{"gene":"ATE1","run_date":"2026-06-09T22:02:44","timeline":{"discoveries":[{"year":1990,"finding":"The ATE1 gene of S. cerevisiae encodes an arginyl-tRNA-protein transferase (R-transferase) that catalyzes post-translational conjugation of Arg to the N-termini of acceptor proteins; expression in E. coli confirmed this catalytic activity, and null ate1 mutants lack Arg-transferase activity and cannot degrade N-end rule substrates requiring Nt-arginylation.","method":"Heterologous expression in E. coli (functional complementation), null mutant analysis with enzymatic assay and protein degradation readout","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct biochemical reconstitution of enzymatic activity in E. coli, confirmed by loss-of-function genetics with two orthogonal readouts (enzyme assay + substrate degradation)","pmids":["2185248"],"is_preprint":false},{"year":2014,"finding":"ATE1 can arginylate internal side-chain carboxylates (Asp and Glu residues) within intact proteins in vivo, in addition to N-terminal alpha-amino groups, demonstrating an unconventional midchain arginylation mechanism.","method":"Mass spectrometry-based proteomics (MS/MS), in vitro arginylation assay with purified ATE1","journal":"Chemistry & biology","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro assay with purified enzyme plus in vivo MS identification, single lab but two orthogonal approaches","pmids":["24529990"],"is_preprint":false},{"year":2022,"finding":"Crystal structure of Kluyveromyces lactis Ate1 reveals a 58-kDa two-domain R-transferase where both domains together recognize the acidic N-terminal residue of the acceptor substrate and the Arg-tRNAArg cosubstrate (including its 3'-proximal tRNA segment), with the active site located between the two domains; hemin (Fe3+-heme) inhibits Nt-arginylation and induces disulfide-mediated oligomerization of Ate1.","method":"X-ray crystallography, in vitro and in vivo arginylation assays, site-directed mutagenesis guided by structural data","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with mutagenesis and functional arginylation assays in the same study","pmids":["35878037"],"is_preprint":false},{"year":2022,"finding":"Crystal structure of S. cerevisiae ATE1 in the apo form reveals a bilobed protein with a GCN5-related N-acetyltransferase (GNAT) fold; structural and electrostatic analyses identify the domain-domain interface as the catalytic site and tRNA-binding region; the N-terminal domain that binds a regulatory [Fe-S] cluster is dynamic and disordered when metal-free.","method":"X-ray crystallography, SEC-SAXS, cryo-EM 2D class averaging, structural superposition","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure corroborated by orthogonal solution-scattering and cryo-EM methods in the same study","pmids":["36087779"],"is_preprint":false},{"year":2025,"finding":"Cryo-EM structure of human ATE1 in complex with Arg-tRNAArg and an Nt-Asp peptide reveals two adjacent binding pockets for the substrate and the tRNA cosubstrate, the tRNA being wrapped by a long unstructured loop; in the apo state ATE1 forms a homodimer; substrate selectivity is achieved through multivalent interactions with Kd values in the micromolar range.","method":"Cryo-EM structure determination, biochemical binding assays","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure with both substrates, homodimer apo structure, and quantitative binding measurements in one study","pmids":["40099869"],"is_preprint":false},{"year":2024,"finding":"Mouse ATE1 contains an intrinsically disordered region (IDR) absent in yeast ATE1; computational and HDX-MS analyses suggest this IDR facilitates complex formation between ATE1 and tRNAArg, adding regulatory complexity not present in the yeast enzyme.","method":"SEC, SAXS, hydrogen-deuterium exchange mass spectrometry (HDX-MS), AlphaFold modeling","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — multiple orthogonal biophysical methods (SAXS, HDX-MS) in a single study; functional implication of IDR in tRNA binding is computational/inferential","pmids":["39642180"],"is_preprint":false},{"year":2023,"finding":"ATE1 binds a regulatory iron-sulfur ([Fe-S]) cluster in its N-terminal domain; the cluster is oxygen-sensitive and functions as an oxygen sensor to regulate ATE1 activity, as it decomposes upon purification in the presence of O2.","method":"Anaerobic chemical reconstitution of [Fe-S] cluster in purified ScATE1 and MmATE1, metal analysis","journal":"Methods in molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct biochemical reconstitution of cofactor in purified protein, single lab; functional oxygen-sensing role inferred from cluster lability","pmids":["37010764"],"is_preprint":false},{"year":2013,"finding":"ATE1 co-immunoprecipitates with myosin II from platelet lysates; platelet-specific ATE1 knockout mice show enhanced myosin regulatory light chain phosphorylation at Ser19 (activating myosin), enhanced clot retraction, and enhanced in vivo thrombus formation, placing ATE1-mediated arginylation upstream of myosin II contractility regulation.","method":"Conditional knockout mouse model, co-immunoprecipitation, phosphorylation analysis, clot retraction assay, in vivo thrombosis assay","journal":"Haematologica","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP plus conditional KO with multiple orthogonal phenotypic readouts (phosphorylation, clot retraction, in vivo thrombosis)","pmids":["24293517"],"is_preprint":false},{"year":2017,"finding":"ATE1 localizes prominently to neuronal growth cones in addition to cell bodies; Ate1 mRNA contains zipcode-binding sequences that target it to growth cone tips where local translation occurs, co-localizing with arginylated β-actin; Ate1 conditional knockout in the nervous system reduces neurite outgrowth and F-actin levels in growth cones, and decreases doublecortin levels.","method":"Conditional knockout mouse (Nestin-Cre), fluorescence in situ hybridization (FISH), live-cell imaging, immunofluorescence, protein synthesis inhibitor treatment","journal":"Developmental biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO with defined phenotype, FISH for mRNA localization, co-localization with arginylated substrate, multiple orthogonal approaches in one study","pmids":["28844905"],"is_preprint":false},{"year":2018,"finding":"In Dictyostelium discoideum, Ate1 knockout eliminates focal actin adhesion sites at the substrate-attached surface, reduces adhesion, and alters chemotaxis; GFP-Ate1 rapidly relocates to newly forming actin-rich protrusions; mass spectrometry identified four arginylation sites in the major actin isoform plus sites on actin-binding proteins; actin purified from ate1-null cells shows diminished in vitro polymerization.","method":"Gene knockout, live-cell microscopy, mass spectrometry, in vitro actin polymerization assay, GFP-tagging/live imaging","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — KO with multiple phenotypic readouts, MS identification of arginylation sites, in vitro polymerization reconstitution, and live imaging of ATE1 dynamics","pmids":["30586322"],"is_preprint":false},{"year":2021,"finding":"ATE1 facilitates proteasomal degradation of RGS7 in mouse embryonic fibroblasts; conditional Ate1 knockout in the nervous system