{"gene":"NAA60","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2011,"finding":"NAA60 (NatF/hNaa60) is an N-terminal acetyltransferase (NAT) with activity targeting Met-Lys- and other Met-starting protein N-termini, as demonstrated by in vitro peptide library acetylation assays using purified recombinant human and Drosophila homologues. In vivo, ectopic expression of hNaa60p in yeast acetylated distinct Met-starting yeast protein N-termini and increased general N-terminal acetylation levels. Knockdown of NAA60 in Drosophila cells induced chromosomal segregation defects.","method":"In vitro peptide library acetylation assay with purified recombinant protein; ectopic expression in yeast followed by N-terminal COFRADIC; RNAi knockdown in Drosophila cells","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzymatic assay with purified recombinant protein plus in vivo validation by two orthogonal methods (yeast N-terminal COFRADIC and human cell knockdown), replicated across species","pmids":["21750686"],"is_preprint":false},{"year":2011,"finding":"HAT4 (NAA60) localizes to the Golgi apparatus and functions as a B-type histone acetyltransferase that preferentially acetylates free (non-nucleosomal) histone H4 at lysine residues K79 and K91 in the globular domain. HAT4 depletion impaired nucleosome assembly, inhibited cell proliferation, sensitized cells to DNA damage, and induced apoptosis.","method":"Subcellular localization by immunofluorescence; in vitro acetyltransferase assay with free histones; siRNA knockdown with cell proliferation, DNA damage, and apoptosis readouts","journal":"Molecular cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct enzymatic assay plus cellular knockdown phenotypes, single lab, multiple orthogonal methods","pmids":["21981917"],"is_preprint":false},{"year":2015,"finding":"NAA60 (Naa60/NatF) localizes exclusively to the cytosolic face of Golgi membranes (established by a new membrane topology assay PROMPT and selective membrane permeabilization). NAA60 specifically acetylates N-termini of transmembrane proteins that face the cytosol, as revealed by Nt-acetylome analysis of NAA60-knockdown cells. NAA60 knockdown causes Golgi fragmentation, indicating a role in maintaining Golgi structural integrity.","method":"Membrane topology assay (PROMPT); selective membrane permeabilization; N-terminal acetylome (COFRADIC) analysis of knockdown cells; immunofluorescence of Golgi morphology in knockdown cells","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (topology assay, proteomics, knockdown phenotype) establishing localization, substrate specificity, and cellular role","pmids":["25732826"],"is_preprint":false},{"year":2016,"finding":"Crystal structures of human Naa60 in complex with Ac-CoA or CoA reveal a GNAT domain followed by an amphipathic helix contributing to Golgi localization. Structural and biochemical studies identified Tyr97 and His138 as key catalytic residues, and Phe34 as influencing cofactor positioning. The non-conserved β3-β4 long loop participates in regulation of hNaa60 activity. Naa60 also harbors lysine Nε-acetyltransferase (KAT) activity toward histone H4 lysine residues.","method":"X-ray crystallography; site-directed mutagenesis; in vitro acetyltransferase activity assays","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure combined with mutagenesis and biochemical activity assays in a single study","pmids":["27550639"],"is_preprint":false},{"year":2017,"finding":"The C-terminal tail of Naa60 anchors it to the Golgi as a peripheral membrane protein via two amphipathic helices that fold into α-helical conformations only in the presence of liposomes and bind membranes in a parallel orientation via hydrophobic and electrostatic interactions. Naa60 shows strong and specific binding preference for phosphatidylinositol PI(4)P-containing membranes, consistent with its primary Golgi residency. Mutational analysis confirmed the hydrophobic face of these helices is critical for membranous localization. Golgi anchoring is proposed to occur post-translationally.","method":"Computational modeling; in vitro liposome binding assays; cellular mutational analysis with localization readout by immunofluorescence","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — combined in vitro biophysical assays, mutagenesis, and cellular localization studies in one study with multiple orthogonal approaches","pmids":["28196861"],"is_preprint":false},{"year":2024,"finding":"Biallelic loss-of-function