{"gene":"NAE1","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2003,"finding":"Crystal structure of the 120 kDa quaternary complex between human APPBP1-UBA3 (NAE1-UBA3), NEDD8, and ATP revealed a bipartite interface for selective NEDD8 recruitment; mutational analysis identified a single conserved arginine in APPBP1-UBA3 as a selectivity gate preventing misactivation of ubiquitin by the NEDD8 E1.","method":"X-ray crystallography of quaternary complex; site-directed mutagenesis and in vitro activity assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional mutagenesis validation, foundational mechanistic study","pmids":["14690597"],"is_preprint":false},{"year":2003,"finding":"Purified human AppBp1-Uba3 (NAE1-UBA3) heterodimer catalyzes NEDD8 activation via a pseudo-ordered mechanism (ATP leading, NEDD8 trailing), forms a stable Nedd8-adenylate/Uba3-thioester ternary complex, and transfers NEDD8 to HsUbc12 with kcat = 3.5 s⁻¹; Ala72 of NEDD8 is a critical specificity determinant—the UbR72L mutant is activated by AppBp1-Uba3 whereas wild-type ubiquitin (Kd > 300 μM) is not.","method":"In vitro enzyme kinetics with radiolabeled substrates (³H-ATP, ¹²⁵I-NEDD8), isotope exchange, transthiolation assays, substrate mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — rigorous in vitro reconstitution with kinetic parameters and mutagenesis, multiple orthogonal methods","pmids":["12740388"],"is_preprint":false},{"year":2000,"finding":"In mammalian cells, APP-BP1 (NAE1) physically interacts with hUBA3 (binding site mapped to APP-BP1 amino acids 443–479), and together they drive the cell cycle through the S-M checkpoint; this function additionally requires the E2 enzyme hUbc12, and overexpression in primary neurons causes caspase-6-dependent apoptosis.","method":"Co-immunoprecipitation in mammalian cells; rescue of ts41 Chinese hamster cell cycle mutation; dominant-negative and overexpression experiments; caspase inhibitor pharmacology","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, genetic rescue, dominant-negative, pharmacological inhibition), replicated across cell types","pmids":["10722740"],"is_preprint":false},{"year":1996,"finding":"APP-BP1 (NAE1) was identified as a protein that directly binds the cytoplasmic (carboxyl-terminal) domain of the amyloid precursor protein (APP); it is a 59-kDa ubiquitously expressed protein related to the ubiquitin-activating enzyme E1.","method":"Yeast two-hybrid screen and cDNA cloning; sequence homology analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — original identification by yeast two-hybrid; replicated in later studies but initial method is single-interaction screen","pmids":["8626687"],"is_preprint":false},{"year":2003,"finding":"APP-BP1 (NAE1) binds the C-terminal 31 amino acids of APP (C31) and colocalizes with APP in lipid rafts; a dominant-negative mutant of hUbc12 blocks APP- and APP(V642I)-induced neuronal apoptosis and DNA synthesis, placing APP-BP1/NEDD8 pathway downstream of APP in neuronal apoptosis signaling.","method":"Co-immunoprecipitation, subcellular fractionation (lipid rafts), coexpression of dominant-negative hUbc12, overexpression/siRNA in primary neurons","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, fractionation, dominant-negative epistasis, siRNA) in the same study","pmids":["14557245"],"is_preprint":false},{"year":2003,"finding":"ASPP2 physically interacts with APP-BP1 (NAE1) through its N-terminal domain (aa 332–483) in non-transfected cells and acts as a negative regulator of the neddylation pathway: ASPP2 reduces NEDD8 conjugation to Cullin-1, inhibits APP-BP1-dependent ts41 cell proliferation, and blocks APP-BP1-induced neuronal apoptosis.","method":"Co-immunoprecipitation in endogenous (non-transfected) cells; ts41 rescue assay; NEDD8-Cullin-1 conjugation assay; neuronal apoptosis assays with overexpression","journal":"Journal of neurochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus functional assays, single lab","pmids":["12694406"],"is_preprint":false},{"year":2008,"finding":"TRIP12, a HECT-domain E3 ubiquitin ligase, specifically interacts with the APP-BP1 (NAE1) monomer (but not the APP-BP1/UBA3 heterodimer) and catalyzes polyubiquitination and proteasomal degradation of APP-BP1; TRIP12 knockdown stabilizes APP-BP1 and increases neddylation of endogenous CUL1.","method":"Yeast two-hybrid; co-immunoprecipitation; in vitro ubiquitination assay; siRNA knockdown with neddylation readout","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro ubiquitination assay plus Co-IP and knockdown, single lab","pmids":["18627766"],"is_preprint":false},{"year":2007,"finding":"APP-BP1 (NAE1) co-precipitates with Presenilin-1 (PS1) in native rat brain extracts, co-migrates with γ-secretase components in glycerol gradient centrifugation, and colocalizes in neurons; siRNA knockdown of APP-BP1 increases cell-associated Aβ42 and APP-CTF levels, while APP-BP1 overexpression decreases Aβ and APP levels, suggesting APP-BP1 negatively regulates Aβ42 production via interaction with PS1.","method":"Co-immunoprecipitation from rat brain; glycerol gradient fractionation; siRNA knockdown; overexpression in primary neurons","journal":"Molecular neurodegeneration","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, gradient fractionation, siRNA, OE), single lab","pmids":["17286867"],"is_preprint":false},{"year":2006,"finding":"Drosophila dAPP-BP1 (ortholog of NAE1) specifically binds the intracellular domain of APPL (Drosophila APP-like protein); loss of dAPP-BP1 blocks NEDD8 conjugation and causes apoptosis in imaginal disc cells; dAPP-BP1 and APPL interact antagonistically—APPL overexpression inhibits the NEDD8 conjugation pathway, and APPL-induced apoptosis is rescued by dAPP-BP1 overexpression.","method":"Genetic loss-of-function (dAPP-BP1 mutation); yeast two-hybrid and Co-IP; NEDD8 conjugation assay; epistasis with overexpression in Drosophila","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis plus biochemical interaction and functional rescue in Drosophila ortholog, single lab","pmids":["16628230"],"is_preprint":false},{"year":2018,"finding":"Using quantitative FRET, only UBA3 subunit of the NAE1-UBA3 E1 heterodimer is required for NEDD8 activation; APPBP1 (NAE1) functions primarily as a scaffold/regulatory subunit that accelerates the activation reaction rate but does not catalyze it independently.","method":"Quantitative FRET assay; computational electrostatic analysis (AESOP); in vitro reconstitution with individual subunits","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with quantitative FRET, single lab, no independent replication","pmids":["29973603"],"is_preprint":false},{"year":2024,"finding":"NAE1 undergoes lysine crotonylation at K238 during cardiac hypertrophy; K238 crotonylation promotes neddylation of gelsolin (GSN) by NAE1, enhancing GSN protein stability; increased GSN promotes actin-severing activity leading to adverse cytoskeletal remodeling and pathological hypertrophy. NAE1 K238R knock-in mice are protected from hypertrophy while K238Q knock-in