{"gene":"NAA40","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2019,"finding":"NAA40 catalyzes N-terminal acetylation of histone H4 serine 1 (N-acH4); loss of NAA40 and N-acH4 reduces H4R3me2s levels through transcriptional downregulation of PRMT5, establishing a functional epistatic link between NAA40-mediated histone acetylation and PRMT5-dependent arginine methylation.","method":"NAA40 knockdown in CRC cells with measurement of H4R3me2s, PRMT5 mRNA/protein levels, and xenograft tumor growth assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KD with defined cellular phenotype and pathway placement, single lab, multiple readouts including in vivo xenograft","pmids":["30858358"],"is_preprint":false},{"year":2016,"finding":"Depletion of NAA40 in colorectal cancer cells activates the mitochondrial caspase-9-mediated apoptotic cascade in a p53-independent manner; treatment with an irreversible caspase-9 inhibitor blocks apoptosis induced by NAA40 knockdown, placing NAA40 upstream of the intrinsic apoptosis pathway.","method":"siRNA knockdown of NAA40 in HCT116 and HT-29 CRC cells (including p53-null HCT116), caspase-9 activity assays, caspase-9 inhibitor rescue experiments","journal":"Apoptosis : an international journal on programmed cell death","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis via inhibitor rescue, p53-null genetic control, single lab with two orthogonal cell models","pmids":["26666750"],"is_preprint":false},{"year":2021,"finding":"NAA40 regulates one-carbon metabolism by controlling expression of the methionine cycle genes and thymidylate synthase (TYMS); mechanistically, NAA40 acetyltransferase activity prevents enrichment of repressive H2A/H4S1ph at the nuclear periphery, thereby activating TYMS transcription and promoting resistance to 5-FU chemotherapy.","method":"Transcriptomic and metabolomic analysis in CRC cells after NAA40 depletion; chromatin fractionation showing H2A/H4S1ph redistribution; in vitro and xenograft chemoresistance assays","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multi-omic approach (transcriptomics + metabolomics + chromatin fractionation) with in vivo validation, single lab","pmids":["34785778"],"is_preprint":false},{"year":2022,"finding":"Depletion of NAA40 in murine hepatocytes leads to increased intracellular acetyl-CoA levels, which associates with enhanced de novo lipogenesis (upregulation of lipogenesis genes, increased diglycerides/triglycerides, cytoplasmic lipid droplet accumulation) and impaired insulin signalling (decreased glucose uptake); the effect on lipid droplet formation is independent of insulin. This effect is replicated in vivo in Drosophila larval fat body.","method":"Metabolomic and lipidomic analysis in NAA40-depleted murine hepatocytes; de novo lipogenesis gene expression; glucose uptake assay; Drosophila in vivo lipid droplet assay","journal":"BMC biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — orthogonal metabolomics + lipidomics + in vivo Drosophila model, single lab","pmids":["35057804"],"is_preprint":false},{"year":2025,"finding":"RNF112 E3 ubiquitin ligase promotes ubiquitin-dependent proteasomal degradation of NAA40 protein; overexpression of NAA40 rescues the anti-tumor effects of RNF112 overexpression in CRC, placing NAA40 downstream of the KLF4-RNF112 axis.","method":"Co-immunoprecipitation, ubiquitination assays, NAA40 overexpression rescue experiments in RNF112-overexpressing CRC cells, in vivo nude mouse tumor formation assay","journal":"Cell biology and toxicology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ubiquitination assay with rescue epistasis, in vivo confirmation, single lab","pmids":["39757327"],"is_preprint":false},{"year":2025,"finding":"NAA40 loss in osteosarcoma cells is associated with increased H4S1ph and H4R3me2a and decreased H4R3me2s, and leads to altered H3K4me3 and H3K27me3 at the AGR2 promoter, reducing AGR2 transcription; AGR2 is identified as a downstream transcriptional target of NAA40 and its knockdown phenocopies NAA40 loss, while AGR2 overexpression partially rescues NAA40-depletion phenotypes.","method":"ChIP-qPCR for histone marks at AGR2 promoter, dual luciferase reporter assay, rescue experiments with AGR2 overexpression, in vivo xenograft assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-qPCR + luciferase + rescue