crystallography, site-directed mutagenesis, in vitro and in vivo arginylation assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with mutagenesis validation and biochemical assays in a single study","pmids":["35878037"],"is_preprint":false},{"year":2022,"finding":"Crystal structure of S. cerevisiae ATE1 (ScATE1) in apo form reveals a bilobed protein with a GCN5-related N-acetyltransferase (GNAT) fold; the domain-domain interface constitutes the catalytic active site and tRNA-binding region. The N-terminal domain that binds a regulatory [Fe-S] cluster is dynamic and disordered in the absence of metal, indicating regulatory influence of this region.","method":"X-ray crystallography, SEC-SAXS, cryo-EM 2D class averaging, structural superposition","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure validated by SAXS and cryo-EM; orthogonal methods in single study","pmids":["36087779"],"is_preprint":false},{"year":2025,"finding":"Cryo-EM structure of human ATE1 in complex with Arg-tRNAArg and an N-terminal Asp peptide shows two adjacent pockets binding the Nt-substrate and Arg-tRNAArg respectively, the tRNA being wrapped by a long unstructured loop. In the apo state, two ATE1 monomers form a homodimer. Substrate selectivity is achieved through multivalent interactions with Kd values in the micromolar range.","method":"Cryo-EM structure determination, binding affinity measurements","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structure with biochemical binding characterization; human enzyme","pmids":["40099869"],"is_preprint":false},{"year":2016,"finding":"ATE1/arginylation promotes cell death and/or growth arrest in response to oxidative, heat, osmotic, heavy metal, and radiation stresses; ATE1 protein levels rise under acute stress, and the elevated ATE1 directly promotes cell death in an arginylation-activity-dependent manner. ATE1 is also required to suppress mutation frequency under DNA-damaging conditions.","method":"ATE1 knockout/knockdown in yeast, mouse, and human cells; genetic complementation with catalytically inactive ATE1; functional cell death and mutagenesis assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — multiple organisms, loss-of-function with defined phenotypes, activity-dependent rescue; single lab","pmids":["27685622"],"is_preprint":false},{"year":2017,"finding":"ATE1 localizes prominently to neuronal growth cones via zipcode-mediated mRNA targeting; Ate1 mRNA co-localizes with arginylated β-actin at growth cone tips, and co-translational arginylation of β-actin at growth cones drives growth cone migration and neurite outgrowth. Conditional Ate1 knockout in the nervous system causes defects in neuronal migration, reduced neurite outgrowth, and decreased doublecortin and F-actin in growth cones.","method":"Conditional mouse knockout (Nestin-Cre), live-cell imaging, FISH, fluorescence microscopy, protein synthesis inhibitor experiments","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 — conditional KO with defined cellular phenotype and direct localization evidence; single lab with orthogonal methods","pmids":["28844905"],"is_preprint":false},{"year":2013,"finding":"ATE1 directly interacts with myosin II in platelets (co-immunoprecipitation); platelet-specific ATE1 knockout leads to enhanced myosin regulatory light chain phosphorylation at Ser19, enhanced clot retraction, and increased in vivo thrombus formation, indicating arginylation modulates myosin contractility.","method":"Conditional platelet/megakaryocyte-specific knockout mouse, co-immunoprecipitation, phosphorylation analysis, in vivo thrombosis assay","journal":"Haematologica","confidence":"Medium","confidence_rationale":"Tier 2 — conditional KO with defined biochemical and in vivo phenotype, direct Co-IP; single lab","pmids":["24293517"],"is_preprint":false},{"year":2018,"finding":"In Dictyostelium discoideum, Ate1 knockout abolishes focal actin adhesion sites and reduces adhesion; mass spectrometry identified four arginylation sites in the major actin isoform plus sites in actin-binding proteins. Actin from ate1-null cells has diminished polymerization capacity in vitro. Ate1-GFP rapidly relocalizes to sites of newly formed actin-rich protrusions.","method":"Gene knockout, live-cell microscopy, mass spectrometry, in vitro actin polymerization assay, chemotaxis assay","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 — KO with multiple orthogonal readouts including in vitro reconstitution; model organism ortholog","pmids":["30586322"],"is_preprint":false},{"year":2021,"finding":"Ate1 regulates RGS7 protein levels by facilitating its proteasomal degradation; conditional Ate1 knockout in the nervous system elevates RGS7 levels in retinal ON bipolar cells, increases light-evoked ON-bipolar response sensitivities, and in cultured MEFs, RGS7 degradation by the proteasome is abolished in Ate1 knockout cells.","method":"Conditional mouse knockout (nervous system), electroretinography, proteasome inhibitor experiments in MEF cells","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 — conditional KO with defined molecular and physiological phenotypes; two cell contexts tested","pmids":["33931669"],"is_preprint":false},{"year":2021,"finding":"ATE1 splicing is controlled by competing RNA secondary structures: five conserved intronic elements (R1–R5) regulate mutual exclusion of exons 7a and 7b, with R1 and R4 competing for base-pairing with R3, and an ultra-long-range R2–R5 interaction spanning 30 kb. Disruption of these RNA structures by mutation or LNA/DNA mixmers abolishes mutually exclusive exon splicing, and co-transcriptional folding links exon 7a inclusion to RNA Pol II elongation rate.","method":"Minigene splicing assay with single/double/compensatory mutations, LNA/DNA mixmer blocking, endogenous pre-mRNA analysis","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 — compensatory mutagenesis in minigenes plus endogenous blocking experiments; orthogonal mechanistic