variants in NAA60 cause autosomal recessive primary familial brain calcification (PFBC) with absent Nt-acetylation activity. In vitro, the phosphate importer SLC20A2 (PiT2) was identified as a direct substrate of NAA60 Nt-acetylation. In cells, NAA60 loss reduced surface levels of SLC20A2 and decreased extracellular phosphate uptake, linking NAA60-mediated Nt-acetylation of transmembrane proteins to phosphate homeostasis.","method":"In vitro Nt-acetylation assay with SLC20A2 as substrate; cell surface biotinylation assay; phosphate uptake assay in NAA60-deficient cells; patient variant functional analysis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro substrate identification plus cellular functional readouts (surface levels, phosphate uptake), replicated across multiple patient families and cell lines","pmids":["38480682"],"is_preprint":false},{"year":2024,"finding":"A homozygous frameshift variant in NAA60 disrupts its Golgi localization and accelerates protein degradation. The mutant NAA60 alters interaction with PFBC-related proteins PiT2 (SLC20A2) and XPR1, affecting intracellular phosphate homeostasis. NAA60 KO in cells also caused decreased expression of multiple brain calcification-associated membrane proteins including RFC (reduced folate carrier), a folate metabolism protein.","method":"Immunofluorescence (Golgi localization); Western blot (protein stability); co-immunoprecipitation (interaction with PiT2/XPR1); mass spectrometry in NAA60 KO cell lines; gene knockout cell lines","journal":"Movement disorders : official journal of the Movement Disorder Society","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — multiple methods (co-IP, IF, MS) in single lab; findings largely corroborate prior work but add novel interactions","pmids":["39229657"],"is_preprint":false},{"year":2025,"finding":"BioID proximity labeling of NAA60 identified over 100 proximal partners enriched on the trans-side of the Golgi, including golgins and GRASP proteins essential for Golgi integrity. Suborganellar localization analysis revealed a more prominent medial/trans-Golgi localization of NAA60. Biotinylated peptide analysis was used to infer the topology of transmembrane protein interactors in the secretory pathway.","method":"BioID proximity labeling; streptavidin affinity purification; mass spectrometry; suborganellar localization analysis","journal":"Open biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — BioID interactomics with MS validation, single lab, establishes proximal interactome and suborganellar localization","pmids":["39965656"],"is_preprint":false},{"year":2025,"finding":"NAA60 facilitates cellular uptake of platinum-based drugs (cis- and carboplatin) by acetylating the N-termini of LRRC8A and LRRC8D (volume-regulated anion channel subunits). Loss of NAA60 decreases drug uptake and causes drug resistance. Introduction of positively charged amino acids at the N-termini of LRRC8A/D (mimicking absence of the acetyl group) similarly decreased platinum sensitivity, functionally validating the role of N-terminal acetylation at these sites.","method":"NAA60 knockout cells; in vitro N-terminal acetylation assay; drug uptake assay; site-directed mutagenesis of LRRC8A/D N-termini; drug sensitivity assays in BRCA1;p53-deficient cells and tumors","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple methods including KO, mutagenesis, and functional drug uptake assays in single lab, novel substrate identification","pmids":["41053424"],"is_preprint":false}],"current_model":"NAA60 (NatF/HAT4) is a Golgi-localized N-terminal acetyltransferase that anchors to the cytosolic face of Golgi membranes via two C-terminal amphipathic helices with preference for PI(4)P-rich membranes, where it post-translationally Nt-acetylates Met-starting N-termini of transmembrane proteins (including SLC20A2/PiT2, LRRC8A, and LRRC8D) that face the cytosol; its catalytic mechanism involves key residues Tyr97 and His138, it also possesses lysine Nε-acetyltransferase activity toward free histone H4, loss of NAA60 causes Golgi fragmentation and chromosome segregation defects, and biallelic loss-of-function variants cause primary familial brain calcification by disrupting Nt-acetylation-dependent surface trafficking and phosphate homeostasis."