mice show enhanced hypertrophic response.","method":"TMT-labeled quantitative crotonylomics; NAE1 K238R and K238Q knock-in mice; co-immunoprecipitation + mass spectrometry; in vitro neddylation assay; actin-severing functional assay","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal methods (proteomics, knock-in mice, in vitro neddylation, functional readout), single lab with rigorous genetic validation","pmids":["39229723"],"is_preprint":false},{"year":2025,"finding":"NAE1 expression in CD8+ T cells is induced by NFATc1 downstream of TCR signaling; NAE1-mediated neddylation is required for CD8+ T cell activation, proliferation, survival, and mitochondrial function—genetic ablation of NAE1 severely compromises all these functions and abolishes antitumor CD8+ T cell activity; overexpression of NAE1 improves tumor-infiltrating CD8+ T cell function.","method":"Genetic ablation (conditional KO); proteomics; mitochondrial function assays; in vivo tumor models; NFATc1 transcription factor binding assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with defined molecular (proteomic) and functional phenotypes, in vivo tumor model, multiple orthogonal methods","pmids":["40030035"],"is_preprint":false},{"year":2025,"finding":"NAE1-mediated neddylation is required for meiotic recombination during spermatogenesis; germ-cell-specific Nae1 knockout mice show late-pachytene arrest, failure of double-strand break repair, excessive recombination intermediate stabilization, and failed crossover formation; ubiquitination regulation coordinates with NAE1-mediated neddylation in this process.","method":"Germ-cell-specific Nae1 knockout mice; chromosome spread fluorescence staining; 10× Genomics single-cell transcriptomics; ubiquitinomics","journal":"Theranostics","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional genetic KO with defined meiotic phenotype, multiple molecular readouts including ubiquitinomics","pmids":["40083933"],"is_preprint":false},{"year":2025,"finding":"Muscle-specific Nae1 (APP-BP1) knockout decreases acetylcholine receptor α (AChRα) stability, reduces AChR clustering, and impairs neuromuscular junction (NMJ) development; double-heterozygous Nae1/rapsyn-C366A mice (rapsyn mutation eliminating its E3 neddylation ligase activity) show NMJ deficits not seen in either single heterozygote, providing genetic evidence that NAE1-mediated neddylation acts via rapsyn E3 activity in NMJ formation.","method":"Muscle-specific conditional Nae1 knockout (Pax7-Cre;Nae1f/f); rapsyn C366A knock-in; double heterozygous genetic epistasis; AChR clustering assays; NMJ morphology analysis","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO plus genetic epistasis with knock-in mutation, multiple functional readouts","pmids":["40659529"],"is_preprint":false},{"year":2026,"finding":"NAE1/UBA3 and UBE2M function as E1 and E2 enzymes, respectively, for the URM1 (ubiquitin-related modifier 1) urmylation pathway in human cells under normal and oxidative stress conditions; pharmacological inhibition of NAE1 with pevonedistat blocks protein urmylation.","method":"Activity-based URM1 probe; proteomic characterization (mass spectrometry); cell-based validation; pharmacological inhibition with pevonedistat","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — activity-based chemical probe with proteomic validation and cell-based functional confirmation, multiple orthogonal methods","pmids":["42056084"],"is_preprint":false},{"year":2023,"finding":"Bi-allelic loss-of-function variants in NAE1 cause decreased NAE1 protein abundance and impaired neddylation; patient-derived fibroblasts show increased cell death upon proteasomal stress (MG132), decreased NF-κB nuclear translocation after stimulation, and decreased lymphocyte counts after CD3/CD28 stimulation; RUNX2 and SOX9 are significantly downregulated in patient fibroblast transcriptomes.","method":"Patient fibroblasts with bi-allelic NAE1 variants; proteasomal stress assay (MG132); NF-κB translocation assay; lymphocyte stimulation assay; transcriptomics","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — human genetic variants with functional cellular validation, multiple phenotypic readouts, single study","pmids":["36608681"],"is_preprint":false},{"year":2025,"finding":"NAE1 mediates neddylation of transferrin receptor 1 (TFR1); H. pylori infection downregulates NAE1, thereby reducing TFR1 neddylation and increasing TFR1 protein stability, which suppresses ferroptosis in gastric cancer cells; overexpression of NAE1 inhibits gastric cancer metastasis and promotes ferroptosis.","method":"In vitro neddylation assay; fluorescence in situ hybridization (co-localization); Western blot; siRNA/overexpression; cell viability and ferroptosis assays","journal":"Critical reviews in eukaryotic gene expression","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro neddylation assay plus cell-based functional validation, single lab","pmids":["41135076"],"is_preprint":false}],"current_model":"NAE1 (APP-BP1) is the regulatory subunit of the heterodimeric NEDD8-activating E1 enzyme (NAE1-UBA3), functioning primarily as a scaffold that enhances UBA3 catalytic activity; together they adenylate NEDD8 and transfer it to the E2 enzyme UBE2M/Ubc12, initiating the neddylation cascade that activates cullin-RING ubiquitin ligases, regulates the cell cycle S-M checkpoint, and controls diverse processes including CD8+ T cell activation, NMJ formation, meiotic recombination, and cardiac hypertrophy (via gelsolin neddylation); NAE1 itself is subject to regulatory crotonylation (K238), ubiquitin-mediated degradation by TRIP12, and transcriptional induction by NFATc1, and it additionally serves as the E1 for the URM1 urmylation pathway and interacts with APP, ASPP2, and Presenilin-1 to modulate neuronal signaling and Aβ processing."},"narrative":{"mechanistic_narrative":"NAE1 (APP-BP1) is the regulatory subunit of the heterodimeric NEDD8-activating E1 enzyme, partnering with the catalytic subunit UBA3 to initiate the neddylation cascade that activates cullin-RING ubiquitin ligases and drives cell cycle progression through the S-M checkpoint [PMID:14690597, PMID:10722740]. The NAE1-UBA3 heterodimer adenylates NEDD8 in a pseudo-ordered ATP-leading mechanism, forms a NEDD8-adenylate/UBA3-thioester intermediate, and transfers NEDD8 to the E2 enzyme UBE2M/Ubc12; selectivity for NEDD8 over ubiquitin is enforced both by a conserved arginine selectivity gate in the E1 and by Ala72 of NEDD8 [PMID:14690597, PMID:12740388]. Within this complex NAE1 acts principally as a scaffold/regulatory subunit—UBA3 carries the catalytic activity while NAE1 accelerates the activation reaction [PMID:29973603]. Through this neddylation activity NAE1 controls a broad range of physiological processes: CD8+ T cell activation, proliferation, survival, and mitochondrial function downstream of NFATc1/TCR signaling [PMID:40030035]; meiotic recombination and double-strand break repair during spermatogenesis [PMID:40083933]; neuromuscular junction formation via the rapsyn E3 neddylation ligase, which