elevates RGS7 protein levels in retinal ON-bipolar cells, leading to increased light-evoked response sensitivities; RGS7 degradation is abolished in Ate1 KO MEFs but is rapid via the proteasome in wildtype cells.","method":"Conditional nervous system knockout mouse, electroretinography, MEF cell-based proteasome inhibitor experiments, immunoblotting","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO with defined biochemical (RGS7 levels) and physiological (ERG) readouts, proteasome pathway confirmed by inhibitor in two cell contexts","pmids":["33931669"],"is_preprint":false},{"year":2021,"finding":"ATE1 inhibits liver cancer progression by regulating turnover of RGS5, which in turn suppresses Wnt/β-catenin signaling by affecting GSK3-β activity; loss- and gain-of-function assays confirmed RGS5 as a key effector downstream of ATE1.","method":"Lentivirus-mediated knockdown/overexpression, loss- and gain-of-function assays, GSK inhibitor treatment, in vitro and in vivo tumor models","journal":"Molecular cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple loss/gain-of-function experiments with pathway rescue, single lab","pmids":["34158395"],"is_preprint":false},{"year":2016,"finding":"ATE1 promotes cell death and growth arrest in response to oxidative, heat, osmotic stress, heavy metals, and radiation; ATE1 protein levels and arginylation activity increase in wild-type cells under acute stress; ATE1-induced cell death requires its arginylation activity; ATE1 is required to suppress mutation frequency under DNA-damaging conditions in yeast and mammalian cells.","method":"Gene deletion/knockdown in yeast, mouse, and human cells; stress viability assays; enzymatic activity assays; mutation frequency assays under UV irradiation","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple organism/cell type models and orthogonal readouts (cell death, mutagenesis, enzymatic activity), single lab","pmids":["27685622"],"is_preprint":false},{"year":2024,"finding":"Mitochondrial translocation of Ate1 is promoted by oxidative stress and is essential for inducing apoptotic cell death; Ate1-induced cell death depends on formation of the mitochondrial permeability transition pore and at least partly on the apoptosis-inducing factor; cytosolic protein degradation pathways (ubiquitin-proteasome, autophagy, ER stress) have negligible impact on Ate1-induced cell death.","method":"Budding yeast model, mitochondrial fractionation, genetic epistasis with permeability pore mutants and AIF deletion, live-cell imaging","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — subcellular fractionation plus genetic epistasis with multiple pathway mutants; preprint, not yet peer-reviewed","pmids":["bio_10.1101_2024.11.22.624728"],"is_preprint":true},{"year":2020,"finding":"The Ligand of Ate1 (Liat1) physically interacts with Ate1 and undergoes liquid-liquid phase separation in the nucleolus via an intrinsically disordered N-terminal region; Jumonji Domain Containing 6 (Jmjd6) hydroxylates Liat1 at its poly-K region, inhibiting nucleolar targeting of Liat1.","method":"Bimolecular fluorescence complementation, immunocytochemistry, phase separation assays, Jmjd6 enzymatic modification assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — bimolecular fluorescence complementation and ICC confirm interaction; phase separation and Jmjd6 modification shown but mechanism connecting back to ATE1 function is indirect","pmids":["33443146"],"is_preprint":false},{"year":2021,"finding":"The mammalian Ate1 gene undergoes alternative splicing controlled by mutually exclusive exons 7a/7b; five conserved intronic RNA structural elements (R1-R5) regulate this splicing via competing base-pair interactions (R1/R3 vs R4/R3) and an ultra-long-range R2/R5 RNA structure (~30 kb); disrupting these interactions by mutation or LNA/DNA mixmers abolishes MXE splicing; exon 7a inclusion responds to RNA Pol II slowdown in a manner dependent on the R2/R5 interaction, indicating co-transcriptional regulation.","method":"Minigene mutagenesis (single, double, compensatory triple mutations), LNA/DNA mixmer blocking in endogenous pre-mRNA, RNA Pol II slowdown experiments","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — compensatory mutation rescue in minigenes plus endogenous LNA blocking, multiple orthogonal approaches establishing RNA structural mechanism","pmids":["33330934"],"is_preprint":false},{"year":2021,"finding":"Targeted proteomics found no evidence of Nt-arginylated β-actin (RDDI-) in wildtype cells or NAA80-knockout cells; only a very minor level of Nt-arginylation of cleaved β-actin (DDDI-) was detectable in NAA80-lacking cells but not in wildtype; the final maturation state of β-actin is Nt-acetylation by NAA80, not arginylation by ATE1 under normal conditions.","method":"State-of-the-art targeted proteomics/mass spectrometry in wildtype and NAA80-KO cells","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — sensitive targeted MS with appropriate controls; negative result contradicting prior claims about β-actin arginylation; single lab","pmids":["34896361"],"is_preprint":false},{"year":2024,"finding":"In vitro, human tRNAArg directly binds the RNA recognition motifs (RRMs) of TDP-43, and the same TDP-43 constructs also bind native fungal tRNAPhe; mouse LIAT1 (Ligand of Ate1) binds human tRNAArg in vitro, identifying LIAT1 as an RNA-binding protein relevant to the arginylation machinery.","method":"In vitro binding assays with recombinant proteins and in vitro-transcribed tRNA","journal":"microPublication biology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single in vitro binding assay, no mutagenesis or functional validation, single lab","pmids":["39081859"],"is_preprint":false},{"year":2025,"finding":"Using isotopic arginine labeling in an ex vivo ATE1 assay on biological lysates, 235 unique arginylation sites were identified in human proteomes, including both N-terminal and midchain sites; representative sites were validated for biological function.","method":"Isotopic arginine labeling, ex vivo ATE1 enzymatic assay, mass spectrometry (bottom-up proteomics)","journal":"Nature chemical biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — novel platform with isotopic controls for bona fide arginylation identification, validated representative sites; single study","pmids":["40855110"],"is_preprint":false},{"year":2022,"finding":"ATE1 depletion in melanoma cells reduces viability, migration, and colony formation; AXIN1 is identified as a putative arginylation substrate of ATE1 in melanoma, suggesting ATE1 may regulate AXIN1 function.","method":"siRNA/shRNA knockdown, cell viability and migration assays, substrate identification","journal":"FEBS letters","confidence":"Low","confidence_rationale":"Tier 3 / Weak — knockdown phenotype without direct mechanistic validation of AXIN1 arginylation; AXIN1 as substrate is described as putative","pmids":["35561126"],"is_preprint":false},{"year":2025,"finding":"ATE1 stabilizes MYC protein in breast cancer cells via ERK-mediated phosphorylation at Ser62; ATE1 depletion impairs MAPK-MYC-CDK6 axis activity, reduces cell cycle progression, and promotes apoptosis; rescue experiments confirmed that ATE1's tumor-promoting activity requires its arginyltransferase catalytic