epistasis + in vivo, single lab","pmids":["40020320"],"is_preprint":false},{"year":2025,"finding":"NAA40 specifically interacts with and N-terminally acetylates histone variant H2A.X (in vitro and in cells); H2A.X N-terminal acetylation is a dynamic modification responsive to UVB-induced DNA damage, and NAA40 affects cell survival upon UVB irradiation.","method":"In silico sequence analysis, biochemical acetyltransferase assays, mass spectrometry detection of Nt-acH2A.X in human cells, Co-IP of NAA40 with H2A.X, UVB cell survival assays","journal":"Epigenetics & chromatin","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution (acetyltransferase assay) plus mass spectrometry detection in cells plus Co-IP, multiple orthogonal methods in single rigorous study","pmids":["40665417"],"is_preprint":false},{"year":2026,"finding":"NAA40 (NatD) acts co-translationally at the ribosomal peptide tunnel exit; cryo-EM structure shows NAA40 coordinates with the Nascent polypeptide-Associated Complex (NAC) for ribosome binding and efficient histone H2A/H4 N-terminal acetylation. The NAA40-NAC interaction is required for efficient ribosome binding and histone acetylation. A multienzyme complex on the ribosome involving METAP1 coordinates methionine removal with subsequent NAA40-mediated acetylation.","method":"Cryo-EM structural determination, biochemical acetyltransferase assays, ribosome binding assays, demonstration that NAA40-NAC interaction is required for histone acetylation","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure combined with biochemical reconstitution and functional validation of NAC interaction requirement","pmids":["41820326"],"is_preprint":false},{"year":2025,"finding":"Affinity purification–mass spectrometry (AP-MS) of NAA40 in the nuclear compartment identifies nuclear protein interaction partners of NAA40, consistent with NAA40 acting within the nucleus in addition to its co-translational ribosome-associated role.","method":"Affinity purification coupled to mass spectrometry (AP-MS) of nuclear NAA40","journal":"Methods in enzymology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single AP-MS dataset identifying interactions, no functional validation of individual interactions reported in abstract","pmids":["40992843"],"is_preprint":false}],"current_model":"NAA40 is a highly specific N-terminal acetyltransferase that co-translationally acetylates the alpha-amino group of serine 1 on histones H4, H2A, and H2A.X at the ribosomal exit tunnel in a process coordinated by the NAC complex and METAP1; this modification suppresses repressive H2A/H4S1 phosphorylation and regulates downstream histone arginine methylation (via PRMT5), gene transcription (including PRMT5, TYMS, and AGR2), one-carbon and acetyl-CoA metabolism, and cell survival, while NAA40 protein levels are negatively regulated by RNF112-mediated ubiquitin-dependent degradation."},"narrative":{"mechanistic_narrative":"NAA40 (NatD) is a histone-specific N-terminal acetyltransferase that acts co-translationally at the ribosomal peptide tunnel exit, where cryo-EM shows it coordinates with the Nascent polypeptide-Associated Complex (NAC) for ribosome binding and with METAP1 in a multienzyme arrangement that couples N-terminal methionine removal to acetylation of histones H4 and H2A [PMID:41820326]. Beyond canonical H4 and H2A, NAA40 specifically interacts with and N-terminally acetylates the histone variant H2A.X, a dynamic modification that responds to UVB-induced DNA damage and influences cell survival [PMID:40665417]. This N-terminal acetylation acts as a master switch over downstream histone marks: it prevents enrichment of repressive H2A/H4S1 phosphorylation and sustains PRMT5-dependent symmetric arginine methylation (H4R3me2s), forming an epistatic link between NAA40 acetylation and arginine methylation [PMID:30858358, PMID:34785778, PMID:40020320]. Through this chromatin control, NAA40 drives transcriptional programs governing one-carbon and methionine-cycle metabolism and thymidylate synthase (TYMS) expression, conferring 5-FU chemoresistance [PMID:34785778], regulates the downstream target AGR2 [PMID:40020320], and constrains intracellular acetyl-CoA levels and de novo lipogenesis in hepatocytes [PMID:35057804]. NAA40 depletion activates the mitochondrial caspase-9 intrinsic apoptotic cascade in a