stabilizes acetylcholine receptors [PMID:40659529]; and cardiac hypertrophy, where K238 crotonylation of NAE1 promotes neddylation and stabilization of gelsolin to drive cytoskeletal remodeling [PMID:39229723]. NAE1 abundance is itself regulated by TRIP12-mediated polyubiquitination and proteasomal degradation of the NAE1 monomer [PMID:18627766]. Beyond NEDD8, NAE1-UBA3 also functions as the E1 for the URM1 urmylation pathway [PMID:42056084]. NAE1 was originally identified through direct binding to the cytoplasmic domain of the amyloid precursor protein (APP), and functions in neuronal apoptosis signaling and Aβ processing through interactions with APP, ASPP2, and Presenilin-1 [PMID:8626687, PMID:14557245, PMID:12694406, PMID:17286867]. Bi-allelic loss-of-function variants in NAE1 cause a human disease characterized by impaired neddylation, immune dysfunction, and skeletal transcriptional defects [PMID:36608681].","teleology":[{"year":1996,"claim":"Established the protein now called NAE1 as a direct binding partner of the amyloid precursor protein, linking it to APP biology and noting its homology to the ubiquitin-activating E1 enzyme.","evidence":"Yeast two-hybrid screen, cDNA cloning, and sequence homology analysis","pmids":["8626687"],"confidence":"Medium","gaps":["Enzymatic activity not yet demonstrated","Functional consequence of APP binding undefined","Single yeast two-hybrid interaction screen"]},{"year":2000,"claim":"Defined NAE1 as a UBA3-binding cell cycle regulator, mapping the UBA3 interaction site and placing NAE1 in the neddylation E1/E2 cascade driving the S-M checkpoint.","evidence":"Co-IP, genetic rescue of the ts41 hamster cell cycle mutant, dominant-negative and overexpression experiments in mammalian cells and neurons","pmids":["10722740"],"confidence":"High","gaps":["Catalytic mechanism of NEDD8 activation not resolved","Substrate ligases downstream not enumerated"]},{"year":2003,"claim":"Resolved the structural and kinetic mechanism of NEDD8 activation and the molecular basis of NEDD8-versus-ubiquitin selectivity, establishing how NAE1-UBA3 charges the E2 Ubc12.","evidence":"X-ray crystallography of the APPBP1-UBA3-NEDD8-ATP quaternary complex, in vitro enzyme kinetics with radiolabeled substrates, transthiolation assays, and substrate mutagenesis","pmids":["14690597","12740388"],"confidence":"High","gaps":["Did not assign relative catalytic contributions of NAE1 versus UBA3 subunits","Regulation of the enzyme in cells not addressed"]},{"year":2003,"claim":"Connected NAE1 neddylation activity to neuronal apoptosis signaling downstream of APP and identified ASPP2 as a negative regulator that suppresses Cullin-1 neddylation.","evidence":"Co-IP, lipid raft fractionation, dominant-negative Ubc12 epistasis, and siRNA in primary neurons (APP/C31 binding); reciprocal endogenous Co-IP and ts41/neddylation/apoptosis assays (ASPP2)","pmids":["14557245","12694406"],"confidence":"High","gaps":["Mechanism by which ASPP2 inhibits the E1 not defined","Physiological relevance of neuronal apoptosis pathway in vivo unresolved"]},{"year":2006,"claim":"Confirmed via the Drosophila ortholog that APP-family proteins antagonistically regulate the NEDD8 conjugation pathway and that NAE1 loss triggers apoptosis, generalizing the APP-neddylation link.","evidence":"Genetic loss-of-function, yeast two-hybrid/Co-IP, NEDD8 conjugation assays, and overexpression epistasis in Drosophila imaginal discs","pmids":["16628230"],"confidence":"Medium","gaps":["Ortholog data may not fully translate to mammalian context","Molecular mechanism of APPL antagonism unresolved"]},{"year":2007,"claim":"Implicated NAE1 in Aβ processing by showing it associates with Presenilin-1/γ-secretase and negatively regulates Aβ42 production.","evidence":"Co-IP from rat brain, glycerol gradient fractionation, siRNA knockdown, and overexpression in primary neurons","pmids":["17286867"],"confidence":"Medium","gaps":["Whether regulation of Aβ requires neddylation catalytic activity unclear","Single-lab study without in vivo confirmation"]},{"year":2008,"claim":"Identified TRIP12 as an E3 ligase that selectively degrades the NAE1 monomer, revealing post-translational control of cellular neddylation capacity.","evidence":"Yeast two-hybrid, Co-IP, in vitro ubiquitination assay, and siRNA knockdown with CUL1 neddylation readout","pmids":["18627766"],"confidence":"Medium","gaps":["Physiological conditions triggering NAE1 turnover not defined","In vivo relevance not established"]},{"year":2018,"claim":"Determined that catalytic activity resides in UBA3 while NAE1 serves as a scaffold/regulatory subunit accelerating the reaction, clarifying the division of labor within the E1 heterodimer.","evidence":"Quantitative FRET, computational electrostatic analysis, and in vitro reconstitution with individual subunits","pmids":["29973603"],"confidence":"Medium","gaps":["Single-lab in vitro result without independent replication","Structural basis of NAE1's rate acceleration not detailed"]},{"year":2023,"claim":"Established NAE1 as a human disease gene, showing bi-allelic loss-of-function impairs neddylation with immune and skeletal transcriptional consequences.","evidence":"Patient fibroblasts with bi-allelic variants, proteasomal stress assay, NF-κB translocation assay, lymphocyte stimulation, and transcriptomics","pmids":["36608681"],"confidence":"Medium","gaps":["Single cohort study","Mechanistic link from neddylation loss to RUNX2/SOX9 downregulation not resolved"]},{"year":2024,"claim":"Revealed that crotonylation of NAE1 at K238 directs neddylation toward gelsolin, defining a PTM-controlled substrate-stabilization mechanism in cardiac hypertrophy.","evidence":"Quantitative crotonylomics, NAE1 K238R/K238Q knock-in mice, Co-IP/MS, in vitro neddylation, and actin-severing assays","pmids":["39229723"],"confidence":"High","gaps":["Crotonyltransferase/decrotonylase enzymes acting on K238 not identified","Whether K238 crotonylation governs other substrates unknown"]},{"year":2025,"claim":"Demonstrated NAE1-mediated neddylation is essential across diverse physiological contexts—CD8+ T cell antitumor function, meiotic recombination, and neuromuscular junction formation—each via distinct downstream effectors.","evidence":"Conditional/germ-cell/muscle-specific Nae1 knockouts, NFATc1 binding assays, single-cell transcriptomics, ubiquitinomics, rapsyn-C366A epistasis, and AChR clustering/NMJ morphology assays; gastric cancer TFR1 neddylation and ferroptosis assays","pmids":["40030035","40083933","40659529","41135076"],"confidence":"High","gaps":["Full substrate repertoire in each tissue incompletely mapped","TFR1 ferroptosis study is single-lab Medium confidence"]},{"year":2026,"claim":"Expanded NAE1's enzymatic scope beyond NEDD8 by showing NAE1-UBA3 also serves as the E1 for the URM1 urmylation pathway under normal and oxidative stress conditions.","evidence":"Activity-based URM1 probe, proteomic characterization, cell-based validation, and pharmacological inhibition with pevonedistat","pmids":["42056084"],"confidence":"High","gaps":["Relative