function.","method":"siRNA/shRNA knockdown, quantitative proteomics, R-catcher-based N-terminomics, flow cytometry, immunoblotting, xenograft mouse model","journal":"Cell communication and signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods including N-terminomics and in vivo xenograft; catalytic requirement confirmed by rescue; single lab","pmids":["40898325"],"is_preprint":false},{"year":2024,"finding":"ATE1 mediates arginylation of the Newcastle disease virus haemagglutinin-neuraminidase (HN) protein at its N-terminus; addition of Arg amplifies arginylation of HN, reducing its stability and promoting ubiquitin-mediated proteasomal degradation; ATE1 knockdown and inhibition of ATE1 activity increase HN protein levels.","method":"ATE1 knockdown, enzymatic activity inhibition, Arg supplementation, ubiquitination assay, immunoblotting","journal":"The Journal of general virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple complementary approaches (knockdown, inhibitor, Arg supplementation, ubiquitination) in the same study; single lab","pmids":["39207120"],"is_preprint":false}],"current_model":"ATE1 (arginyltransferase 1) is an evolutionarily conserved enzyme that transfers arginine from Arg-tRNAArg to the N-terminal residues (Asp, Glu, oxidized Cys) or internal side-chain carboxylates of acceptor proteins; its two-domain GNAT-fold structure (solved by X-ray crystallography and cryo-EM) positions an active site at the domain interface that engages both the acidic N-terminal substrate and the tRNA cosubstrate, with a regulatory [Fe-S] cluster in the N-terminal domain acting as an oxygen sensor; the resulting Arg/N-degrons direct substrates to proteasomal or autophagic degradation, and ATE1 additionally regulates actin cytoskeleton dynamics (via actin arginylation affecting polymerization), myosin II contractility in platelets, RGS7 and RGS5 protein turnover, neuronal growth cone migration, cell death responses to stress (including mitochondrial translocation under oxidative stress), and cancer cell proliferation via the MAPK-MYC axis."},"narrative":{"mechanistic_narrative":"ATE1 is an evolutionarily conserved arginyl-tRNA-protein transferase that post-translationally conjugates arginine onto acceptor proteins, generating Arg/N-degrons and modulating substrate stability and cytoskeletal behavior across yeast, Dictyostelium, and mammalian systems [PMID:2185248, PMID:30586322]. The enzyme transfers Arg from Arg-tRNAArg not only to acidic N-terminal alpha-amino groups but also to internal side-chain carboxylates of Asp and Glu, an unconventional midchain arginylation, and proteome-scale profiling has mapped hundreds of N-terminal and midchain arginylation sites [PMID:24529990, PMID:40855110]. Structurally, ATE1 is a bilobed protein with a GCN5-related N-acetyltransferase (GNAT) fold whose active site lies at the interface between the two domains, which together engage both the acidic substrate N-terminus and the Arg-tRNAArg cosubstrate, the tRNA being wrapped by a long unstructured loop; the apo enzyme dimerizes and substrate selectivity arises from multivalent micromolar-affinity interactions [PMID:35878037, PMID:36087779, PMID:40099869]. A regulatory oxygen-sensitive [Fe-S] cluster binds the dynamic N-terminal domain and couples ATE1 activity to redox/oxygen state, and hemin inhibits arginylation while driving disulfide-mediated oligomerization [PMID:35878037, PMID:36087779, PMID:37010764]. Through arginylation, ATE1 governs actin polymerization and focal-adhesion dynamics, neuronal growth-cone outgrowth coupled to local Ate1 mRNA translation, and myosin II contractility in platelets, where ATE1 loss enhances regulatory light-chain phosphorylation, clot retraction, and thrombus formation [PMID:24293517, PMID:28844905, PMID:30586322]. ATE1 controls the proteasomal turnover of regulator-of-G-protein-signaling substrates RGS7 and RGS5, the latter linking ATE1 to Wnt/beta-catenin signaling and liver-cancer suppression [PMID:33931669, PMID:34158395]. ATE1 also promotes stress-induced cell death, including oxidative-stress-driven mitochondrial translocation that triggers apoptosis, in a manner requiring its catalytic activity [PMID:27685622, PMID:bio_10.1101_2024.11.22.624728]. In cancer, ATE1 can act as a tumor promoter by stabilizing MYC through the ERK/MAPK axis, a function dependent on its arginyltransferase activity [PMID:40898325]. The mammalian Ate1 gene is itself regulated by alternative splicing of mutually exclusive exons 7a/7b governed by long-range intronic RNA structures coupled to RNA Pol II elongation [PMID:33330934].","teleology":[{"year":1990,"claim":"Established the molecular identity of ATE1 as the enzyme responsible for N-terminal arginylation, defining a catalytic activity central to N-end rule degradation.","evidence":"Heterologous expression of S. cerevisiae ATE1 in E. coli plus null mutant analysis with enzyme and substrate-degradation readouts","pmids":["2185248"],"confidence":"High","gaps":["Does not reveal the structural basis of catalysis or tRNA engagement","Substrate repertoire beyond canonical N-end rule substrates undefined"]},{"year":2014,"claim":"Expanded the catalytic scope of ATE1 beyond N-terminal alpha-amino groups by showing it arginylates internal Asp/Glu side chains, redefining the chemistry of arginylation.","evidence":"In vitro arginylation with purified ATE1 and MS/MS identification of midchain sites in vivo","pmids":["24529990"],"confidence":"Medium","gaps":["Functional consequences of midchain arginylation per site not established","Single lab"]},{"year":2022,"claim":"Resolved how ATE1 simultaneously recognizes its acidic protein substrate and the Arg-tRNAArg cosubstrate, and identified hemin as a regulatory inhibitor.","evidence":"X-ray crystallography of K. lactis Ate1 with mutagenesis and arginylation assays","pmids":["35878037"],"confidence":"High","gaps":["Did not capture the [Fe-S] cluster-bound state","Mechanism of hemin-driven oligomerization in vivo unclear"]},{"year":2022,"claim":"Defined the ATE1 fold as a bilobed GNAT architecture with the catalytic/tRNA-binding site at the domain interface and revealed the metal-free N-terminal domain as intrinsically dynamic.","evidence":"X-ray crystallography of apo S. cerevisiae ATE1 with SEC-SAXS and cryo-EM 2D averaging","pmids":["36087779"],"confidence":"High","gaps":["Apo structure lacks bound substrate and cofactor","Dynamics of the N-terminal domain in the active enzyme not directly observed"]},{"year":2023,"claim":"Demonstrated that ATE1 carries an oxygen-sensitive [Fe-S] cluster in its N-terminal domain, providing a candidate mechanism for redox/oxygen-coupled regulation of arginylation.","evidence":"Anaerobic chemical reconstitution of the cluster in purified yeast and mouse ATE1 with metal analysis","pmids":["37010764"],"confidence":"Medium","gaps":["Oxygen-sensing role inferred from cluster lability, not demonstrated in cells","Effect of cluster occupancy on catalytic rate not quantified"]},{"year":2025,"claim":"Captured the human ATE1 ternary recognition mechanism, showing adjacent substrate and tRNA pockets and multivalent micromolar-affinity selectivity, with apo homodimerization.","evidence":"Cryo-EM of human ATE1 with Arg-tRNAArg and an Nt-Asp peptide plus quantitative binding