p53-independent manner [PMID:26666750], and NAA40 protein abundance is negatively controlled by RNF112-mediated ubiquitin-dependent proteasomal degradation within the KLF4-RNF112 axis [PMID:39757327].","teleology":[{"year":2016,"claim":"Establishing that NAA40 is required for cancer cell survival placed it functionally upstream of the intrinsic apoptotic machinery rather than as an incidental modifier.","evidence":"siRNA knockdown in HCT116 and HT-29 CRC cells with caspase-9 activity assays and caspase-9 inhibitor rescue, including p53-null controls","pmids":["26666750"],"confidence":"Medium","gaps":["Does not connect the survival phenotype to NAA40's catalytic histone acetylation activity","Mechanism linking histone modification to caspase-9 activation unresolved"]},{"year":2019,"claim":"Defining N-terminal acetylation of histone H4 serine 1 and its control of PRMT5 expression revealed that NAA40 acetylation governs a downstream arginine-methylation axis.","evidence":"NAA40 knockdown in CRC cells measuring H4R3me2s, PRMT5 levels, and xenograft growth","pmids":["30858358"],"confidence":"Medium","gaps":["Whether PRMT5 downregulation is direct or secondary to chromatin changes not fully resolved","Single lab, single cancer context"]},{"year":2021,"claim":"Linking NAA40 to one-carbon metabolism showed that the acetylation mark actively prevents repressive H2A/H4S1 phosphorylation to keep metabolic genes such as TYMS transcribed, explaining a chemoresistance phenotype.","evidence":"Transcriptomic/metabolomic profiling, chromatin fractionation showing H2A/H4S1ph redistribution, and xenograft chemoresistance assays in CRC","pmids":["34785778"],"confidence":"Medium","gaps":["Direct chromatin occupancy of NAA40 at metabolic gene promoters not shown","How acetylation antagonizes S1 phosphorylation mechanistically unclear"]},{"year":2022,"claim":"Showing that NAA40 loss raises acetyl-CoA and drives lipogenesis extended its role beyond cancer to systemic metabolic homeostasis, conserved from mouse hepatocytes to Drosophila.","evidence":"Metabolomics, lipidomics, lipogenesis gene expression and glucose uptake in NAA40-depleted hepatocytes plus Drosophila fat body assay","pmids":["35057804"],"confidence":"Medium","gaps":["Causal chain from histone acetylation to acetyl-CoA accumulation not delineated","Insulin-independent lipid droplet mechanism unexplained"]},{"year":2025,"claim":"Identifying AGR2 as a downstream transcriptional target connected NAA40-dependent histone mark balance (H4S1ph, H4R3me2a/s, H3K4me3/H3K27me3) at a specific promoter to a phenocopying effector gene in osteosarcoma.","evidence":"ChIP-qPCR for histone marks at AGR2 promoter, luciferase reporter, and AGR2 rescue/knockdown with xenograft validation","pmids":["40020320"],"confidence":"Medium","gaps":["Only partial rescue by AGR2 indicates additional effectors","Generality beyond osteosarcoma not tested"]},{"year":2025,"claim":"Demonstrating that RNF112 ubiquitinates and degrades NAA40 defined a post-translational control point and embedded NAA40 in the KLF4-RNF112 tumor-suppressive axis.","evidence":"Co-IP, ubiquitination assays, and NAA40 overexpression rescue in RNF112-overexpressing CRC cells with nude mouse tumor assays","pmids":["39757327"],"confidence":"Medium","gaps":["Ubiquitination site(s) on NAA40 not mapped","Single Co-IP context for the RNF112-NAA40 interaction"]},{"year":2025,"claim":"Establishing direct N-terminal acetylation of histone variant H2A.X and its DNA-damage responsiveness expanded NAA40's substrate range and tied it to the genotoxic stress response.","evidence":"In vitro acetyltransferase assays, mass spectrometry detection of Nt-acH2A.X in cells, Co-IP, and UVB survival assays","pmids":["40665417"],"confidence":"High","gaps":["How H2A.X N-terminal acetylation is dynamically removed/regulated not defined","Functional consequence for DNA repair signaling not mechanistically resolved"]},{"year":2026,"claim":"Solving the cryo-EM structure of NAA40 on the ribosome resolved how it achieves co-translational, histone-specific acetylation by coordinating with NAC and METAP1, providing the molecular basis for its substrate engagement.","evidence":"Cryo-EM structure with biochemical acetyltransferase and ribosome binding assays demonstrating the NAA40-NAC interaction requirement","pmids":["41820326"],"confidence":"High","gaps":["Structural