cellular flux through NEDD8 versus URM1 pathways unquantified","Determinants of NAE1 substrate selection between modifiers undefined"]},{"year":null,"claim":"How NAE1 substrate/effector specificity is tuned across tissues and between the NEDD8 and URM1 pathways, and how its post-translational regulation integrates with disease, remains open.","evidence":"","pmids":[],"confidence":"High","gaps":["No unified model linking PTM state, partner availability, and substrate choice","Mechanism routing NAE1 between neddylation and urmylation unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,10,16]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[1]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[9]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[1,14]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,2]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[2]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[11,15]},{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[12]}],"complexes":["NEDD8-activating E1 enzyme (NAE1-UBA3 heterodimer)"],"partners":["UBA3","UBE2M","APP","ASPP2","TRIP12","PSEN1","GSN","TFRC"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q13564","full_name":"NEDD8-activating enzyme E1 regulatory subunit","aliases":["Amyloid beta precursor protein-binding protein 1, 59 kDa","APP-BP1","Amyloid protein-binding protein 1","Proto-oncogene protein 1"],"length_aa":534,"mass_kda":60.2,"function":"Regulatory subunit of the dimeric UBA3-NAE1 E1 enzyme. E1 activates NEDD8 by first adenylating its C-terminal glycine residue with ATP, thereafter linking this residue to the side chain of the catalytic cysteine, yielding a NEDD8-UBA3 thioester and free AMP. E1 finally transfers NEDD8 to the catalytic cysteine of UBE2M. Necessary for cell cycle progression through the S-M checkpoint. Overexpression of NAE1 causes apoptosis through deregulation of NEDD8 conjugation. The covalent attachment of NEDD8 to target proteins is known as 'neddylation' and the process is involved in the regulation of cell growth, viability and development","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q13564/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/NAE1","classification":"Common Essential","n_dependent_lines":1026,"n_total_lines":1208,"dependency_fraction":0.8493377483443708},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"SAR1B","stoichiometry":0.2},{"gene":"UBE2M","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/NAE1","total_profiled":1310},"omim":[{"mim_id":"620210","title":"NEURODEVELOPMENTAL DISORDER WITH DYSMORPHIC FACIES AND ISCHIOPUBIC HYPOPLASIA; NEDFIH","url":"https://www.omim.org/entry/620210"},{"mim_id":"617700","title":"UBIQUITIN-CONJUGATING ENZYME E2 F; UBE2F","url":"https://www.omim.org/entry/617700"},{"mim_id":"616522","title":"DCN1 DOMAIN-CONTAINING PROTEIN 5; DCUN1D5","url":"https://www.omim.org/entry/616522"},{"mim_id":"603385","title":"NEDD8-ACTIVATING ENZYME E1, SUBUNIT 1; NAE1","url":"https://www.omim.org/entry/603385"},{"mim_id":"603172","title":"UBIQUITIN-LIKE MODIFIER-ACTIVATING ENZYME 3; UBA3","url":"https://www.omim.org/entry/603172"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Microtubules","reliability":"Additional"},{"location":"Centrosome","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/NAE1"},"hgnc":{"alias_symbol":["ula-1","APP-BP1"],"prev_symbol":["APPBP1"]},"alphafold":{"accession":"Q13564","domains":[{"cath_id":"3.40.50.720","chopping":"2-173_490-533","consensus_level":"medium","plddt":97.2579,"start":2,"end":533},{"cath_id":"-","chopping":"191-277","consensus_level":"high","plddt":93.4495,"start":191,"end":277},{"cath_id":"-","chopping":"284-394","consensus_level":"high","plddt":96.6394,"start":284,"end":394},{"cath_id":"1.10.10","chopping":"409-486","consensus_level":"medium","plddt":97.5167,"start":409,"end":486}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13564","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q13564-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q13564-F1-predicted_aligned_error_v6.png","plddt_mean":96.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NAE1","jax_strain_url":"https://www.jax.org/strain/search?query=NAE1"},"sequence":{"accession":"Q13564","fasta_url":"https://rest.uniprot.org/uniprotkb/Q13564.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q13564/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13564"}},"corpus_meta":[{"pmid":"14690597","id":"PMC_14690597","title":"The structure of the APPBP1-UBA3-NEDD8-ATP complex reveals the basis for selective ubiquitin-like protein activation by an E1.","date":"2003","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/14690597","citation_count":242,"is_preprint":false},{"pmid":"10722740","id":"PMC_10722740","title":"The amyloid precursor protein-binding protein APP-BP1 drives the cell cycle through the S-M checkpoint and causes apoptosis in neurons.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10722740","citation_count":123,"is_preprint":false},{"pmid":"8626687","id":"PMC_8626687","title":"APP-BP1, a novel protein that binds to the carboxyl-terminal region of the amyloid precursor protein.","date":"1996","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8626687","citation_count":119,"is_preprint":false},{"pmid":"12740388","id":"PMC_12740388","title":"Conservation in the mechanism of Nedd8 activation by the human AppBp1-Uba3 heterodimer.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12740388","citation_count":110,"is_preprint":false},{"pmid":"14557245","id":"PMC_14557245","title":"APP-BP1 mediates APP-induced apoptosis and DNA synthesis and is increased in Alzheimer's disease brain.","date":"2003","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/14557245","citation_count":77,"is_preprint":false},{"pmid":"35217064","id":"PMC_35217064","title":"Targeting NAE1-mediated protein hyper-NEDDylation halts cholangiocarcinogenesis and impacts on tumor-stroma crosstalk in experimental models.","date":"2022","source":"Journal of hepatology","url":"https://pubmed.ncbi.nlm.nih.gov/35217064","citation_count":39,"is_preprint":false},{"pmid":"15192323","id":"PMC_15192323","title":"APP induces neuronal apoptosis through APP-BP1-mediated downregulation of beta-catenin.","date":"2004","source":"Apoptosis : an international journal on programmed cell death","url":"https://pubmed.ncbi.nlm.nih.gov/15192323","citation_count":32,"is_preprint":false},{"pmid":"12694406","id":"PMC_12694406","title":"ASPP2 inhibits APP-BP1-mediated NEDD8 conjugation to cullin-1 and decreases APP-BP1-induced cell proliferation and neuronal apoptosis.","date":"2003","source":"Journal of neurochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12694406","citation_count":32,"is_preprint":false},{"pmid":"18627766","id":"PMC_18627766","title":"TRIP12 functions as an E3 ubiquitin ligase of APP-BP1.","date":"2008","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/18627766","citation_count":30,"is_preprint":false},{"pmid":"16275315","id":"PMC_16275315","title":"Expression, purification, and characterization