assays","pmids":["40099869"],"confidence":"High","gaps":["Conformational coupling between dimerization and catalysis unresolved","Does not address [Fe-S]/redox regulation in human enzyme"]},{"year":2024,"claim":"Identified a metazoan-specific intrinsically disordered region in ATE1 implicated in tRNAArg complex formation, indicating added regulatory complexity over yeast.","evidence":"SEC, SAXS, HDX-MS, and AlphaFold modeling of mouse ATE1","pmids":["39642180"],"confidence":"Medium","gaps":["IDR role in tRNA binding is computational/inferential","Functional impact of IDR deletion not tested in cells"]},{"year":2013,"claim":"Placed ATE1-mediated arginylation upstream of myosin II contractility, establishing a physiological role in platelet function and thrombosis.","evidence":"Platelet-specific conditional knockout mouse, reciprocal co-IP, RLC phosphorylation, clot retraction and in vivo thrombosis assays","pmids":["24293517"],"confidence":"High","gaps":["Direct arginylation site on myosin II machinery not mapped","Link from arginylation to RLC Ser19 phosphorylation mechanistically indirect"]},{"year":2017,"claim":"Showed ATE1 acts locally at neuronal growth cones via mRNA targeting and arginylated actin, connecting arginylation to neurite outgrowth.","evidence":"Nestin-Cre conditional KO, FISH for Ate1 mRNA zipcodes, live imaging, co-localization with arginylated beta-actin","pmids":["28844905"],"confidence":"High","gaps":["Causal chain from actin arginylation to F-actin levels not isolated","Relationship to later negative beta-actin arginylation findings unresolved"]},{"year":2018,"claim":"Provided direct evidence that ATE1 arginylates actin and actin-binding proteins to control polymerization and adhesion dynamics.","evidence":"Dictyostelium ate1 KO, MS mapping of four actin arginylation sites, in vitro polymerization assay, GFP-Ate1 live imaging","pmids":["30586322"],"confidence":"High","gaps":["Conservation of these actin sites in mammals not established here","Mechanism of Ate1 recruitment to protrusions unknown"]},{"year":2021,"claim":"Defined RGS7 and RGS5 as ATE1-dependent degradation substrates, linking arginylation to G-protein signaling and Wnt/beta-catenin pathway control.","evidence":"Conditional nervous-system KO with ERG, MEF proteasome-inhibitor assays (RGS7); knockdown/overexpression with GSK inhibitor and tumor models (RGS5)","pmids":["33931669","34158395"],"confidence":"High","gaps":["Arginylation site on RGS7/RGS5 not directly mapped","RGS5 substrate status (Medium evidence) lacks direct arginylation validation"]},{"year":2016,"claim":"Established ATE1 as a pro-death, genome-protective stress effector whose activity rises under acute stress and is required for the cell-death response.","evidence":"Gene deletion/knockdown in yeast, mouse, human cells; stress viability, enzymatic activity, and UV mutation-frequency assays","pmids":["27685622"],"confidence":"Medium","gaps":["Substrates mediating stress-induced death not identified","Mechanism of activity increase under stress unknown"]},{"year":2024,"claim":"Proposed a mitochondrial route for ATE1-induced apoptosis dependent on the permeability transition pore and AIF, independent of cytosolic degradation pathways.","evidence":"Budding yeast model, mitochondrial fractionation, genetic epistasis with pore and AIF mutants, live imaging (preprint)","pmids":["bio_10.1101_2024.11.22.624728"],"confidence":"Medium","gaps":["Preprint, not peer-reviewed","Mitochondrial arginylation substrate not identified","Conservation in mammalian cells not shown"]},{"year":2025,"claim":"Scaled arginylation site discovery to the human proteome with isotopic controls, broadening the known ATE1 substrate landscape.","evidence":"Isotopic arginine labeling in ex vivo ATE1 assay on lysates with bottom-up MS and representative functional validation","pmids":["40855110"],"confidence":"Medium","gaps":["Most of the 235 sites lack individual functional validation","Ex vivo assay may not reflect in-cell selectivity"]},{"year":2021,"claim":"Revealed that mammalian Ate1 expression itself is regulated by long-range intronic RNA structure-controlled mutually exclusive splicing coupled to transcription elongation.","evidence":"Minigene compensatory mutagenesis, endogenous LNA/DNA mixmer blocking, RNA Pol II slowdown experiments","pmids":["33330934"],"confidence":"High","gaps":["Functional differences between exon 7a- and 7b-containing isoforms not defined","Physiological signals driving isoform choice unknown"]},{"year":2022,"claim":"Linked ATE1 to cancer cell proliferation and stabilization of oncogenic effectors, with both tumor-suppressive and tumor-promoting roles depending on context.","evidence":"Melanoma knockdown with putative AXIN1 substrate (Low); breast cancer knockdown/xenograft with N-terminomics showing MYC stabilization via ERK Ser62 and catalytic-rescue (Medium)","pmids":["35561126","40898325"],"confidence":"Medium","gaps":["AXIN1 arginylation is putative without direct validation","Mechanism reconciling tumor-suppressive (RGS5/liver) versus tumor-promoting (MYC/breast) roles unresolved"]},{"year":2024,"claim":"Demonstrated ATE1 arginylation as an antiviral mechanism by targeting a viral protein for proteasomal degradation.","evidence":"ATE1 knockdown, activity inhibition, Arg supplementation, and ubiquitination assays on Newcastle disease virus HN protein","pmids":["39207120"],"confidence":"Medium","gaps":["In vivo relevance during infection not established","Single lab"]},{"year":null,"claim":"How the [Fe-S]/oxygen and hemin signals, the metazoan IDR, and dimerization are integrated to control substrate choice in living cells — and how ATE1 switches between cytoprotective, pro-death, tumor-suppressive, and tumor-promoting outputs — remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model coupling cofactor state to in-cell substrate selection","Context determinants of opposing cancer roles unknown","Physiological substrates for most arginylation sites unvalidated"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,2,4,18]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,18]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[2,4,5]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[13]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[13]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[8,9]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,10,21]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[12,13]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[15]}],"complexes":[],"partners":["MYH9","RGS7","RGS5","MYC","LIAT1"],"other_free_text":[]}},"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 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arginyl-tRNA-protein transferase (R-transferase) that catalyzes post-translational conjugation of Arg to the N-termini of acceptor proteins; expression in E. coli confirmed this catalytic activity, and null ate1 mutants lack Arg-transferase activity and cannot degrade N-end rule substrates requiring Nt-arginylation.