evidence","pmids":["33330934"],"is_preprint":false},{"year":2020,"finding":"Liat1 (Ligand of Ate1) physically interacts with Ate1 and undergoes liquid-liquid phase separation in the nucleolus via an intrinsically disordered N-terminal region; Jmjd6 lysyl-hydroxylase modifies Liat1's poly-K region and inhibits its nucleolar targeting.","method":"Bimolecular fluorescence complementation, immunocytochemistry, IDR characterization, phase separation assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2–3 — direct protein interaction shown by BiFC with functional nucleolar localization consequence; single lab","pmids":["33443146"],"is_preprint":false},{"year":2023,"finding":"ATE1 binds a regulatory iron-sulfur ([Fe-S]) cluster in its N-terminal domain that functions as an oxygen sensor to regulate ATE1 activity; chemical reconstitution under anoxic conditions restores [Fe-S] cluster binding in both yeast and mouse ATE1.","method":"Anaerobic [Fe-S] cluster reconstitution, spectroscopic characterization","journal":"Methods in molecular biology","confidence":"Medium","confidence_rationale":"Tier 1 in vitro reconstitution of cofactor; single lab but biochemically defined","pmids":["37010764"],"is_preprint":false},{"year":2024,"finding":"Mouse ATE1 contains an intrinsically disordered region (IDR) absent in yeast ATE1; HDX-MS and SAXS demonstrate this IDR is present in all mouse ATE1 splice variants and is predicted to facilitate complex formation with tRNAArg.","method":"SEC, SAXS, HDX-MS, AlphaFold modeling, bioinformatics","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal biophysical methods; single lab; functional role is computational inference","pmids":["39642180"],"is_preprint":false},{"year":2024,"finding":"tRNAArg directly binds in vitro to TDP-43 RNA recognition motifs and stabilizes binding via TDP-43 dimerization (promoted by the N-terminal domain and NLS); the arginylation cofactor LIAT1 also binds tRNAArg in vitro, identifying LIAT1 as an RNA-binding protein.","method":"In vitro binding assays with recombinant proteins and in vitro-transcribed tRNA","journal":"microPublication biology","confidence":"Low","confidence_rationale":"Tier 3 — in vitro binding only, single study, no functional consequence directly tested for LIAT1-tRNA interaction","pmids":["39081859"],"is_preprint":false},{"year":2021,"finding":"ATE1 inhibits liver cancer growth by facilitating proteasomal degradation of RGS5, which in turn maintains GSK3-β activity and promotes β-catenin degradation, thereby suppressing Wnt/β-catenin signaling; loss- and gain-of-function assays with RGS5 and GSK3-β inhibitor confirmed RGS5 as a key effector downstream of ATE1.","method":"Lentivirus-mediated knockdown/overexpression, loss- and gain-of-function, GSK3-β inhibitor rescue experiments, in vitro and in vivo tumor assays","journal":"Molecular cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — epistasis-type rescue experiments placing ATE1 upstream of RGS5 and GSK3-β/β-catenin; single lab","pmids":["34158395"],"is_preprint":false},{"year":2024,"finding":"Mitochondrial translocation of Ate1 is promoted by oxidative stressors and is essential for inducing apoptotic cell death; Ate1-induced cell death depends on mitochondrial permeability pore formation and apoptosis-inducing factor, but not on electron transport chain activity or ROS, and is independent of cytosolic ubiquitin-proteasome, autophagy, or ER stress pathways.","method":"Yeast genetic and cell biology experiments, mitochondrial fractionation, mitochondrial permeability pore assays, AIF analysis","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — multiple genetic and cell biology approaches; preprint, not yet peer-reviewed","pmids":["bio_10.1101_2024.11.22.624728"],"is_preprint":true}],"current_model":"ATE1 is an arginyl-tRNA-protein transferase that post-translationally transfers Arg from Arg-tRNAArg to N-terminal acidic residues (Asp, Glu, oxidized Cys) and internal side-chain carboxylates of substrate proteins via a bilobed GNAT-fold enzyme whose active site sits at the domain-domain interface; this N-terminal arginylation creates Arg/N-degrons that direct substrates to proteasomal or autophagic degradation, regulates actin cytoskeleton dynamics, myosin contractility, neuronal growth cone migration, and stress-induced cell death (including via mitochondrial translocation), and its activity is regulated by an [Fe-S] cluster oxygen sensor and by alternative splicing of mutually exclusive exons controlled by competing RNA secondary structures."},"narrative":{"teleology":[{"year":1990,"claim":"Establishing the molecular identity of ATE1 as the gene encoding arginyl-tRNA-protein transferase resolved how the N-end rule pathway activates degradation signals on protein substrates.","evidence":"Heterologous expression of yeast ATE1 in E. coli conferred Arg-transferase activity; ate1-null yeast lacked this activity and failed to degrade N-end rule substrates.","pmids":["2185248"],"confidence":"High","gaps":["Substrate scope beyond N-end rule model peptides unknown","Mammalian ATE1 not yet characterized","Catalytic mechanism unresolved"]},{"year":2013,"claim":"Demonstrating that ATE1 directly interacts with myosin II and modulates its regulatory light chain phosphorylation extended arginylation beyond protein degradation to actomyosin contractility regulation.","evidence":"Platelet-specific Ate1 knockout mice showed enhanced MLC phosphorylation, increased clot retraction, and in vivo thrombus formation; Co-IP confirmed direct ATE1–myosin II interaction.","pmids":["24293517"],"confidence":"Medium","gaps":["Whether myosin II is directly arginylated was not determined","Mechanism linking arginylation to MLC phosphorylation state unclear","Single Co-IP without reciprocal validation"]},{"year":2014,"claim":"Discovery of midchain arginylation at internal Asp/Glu side-chain carboxylates revealed that ATE1 catalytic scope extends beyond N-terminal modification, fundamentally