of the E1 for human NEDD8, the heterodimeric APPBP1-UBA3 complex.","date":"2005","source":"Methods in enzymology","url":"https://pubmed.ncbi.nlm.nih.gov/16275315","citation_count":29,"is_preprint":false},{"pmid":"17286867","id":"PMC_17286867","title":"APP-BP1 inhibits Abeta42 levels by interacting with Presenilin-1.","date":"2007","source":"Molecular neurodegeneration","url":"https://pubmed.ncbi.nlm.nih.gov/17286867","citation_count":24,"is_preprint":false},{"pmid":"16628230","id":"PMC_16628230","title":"Drosophila homolog of APP-BP1 (dAPP-BP1) interacts antagonistically with APPL during Drosophila development.","date":"2006","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/16628230","citation_count":21,"is_preprint":false},{"pmid":"39229723","id":"PMC_39229723","title":"Crotonylation of NAE1 Modulates Cardiac Hypertrophy via Gelsolin Neddylation.","date":"2024","source":"Circulation research","url":"https://pubmed.ncbi.nlm.nih.gov/39229723","citation_count":20,"is_preprint":false},{"pmid":"29973603","id":"PMC_29973603","title":"Dissecting Distinct Roles of NEDDylation E1 Ligase Heterodimer APPBP1 and UBA3 Reveals Potential Evolution Process for Activation of Ubiquitin-related Pathways.","date":"2018","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/29973603","citation_count":16,"is_preprint":false},{"pmid":"38984049","id":"PMC_38984049","title":"RBM15 Protects From Myocardial Infarction by Stabilizing NAE1.","date":"2024","source":"JACC. Basic to translational science","url":"https://pubmed.ncbi.nlm.nih.gov/38984049","citation_count":15,"is_preprint":false},{"pmid":"40030035","id":"PMC_40030035","title":"The NAE1-mediated neddylation operates as an essential post-translational modification checkpoint for effector CD8+ T cells.","date":"2025","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/40030035","citation_count":8,"is_preprint":false},{"pmid":"25568892","id":"PMC_25568892","title":"Cycle on Wheels: Is APP Key to the AppBp1 Pathway?","date":"2014","source":"Austin Alzheimer's and Parkinson's disease","url":"https://pubmed.ncbi.nlm.nih.gov/25568892","citation_count":8,"is_preprint":false},{"pmid":"36608681","id":"PMC_36608681","title":"Bi-allelic variants in NAE1 cause intellectual disability, ischiopubic hypoplasia, stress-mediated lymphopenia and neurodegeneration.","date":"2023","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/36608681","citation_count":7,"is_preprint":false},{"pmid":"21386696","id":"PMC_21386696","title":"Focal transient ischemia increases APP-BP1 expression in neural progenitor cells.","date":"2011","source":"Neuroreport","url":"https://pubmed.ncbi.nlm.nih.gov/21386696","citation_count":3,"is_preprint":false},{"pmid":"40083933","id":"PMC_40083933","title":"NAE1-mediated neddylation coordinates ubiquitination regulation of meiotic recombination during spermatogenesis.","date":"2025","source":"Theranostics","url":"https://pubmed.ncbi.nlm.nih.gov/40083933","citation_count":2,"is_preprint":false},{"pmid":"39296043","id":"PMC_39296043","title":"Dual role of targeting NAE1 in nasopharyngeal carcinoma: Antitumor effects yet inducing radiotherapy resistance.","date":"2024","source":"Heliyon","url":"https://pubmed.ncbi.nlm.nih.gov/39296043","citation_count":1,"is_preprint":false},{"pmid":"40659529","id":"PMC_40659529","title":"Neddylation E1 Obligatory Subunit Nae1 Is Critical to Neuromuscular Junction Development and Maintenance.","date":"2025","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/40659529","citation_count":0,"is_preprint":false},{"pmid":"42144028","id":"PMC_42144028","title":"Neocryptomerin targets NAE1 to induce DNA re-replication and DNA damage by inhibiting neddylation-mediated CDT1 degradation in gastric cancer.","date":"2026","source":"Pharmacological research","url":"https://pubmed.ncbi.nlm.nih.gov/42144028","citation_count":0,"is_preprint":false},{"pmid":"42056084","id":"PMC_42056084","title":"NAE1/UBA3-UBE2M are E1 and E2 enzymes for the URM1 modification.","date":"2026","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/42056084","citation_count":0,"is_preprint":false},{"pmid":"41135076","id":"PMC_41135076","title":"Helicobacter pylori Suppress the Ferroptosis of Gastric Cancer via Inhibiting NAE1-Mediated Neddylation of TFR1.","date":"2025","source":"Critical reviews in eukaryotic gene expression","url":"https://pubmed.ncbi.nlm.nih.gov/41135076","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":13989,"output_tokens":4626,"usd":0.055678,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12751,"output_tokens":4458,"usd":0.087602,"stage2_stop_reason":"end_turn"},"total_usd":0.14328,"stage1_batch_id":"msgbatch_013a8chtv3AWyTqv4z4sEF4C","stage2_batch_id":"msgbatch_01AHsuW63vinsvuTgzmQ3n62","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2003,\n      \"finding\": \"Crystal structure of the 120 kDa quaternary complex between human APPBP1-UBA3 (NAE1-UBA3), NEDD8, and ATP revealed a bipartite interface for selective NEDD8 recruitment; mutational analysis identified a single conserved arginine in APPBP1-UBA3 as a selectivity gate preventing misactivation of ubiquitin by the NEDD8 E1.\",\n      \"method\": \"X-ray crystallography of quaternary complex; site-directed mutagenesis and in vitro activity assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional mutagenesis validation, foundational mechanistic study\",\n      \"pmids\": [\"14690597\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Purified human AppBp1-Uba3 (NAE1-UBA3) heterodimer catalyzes NEDD8 activation via a pseudo-ordered mechanism (ATP leading, NEDD8 trailing), forms a stable Nedd8-adenylate/Uba3-thioester ternary complex, and transfers NEDD8 to HsUbc12 with kcat = 3.5 s⁻¹; Ala72 of NEDD8 is a critical specificity determinant—the UbR72L mutant is activated by AppBp1-Uba3 whereas wild-type ubiquitin (Kd > 300 μM) is not.\",\n      \"method\": \"In vitro enzyme kinetics with radiolabeled substrates (³H-ATP, ¹²⁵I-NEDD8), isotope exchange, transthiolation assays, substrate mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — rigorous in vitro reconstitution with kinetic parameters and mutagenesis, multiple orthogonal methods\",\n      \"pmids\": [\"12740388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"In mammalian cells, APP-BP1 (NAE1) physically interacts with hUBA3 (binding site mapped to APP-BP1 amino acids 443–479), and together they drive the cell cycle through the S-M checkpoint; this function additionally requires the E2 enzyme hUbc12, and overexpression in primary neurons causes caspase-6-dependent apoptosis.\",\n      \"method\": \"Co-immunoprecipitation in mammalian cells; rescue of ts41 Chinese hamster cell cycle mutation; dominant-negative and overexpression experiments; caspase inhibitor pharmacology\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, genetic rescue, dominant-negative, pharmacological inhibition), replicated across cell types\",\n      \"pmids\": [\"10722740\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"APP-BP1 (NAE1) was identified as a protein that directly binds the cytoplasmic (carboxyl-terminal) domain of the amyloid precursor protein (APP); it is a 59-kDa ubiquitously expressed protein related to the ubiquitin-activating enzyme E1.