basis for nuclear (non-ribosomal) NAA40 activity not addressed","Relative contributions of co-translational vs post-translational acetylation in vivo unquantified"]},{"year":null,"claim":"The nuclear interactome and non-ribosomal functions of NAA40 remain to be defined and connected to its established chromatin and metabolic roles.","evidence":"AP-MS of nuclear NAA40 identifies partners but without functional validation","pmids":[],"confidence":"Low","gaps":["Single AP-MS dataset without validation of individual interactions","No functional role assigned to nuclear NAA40 partners","Whether nuclear NAA40 acetylates non-histone substrates unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,6,7]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[6,7]},{"term_id":"GO:0042393","term_label":"histone binding","supporting_discovery_ids":[6,7]}],"localization":[{"term_id":"GO:0005840","term_label":"ribosome","supporting_discovery_ids":[7]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[0,2,5]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[7]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[1]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[2,3]}],"complexes":["ribosome-associated NAA40-NAC co-translational complex"],"partners":["NAC","METAP1","H2AFX","RNF112"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q86UY6","full_name":"N-alpha-acetyltransferase 40","aliases":["N-acetyltransferase 11","N-alpha-acetyltransferase D","NatD","hNatD","Protein acetyltransferase 1"],"length_aa":237,"mass_kda":27.2,"function":"N-alpha-acetyltransferase that specifically mediates the acetylation of the N-terminal residues of histones H4 and H2A (PubMed:21935442, PubMed:25619998). In contrast to other N-alpha-acetyltransferase, has a very specific selectivity for histones H4 and H2A N-terminus and specifically recognizes the 'Ser-Gly-Arg-Gly sequence' (PubMed:21935442, PubMed:25619998). Acts as a negative regulator of apoptosis (PubMed:26666750). May play a role in hepatic lipid metabolism (By similarity)","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q86UY6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NAA40","classification":"Not Classified","n_dependent_lines":89,"n_total_lines":1208,"dependency_fraction":0.07367549668874172},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"HMGN2","stoichiometry":10.0},{"gene":"KLC4","stoichiometry":4.0},{"gene":"HIST2H2BE","stoichiometry":0.2},{"gene":"HMGA1","stoichiometry":0.2},{"gene":"HMGN5","stoichiometry":0.2},{"gene":"KIF5B","stoichiometry":0.2},{"gene":"KLC2","stoichiometry":0.2},{"gene":"PSPC1","stoichiometry":0.2},{"gene":"RPS16","stoichiometry":0.2},{"gene":"SSRP1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/NAA40","total_profiled":1310},"omim":[{"mim_id":"619999","title":"N-ALPHA-ACETYLTRANSFERASE 40, NatD CATALYTIC SUBUNIT; NAA40","url":"https://www.omim.org/entry/619999"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Centriolar satellite","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/NAA40"},"hgnc":{"alias_symbol":["FLJ13848"],"prev_symbol":["NAT11"]},"alphafold":{"accession":"Q86UY6","domains":[{"cath_id":"3.40.630.30","chopping":"35-216","consensus_level":"high","plddt":96.796,"start":35,"end":216}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86UY6","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q86UY6-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q86UY6-F1-predicted_aligned_error_v6.png","plddt_mean":92.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NAA40","jax_strain_url":"https://www.jax.org/strain/search?query=NAA40"},"sequence":{"accession":"Q86UY6","fasta_url":"https://rest.uniprot.org/uniprotkb/Q86UY6.