},"narrative":{"mechanistic_narrative":"NAA60 (NatF/HAT4) is an N-terminal acetyltransferase that post-translationally Nt-acetylates Met-starting protein N-termini, with a substrate preference for the cytosol-facing N-termini of transmembrane proteins in the secretory pathway [PMID:21750686, PMID:25732826]. It anchors as a peripheral membrane protein to the cytosolic face of Golgi membranes through two C-terminal amphipathic helices that fold only upon membrane contact and bind preferentially to PI(4)P-rich membranes, accounting for its medial/trans-Golgi residency among golgins and GRASP proteins [PMID:28196861, PMID:39965656]. Crystal structures define a GNAT catalytic fold using Tyr97 and His138 as key catalytic residues, and the enzyme additionally displays lysine Nε-acetyltransferase activity toward free histone H4 at K79 and K91 [PMID:21981917, PMID:27550639]. Through Nt-acetylation of transmembrane substrates including the phosphate importer SLC20A2/PiT2 and the volume-regulated anion channel subunits LRRC8A and LRRC8D, NAA60 controls their surface trafficking, thereby governing extracellular phosphate uptake and cellular uptake of platinum-based drugs [PMID:38480682, PMID:41053424]. Loss of NAA60 causes Golgi fragmentation and chromosome segregation defects [PMID:21750686, PMID:25732826], and biallelic loss-of-function variants cause autosomal recessive primary familial brain calcification via disrupted Nt-acetylation-dependent surface trafficking and phosphate homeostasis [PMID:38480682, PMID:39229657].","teleology":[{"year":2011,"claim":"Established that NAA60 is a bona fide N-terminal acetyltransferase with a distinct substrate preference for Met-starting N-termini, defining a new NAT (NatF) and linking it to chromosome segregation.","evidence":"In vitro peptide library acetylation with purified recombinant human and Drosophila enzyme, ectopic expression in yeast with N-terminal COFRADIC, and RNAi knockdown in Drosophila cells","pmids":["21750686"],"confidence":"High","gaps":["Subcellular site of activity not yet defined","Physiological transmembrane substrates not identified","Mechanism linking loss to segregation defects unresolved"]},{"year":2011,"claim":"Showed NAA60 (HAT4) localizes to the Golgi and additionally acts as a lysine acetyltransferase toward free histone H4 at K79/K91, connecting it to nucleosome assembly and cell survival.","evidence":"Immunofluorescence, in vitro acetyltransferase assay with free histones, and siRNA knockdown with proliferation/DNA-damage/apoptosis readouts","pmids":["21981917"],"confidence":"Medium","gaps":["How a Golgi-anchored enzyme accesses free histones unresolved","Relative physiological importance of KAT versus NAT activity unclear"]},{"year":2015,"claim":"Resolved that NAA60 sits on the cytosolic face of Golgi membranes and selectively acetylates cytosol-facing transmembrane protein N-termini, and that its loss fragments the Golgi—tying the enzyme to organelle integrity.","evidence":"PROMPT membrane topology assay, selective permeabilization, N-terminal acetylome (COFRADIC) of knockdown cells, and Golgi morphology imaging","pmids":["25732826"],"confidence":"High","gaps":["Membrane anchoring mechanism not yet defined","Causal link between substrate acetylation and Golgi integrity not established"]},{"year":2016,"claim":"Defined the catalytic architecture by solving Naa60 crystal structures and identifying Tyr97/His138 as catalytic residues plus a regulatory β3-β4 loop and an amphipathic helix for Golgi localization.","evidence":"X-ray crystallography with Ac-CoA/CoA, site-directed mutagenesis, and in vitro activity assays","pmids":["27550639"],"confidence":"High","gaps":["Structure of the membrane-bound enzyme not captured","Regulatory function of the β3-β4 loop not mechanistically resolved"]},{"year":2017,"claim":"Explained how NAA60 reaches the Golgi: two C-terminal amphipathic helices fold on membrane contact and bind with strong preference for PI(4)P, defining a lipid-dependent post-translational anchoring mode.","evidence":"Computational modeling, in vitro liposome binding assays, and cellular mutational analysis of helix hydrophobic face with localization readout","pmids":["28196861"],"confidence":"High","gaps":["Regulation of membrane targeting in vivo unclear","Whether lipid binding modulates catalytic activity not addressed"]},{"year":2024,"claim":"Connected NAA60 to human disease and to phosphate physiology by showing biallelic loss-of-function causes primary familial brain calcification and that NAA60 Nt-acetylates SLC20A2 to control its surface levels and phosphate uptake.","evidence":"Patient variant functional analysis, in vitro Nt-acetylation with SLC20A2, cell surface biotinylation, and