\",\n      \"method\": \"Yeast two-hybrid screen and cDNA cloning; sequence homology analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — original identification by yeast two-hybrid; replicated in later studies but initial method is single-interaction screen\",\n      \"pmids\": [\"8626687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"APP-BP1 (NAE1) binds the C-terminal 31 amino acids of APP (C31) and colocalizes with APP in lipid rafts; a dominant-negative mutant of hUbc12 blocks APP- and APP(V642I)-induced neuronal apoptosis and DNA synthesis, placing APP-BP1/NEDD8 pathway downstream of APP in neuronal apoptosis signaling.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation (lipid rafts), coexpression of dominant-negative hUbc12, overexpression/siRNA in primary neurons\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, fractionation, dominant-negative epistasis, siRNA) in the same study\",\n      \"pmids\": [\"14557245\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"ASPP2 physically interacts with APP-BP1 (NAE1) through its N-terminal domain (aa 332–483) in non-transfected cells and acts as a negative regulator of the neddylation pathway: ASPP2 reduces NEDD8 conjugation to Cullin-1, inhibits APP-BP1-dependent ts41 cell proliferation, and blocks APP-BP1-induced neuronal apoptosis.\",\n      \"method\": \"Co-immunoprecipitation in endogenous (non-transfected) cells; ts41 rescue assay; NEDD8-Cullin-1 conjugation assay; neuronal apoptosis assays with overexpression\",\n      \"journal\": \"Journal of neurochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus functional assays, single lab\",\n      \"pmids\": [\"12694406\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"TRIP12, a HECT-domain E3 ubiquitin ligase, specifically interacts with the APP-BP1 (NAE1) monomer (but not the APP-BP1/UBA3 heterodimer) and catalyzes polyubiquitination and proteasomal degradation of APP-BP1; TRIP12 knockdown stabilizes APP-BP1 and increases neddylation of endogenous CUL1.\",\n      \"method\": \"Yeast two-hybrid; co-immunoprecipitation; in vitro ubiquitination assay; siRNA knockdown with neddylation readout\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro ubiquitination assay plus Co-IP and knockdown, single lab\",\n      \"pmids\": [\"18627766\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"APP-BP1 (NAE1) co-precipitates with Presenilin-1 (PS1) in native rat brain extracts, co-migrates with γ-secretase components in glycerol gradient centrifugation, and colocalizes in neurons; siRNA knockdown of APP-BP1 increases cell-associated Aβ42 and APP-CTF levels, while APP-BP1 overexpression decreases Aβ and APP levels, suggesting APP-BP1 negatively regulates Aβ42 production via interaction with PS1.\",\n      \"method\": \"Co-immunoprecipitation from rat brain; glycerol gradient fractionation; siRNA knockdown; overexpression in primary neurons\",\n      \"journal\": \"Molecular neurodegeneration\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, gradient fractionation, siRNA, OE), single lab\",\n      \"pmids\": [\"17286867\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Drosophila dAPP-BP1 (ortholog of NAE1) specifically binds the intracellular domain of APPL (Drosophila APP-like protein); loss of dAPP-BP1 blocks NEDD8 conjugation and causes apoptosis in imaginal disc cells; dAPP-BP1 and APPL interact antagonistically—APPL overexpression inhibits the NEDD8 conjugation pathway, and APPL-induced apoptosis is rescued by dAPP-BP1 overexpression.\",\n      \"method\": \"Genetic loss-of-function (dAPP-BP1 mutation); yeast two-hybrid and Co-IP; NEDD8 conjugation assay; epistasis with overexpression in Drosophila\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis plus biochemical interaction and functional rescue in Drosophila ortholog, single lab\",\n      \"pmids\": [\"16628230\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Using quantitative FRET, only UBA3 subunit of the NAE1-UBA3 E1 heterodimer is required for NEDD8 activation; APPBP1 (NAE1) functions primarily as a scaffold/regulatory subunit that accelerates the activation reaction rate but does not catalyze it independently.\",\n      \"method\": \"Quantitative FRET assay; computational electrostatic analysis (AESOP); in vitro reconstitution with individual subunits\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with quantitative FRET, single lab, no independent replication\",\n      \"pmids\": [\"29973603\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NAE1 undergoes lysine crotonylation at K238 during cardiac hypertrophy; K238 crotonylation promotes neddylation of gelsolin (GSN) by NAE1, enhancing GSN protein stability; increased GSN promotes actin-severing activity leading to adverse cytoskeletal remodeling and pathological hypertrophy. NAE1 K238R knock-in mice are protected from hypertrophy while K238Q knock-in mice show enhanced hypertrophic response.\",\n      \"method\": \"TMT-labeled quantitative crotonylomics; NAE1 K238R and K238Q knock-in mice; co-immunoprecipitation + mass spectrometry; in vitro neddylation assay; actin-severing functional assay\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal methods (proteomics, knock-in mice, in vitro neddylation, functional readout), single lab with rigorous genetic validation\",\n      \"pmids\": [\"39229723\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NAE1 expression in CD8+ T cells is induced by NFATc1 downstream of TCR signaling; NAE1-mediated neddylation is required for CD8+ T cell activation, proliferation, survival, and mitochondrial function—genetic ablation of NAE1 severely compromises all these functions and abolishes antitumor CD8+ T cell activity; overexpression of NAE1 improves tumor-infiltrating CD8+ T cell function.\",\n      \"method\": \"Genetic ablation (conditional KO); proteomics; mitochondrial function assays; in vivo tumor models; NFATc1 transcription factor binding assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with defined molecular (proteomic) and functional phenotypes, in vivo tumor model, multiple orthogonal methods\",\n      \"pmids\": [\"40030035\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NAE1-mediated neddylation is required for meiotic recombination during spermatogenesis; germ-cell-specific Nae1 knockout mice show late-pachytene arrest, failure of double-strand break repair, excessive recombination intermediate stabilization, and failed crossover formation; ubiquitination regulation coordinates with NAE1-mediated neddylation in this process.