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q86UY6/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86UY6"}},"corpus_meta":[{"pmid":"30858358","id":"PMC_30858358","title":"NAA40 contributes to colorectal cancer growth by controlling PRMT5 expression.","date":"2019","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/30858358","citation_count":52,"is_preprint":false},{"pmid":"26666750","id":"PMC_26666750","title":"Depletion of histone N-terminal-acetyltransferase Naa40 induces p53-independent apoptosis in colorectal cancer cells via the mitochondrial pathway.","date":"2016","source":"Apoptosis : an international journal on programmed cell death","url":"https://pubmed.ncbi.nlm.nih.gov/26666750","citation_count":45,"is_preprint":false},{"pmid":"34785778","id":"PMC_34785778","title":"Histone N-terminal acetyltransferase NAA40 links one-carbon metabolism to chemoresistance.","date":"2021","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/34785778","citation_count":25,"is_preprint":false},{"pmid":"35057804","id":"PMC_35057804","title":"Histone acetyltransferase NAA40 modulates acetyl-CoA levels and lipid synthesis.","date":"2022","source":"BMC biology","url":"https://pubmed.ncbi.nlm.nih.gov/35057804","citation_count":23,"is_preprint":false},{"pmid":"39757327","id":"PMC_39757327","title":"RNF112, whose transcription is regulated by KLF4, inhibits colorectal cancer growth via promoting ubiquitin-dependent degradation of NAA40.","date":"2025","source":"Cell biology and toxicology","url":"https://pubmed.ncbi.nlm.nih.gov/39757327","citation_count":4,"is_preprint":false},{"pmid":"40020320","id":"PMC_40020320","title":"N-terminal histone acetyltransferase NAA40 modulates osteosarcoma progression by controlling AGR2 expression.","date":"2025","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/40020320","citation_count":2,"is_preprint":false},{"pmid":"40665417","id":"PMC_40665417","title":"H2A.X N-terminal acetylation is a newly identified NAA40-mediated modification that is responsive to UV irradiation.","date":"2025","source":"Epigenetics & chromatin","url":"https://pubmed.ncbi.nlm.nih.gov/40665417","citation_count":2,"is_preprint":false},{"pmid":"41820326","id":"PMC_41820326","title":"NAA40 and NAC cooperate in co-translational histone acetylation in humans.","date":"2026","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/41820326","citation_count":0,"is_preprint":false},{"pmid":"40992843","id":"PMC_40992843","title":"Affinity purification-mass spectrometry to identify nuclear protein interactions of N-terminal acetyltransferase NAA40.","date":"2025","source":"Methods in enzymology","url":"https://pubmed.ncbi.nlm.nih.gov/40992843","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":6635,"output_tokens":2486,"usd":0.028598,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9589,"output_tokens":3256,"usd":0.064672,"stage2_stop_reason":"end_turn"},"total_usd":0.09327,"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\": 2019,\n      \"finding\": \"NAA40 catalyzes N-terminal acetylation of histone H4 serine 1 (N-acH4); loss of NAA40 and N-acH4 reduces H4R3me2s levels through transcriptional downregulation of PRMT5, establishing a functional epistatic link between NAA40-mediated histone acetylation and PRMT5-dependent arginine methylation.\",\n      \"method\": \"NAA40 knockdown in CRC cells with measurement of H4R3me2s, PRMT5 mRNA/protein levels, and xenograft tumor growth assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KD with defined cellular phenotype and pathway placement, single lab, multiple readouts including in vivo xenograft\",\n      \"pmids\": [\"30858358\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Depletion of NAA40 in colorectal cancer cells activates the mitochondrial caspase-9-mediated apoptotic cascade in a p53-independent manner; treatment with an irreversible caspase-9 inhibitor blocks apoptosis induced by NAA40 knockdown, placing NAA40 upstream of the intrinsic apoptosis pathway.\",\n      \"method\": \"siRNA knockdown of NAA40 in HCT116 and HT-29 CRC cells (including p53-null HCT116), caspase-9 activity assays, caspase-9 inhibitor rescue experiments\",\n      \"journal\": \"Apoptosis : an international journal on programmed cell death\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis via inhibitor rescue, p53-null genetic control, single lab with two orthogonal cell models\",\n      \"pmids\": [\"26666750\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NAA40 regulates one-carbon metabolism by controlling expression of the methionine cycle genes and thymidylate synthase (TYMS); mechanistically, NAA40 acetyltransferase activity prevents enrichment of repressive H2A/H4S1ph at the nuclear periphery, thereby activating TYMS transcription and promoting resistance to 5-FU chemotherapy.