broadening the substrate landscape.","evidence":"In vivo mass spectrometry identified internal arginylation sites on intact proteins; in vitro assays with purified ATE1 confirmed midchain conjugation.","pmids":["24529990"],"confidence":"High","gaps":["Functional consequences of specific midchain arginylation events not established","Structural basis for midchain versus N-terminal selectivity unknown"]},{"year":2016,"claim":"Showing that ATE1 protein accumulates under diverse stresses and promotes arginylation-dependent cell death established ATE1 as a stress-response effector rather than solely a constitutive housekeeping enzyme.","evidence":"ATE1 KO/KD across yeast, mouse, and human cells reduced stress-induced cell death; complementation with catalytically inactive ATE1 failed to rescue.","pmids":["27685622"],"confidence":"Medium","gaps":["Critical stress-induced arginylation substrates not identified","Mechanism of ATE1 protein stabilization under stress unknown"]},{"year":2017,"claim":"Localizing Ate1 mRNA to neuronal growth cones and showing co-translational arginylation of β-actin there connected ATE1 to local cytoskeletal remodeling required for neuronal migration and outgrowth.","evidence":"Conditional Ate1 KO in mouse nervous system caused growth cone defects, reduced neurite outgrowth, and decreased F-actin; FISH showed co-localization of Ate1 mRNA with arginylated β-actin at growth cone tips.","pmids":["28844905"],"confidence":"Medium","gaps":["Zipcode elements in Ate1 mRNA not mapped","Whether arginylation of actin is sufficient to rescue growth cone defects not tested"]},{"year":2018,"claim":"Demonstrating that Ate1 knockout abolishes actin focal adhesions and reduces actin polymerization capacity in Dictyostelium confirmed a conserved role for arginylation in actin dynamics beyond vertebrates.","evidence":"Ate1-null Dictyostelium lacked focal adhesion sites; MS identified arginylation sites on actin; in vitro polymerization of ate1-null actin was diminished; Ate1-GFP relocated to actin-rich protrusions.","pmids":["30586322"],"confidence":"Medium","gaps":["Whether individual arginylation sites on actin are functionally distinct not resolved","Direct structural effect of arginylation on actin filament properties unknown"]},{"year":2021,"claim":"Identifying RGS7 and RGS5 as ATE1-dependent degradation substrates connected arginylation to G-protein signaling modulation in retina and Wnt/β-catenin suppression in liver, respectively.","evidence":"Conditional Ate1 KO elevated RGS7 in retinal ON bipolar cells altering electroretinogram responses; ATE1 knockdown/overexpression in liver cancer cells showed RGS5 accumulation activating β-catenin via GSK3-β inhibition.","pmids":["33931669","34158395"],"confidence":"Medium","gaps":["Whether RGS7/RGS5 are directly arginylated by ATE1 not shown","Generalizability of RGS family as ATE1 substrates not tested"]},{"year":2021,"claim":"Elucidating the RNA structural mechanism controlling ATE1 mutually exclusive exon splicing revealed how alternative ATE1 isoforms are generated through competing intronic secondary structures spanning 30 kb.","evidence":"Compensatory mutagenesis in minigenes and LNA/DNA mixmer blocking of endogenous pre-mRNA demonstrated R1–R4 competition for R3 base-pairing and ultra-long-range R2–R5 interaction.","pmids":["33330934"],"confidence":"High","gaps":["Functional differences between exon 7a and 7b isoforms not biochemically defined","Regulation by RNA Pol II elongation rate not tested endogenously"]},{"year":2022,"claim":"Crystal structures of yeast and Kluyveromyces Ate1 resolved the bilobed GNAT-fold architecture, revealed the active site at the domain interface, and identified hemin inhibition and an [Fe-S] cluster regulatory domain, providing the first structural framework for the enzyme.","evidence":"X-ray crystallography of KlAte1 and ScAte1 with mutagenesis validation; hemin-induced oligomerization; SAXS and cryo-EM of apo ScAte1 showing N-terminal domain disorder.","pmids":["35878037","36087779"],"confidence":"High","gaps":["No structure with both tRNA and substrate bound at this point","Mechanism by which [Fe-S] cluster senses oxygen not determined","Human enzyme structure not yet available"]},{"year":2023,"claim":"Biochemical reconstitution of the [Fe-S] cluster under anoxic conditions in both yeast and mouse ATE1 confirmed it as a bona fide oxygen-sensing cofactor regulating enzymatic activity.","evidence":"Anaerobic [Fe-S] cluster reconstitution with spectroscopic characterization of yeast and mouse ATE1.","pmids":["37010764"],"confidence":"Medium","gaps":["Oxygen-dependent switching mechanism not structurally resolved","Physiological oxygen tension thresholds for ATE1 activity modulation unknown"]},{"year":2025,"claim":"The cryo-EM structure of human ATE1 bound to Arg-tRNAArg and an Nt-Asp peptide provided the first ternary complex view, explaining substrate selectivity through multivalent micromolar-affinity interactions and showing tRNA wrapping by an unstructured loop.","evidence":"Cryo-EM of human ATE1–Arg-tRNAArg–peptide complex with binding affinity measurements.","pmids":["40099869"],"confidence":"High","gaps":["Catalytic mechanism (transition state) not captured","How midchain substrates are accommodated in the active site remains structurally unresolved"]},{"year":null,"claim":"Key unresolved questions include the structural basis for midchain arginylation, the identity of critical stress-induced substrates mediating cell death, the biochemical distinction between ATE1 splice isoforms, and the physiological oxygen thresholds governing [Fe-S]-cluster-dependent activity regulation.","evidence":"","pmids":[],"confidence":"High","gaps":["No structure of ATE1 with a midchain substrate","Stress-induced arginylation substrates driving apoptosis unidentified","Functional biochemistry of exon 7a vs 7b isoforms not