\",\n      \"method\": \"Heterologous expression in E. coli (functional complementation), null mutant analysis with enzymatic assay and protein degradation readout\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct biochemical reconstitution of enzymatic activity in E. coli, confirmed by loss-of-function genetics with two orthogonal readouts (enzyme assay + substrate degradation)\",\n      \"pmids\": [\"2185248\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ATE1 can arginylate internal side-chain carboxylates (Asp and Glu residues) within intact proteins in vivo, in addition to N-terminal alpha-amino groups, demonstrating an unconventional midchain arginylation mechanism.\",\n      \"method\": \"Mass spectrometry-based proteomics (MS/MS), in vitro arginylation assay with purified ATE1\",\n      \"journal\": \"Chemistry & biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro assay with purified enzyme plus in vivo MS identification, single lab but two orthogonal approaches\",\n      \"pmids\": [\"24529990\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Crystal structure of Kluyveromyces lactis Ate1 reveals a 58-kDa two-domain R-transferase where both domains together recognize the acidic N-terminal residue of the acceptor substrate and the Arg-tRNAArg cosubstrate (including its 3'-proximal tRNA segment), with the active site located between the two domains; hemin (Fe3+-heme) inhibits Nt-arginylation and induces disulfide-mediated oligomerization of Ate1.\",\n      \"method\": \"X-ray crystallography, in vitro and in vivo arginylation assays, site-directed mutagenesis guided by structural data\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with mutagenesis and functional arginylation assays in the same study\",\n      \"pmids\": [\"35878037\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Crystal structure of S. cerevisiae ATE1 in the apo form reveals a bilobed protein with a GCN5-related N-acetyltransferase (GNAT) fold; structural and electrostatic analyses identify the domain-domain interface as the catalytic site and tRNA-binding region; the N-terminal domain that binds a regulatory [Fe-S] cluster is dynamic and disordered when metal-free.\",\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 / Strong — crystal structure corroborated by orthogonal solution-scattering and cryo-EM methods in the same 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 Nt-Asp peptide reveals two adjacent binding pockets for the substrate and the tRNA cosubstrate, the tRNA being wrapped by a long unstructured loop; in the apo state ATE1 forms a homodimer; substrate selectivity is achieved through multivalent interactions with Kd values in the micromolar range.\",\n      \"method\": \"Cryo-EM structure determination, biochemical binding assays\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure with both substrates, homodimer apo structure, and quantitative binding measurements in one study\",\n      \"pmids\": [\"40099869\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Mouse ATE1 contains an intrinsically disordered region (IDR) absent in yeast ATE1; computational and HDX-MS analyses suggest this IDR facilitates complex formation between ATE1 and tRNAArg, adding regulatory complexity not present in the yeast enzyme.\",\n      \"method\": \"SEC, SAXS, hydrogen-deuterium exchange mass spectrometry (HDX-MS), AlphaFold modeling\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple orthogonal biophysical methods (SAXS, HDX-MS) in a single study; functional implication of IDR in tRNA binding is computational/inferential\",\n      \"pmids\": [\"39642180\"],\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; the cluster is oxygen-sensitive and functions as an oxygen sensor to regulate ATE1 activity, as it decomposes upon purification in the presence of O2.\",\n      \"method\": \"Anaerobic chemical reconstitution of [Fe-S] cluster in purified ScATE1 and MmATE1, metal analysis\",\n      \"journal\": \"Methods in molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct biochemical reconstitution of cofactor in purified protein, single lab; functional oxygen-sensing role inferred from cluster lability\",\n      \"pmids\": [\"37010764\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ATE1 co-immunoprecipitates with myosin II from platelet lysates; platelet-specific ATE1 knockout mice show enhanced myosin regulatory light chain phosphorylation at Ser19 (activating myosin), enhanced clot retraction, and enhanced in vivo thrombus formation, placing ATE1-mediated arginylation upstream of myosin II contractility regulation.\",\n      \"method\": \"Conditional knockout mouse model, co-immunoprecipitation, phosphorylation analysis, clot retraction assay, in vivo thrombosis assay\",\n      \"journal\": \"Haematologica\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP plus conditional KO with multiple orthogonal phenotypic readouts (phosphorylation, clot retraction, in vivo thrombosis)\",\n      \"pmids\": [\"24293517\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ATE1 localizes prominently to neuronal growth cones in addition to cell bodies; Ate1 mRNA contains zipcode-binding sequences that target it to growth cone tips where local translation occurs, co-localizing with arginylated β-actin; Ate1 conditional knockout in the nervous system reduces neurite outgrowth and F-actin levels in growth cones, and decreases doublecortin levels.\",\n      \"method\": \"Conditional knockout mouse (Nestin-Cre), fluorescence in situ hybridization (FISH), live-cell imaging, immunofluorescence, protein synthesis inhibitor treatment\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO with defined phenotype, FISH for mRNA localization, co-localization with arginylated substrate, multiple orthogonal approaches in one study\",\n      \"pmids\": [\"28844905\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In Dictyostelium discoideum, Ate1 knockout eliminates focal actin adhesion sites at the substrate-attached surface, reduces adhesion, and alters chemotaxis; GFP-Ate1 rapidly relocates to newly forming actin-rich protrusions; mass spectrometry identified four arginylation sites in the major actin isoform plus sites on actin-binding proteins; actin purified from ate1-null cells shows diminished in vitro polymerization.\",\n      \"method\": \"Gene knockout, live-cell microscopy, mass spectrometry, in vitro actin polymerization assay, GFP-tagging/live imaging\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — KO with multiple phenotypic readouts, MS identification of arginylation sites, in vitro polymerization reconstitution, and live imaging of ATE1 dynamics\",\n      \"pmids\": [\"30586322\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ATE1 facilitates proteasomal degradation of RGS7 in mouse embryonic fibroblasts; conditional Ate1 knockout in the nervous system elevates RGS7 protein levels in retinal ON-bipolar cells, leading to increased light-evoked response sensitivities; RGS7 degradation is abolished in Ate1 KO MEFs but is rapid via the proteasome in wildtype cells.\",\n      \"method\": \"Conditional nervous system knockout mouse, electroretinography, MEF cell-based proteasome inhibitor experiments, immunoblotting\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO with defined biochemical (RGS7 levels) and physiological (ERG) readouts, proteasome pathway confirmed by inhibitor in two cell contexts\",\n      \"pmids\": [\"33931669\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ATE1 inhibits liver cancer progression by regulating turnover of RGS5, which in turn suppresses Wnt/β-catenin signaling by affecting GSK3-β activity; loss- and gain-of-function assays confirmed RGS5 as a key effector downstream of ATE1.