phosphate uptake assays in NAA60-deficient cells","pmids":["38480682","39229657"],"confidence":"High","gaps":["Precise mechanism linking acetylation to trafficking not fully defined","Role of additional calcification-associated substrates (e.g., XPR1, RFC) not mechanistically resolved"]},{"year":2025,"claim":"Mapped the NAA60 proximal interactome to the medial/trans-Golgi, enriched for golgins and GRASP proteins, refining its suborganellar position relative to Golgi-integrity machinery.","evidence":"BioID proximity labeling with streptavidin purification and mass spectrometry, plus suborganellar localization analysis","pmids":["39965656"],"confidence":"Medium","gaps":["Direct versus proximal interactions not distinguished","Functional significance of golgin/GRASP proximity untested"]},{"year":2025,"claim":"Extended NAA60 substrate scope and clinical relevance by showing it acetylates LRRC8A/D N-termini to enable cellular uptake of platinum drugs, with loss conferring drug resistance.","evidence":"NAA60 knockout cells, in vitro Nt-acetylation, drug uptake and sensitivity assays, and charge-mimic N-terminal mutagenesis of LRRC8A/D","pmids":["41053424"],"confidence":"Medium","gaps":["Single lab; reciprocal in vivo validation limited","Mechanism by which N-terminal acetylation alters channel function not resolved"]},{"year":null,"claim":"How NAA60-mediated N-terminal acetylation mechanistically controls transmembrane protein surface trafficking, and how its Golgi/histone activities are coordinated, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of the membrane-anchored enzyme engaging a transmembrane substrate","Mechanistic basis linking acetylation to trafficking competence unknown","Integration of cytosolic NAT activity with reported histone KAT activity unexplained"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,2,3,5,8]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,5,8]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[4]}],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[1,2,4,7]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,2,5]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[5,6]}],"complexes":[],"partners":["SLC20A2","XPR1","LRRC8A","LRRC8D"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9H7X0","full_name":"N-alpha-acetyltransferase 60","aliases":["Histone acetyltransferase type B protein 4","HAT4","N-acetyltransferase 15","N-alpha-acetyltransferase F","NatF"],"length_aa":242,"mass_kda":27.5,"function":"N-alpha-acetyltransferase that specifically mediates the acetylation of N-terminal residues of the transmembrane proteins, with a strong preference for N-termini facing the cytosol (PubMed:25732826, PubMed:38480682). Displays N-terminal acetyltransferase activity towards a range of N-terminal sequences including those starting with Met-Lys, Met-Val, Met-Ala and Met-Met (PubMed:21750686, PubMed:25732826, PubMed:27320834, PubMed:27550639). Required for normal chromosomal segregation during anaphase (PubMed:21750686). May also show histone acetyltransferase activity; such results are however unclear in vivo and would require additional experimental evidences (PubMed:21981917)","subcellular_location":"Golgi apparatus membrane","url":"https://www.uniprot.org/uniprotkb/Q9H7X0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NAA60","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/NAA60","total_profiled":1310},"omim":[{"mim_id":"620786","title":"BASAL GANGLIA CALCIFICATION, IDIOPATHIC, 9, AUTOSOMAL RECESSIVE; IBGC9","url":"https://www.omim.org/entry/620786"},{"mim_id":"614686","title":"FAMILY WITH SEQUENCE SIMILARITY 50, MEMBER B; FAM50B","url":"https://www.omim.org/entry/614686"},{"mim_id":"614685","title":"ZINC FINGER PROTEIN 597; ZNF597","url":"https://www.omim.org/entry/614685"},{"mim_id":"614246","title":"N-ALPHA-ACETYLTRANSFERASE 60, NatF CATALYTIC SUBUNIT; NAA60","url":"https://www.omim.org/entry/614246"},{"mim_id":"213600","title":"BASAL GANGLIA CALCIFICATION, IDIOPATHIC, 1; IBGC1","url":"https://www.omim.org/entry/213600"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Uncertain","locations":[{"location":"Actin filaments","reliability":"Uncertain"},{"location":"Cytosol","reliability":"Uncertain"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/NAA60"},"hgnc":{"alias_symbol":["FLJ14154","HAT4","NatF","hNaa60"],"prev_symbol":["NAT15"]},"alphafold":{"accession":"Q9H7X0","domains":[{"cath_id":"3.40.630.30","chopping":"8-186","consensus_level":"high","plddt":96.9451,"start":8,"end":186}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H7X0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H7X0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H7X0-F1-predicted_aligned_error_v6.png","plddt_mean":88.