compared"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,2,4]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[4,13]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,5,8]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[16]}],"pathway":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[6,7,8]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,9,15]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[10]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[5,16]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[5,12]}],"complexes":[],"partners":["MYH9","LIAT1","RGS7","RGS5","ACTB"],"other_free_text":[]},"mechanistic_narrative":"ATE1 encodes the arginyl-tRNA-protein transferase (R-transferase) that post-translationally conjugates arginine from Arg-tRNAArg to N-terminal acidic residues and internal side-chain carboxylates of substrate proteins, thereby creating Arg/N-degrons that target substrates for proteasomal degradation [PMID:2185248, PMID:24529990]. Structural studies reveal a bilobed GNAT-fold enzyme whose active site resides at the domain–domain interface, with substrate and tRNA bound in adjacent pockets; the N-terminal domain harbors a regulatory [Fe-S] cluster that functions as an oxygen sensor, and hemin inhibits activity through disulfide-mediated oligomerization [PMID:35878037, PMID:36087779, PMID:40099869, PMID:37010764]. ATE1-mediated arginylation regulates actin polymerization and focal adhesion formation, modulates myosin contractility in platelets, drives neuronal growth cone migration through co-translational arginylation of β-actin, and controls RGS protein turnover affecting retinal signaling and Wnt/β-catenin pathway activity [PMID:30586322, PMID:24293517, PMID:28844905, PMID:33931669, PMID:34158395]. ATE1 alternative splicing of mutually exclusive exons is governed by competing intronic RNA secondary structures including an ultra-long-range interaction, and ATE1 protein levels increase under diverse stresses to promote arginylation-dependent cell death [PMID:33330934, PMID:27685622]."},"prefetch_data":{"uniprot":{"accession":"O95260","full_name":"Arginyl-tRNA--protein transferase 1","aliases":["Arginine-tRNA--protein transferase 1"],"length_aa":518,"mass_kda":59.1,"function":"Involved in the post-translational conjugation of arginine to the N-terminal aspartate or glutamate of a protein (PubMed:34893540). This arginylation is required for degradation of the protein via the ubiquitin pathway (PubMed:34893540). Does not arginylate cysteine residues (By similarity)","subcellular_location":"Nucleus; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/O95260/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ATE1","classification":"Not Classified","n_dependent_lines":7,"n_total_lines":1208,"dependency_fraction":0.005794701986754967},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ATE1","total_profiled":1310},"omim":[{"mim_id":"620846","title":"N-TERMINAL GLUTAMINE AMIDASE 1; NTAQ1","url":"https://www.omim.org/entry/620846"},{"mim_id":"615367","title":"N-TERMINAL ASPARAGINE AMIDASE; NTAN1","url":"https://www.omim.org/entry/615367"},{"mim_id":"609134","title":"UBIQUITIN-PROTEIN LIGASE E3 COMPONENT N-RECOGNIN 2; UBR2","url":"https://www.omim.org/entry/609134"},{"mim_id":"607103","title":"ARGINYLTRANSFERASE 1; ATE1","url":"https://www.omim.org/entry/607103"},{"mim_id":"605981","title":"UBIQUITIN-PROTEIN LIGASE E3 COMPONENT N-RECOGNIN 1; UBR1","url":"https://www.omim.org/entry/605981"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ATE1"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"O95260","domains":[{"cath_id":"-","chopping":"12-152_257-463","consensus_level":"medium","plddt":89.1541,"start":12,"end":463}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O95260","model_url":"https://alphafold.ebi.ac.uk/files/AF-O95260-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O95260-F1-predicted_aligned_error_v6.png","plddt_mean":79.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ATE1","jax_strain_url":"https://www.jax.org/strain/search?query=ATE1"},"sequence":{"accession":"O95260","fasta_url":"https://rest.uniprot.org/uniprotkb/O95260.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O95260/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O95260"}},"corpus_meta":[{"pmid":"2185248","id":"PMC_2185248","title":"Cloning and functional analysis of the arginyl-tRNA-protein transferase gene ATE1 of Saccharomyces cerevisiae.","date":"1990","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/2185248","citation_count":140,"is_preprint":false},{"pmid":"24529990","id":"PMC_24529990","title":"Arginyltransferase ATE1 catalyzes midchain arginylation of proteins at side chain carboxylates in vivo.","date":"2014","source":"Chemistry & biology","url":"https://pubmed.ncbi.nlm.nih.gov/24529990","citation_count":77,"is_preprint":false},{"pmid":"27685622","id":"PMC_27685622","title":"Posttranslational arginylation enzyme Ate1 affects DNA mutagenesis by regulating stress response.","date":"2016","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/27685622","citation_count":38,"is_preprint":false},{"pmid":"28844905","id":"PMC_28844905","title":"Arginyltransferase ATE1 is targeted to the neuronal growth cones and regulates neurite outgrowth during brain 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England)","url":"https://pubmed.ncbi.nlm.nih.gov/1882552","citation_count":17,"is_preprint":false},{"pmid":"30586322","id":"PMC_30586322","title":"Ate1-mediated posttranslational arginylation affects substrate adhesion and cell migration in Dictyostelium discoideum.","date":"2018","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/30586322","citation_count":16,"is_preprint":false},{"pmid":"34896361","id":"PMC_34896361","title":"The Final Maturation State of β-actin Involves N-terminal Acetylation by NAA80, not N-terminal Arginylation by ATE1.","date":"2021","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/34896361","citation_count":16,"is_preprint":false},{"pmid":"36087779","id":"PMC_36087779","title":"The