\",\n      \"method\": \"Germ-cell-specific Nae1 knockout mice; chromosome spread fluorescence staining; 10× Genomics single-cell transcriptomics; ubiquitinomics\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional genetic KO with defined meiotic phenotype, multiple molecular readouts including ubiquitinomics\",\n      \"pmids\": [\"40083933\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Muscle-specific Nae1 (APP-BP1) knockout decreases acetylcholine receptor α (AChRα) stability, reduces AChR clustering, and impairs neuromuscular junction (NMJ) development; double-heterozygous Nae1/rapsyn-C366A mice (rapsyn mutation eliminating its E3 neddylation ligase activity) show NMJ deficits not seen in either single heterozygote, providing genetic evidence that NAE1-mediated neddylation acts via rapsyn E3 activity in NMJ formation.\",\n      \"method\": \"Muscle-specific conditional Nae1 knockout (Pax7-Cre;Nae1f/f); rapsyn C366A knock-in; double heterozygous genetic epistasis; AChR clustering assays; NMJ morphology analysis\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO plus genetic epistasis with knock-in mutation, multiple functional readouts\",\n      \"pmids\": [\"40659529\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"NAE1/UBA3 and UBE2M function as E1 and E2 enzymes, respectively, for the URM1 (ubiquitin-related modifier 1) urmylation pathway in human cells under normal and oxidative stress conditions; pharmacological inhibition of NAE1 with pevonedistat blocks protein urmylation.\",\n      \"method\": \"Activity-based URM1 probe; proteomic characterization (mass spectrometry); cell-based validation; pharmacological inhibition with pevonedistat\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — activity-based chemical probe with proteomic validation and cell-based functional confirmation, multiple orthogonal methods\",\n      \"pmids\": [\"42056084\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Bi-allelic loss-of-function variants in NAE1 cause decreased NAE1 protein abundance and impaired neddylation; patient-derived fibroblasts show increased cell death upon proteasomal stress (MG132), decreased NF-κB nuclear translocation after stimulation, and decreased lymphocyte counts after CD3/CD28 stimulation; RUNX2 and SOX9 are significantly downregulated in patient fibroblast transcriptomes.\",\n      \"method\": \"Patient fibroblasts with bi-allelic NAE1 variants; proteasomal stress assay (MG132); NF-κB translocation assay; lymphocyte stimulation assay; transcriptomics\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — human genetic variants with functional cellular validation, multiple phenotypic readouts, single study\",\n      \"pmids\": [\"36608681\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NAE1 mediates neddylation of transferrin receptor 1 (TFR1); H. pylori infection downregulates NAE1, thereby reducing TFR1 neddylation and increasing TFR1 protein stability, which suppresses ferroptosis in gastric cancer cells; overexpression of NAE1 inhibits gastric cancer metastasis and promotes ferroptosis.\",\n      \"method\": \"In vitro neddylation assay; fluorescence in situ hybridization (co-localization); Western blot; siRNA/overexpression; cell viability and ferroptosis assays\",\n      \"journal\": \"Critical reviews in eukaryotic gene expression\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro neddylation assay plus cell-based functional validation, single lab\",\n      \"pmids\": [\"41135076\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NAE1 (APP-BP1) is the regulatory subunit of the heterodimeric NEDD8-activating E1 enzyme (NAE1-UBA3), functioning primarily as a scaffold that enhances UBA3 catalytic activity; together they adenylate NEDD8 and transfer it to the E2 enzyme UBE2M/Ubc12, initiating the neddylation cascade that activates cullin-RING ubiquitin ligases, regulates the cell cycle S-M checkpoint, and controls diverse processes including CD8+ T cell activation, NMJ formation, meiotic recombination, and cardiac hypertrophy (via gelsolin neddylation); NAE1 itself is subject to regulatory crotonylation (K238), ubiquitin-mediated degradation by TRIP12, and transcriptional induction by NFATc1, and it additionally serves as the E1 for the URM1 urmylation pathway and interacts with APP, ASPP2, and Presenilin-1 to modulate neuronal signaling and Aβ processing.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NAE1 (APP-BP1) is the regulatory subunit of the heterodimeric NEDD8-activating E1 enzyme, partnering with the catalytic subunit UBA3 to initiate the neddylation cascade that activates cullin-RING ubiquitin ligases and drives cell cycle progression through the S-M checkpoint [#0, #2]. The NAE1-UBA3 heterodimer adenylates NEDD8 in a pseudo-ordered ATP-leading mechanism, forms a NEDD8-adenylate/UBA3-thioester intermediate, and transfers NEDD8 to the E2 enzyme UBE2M/Ubc12; selectivity for NEDD8 over ubiquitin is enforced both by a conserved arginine selectivity gate in the E1 and by Ala72 of NEDD8 [#0, #1]. Within this complex NAE1 acts principally as a scaffold/regulatory subunit—UBA3 carries the catalytic activity while NAE1 accelerates the activation reaction [#9]. Through this neddylation activity NAE1 controls a broad range of physiological processes: CD8+ T cell activation, proliferation, survival, and mitochondrial function downstream of NFATc1/TCR signaling [#11]; meiotic recombination and double-strand break repair during spermatogenesis [#12]; neuromuscular junction formation via the rapsyn E3 neddylation ligase, which stabilizes acetylcholine receptors [#13]; and cardiac hypertrophy, where K238 crotonylation of NAE1 promotes neddylation and stabilization of gelsolin to drive cytoskeletal remodeling [#10]. NAE1 abundance is itself regulated by TRIP12-mediated polyubiquitination and proteasomal degradation of the NAE1 monomer [#6]. Beyond NEDD8, NAE1-UBA3 also functions as the E1 for the URM1 urmylation pathway [#14]. NAE1 was originally identified through direct binding to the cytoplasmic domain of the amyloid precursor protein (APP), and functions in neuronal apoptosis signaling and Aβ processing through interactions with APP, ASPP2, and Presenilin-1 [#3, #4, #5, #7]. Bi-allelic loss-of-function variants in NAE1 cause a human disease characterized by impaired neddylation, immune dysfunction, and skeletal transcriptional defects [#15].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Established the protein now called NAE1 as a direct binding partner of the amyloid precursor protein, linking it to APP biology and noting its homology to the ubiquitin-activating E1 enzyme.\",\n      \"evidence\": \"Yeast two-hybrid screen, cDNA cloning, and sequence homology analysis\",\n      \"pmids\": [\"8626687\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Enzymatic activity not yet demonstrated\", \"Functional consequence of APP binding undefined\", \"Single yeast two-hybrid interaction screen\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Defined NAE1 as a UBA3-binding cell cycle regulator, mapping the UBA3 interaction site and placing NAE1 in the neddylation E1/E2 cascade driving the S-M checkpoint.