\",\n      \"method\": \"Transcriptomic and metabolomic analysis in CRC cells after NAA40 depletion; chromatin fractionation showing H2A/H4S1ph redistribution; in vitro and xenograft chemoresistance assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multi-omic approach (transcriptomics + metabolomics + chromatin fractionation) with in vivo validation, single lab\",\n      \"pmids\": [\"34785778\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Depletion of NAA40 in murine hepatocytes leads to increased intracellular acetyl-CoA levels, which associates with enhanced de novo lipogenesis (upregulation of lipogenesis genes, increased diglycerides/triglycerides, cytoplasmic lipid droplet accumulation) and impaired insulin signalling (decreased glucose uptake); the effect on lipid droplet formation is independent of insulin. This effect is replicated in vivo in Drosophila larval fat body.\",\n      \"method\": \"Metabolomic and lipidomic analysis in NAA40-depleted murine hepatocytes; de novo lipogenesis gene expression; glucose uptake assay; Drosophila in vivo lipid droplet assay\",\n      \"journal\": \"BMC biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — orthogonal metabolomics + lipidomics + in vivo Drosophila model, single lab\",\n      \"pmids\": [\"35057804\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RNF112 E3 ubiquitin ligase promotes ubiquitin-dependent proteasomal degradation of NAA40 protein; overexpression of NAA40 rescues the anti-tumor effects of RNF112 overexpression in CRC, placing NAA40 downstream of the KLF4-RNF112 axis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, NAA40 overexpression rescue experiments in RNF112-overexpressing CRC cells, in vivo nude mouse tumor formation assay\",\n      \"journal\": \"Cell biology and toxicology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ubiquitination assay with rescue epistasis, in vivo confirmation, single lab\",\n      \"pmids\": [\"39757327\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NAA40 loss in osteosarcoma cells is associated with increased H4S1ph and H4R3me2a and decreased H4R3me2s, and leads to altered H3K4me3 and H3K27me3 at the AGR2 promoter, reducing AGR2 transcription; AGR2 is identified as a downstream transcriptional target of NAA40 and its knockdown phenocopies NAA40 loss, while AGR2 overexpression partially rescues NAA40-depletion phenotypes.\",\n      \"method\": \"ChIP-qPCR for histone marks at AGR2 promoter, dual luciferase reporter assay, rescue experiments with AGR2 overexpression, in vivo xenograft assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-qPCR + luciferase + rescue epistasis + in vivo, single lab\",\n      \"pmids\": [\"40020320\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NAA40 specifically interacts with and N-terminally acetylates histone variant H2A.X (in vitro and in cells); H2A.X N-terminal acetylation is a dynamic modification responsive to UVB-induced DNA damage, and NAA40 affects cell survival upon UVB irradiation.\",\n      \"method\": \"In silico sequence analysis, biochemical acetyltransferase assays, mass spectrometry detection of Nt-acH2A.X in human cells, Co-IP of NAA40 with H2A.X, UVB cell survival assays\",\n      \"journal\": \"Epigenetics & chromatin\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution (acetyltransferase assay) plus mass spectrometry detection in cells plus Co-IP, multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"40665417\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"NAA40 (NatD) acts co-translationally at the ribosomal peptide tunnel exit; cryo-EM structure shows NAA40 coordinates with the Nascent polypeptide-Associated Complex (NAC) for ribosome binding and efficient histone H2A/H4 N-terminal acetylation. The NAA40-NAC interaction is required for efficient ribosome binding and histone acetylation. A multienzyme complex on the ribosome involving METAP1 coordinates methionine removal with subsequent NAA40-mediated acetylation.\",\n      \"method\": \"Cryo-EM structural determination, biochemical acetyltransferase assays, ribosome binding assays, demonstration that NAA40-NAC interaction is required for histone acetylation\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure combined with biochemical reconstitution and functional validation of NAC interaction requirement\",\n      \"pmids\": [\"41820326\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Affinity purification–mass spectrometry (AP-MS) of NAA40 in the nuclear compartment identifies nuclear protein interaction partners of NAA40, consistent with NAA40 acting within the nucleus in addition to its co-translational ribosome-associated role.