Structure of Saccharomyces cerevisiae Arginyltransferase 1 (ATE1).","date":"2022","source":"Journal of molecular 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Arginylation","date":"2024-08-08","source":"bioRxiv","url":"https://doi.org/10.1101/2024.08.08.607245","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.04.09.646507","title":"Implementing N-terminomics and machine learning to probe in vivo Nt-arginylation","date":"2025-04-09","source":"bioRxiv","url":"https://doi.org/10.1101/2025.04.09.646507","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":20759,"output_tokens":4113,"usd":0.061986},"stage2":{"model":"claude-opus-4-6","input_tokens":7588,"output_tokens":3053,"usd":0.171398},"total_usd":0.233384,"stage1_batch_id":"msgbatch_01TqNM6p6FsoMBT2jzQTwKBr","stage2_batch_id":"msgbatch_01Ya473eco8sTHbvRymQaU8o","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1990,\n      \"finding\": \"ATE1 encodes arginyl-tRNA-protein transferase (R-transferase) that catalyzes post-translational conjugation of Arg to N-terminal residues of acceptor proteins; expression of yeast ATE1 in E. coli (which lacks R-transferases) conferred Arg-transferase activity, and null ate1 mutants lack this activity and cannot degrade N-end rule substrates requiring Arg-transferase action.\",\n      \"method\": \"Heterologous expression in E. coli, null mutant analysis, in vitro enzymatic assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted activity in heterologous host, functional null mutant phenotype, foundational paper with 140 citations\",\n      \"pmids\": [\"2185248\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ATE1 catalyzes arginylation not only at N-terminal alpha-amino groups but also at internal side-chain carboxylates of Asp and Glu residues within intact proteins in vivo, revealing an unconventional midchain arginylation mechanism.\",\n      \"method\": \"Mass spectrometry (in vivo arginylation profiling), in vitro ATE1 arginylation assay with purified components\",\n      \"journal\": \"Chemistry & biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro assay with purified enzyme plus in vivo MS identification; replicated across multiple protein targets\",\n      \"pmids\": [\"24529990\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Crystal structure of K. lactis Ate1 R-transferase at 58 kDa reveals two domains that together recognize an acidic N-terminal residue of the acceptor substrate, the Arg residue of Arg-tRNAArg, and the 3'-proximal tRNAArg segment; the active site is located between the two domains. Hemin (Fe3+-heme) inhibits Nt-arginylation activity and induces disulfide-mediated oligomerization of Ate1. Site-directed mutagenesis guided by the structure identified specific binding residues.\",\n      \"method\": \"X-ray crystallography, site-directed mutagenesis, in vitro and in vivo arginylation assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with mutagenesis validation and biochemical assays in a single study\",\n      \"pmids\": [\"35878037\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Crystal structure of S. cerevisiae ATE1 (ScATE1) in apo form reveals a bilobed protein with a GCN5-related N-acetyltransferase (GNAT) fold; the domain-domain interface constitutes the catalytic active site and tRNA-binding region. The N-terminal domain that binds a regulatory [Fe-S] cluster is dynamic and disordered in the absence of metal, indicating regulatory influence of this region.\",\n      \"method\": \"X-ray crystallography, SEC-SAXS, cryo-EM 2D class averaging, structural superposition\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure validated by SAXS and cryo-EM; orthogonal methods in single study\",\n      \"pmids\": [\"36087779\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM structure of human ATE1 in complex with Arg-tRNAArg and an N-terminal Asp peptide shows two adjacent pockets binding the Nt-substrate and Arg-tRNAArg respectively, the tRNA being wrapped by a long unstructured loop. In the apo state, two ATE1 monomers form a homodimer. Substrate selectivity is achieved through multivalent interactions with Kd values in the micromolar range.\",\n      \"method\": \"Cryo-EM structure determination, binding affinity measurements\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure with biochemical binding characterization; human enzyme\",\n      \"pmids\": [\"40099869\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ATE1/arginylation promotes cell death and/or growth arrest in response to oxidative, heat, osmotic, heavy metal, and radiation stresses; ATE1 protein levels rise under acute stress, and the elevated ATE1 directly promotes cell death in an arginylation-activity-dependent manner. ATE1 is also required to suppress mutation frequency under DNA-damaging conditions.\",\n      \"method\": \"ATE1 knockout/knockdown in yeast, mouse, and human cells; genetic complementation with catalytically inactive ATE1; functional cell death and mutagenesis assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple organisms, loss-of-function with defined phenotypes, activity-dependent rescue; single lab\",\n      \"pmids\": [\"27685622\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ATE1 localizes prominently to neuronal growth cones via zipcode-mediated mRNA targeting; Ate1 mRNA co-localizes with arginylated β-actin at growth cone tips, and co-translational arginylation of β-actin at growth cones drives growth cone migration and neurite outgrowth. Conditional Ate1 knockout in the nervous system causes defects in neuronal migration, reduced neurite outgrowth, and decreased doublecortin and F-actin in growth cones.