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NAA60","jax_strain_url":"https://www.jax.org/strain/search?query=NAA60"},"sequence":{"accession":"Q9H7X0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9H7X0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9H7X0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H7X0"}},"corpus_meta":[{"pmid":"21750686","id":"PMC_21750686","title":"NatF contributes to an evolutionary shift in protein N-terminal acetylation and is important for normal chromosome segregation.","date":"2011","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/21750686","citation_count":161,"is_preprint":false},{"pmid":"25732826","id":"PMC_25732826","title":"An organellar nα-acetyltransferase, naa60, acetylates cytosolic N termini of transmembrane proteins and maintains Golgi integrity.","date":"2015","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/25732826","citation_count":107,"is_preprint":false},{"pmid":"21981917","id":"PMC_21981917","title":"HAT4, a Golgi apparatus-anchored B-type histone acetyltransferase, acetylates free histone H4 and facilitates chromatin assembly.","date":"2011","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/21981917","citation_count":67,"is_preprint":false},{"pmid":"38480682","id":"PMC_38480682","title":"Biallelic NAA60 variants with impaired n-terminal acetylation capacity cause autosomal recessive primary familial brain calcifications.","date":"2024","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/38480682","citation_count":41,"is_preprint":false},{"pmid":"30952669","id":"PMC_30952669","title":"NATF (Native and Tissue-Specific Fluorescence): A Strategy for Bright, Tissue-Specific GFP Labeling of Native Proteins in Caenorhabditis elegans.","date":"2019","source":"Genetics","url":"https://pubmed.ncbi.nlm.nih.gov/30952669","citation_count":39,"is_preprint":false},{"pmid":"28196861","id":"PMC_28196861","title":"Molecular determinants of the N-terminal acetyltransferase Naa60 anchoring to the Golgi membrane.","date":"2017","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/28196861","citation_count":36,"is_preprint":false},{"pmid":"27550639","id":"PMC_27550639","title":"Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase.","date":"2016","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/27550639","citation_count":30,"is_preprint":false},{"pmid":"22016570","id":"PMC_22016570","title":"Histone H4 lysine 14 acetylation in Leishmania donovani is mediated by the MYST-family protein HAT4.","date":"2011","source":"Microbiology (Reading, England)","url":"https://pubmed.ncbi.nlm.nih.gov/22016570","citation_count":22,"is_preprint":false},{"pmid":"27272906","id":"PMC_27272906","title":"Histone acetyltransferase HAT4 modulates navigation across G2/M and re-entry into G1 in Leishmania donovani.","date":"2016","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/27272906","citation_count":18,"is_preprint":false},{"pmid":"32362349","id":"PMC_32362349","title":"Extended Venous Thromboembolism Prophylaxis in Medically Ill Patients: An NATF Anticoagulation Action Initiative.","date":"2020","source":"The American journal of medicine","url":"https://pubmed.ncbi.nlm.nih.gov/32362349","citation_count":17,"is_preprint":false},{"pmid":"39229657","id":"PMC_39229657","title":"A Homozygous Variant in NAA60 Is Associated with Primary Familial Brain Calcification.","date":"2024","source":"Movement disorders : official journal of the Movement Disorder Society","url":"https://pubmed.ncbi.nlm.nih.gov/39229657","citation_count":8,"is_preprint":false},{"pmid":"36539894","id":"PMC_36539894","title":"NAA60 (HAT4): the newly discovered bi-functional Golgi member of the acetyltransferase family.","date":"2022","source":"Clinical epigenetics","url":"https://pubmed.ncbi.nlm.nih.gov/36539894","citation_count":5,"is_preprint":false},{"pmid":"39965656","id":"PMC_39965656","title":"Proximal partners of the organellar N-terminal acetyltransferase NAA60: insights into Golgi structure and transmembrane protein topology.","date":"2025","source":"Open biology","url":"https://pubmed.ncbi.nlm.nih.gov/39965656","citation_count":4,"is_preprint":false},{"pmid":"41053424","id":"PMC_41053424","title":"NAA60 facilitates LRRC8A- and LRRC8D-mediated platinum drug