\",\n      \"evidence\": \"Co-IP, genetic rescue of the ts41 hamster cell cycle mutant, dominant-negative and overexpression experiments in mammalian cells and neurons\",\n      \"pmids\": [\"10722740\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Catalytic mechanism of NEDD8 activation not resolved\", \"Substrate ligases downstream not enumerated\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Resolved the structural and kinetic mechanism of NEDD8 activation and the molecular basis of NEDD8-versus-ubiquitin selectivity, establishing how NAE1-UBA3 charges the E2 Ubc12.\",\n      \"evidence\": \"X-ray crystallography of the APPBP1-UBA3-NEDD8-ATP quaternary complex, in vitro enzyme kinetics with radiolabeled substrates, transthiolation assays, and substrate mutagenesis\",\n      \"pmids\": [\"14690597\", \"12740388\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not assign relative catalytic contributions of NAE1 versus UBA3 subunits\", \"Regulation of the enzyme in cells not addressed\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Connected NAE1 neddylation activity to neuronal apoptosis signaling downstream of APP and identified ASPP2 as a negative regulator that suppresses Cullin-1 neddylation.\",\n      \"evidence\": \"Co-IP, lipid raft fractionation, dominant-negative Ubc12 epistasis, and siRNA in primary neurons (APP/C31 binding); reciprocal endogenous Co-IP and ts41/neddylation/apoptosis assays (ASPP2)\",\n      \"pmids\": [\"14557245\", \"12694406\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which ASPP2 inhibits the E1 not defined\", \"Physiological relevance of neuronal apoptosis pathway in vivo unresolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Confirmed via the Drosophila ortholog that APP-family proteins antagonistically regulate the NEDD8 conjugation pathway and that NAE1 loss triggers apoptosis, generalizing the APP-neddylation link.\",\n      \"evidence\": \"Genetic loss-of-function, yeast two-hybrid/Co-IP, NEDD8 conjugation assays, and overexpression epistasis in Drosophila imaginal discs\",\n      \"pmids\": [\"16628230\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ortholog data may not fully translate to mammalian context\", \"Molecular mechanism of APPL antagonism unresolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Implicated NAE1 in Aβ processing by showing it associates with Presenilin-1/γ-secretase and negatively regulates Aβ42 production.\",\n      \"evidence\": \"Co-IP from rat brain, glycerol gradient fractionation, siRNA knockdown, and overexpression in primary neurons\",\n      \"pmids\": [\"17286867\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether regulation of Aβ requires neddylation catalytic activity unclear\", \"Single-lab study without in vivo confirmation\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identified TRIP12 as an E3 ligase that selectively degrades the NAE1 monomer, revealing post-translational control of cellular neddylation capacity.\",\n      \"evidence\": \"Yeast two-hybrid, Co-IP, in vitro ubiquitination assay, and siRNA knockdown with CUL1 neddylation readout\",\n      \"pmids\": [\"18627766\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological conditions triggering NAE1 turnover not defined\", \"In vivo relevance not established\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Determined that catalytic activity resides in UBA3 while NAE1 serves as a scaffold/regulatory subunit accelerating the reaction, clarifying the division of labor within the E1 heterodimer.\",\n      \"evidence\": \"Quantitative FRET, computational electrostatic analysis, and in vitro reconstitution with individual subunits\",\n      \"pmids\": [\"29973603\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab in vitro result without independent replication\", \"Structural basis of NAE1's rate acceleration not detailed\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Established NAE1 as a human disease gene, showing bi-allelic loss-of-function impairs neddylation with immune and skeletal transcriptional consequences.\",\n      \"evidence\": \"Patient fibroblasts with bi-allelic variants, proteasomal stress assay, NF-κB translocation assay, lymphocyte stimulation, and transcriptomics\",\n      \"pmids\": [\"36608681\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single cohort study\", \"Mechanistic link from neddylation loss to RUNX2/SOX9 downregulation not resolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealed that crotonylation of NAE1 at K238 directs neddylation toward gelsolin, defining a PTM-controlled substrate-stabilization mechanism in cardiac hypertrophy.\",\n      \"evidence\": \"Quantitative crotonylomics, NAE1 K238R/K238Q knock-in mice, Co-IP/MS, in vitro neddylation, and actin-severing assays\",\n      \"pmids\": [\"39229723\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Crotonyltransferase/decrotonylase enzymes acting on K238 not identified\", \"Whether K238 crotonylation governs other substrates unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated NAE1-mediated neddylation is essential across diverse physiological contexts—CD8+ T cell antitumor function, meiotic recombination, and neuromuscular junction formation—each via distinct downstream effectors.\",\n      \"evidence\": \"Conditional/germ-cell/muscle-specific Nae1 knockouts, NFATc1 binding assays, single-cell transcriptomics, ubiquitinomics, rapsyn-C366A epistasis, and AChR clustering/NMJ morphology assays; gastric cancer TFR1 neddylation and ferroptosis assays\",\n      \"pmids\": [\"40030035\", \"40083933\", \"40659529\", \"41135076\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full substrate repertoire in each tissue incompletely mapped\", \"TFR1 ferroptosis study is single-lab Medium confidence\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Expanded NAE1's enzymatic scope beyond NEDD8 by showing NAE1-UBA3 also serves as the E1 for the URM1 urmylation pathway under normal and oxidative stress conditions.\",\n      \"evidence\": \"Activity-based URM1 probe, proteomic characterization, cell-based validation, and pharmacological inhibition with pevonedistat\",\n      \"pmids\": [\"42056084\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative cellular flux through NEDD8 versus URM1 pathways unquantified\", \"Determinants of NAE1 substrate selection between modifiers undefined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How NAE1 substrate/effector specificity is tuned across tissues and between the NEDD8 and URM1 pathways, and how its post-translational regulation integrates with disease, remains open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No unified model linking PTM state, partner availability, and substrate choice\", \"Mechanism routing NAE1 between neddylation and urmylation unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 10, 16]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [9]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [1, 14]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [11, 15]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [12]}\n    ],\n    \"complexes\": [\"NEDD8-activating E1 enzyme (NAE1-UBA3 heterodimer)\"],\n    \"partners\": [\"UBA3\", \"UBE2M\", \"APP\", \"ASPP2\", \"TRIP12\", \"PSEN1\", \"GSN\", \"TFRC\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}