\",\n      \"method\": \"Conditional mouse knockout (Nestin-Cre), live-cell imaging, FISH, fluorescence microscopy, protein synthesis inhibitor experiments\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with defined cellular phenotype and direct localization evidence; single lab with orthogonal methods\",\n      \"pmids\": [\"28844905\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ATE1 directly interacts with myosin II in platelets (co-immunoprecipitation); platelet-specific ATE1 knockout leads to enhanced myosin regulatory light chain phosphorylation at Ser19, enhanced clot retraction, and increased in vivo thrombus formation, indicating arginylation modulates myosin contractility.\",\n      \"method\": \"Conditional platelet/megakaryocyte-specific knockout mouse, co-immunoprecipitation, phosphorylation analysis, in vivo thrombosis assay\",\n      \"journal\": \"Haematologica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with defined biochemical and in vivo phenotype, direct Co-IP; single lab\",\n      \"pmids\": [\"24293517\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In Dictyostelium discoideum, Ate1 knockout abolishes focal actin adhesion sites and reduces adhesion; mass spectrometry identified four arginylation sites in the major actin isoform plus sites in actin-binding proteins. Actin from ate1-null cells has diminished polymerization capacity in vitro. Ate1-GFP rapidly relocalizes to sites of newly formed actin-rich protrusions.\",\n      \"method\": \"Gene knockout, live-cell microscopy, mass spectrometry, in vitro actin polymerization assay, chemotaxis assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO with multiple orthogonal readouts including in vitro reconstitution; model organism ortholog\",\n      \"pmids\": [\"30586322\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Ate1 regulates RGS7 protein levels by facilitating its proteasomal degradation; conditional Ate1 knockout in the nervous system elevates RGS7 levels in retinal ON bipolar cells, increases light-evoked ON-bipolar response sensitivities, and in cultured MEFs, RGS7 degradation by the proteasome is abolished in Ate1 knockout cells.\",\n      \"method\": \"Conditional mouse knockout (nervous system), electroretinography, proteasome inhibitor experiments in MEF cells\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with defined molecular and physiological phenotypes; two cell contexts tested\",\n      \"pmids\": [\"33931669\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ATE1 splicing is controlled by competing RNA secondary structures: five conserved intronic elements (R1–R5) regulate mutual exclusion of exons 7a and 7b, with R1 and R4 competing for base-pairing with R3, and an ultra-long-range R2–R5 interaction spanning 30 kb. Disruption of these RNA structures by mutation or LNA/DNA mixmers abolishes mutually exclusive exon splicing, and co-transcriptional folding links exon 7a inclusion to RNA Pol II elongation rate.\",\n      \"method\": \"Minigene splicing assay with single/double/compensatory mutations, LNA/DNA mixmer blocking, endogenous pre-mRNA analysis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — compensatory mutagenesis in minigenes plus endogenous blocking experiments; orthogonal mechanistic evidence\",\n      \"pmids\": [\"33330934\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Liat1 (Ligand of Ate1) physically interacts with Ate1 and undergoes liquid-liquid phase separation in the nucleolus via an intrinsically disordered N-terminal region; Jmjd6 lysyl-hydroxylase modifies Liat1's poly-K region and inhibits its nucleolar targeting.\",\n      \"method\": \"Bimolecular fluorescence complementation, immunocytochemistry, IDR characterization, phase separation assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — direct protein interaction shown by BiFC with functional nucleolar localization consequence; single lab\",\n      \"pmids\": [\"33443146\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ATE1 binds a regulatory iron-sulfur ([Fe-S]) cluster in its N-terminal domain that functions as an oxygen sensor to regulate ATE1 activity; chemical reconstitution under anoxic conditions restores [Fe-S] cluster binding in both yeast and mouse ATE1.\",\n      \"method\": \"Anaerobic [Fe-S] cluster reconstitution, spectroscopic characterization\",\n      \"journal\": \"Methods in molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 in vitro reconstitution of cofactor; single lab but biochemically defined\",\n      \"pmids\": [\"37010764\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Mouse ATE1 contains an intrinsically disordered region (IDR) absent in yeast ATE1; HDX-MS and SAXS demonstrate this IDR is present in all mouse ATE1 splice variants and is predicted to facilitate complex formation with tRNAArg.\",\n      \"method\": \"SEC, SAXS, HDX-MS, AlphaFold modeling, bioinformatics\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal biophysical methods; single lab; functional role is computational inference\",\n      \"pmids\": [\"39642180\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"tRNAArg directly binds in vitro to TDP-43 RNA recognition motifs and stabilizes binding via TDP-43 dimerization (promoted by the N-terminal domain and NLS); the arginylation cofactor LIAT1 also binds tRNAArg in vitro, identifying LIAT1 as an RNA-binding protein.\",\n      \"method\": \"In vitro binding assays with recombinant proteins and in vitro-transcribed tRNA\",\n      \"journal\": \"microPublication biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — in vitro binding only, single study, no functional consequence directly tested for LIAT1-tRNA interaction\",\n      \"pmids\": [\"39081859\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ATE1 inhibits liver cancer growth by facilitating proteasomal degradation of RGS5, which in turn maintains GSK3-β activity and promotes β-catenin degradation, thereby suppressing Wnt/β-catenin signaling; loss- and gain-of-function assays with RGS5 and GSK3-β inhibitor confirmed RGS5 as a key effector downstream of ATE1.