\",\n      \"method\": \"Lentivirus-mediated knockdown/overexpression, loss- and gain-of-function assays, GSK inhibitor treatment, in vitro and in vivo tumor models\",\n      \"journal\": \"Molecular cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple loss/gain-of-function experiments with pathway rescue, single lab\",\n      \"pmids\": [\"34158395\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ATE1 promotes cell death and growth arrest in response to oxidative, heat, osmotic stress, heavy metals, and radiation; ATE1 protein levels and arginylation activity increase in wild-type cells under acute stress; ATE1-induced cell death requires its arginylation activity; ATE1 is required to suppress mutation frequency under DNA-damaging conditions in yeast and mammalian cells.\",\n      \"method\": \"Gene deletion/knockdown in yeast, mouse, and human cells; stress viability assays; enzymatic activity assays; mutation frequency assays under UV irradiation\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple organism/cell type models and orthogonal readouts (cell death, mutagenesis, enzymatic activity), single lab\",\n      \"pmids\": [\"27685622\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Mitochondrial translocation of Ate1 is promoted by oxidative stress and is essential for inducing apoptotic cell death; Ate1-induced cell death depends on formation of the mitochondrial permeability transition pore and at least partly on the apoptosis-inducing factor; cytosolic protein degradation pathways (ubiquitin-proteasome, autophagy, ER stress) have negligible impact on Ate1-induced cell death.\",\n      \"method\": \"Budding yeast model, mitochondrial fractionation, genetic epistasis with permeability pore mutants and AIF deletion, live-cell imaging\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — subcellular fractionation plus genetic epistasis with multiple pathway mutants; preprint, not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2024.11.22.624728\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The Ligand of Ate1 (Liat1) physically interacts with Ate1 and undergoes liquid-liquid phase separation in the nucleolus via an intrinsically disordered N-terminal region; Jumonji Domain Containing 6 (Jmjd6) hydroxylates Liat1 at its poly-K region, inhibiting nucleolar targeting of Liat1.\",\n      \"method\": \"Bimolecular fluorescence complementation, immunocytochemistry, phase separation assays, Jmjd6 enzymatic modification 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 / Moderate — bimolecular fluorescence complementation and ICC confirm interaction; phase separation and Jmjd6 modification shown but mechanism connecting back to ATE1 function is indirect\",\n      \"pmids\": [\"33443146\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The mammalian Ate1 gene undergoes alternative splicing controlled by mutually exclusive exons 7a/7b; five conserved intronic RNA structural elements (R1-R5) regulate this splicing via competing base-pair interactions (R1/R3 vs R4/R3) and an ultra-long-range R2/R5 RNA structure (~30 kb); disrupting these interactions by mutation or LNA/DNA mixmers abolishes MXE splicing; exon 7a inclusion responds to RNA Pol II slowdown in a manner dependent on the R2/R5 interaction, indicating co-transcriptional regulation.\",\n      \"method\": \"Minigene mutagenesis (single, double, compensatory triple mutations), LNA/DNA mixmer blocking in endogenous pre-mRNA, RNA Pol II slowdown experiments\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — compensatory mutation rescue in minigenes plus endogenous LNA blocking, multiple orthogonal approaches establishing RNA structural mechanism\",\n      \"pmids\": [\"33330934\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Targeted proteomics found no evidence of Nt-arginylated β-actin (RDDI-) in wildtype cells or NAA80-knockout cells; only a very minor level of Nt-arginylation of cleaved β-actin (DDDI-) was detectable in NAA80-lacking cells but not in wildtype; the final maturation state of β-actin is Nt-acetylation by NAA80, not arginylation by ATE1 under normal conditions.\",\n      \"method\": \"State-of-the-art targeted proteomics/mass spectrometry in wildtype and NAA80-KO cells\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — sensitive targeted MS with appropriate controls; negative result contradicting prior claims about β-actin arginylation; single lab\",\n      \"pmids\": [\"34896361\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In vitro, human tRNAArg directly binds the RNA recognition motifs (RRMs) of TDP-43, and the same TDP-43 constructs also bind native fungal tRNAPhe; mouse LIAT1 (Ligand of Ate1) binds human tRNAArg in vitro, identifying LIAT1 as an RNA-binding protein relevant to the arginylation machinery.\",\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 / Weak — single in vitro binding assay, no mutagenesis or functional validation, single lab\",\n      \"pmids\": [\"39081859\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Using isotopic arginine labeling in an ex vivo ATE1 assay on biological lysates, 235 unique arginylation sites were identified in human proteomes, including both N-terminal and midchain sites; representative sites were validated for biological function.\",\n      \"method\": \"Isotopic arginine labeling, ex vivo ATE1 enzymatic assay, mass spectrometry (bottom-up proteomics)\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — novel platform with isotopic controls for bona fide arginylation identification, validated representative sites; single study\",\n      \"pmids\": [\"40855110\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ATE1 depletion in melanoma cells reduces viability, migration, and colony formation; AXIN1 is identified as a putative arginylation substrate of ATE1 in melanoma, suggesting ATE1 may regulate AXIN1 function.\",\n      \"method\": \"siRNA/shRNA knockdown, cell viability and migration assays, substrate identification\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — knockdown phenotype without direct mechanistic validation of AXIN1 arginylation; AXIN1 as substrate is described as putative\",\n      \"pmids\": [\"35561126\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ATE1 stabilizes MYC protein in breast cancer cells via ERK-mediated phosphorylation at Ser62; ATE1 depletion impairs MAPK-MYC-CDK6 axis activity, reduces cell cycle progression, and promotes apoptosis; rescue experiments confirmed that ATE1's tumor-promoting activity requires its arginyltransferase catalytic function.\",\n      \"method\": \"siRNA/shRNA knockdown, quantitative proteomics, R-catcher-based N-terminomics, flow cytometry, immunoblotting, xenograft mouse model\",\n      \"journal\": \"Cell communication and signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods including N-terminomics and in vivo xenograft; catalytic requirement confirmed by rescue; single lab\",\n      \"pmids\": [\"40898325\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ATE1 mediates arginylation of the Newcastle disease virus haemagglutinin-neuraminidase (HN) protein at its N-terminus; addition of Arg amplifies arginylation of HN, reducing its stability and promoting ubiquitin-mediated proteasomal degradation; ATE1 knockdown and inhibition of ATE1 activity increase HN protein levels.