uptake.","date":"2025","source":"Communications biology","url":"https://pubmed.ncbi.nlm.nih.gov/41053424","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8773,"output_tokens":2818,"usd":0.034294,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10045,"output_tokens":3082,"usd":0.063637,"stage2_stop_reason":"end_turn"},"total_usd":0.097931,"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\": 2011,\n      \"finding\": \"NAA60 (NatF/hNaa60) is an N-terminal acetyltransferase (NAT) with activity targeting Met-Lys- and other Met-starting protein N-termini, as demonstrated by in vitro peptide library acetylation assays using purified recombinant human and Drosophila homologues. In vivo, ectopic expression of hNaa60p in yeast acetylated distinct Met-starting yeast protein N-termini and increased general N-terminal acetylation levels. Knockdown of NAA60 in Drosophila cells induced chromosomal segregation defects.\",\n      \"method\": \"In vitro peptide library acetylation assay with purified recombinant protein; ectopic expression in yeast followed by N-terminal COFRADIC; RNAi knockdown in Drosophila cells\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzymatic assay with purified recombinant protein plus in vivo validation by two orthogonal methods (yeast N-terminal COFRADIC and human cell knockdown), replicated across species\",\n      \"pmids\": [\"21750686\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"HAT4 (NAA60) localizes to the Golgi apparatus and functions as a B-type histone acetyltransferase that preferentially acetylates free (non-nucleosomal) histone H4 at lysine residues K79 and K91 in the globular domain. HAT4 depletion impaired nucleosome assembly, inhibited cell proliferation, sensitized cells to DNA damage, and induced apoptosis.\",\n      \"method\": \"Subcellular localization by immunofluorescence; in vitro acetyltransferase assay with free histones; siRNA knockdown with cell proliferation, DNA damage, and apoptosis readouts\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct enzymatic assay plus cellular knockdown phenotypes, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"21981917\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NAA60 (Naa60/NatF) localizes exclusively to the cytosolic face of Golgi membranes (established by a new membrane topology assay PROMPT and selective membrane permeabilization). NAA60 specifically acetylates N-termini of transmembrane proteins that face the cytosol, as revealed by Nt-acetylome analysis of NAA60-knockdown cells. NAA60 knockdown causes Golgi fragmentation, indicating a role in maintaining Golgi structural integrity.\",\n      \"method\": \"Membrane topology assay (PROMPT); selective membrane permeabilization; N-terminal acetylome (COFRADIC) analysis of knockdown cells; immunofluorescence of Golgi morphology in knockdown cells\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (topology assay, proteomics, knockdown phenotype) establishing localization, substrate specificity, and cellular role\",\n      \"pmids\": [\"25732826\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Crystal structures of human Naa60 in complex with Ac-CoA or CoA reveal a GNAT domain followed by an amphipathic helix contributing to Golgi localization. Structural and biochemical studies identified Tyr97 and His138 as key catalytic residues, and Phe34 as influencing cofactor positioning. The non-conserved β3-β4 long loop participates in regulation of hNaa60 activity. Naa60 also harbors lysine Nε-acetyltransferase (KAT) activity toward histone H4 lysine residues.\",\n      \"method\": \"X-ray crystallography; site-directed mutagenesis; in vitro acetyltransferase activity assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure combined with mutagenesis and biochemical activity assays in a single study\",\n      \"pmids\": [\"27550639\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The C-terminal tail of Naa60 anchors it to the Golgi as a peripheral membrane protein via two amphipathic helices that fold into α-helical conformations only in the presence of liposomes and bind membranes in a parallel orientation via hydrophobic and electrostatic interactions. Naa60 shows strong and specific binding preference for phosphatidylinositol PI(4)P-containing membranes, consistent with its primary Golgi residency. Mutational analysis confirmed the hydrophobic face of these helices is critical for membranous localization. Golgi anchoring is proposed to occur post-translationally.