\",\n      \"method\": \"Lentivirus-mediated knockdown/overexpression, loss- and gain-of-function, GSK3-β inhibitor rescue experiments, in vitro and in vivo tumor assays\",\n      \"journal\": \"Molecular cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — epistasis-type rescue experiments placing ATE1 upstream of RGS5 and GSK3-β/β-catenin; single lab\",\n      \"pmids\": [\"34158395\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Mitochondrial translocation of Ate1 is promoted by oxidative stressors and is essential for inducing apoptotic cell death; Ate1-induced cell death depends on mitochondrial permeability pore formation and apoptosis-inducing factor, but not on electron transport chain activity or ROS, and is independent of cytosolic ubiquitin-proteasome, autophagy, or ER stress pathways.\",\n      \"method\": \"Yeast genetic and cell biology experiments, mitochondrial fractionation, mitochondrial permeability pore assays, AIF analysis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple genetic and cell biology approaches; preprint, not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2024.11.22.624728\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"ATE1 is an arginyl-tRNA-protein transferase that post-translationally transfers Arg from Arg-tRNAArg to N-terminal acidic residues (Asp, Glu, oxidized Cys) and internal side-chain carboxylates of substrate proteins via a bilobed GNAT-fold enzyme whose active site sits at the domain-domain interface; this N-terminal arginylation creates Arg/N-degrons that direct substrates to proteasomal or autophagic degradation, regulates actin cytoskeleton dynamics, myosin contractility, neuronal growth cone migration, and stress-induced cell death (including via mitochondrial translocation), and its activity is regulated by an [Fe-S] cluster oxygen sensor and by alternative splicing of mutually exclusive exons controlled by competing RNA secondary structures.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"ATE1 encodes the arginyl-tRNA-protein transferase (R-transferase) that post-translationally conjugates arginine from Arg-tRNAArg to N-terminal acidic residues and internal side-chain carboxylates of substrate proteins, thereby creating Arg/N-degrons that target substrates for proteasomal degradation [PMID:2185248, PMID:24529990]. Structural studies reveal a bilobed GNAT-fold enzyme whose active site resides at the domain–domain interface, with substrate and tRNA bound in adjacent pockets; the N-terminal domain harbors a regulatory [Fe-S] cluster that functions as an oxygen sensor, and hemin inhibits activity through disulfide-mediated oligomerization [PMID:35878037, PMID:36087779, PMID:40099869, PMID:37010764]. ATE1-mediated arginylation regulates actin polymerization and focal adhesion formation, modulates myosin contractility in platelets, drives neuronal growth cone migration through co-translational arginylation of β-actin, and controls RGS protein turnover affecting retinal signaling and Wnt/β-catenin pathway activity [PMID:30586322, PMID:24293517, PMID:28844905, PMID:33931669, PMID:34158395]. ATE1 alternative splicing of mutually exclusive exons is governed by competing intronic RNA secondary structures including an ultra-long-range interaction, and ATE1 protein levels increase under diverse stresses to promote arginylation-dependent cell death [PMID:33330934, PMID:27685622].\",\n  \"teleology\": [\n    {\n      \"year\": 1990,\n      \"claim\": \"Establishing the molecular identity of ATE1 as the gene encoding arginyl-tRNA-protein transferase resolved how the N-end rule pathway activates degradation signals on protein substrates.\",\n      \"evidence\": \"Heterologous expression of yeast ATE1 in E. coli conferred Arg-transferase activity; ate1-null yeast lacked this activity and failed to degrade N-end rule substrates.\",\n      \"pmids\": [\"2185248\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Substrate scope beyond N-end rule model peptides unknown\", \"Mammalian ATE1 not yet characterized\", \"Catalytic mechanism unresolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstrating that ATE1 directly interacts with myosin II and modulates its regulatory light chain phosphorylation extended arginylation beyond protein degradation to actomyosin contractility regulation.\",\n      \"evidence\": \"Platelet-specific Ate1 knockout mice showed enhanced MLC phosphorylation, increased clot retraction, and in vivo thrombus formation; Co-IP confirmed direct ATE1–myosin II interaction.\",\n      \"pmids\": [\"24293517\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether myosin II is directly arginylated was not determined\", \"Mechanism linking arginylation to MLC phosphorylation state unclear\", \"Single Co-IP without reciprocal validation\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Discovery of midchain arginylation at internal Asp/Glu side-chain carboxylates revealed that ATE1 catalytic scope extends beyond N-terminal modification, fundamentally broadening the substrate landscape.\",\n      \"evidence\": \"In vivo mass spectrometry identified internal arginylation sites on intact proteins; in vitro assays with purified ATE1 confirmed midchain conjugation.\",\n      \"pmids\": [\"24529990\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequences of specific midchain arginylation events not established\", \"Structural basis for midchain versus N-terminal selectivity unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showing that ATE1 protein accumulates under diverse stresses and promotes arginylation-dependent cell death established ATE1 as a stress-response effector rather than solely a constitutive housekeeping enzyme.\",\n      \"evidence\": \"ATE1 KO/KD across yeast, mouse, and human cells reduced stress-induced cell death; complementation with catalytically inactive ATE1 failed to rescue.\",\n      \"pmids\": [\"27685622\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Critical stress-induced arginylation substrates not identified\", \"Mechanism of ATE1 protein stabilization under stress unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Localizing Ate1 mRNA to neuronal growth cones and showing co-translational arginylation of β-actin there connected ATE1 to local cytoskeletal remodeling required for neuronal migration and outgrowth.