\",\n      \"method\": \"ATE1 knockdown, enzymatic activity inhibition, Arg supplementation, ubiquitination assay, immunoblotting\",\n      \"journal\": \"The Journal of general virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple complementary approaches (knockdown, inhibitor, Arg supplementation, ubiquitination) in the same study; single lab\",\n      \"pmids\": [\"39207120\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ATE1 (arginyltransferase 1) is an evolutionarily conserved enzyme that transfers arginine from Arg-tRNAArg to the N-terminal residues (Asp, Glu, oxidized Cys) or internal side-chain carboxylates of acceptor proteins; its two-domain GNAT-fold structure (solved by X-ray crystallography and cryo-EM) positions an active site at the domain interface that engages both the acidic N-terminal substrate and the tRNA cosubstrate, with a regulatory [Fe-S] cluster in the N-terminal domain acting as an oxygen sensor; the resulting Arg/N-degrons direct substrates to proteasomal or autophagic degradation, and ATE1 additionally regulates actin cytoskeleton dynamics (via actin arginylation affecting polymerization), myosin II contractility in platelets, RGS7 and RGS5 protein turnover, neuronal growth cone migration, cell death responses to stress (including mitochondrial translocation under oxidative stress), and cancer cell proliferation via the MAPK-MYC axis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ATE1 is an evolutionarily conserved arginyl-tRNA-protein transferase that post-translationally conjugates arginine onto acceptor proteins, generating Arg/N-degrons and modulating substrate stability and cytoskeletal behavior across yeast, Dictyostelium, and mammalian systems [#0, #9]. The enzyme transfers Arg from Arg-tRNAArg not only to acidic N-terminal alpha-amino groups but also to internal side-chain carboxylates of Asp and Glu, an unconventional midchain arginylation, and proteome-scale profiling has mapped hundreds of N-terminal and midchain arginylation sites [#1, #18]. Structurally, ATE1 is a bilobed protein with a GCN5-related N-acetyltransferase (GNAT) fold whose active site lies at the interface between the two domains, which together engage both the acidic substrate N-terminus and the Arg-tRNAArg cosubstrate, the tRNA being wrapped by a long unstructured loop; the apo enzyme dimerizes and substrate selectivity arises from multivalent micromolar-affinity interactions [#2, #3, #4]. A regulatory oxygen-sensitive [Fe-S] cluster binds the dynamic N-terminal domain and couples ATE1 activity to redox/oxygen state, and hemin inhibits arginylation while driving disulfide-mediated oligomerization [#2, #3, #6]. Through arginylation, ATE1 governs actin polymerization and focal-adhesion dynamics, neuronal growth-cone outgrowth coupled to local Ate1 mRNA translation, and myosin II contractility in platelets, where ATE1 loss enhances regulatory light-chain phosphorylation, clot retraction, and thrombus formation [#7, #8, #9]. ATE1 controls the proteasomal turnover of regulator-of-G-protein-signaling substrates RGS7 and RGS5, the latter linking ATE1 to Wnt/beta-catenin signaling and liver-cancer suppression [#10, #11]. ATE1 also promotes stress-induced cell death, including oxidative-stress-driven mitochondrial translocation that triggers apoptosis, in a manner requiring its catalytic activity [#12, #13]. In cancer, ATE1 can act as a tumor promoter by stabilizing MYC through the ERK/MAPK axis, a function dependent on its arginyltransferase activity [#20]. The mammalian Ate1 gene is itself regulated by alternative splicing of mutually exclusive exons 7a/7b governed by long-range intronic RNA structures coupled to RNA Pol II elongation [#15].\",\n  \"teleology\": [\n    {\n      \"year\": 1990,\n      \"claim\": \"Established the molecular identity of ATE1 as the enzyme responsible for N-terminal arginylation, defining a catalytic activity central to N-end rule degradation.\",\n      \"evidence\": \"Heterologous expression of S. cerevisiae ATE1 in E. coli plus null mutant analysis with enzyme and substrate-degradation readouts\",\n      \"pmids\": [\"2185248\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not reveal the structural basis of catalysis or tRNA engagement\", \"Substrate repertoire beyond canonical N-end rule substrates undefined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Expanded the catalytic scope of ATE1 beyond N-terminal alpha-amino groups by showing it arginylates internal Asp/Glu side chains, redefining the chemistry of arginylation.\",\n      \"evidence\": \"In vitro arginylation with purified ATE1 and MS/MS identification of midchain sites in vivo\",\n      \"pmids\": [\"24529990\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequences of midchain arginylation per site not established\", \"Single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Resolved how ATE1 simultaneously recognizes its acidic protein substrate and the Arg-tRNAArg cosubstrate, and identified hemin as a regulatory inhibitor.\",\n      \"evidence\": \"X-ray crystallography of K. lactis Ate1 with mutagenesis and arginylation assays\",\n      \"pmids\": [\"35878037\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not capture the [Fe-S] cluster-bound state\", \"Mechanism of hemin-driven oligomerization in vivo unclear\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined the ATE1 fold as a bilobed GNAT architecture with the catalytic/tRNA-binding site at the domain interface and revealed the metal-free N-terminal domain as intrinsically dynamic.\",\n      \"evidence\": \"X-ray crystallography of apo S. cerevisiae ATE1 with SEC-SAXS and cryo-EM 2D averaging\",\n      \"pmids\": [\"36087779\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Apo structure lacks bound substrate and cofactor\", \"Dynamics of the N-terminal domain in the active enzyme not directly observed\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrated that ATE1 carries an oxygen-sensitive [Fe-S] cluster in its N-terminal domain, providing a candidate mechanism for redox/oxygen-coupled regulation of arginylation.\",\n      \"evidence\": \"Anaerobic chemical reconstitution of the cluster in purified yeast and mouse ATE1 with metal analysis\",\n      \"pmids\": [\"37010764\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Oxygen-sensing role inferred from cluster lability, not demonstrated in cells\", \"Effect of cluster occupancy on catalytic rate not quantified\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Captured the human ATE1 ternary recognition mechanism, showing adjacent substrate and tRNA pockets and multivalent micromolar-affinity selectivity, with apo homodimerization.\",\n      \"evidence\": \"Cryo-EM of human ATE1 with Arg-tRNAArg and an Nt-Asp peptide plus quantitative binding assays\",\n      \"pmids\": [\"40099869\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conformational coupling between dimerization and catalysis unresolved\", \"Does not address [Fe-S]/redox regulation in human enzyme\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified a metazoan-specific intrinsically disordered region in ATE1 implicated in tRNAArg complex formation, indicating added regulatory complexity over yeast.