\",\n      \"method\": \"Affinity purification coupled to mass spectrometry (AP-MS) of nuclear NAA40\",\n      \"journal\": \"Methods in enzymology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single AP-MS dataset identifying interactions, no functional validation of individual interactions reported in abstract\",\n      \"pmids\": [\"40992843\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NAA40 is a highly specific N-terminal acetyltransferase that co-translationally acetylates the alpha-amino group of serine 1 on histones H4, H2A, and H2A.X at the ribosomal exit tunnel in a process coordinated by the NAC complex and METAP1; this modification suppresses repressive H2A/H4S1 phosphorylation and regulates downstream histone arginine methylation (via PRMT5), gene transcription (including PRMT5, TYMS, and AGR2), one-carbon and acetyl-CoA metabolism, and cell survival, while NAA40 protein levels are negatively regulated by RNF112-mediated ubiquitin-dependent degradation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NAA40 (NatD) is a histone-specific N-terminal acetyltransferase that acts co-translationally at the ribosomal peptide tunnel exit, where cryo-EM shows it coordinates with the Nascent polypeptide-Associated Complex (NAC) for ribosome binding and with METAP1 in a multienzyme arrangement that couples N-terminal methionine removal to acetylation of histones H4 and H2A [#7]. Beyond canonical H4 and H2A, NAA40 specifically interacts with and N-terminally acetylates the histone variant H2A.X, a dynamic modification that responds to UVB-induced DNA damage and influences cell survival [#6]. This N-terminal acetylation acts as a master switch over downstream histone marks: it prevents enrichment of repressive H2A/H4S1 phosphorylation and sustains PRMT5-dependent symmetric arginine methylation (H4R3me2s), forming an epistatic link between NAA40 acetylation and arginine methylation [#0, #2, #5]. Through this chromatin control, NAA40 drives transcriptional programs governing one-carbon and methionine-cycle metabolism and thymidylate synthase (TYMS) expression, conferring 5-FU chemoresistance [#2], regulates the downstream target AGR2 [#5], and constrains intracellular acetyl-CoA levels and de novo lipogenesis in hepatocytes [#3]. NAA40 depletion activates the mitochondrial caspase-9 intrinsic apoptotic cascade in a p53-independent manner [#1], and NAA40 protein abundance is negatively controlled by RNF112-mediated ubiquitin-dependent proteasomal degradation within the KLF4-RNF112 axis [#4].\",\n  \"teleology\": [\n    {\n      \"year\": 2016,\n      \"claim\": \"Establishing that NAA40 is required for cancer cell survival placed it functionally upstream of the intrinsic apoptotic machinery rather than as an incidental modifier.\",\n      \"evidence\": \"siRNA knockdown in HCT116 and HT-29 CRC cells with caspase-9 activity assays and caspase-9 inhibitor rescue, including p53-null controls\",\n      \"pmids\": [\"26666750\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not connect the survival phenotype to NAA40's catalytic histone acetylation activity\", \"Mechanism linking histone modification to caspase-9 activation unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defining N-terminal acetylation of histone H4 serine 1 and its control of PRMT5 expression revealed that NAA40 acetylation governs a downstream arginine-methylation axis.\",\n      \"evidence\": \"NAA40 knockdown in CRC cells measuring H4R3me2s, PRMT5 levels, and xenograft growth\",\n      \"pmids\": [\"30858358\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether PRMT5 downregulation is direct or secondary to chromatin changes not fully resolved\", \"Single lab, single cancer context\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Linking NAA40 to one-carbon metabolism showed that the acetylation mark actively prevents repressive H2A/H4S1 phosphorylation to keep metabolic genes such as TYMS transcribed, explaining a chemoresistance phenotype.