\",\n      \"evidence\": \"Conditional Ate1 KO in mouse nervous system caused growth cone defects, reduced neurite outgrowth, and decreased F-actin; FISH showed co-localization of Ate1 mRNA with arginylated β-actin at growth cone tips.\",\n      \"pmids\": [\"28844905\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Zipcode elements in Ate1 mRNA not mapped\", \"Whether arginylation of actin is sufficient to rescue growth cone defects not tested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrating that Ate1 knockout abolishes actin focal adhesions and reduces actin polymerization capacity in Dictyostelium confirmed a conserved role for arginylation in actin dynamics beyond vertebrates.\",\n      \"evidence\": \"Ate1-null Dictyostelium lacked focal adhesion sites; MS identified arginylation sites on actin; in vitro polymerization of ate1-null actin was diminished; Ate1-GFP relocated to actin-rich protrusions.\",\n      \"pmids\": [\"30586322\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether individual arginylation sites on actin are functionally distinct not resolved\", \"Direct structural effect of arginylation on actin filament properties unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identifying RGS7 and RGS5 as ATE1-dependent degradation substrates connected arginylation to G-protein signaling modulation in retina and Wnt/β-catenin suppression in liver, respectively.\",\n      \"evidence\": \"Conditional Ate1 KO elevated RGS7 in retinal ON bipolar cells altering electroretinogram responses; ATE1 knockdown/overexpression in liver cancer cells showed RGS5 accumulation activating β-catenin via GSK3-β inhibition.\",\n      \"pmids\": [\"33931669\", \"34158395\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether RGS7/RGS5 are directly arginylated by ATE1 not shown\", \"Generalizability of RGS family as ATE1 substrates not tested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Elucidating the RNA structural mechanism controlling ATE1 mutually exclusive exon splicing revealed how alternative ATE1 isoforms are generated through competing intronic secondary structures spanning 30 kb.\",\n      \"evidence\": \"Compensatory mutagenesis in minigenes and LNA/DNA mixmer blocking of endogenous pre-mRNA demonstrated R1–R4 competition for R3 base-pairing and ultra-long-range R2–R5 interaction.\",\n      \"pmids\": [\"33330934\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional differences between exon 7a and 7b isoforms not biochemically defined\", \"Regulation by RNA Pol II elongation rate not tested endogenously\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Crystal structures of yeast and Kluyveromyces Ate1 resolved the bilobed GNAT-fold architecture, revealed the active site at the domain interface, and identified hemin inhibition and an [Fe-S] cluster regulatory domain, providing the first structural framework for the enzyme.\",\n      \"evidence\": \"X-ray crystallography of KlAte1 and ScAte1 with mutagenesis validation; hemin-induced oligomerization; SAXS and cryo-EM of apo ScAte1 showing N-terminal domain disorder.\",\n      \"pmids\": [\"35878037\", \"36087779\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure with both tRNA and substrate bound at this point\", \"Mechanism by which [Fe-S] cluster senses oxygen not determined\", \"Human enzyme structure not yet available\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Biochemical reconstitution of the [Fe-S] cluster under anoxic conditions in both yeast and mouse ATE1 confirmed it as a bona fide oxygen-sensing cofactor regulating enzymatic activity.\",\n      \"evidence\": \"Anaerobic [Fe-S] cluster reconstitution with spectroscopic characterization of yeast and mouse ATE1.\",\n      \"pmids\": [\"37010764\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Oxygen-dependent switching mechanism not structurally resolved\", \"Physiological oxygen tension thresholds for ATE1 activity modulation unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"The cryo-EM structure of human ATE1 bound to Arg-tRNAArg and an Nt-Asp peptide provided the first ternary complex view, explaining substrate selectivity through multivalent micromolar-affinity interactions and showing tRNA wrapping by an unstructured loop.\",\n      \"evidence\": \"Cryo-EM of human ATE1–Arg-tRNAArg–peptide complex with binding affinity measurements.\",\n      \"pmids\": [\"40099869\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Catalytic mechanism (transition state) not captured\", \"How midchain substrates are accommodated in the active site remains structurally unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis for midchain arginylation, the identity of critical stress-induced substrates mediating cell death, the biochemical distinction between ATE1 splice isoforms, and the physiological oxygen thresholds governing [Fe-S]-cluster-dependent activity regulation.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure of ATE1 with a midchain substrate\", \"Stress-induced arginylation substrates driving apoptosis unidentified\", \"Functional biochemistry of exon 7a vs 7b isoforms not compared\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 2, 4]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [4, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 5, 8]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [16]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [6, 7, 8]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 9, 15]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [5, 16]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [5, 12]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"MYH9\",\n      \"LIAT1\",\n      \"RGS7\",\n      \"RGS5\",\n      \"ACTB\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}