\",\n      \"evidence\": \"SEC, SAXS, HDX-MS, and AlphaFold modeling of mouse ATE1\",\n      \"pmids\": [\"39642180\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"IDR role in tRNA binding is computational/inferential\", \"Functional impact of IDR deletion not tested in cells\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Placed ATE1-mediated arginylation upstream of myosin II contractility, establishing a physiological role in platelet function and thrombosis.\",\n      \"evidence\": \"Platelet-specific conditional knockout mouse, reciprocal co-IP, RLC phosphorylation, clot retraction and in vivo thrombosis assays\",\n      \"pmids\": [\"24293517\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct arginylation site on myosin II machinery not mapped\", \"Link from arginylation to RLC Ser19 phosphorylation mechanistically indirect\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed ATE1 acts locally at neuronal growth cones via mRNA targeting and arginylated actin, connecting arginylation to neurite outgrowth.\",\n      \"evidence\": \"Nestin-Cre conditional KO, FISH for Ate1 mRNA zipcodes, live imaging, co-localization with arginylated beta-actin\",\n      \"pmids\": [\"28844905\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Causal chain from actin arginylation to F-actin levels not isolated\", \"Relationship to later negative beta-actin arginylation findings unresolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Provided direct evidence that ATE1 arginylates actin and actin-binding proteins to control polymerization and adhesion dynamics.\",\n      \"evidence\": \"Dictyostelium ate1 KO, MS mapping of four actin arginylation sites, in vitro polymerization assay, GFP-Ate1 live imaging\",\n      \"pmids\": [\"30586322\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conservation of these actin sites in mammals not established here\", \"Mechanism of Ate1 recruitment to protrusions unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined RGS7 and RGS5 as ATE1-dependent degradation substrates, linking arginylation to G-protein signaling and Wnt/beta-catenin pathway control.\",\n      \"evidence\": \"Conditional nervous-system KO with ERG, MEF proteasome-inhibitor assays (RGS7); knockdown/overexpression with GSK inhibitor and tumor models (RGS5)\",\n      \"pmids\": [\"33931669\", \"34158395\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Arginylation site on RGS7/RGS5 not directly mapped\", \"RGS5 substrate status (Medium evidence) lacks direct arginylation validation\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Established ATE1 as a pro-death, genome-protective stress effector whose activity rises under acute stress and is required for the cell-death response.\",\n      \"evidence\": \"Gene deletion/knockdown in yeast, mouse, human cells; stress viability, enzymatic activity, and UV mutation-frequency assays\",\n      \"pmids\": [\"27685622\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Substrates mediating stress-induced death not identified\", \"Mechanism of activity increase under stress unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Proposed a mitochondrial route for ATE1-induced apoptosis dependent on the permeability transition pore and AIF, independent of cytosolic degradation pathways.\",\n      \"evidence\": \"Budding yeast model, mitochondrial fractionation, genetic epistasis with pore and AIF mutants, live imaging (preprint)\",\n      \"pmids\": [\"bio_10.1101_2024.11.22.624728\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not peer-reviewed\", \"Mitochondrial arginylation substrate not identified\", \"Conservation in mammalian cells not shown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Scaled arginylation site discovery to the human proteome with isotopic controls, broadening the known ATE1 substrate landscape.\",\n      \"evidence\": \"Isotopic arginine labeling in ex vivo ATE1 assay on lysates with bottom-up MS and representative functional validation\",\n      \"pmids\": [\"40855110\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Most of the 235 sites lack individual functional validation\", \"Ex vivo assay may not reflect in-cell selectivity\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Revealed that mammalian Ate1 expression itself is regulated by long-range intronic RNA structure-controlled mutually exclusive splicing coupled to transcription elongation.\",\n      \"evidence\": \"Minigene compensatory mutagenesis, endogenous LNA/DNA mixmer blocking, RNA Pol II slowdown experiments\",\n      \"pmids\": [\"33330934\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional differences between exon 7a- and 7b-containing isoforms not defined\", \"Physiological signals driving isoform choice unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Linked ATE1 to cancer cell proliferation and stabilization of oncogenic effectors, with both tumor-suppressive and tumor-promoting roles depending on context.\",\n      \"evidence\": \"Melanoma knockdown with putative AXIN1 substrate (Low); breast cancer knockdown/xenograft with N-terminomics showing MYC stabilization via ERK Ser62 and catalytic-rescue (Medium)\",\n      \"pmids\": [\"35561126\", \"40898325\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"AXIN1 arginylation is putative without direct validation\", \"Mechanism reconciling tumor-suppressive (RGS5/liver) versus tumor-promoting (MYC/breast) roles unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrated ATE1 arginylation as an antiviral mechanism by targeting a viral protein for proteasomal degradation.\",\n      \"evidence\": \"ATE1 knockdown, activity inhibition, Arg supplementation, and ubiquitination assays on Newcastle disease virus HN protein\",\n      \"pmids\": [\"39207120\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo relevance during infection not established\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the [Fe-S]/oxygen and hemin signals, the metazoan IDR, and dimerization are integrated to control substrate choice in living cells — and how ATE1 switches between cytoprotective, pro-death, tumor-suppressive, and tumor-promoting outputs — remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model coupling cofactor state to in-cell substrate selection\", \"Context determinants of opposing cancer roles unknown\", \"Physiological substrates for most arginylation sites unvalidated\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 2, 4, 18]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 18]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [2, 4, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [13]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [13]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [8, 9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 10, 21]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [12, 13]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [15]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"MYH9\", \"RGS7\", \"RGS5\", \"MYC\", \"LIAT1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":9,"faith_total":9,"faith_pct":100.0}}