\",\n      \"evidence\": \"Transcriptomic/metabolomic profiling, chromatin fractionation showing H2A/H4S1ph redistribution, and xenograft chemoresistance assays in CRC\",\n      \"pmids\": [\"34785778\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct chromatin occupancy of NAA40 at metabolic gene promoters not shown\", \"How acetylation antagonizes S1 phosphorylation mechanistically unclear\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showing that NAA40 loss raises acetyl-CoA and drives lipogenesis extended its role beyond cancer to systemic metabolic homeostasis, conserved from mouse hepatocytes to Drosophila.\",\n      \"evidence\": \"Metabolomics, lipidomics, lipogenesis gene expression and glucose uptake in NAA40-depleted hepatocytes plus Drosophila fat body assay\",\n      \"pmids\": [\"35057804\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal chain from histone acetylation to acetyl-CoA accumulation not delineated\", \"Insulin-independent lipid droplet mechanism unexplained\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identifying AGR2 as a downstream transcriptional target connected NAA40-dependent histone mark balance (H4S1ph, H4R3me2a/s, H3K4me3/H3K27me3) at a specific promoter to a phenocopying effector gene in osteosarcoma.\",\n      \"evidence\": \"ChIP-qPCR for histone marks at AGR2 promoter, luciferase reporter, and AGR2 rescue/knockdown with xenograft validation\",\n      \"pmids\": [\"40020320\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Only partial rescue by AGR2 indicates additional effectors\", \"Generality beyond osteosarcoma not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrating that RNF112 ubiquitinates and degrades NAA40 defined a post-translational control point and embedded NAA40 in the KLF4-RNF112 tumor-suppressive axis.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, and NAA40 overexpression rescue in RNF112-overexpressing CRC cells with nude mouse tumor assays\",\n      \"pmids\": [\"39757327\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitination site(s) on NAA40 not mapped\", \"Single Co-IP context for the RNF112-NAA40 interaction\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Establishing direct N-terminal acetylation of histone variant H2A.X and its DNA-damage responsiveness expanded NAA40's substrate range and tied it to the genotoxic stress response.\",\n      \"evidence\": \"In vitro acetyltransferase assays, mass spectrometry detection of Nt-acH2A.X in cells, Co-IP, and UVB survival assays\",\n      \"pmids\": [\"40665417\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How H2A.X N-terminal acetylation is dynamically removed/regulated not defined\", \"Functional consequence for DNA repair signaling not mechanistically resolved\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Solving the cryo-EM structure of NAA40 on the ribosome resolved how it achieves co-translational, histone-specific acetylation by coordinating with NAC and METAP1, providing the molecular basis for its substrate engagement.\",\n      \"evidence\": \"Cryo-EM structure with biochemical acetyltransferase and ribosome binding assays demonstrating the NAA40-NAC interaction requirement\",\n      \"pmids\": [\"41820326\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for nuclear (non-ribosomal) NAA40 activity not addressed\", \"Relative contributions of co-translational vs post-translational acetylation in vivo unquantified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The nuclear interactome and non-ribosomal functions of NAA40 remain to be defined and connected to its established chromatin and metabolic roles.\",\n      \"evidence\": \"AP-MS of nuclear NAA40 identifies partners but without functional validation\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single AP-MS dataset without validation of individual interactions\", \"No functional role assigned to nuclear NAA40 partners\", \"Whether nuclear NAA40 acetylates non-histone substrates unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 6, 7]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [6, 7]},\n      {\"term_id\": \"GO:0042393\", \"supporting_discovery_ids\": [6, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005840\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [0, 2, 5]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"complexes\": [\"ribosome-associated NAA40-NAC co-translational complex\"],\n    \"partners\": [\"NAC\", \"METAP1\", \"H2AFX\", \"RNF112\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}