\",\n      \"method\": \"Computational modeling; in vitro liposome binding assays; cellular mutational analysis with localization readout by immunofluorescence\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — combined in vitro biophysical assays, mutagenesis, and cellular localization studies in one study with multiple orthogonal approaches\",\n      \"pmids\": [\"28196861\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Biallelic loss-of-function variants in NAA60 cause autosomal recessive primary familial brain calcification (PFBC) with absent Nt-acetylation activity. In vitro, the phosphate importer SLC20A2 (PiT2) was identified as a direct substrate of NAA60 Nt-acetylation. In cells, NAA60 loss reduced surface levels of SLC20A2 and decreased extracellular phosphate uptake, linking NAA60-mediated Nt-acetylation of transmembrane proteins to phosphate homeostasis.\",\n      \"method\": \"In vitro Nt-acetylation assay with SLC20A2 as substrate; cell surface biotinylation assay; phosphate uptake assay in NAA60-deficient cells; patient variant functional analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro substrate identification plus cellular functional readouts (surface levels, phosphate uptake), replicated across multiple patient families and cell lines\",\n      \"pmids\": [\"38480682\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"A homozygous frameshift variant in NAA60 disrupts its Golgi localization and accelerates protein degradation. The mutant NAA60 alters interaction with PFBC-related proteins PiT2 (SLC20A2) and XPR1, affecting intracellular phosphate homeostasis. NAA60 KO in cells also caused decreased expression of multiple brain calcification-associated membrane proteins including RFC (reduced folate carrier), a folate metabolism protein.\",\n      \"method\": \"Immunofluorescence (Golgi localization); Western blot (protein stability); co-immunoprecipitation (interaction with PiT2/XPR1); mass spectrometry in NAA60 KO cell lines; gene knockout cell lines\",\n      \"journal\": \"Movement disorders : official journal of the Movement Disorder Society\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — multiple methods (co-IP, IF, MS) in single lab; findings largely corroborate prior work but add novel interactions\",\n      \"pmids\": [\"39229657\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"BioID proximity labeling of NAA60 identified over 100 proximal partners enriched on the trans-side of the Golgi, including golgins and GRASP proteins essential for Golgi integrity. Suborganellar localization analysis revealed a more prominent medial/trans-Golgi localization of NAA60. Biotinylated peptide analysis was used to infer the topology of transmembrane protein interactors in the secretory pathway.\",\n      \"method\": \"BioID proximity labeling; streptavidin affinity purification; mass spectrometry; suborganellar localization analysis\",\n      \"journal\": \"Open biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — BioID interactomics with MS validation, single lab, establishes proximal interactome and suborganellar localization\",\n      \"pmids\": [\"39965656\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NAA60 facilitates cellular uptake of platinum-based drugs (cis- and carboplatin) by acetylating the N-termini of LRRC8A and LRRC8D (volume-regulated anion channel subunits). Loss of NAA60 decreases drug uptake and causes drug resistance. Introduction of positively charged amino acids at the N-termini of LRRC8A/D (mimicking absence of the acetyl group) similarly decreased platinum sensitivity, functionally validating the role of N-terminal acetylation at these sites.\",\n      \"method\": \"NAA60 knockout cells; in vitro N-terminal acetylation assay; drug uptake assay; site-directed mutagenesis of LRRC8A/D N-termini; drug sensitivity assays in BRCA1;p53-deficient cells and tumors\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple methods including KO, mutagenesis, and functional drug uptake assays in single lab, novel substrate identification\",\n      \"pmids\": [\"41053424\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NAA60 (NatF/HAT4) is a Golgi-localized N-terminal acetyltransferase that anchors to the cytosolic face of Golgi membranes via two C-terminal amphipathic helices with preference for PI(4)P-rich membranes, where it post-translationally Nt-acetylates Met-starting N-termini of transmembrane proteins (including SLC20A2/PiT2, LRRC8A, and LRRC8D) that face the cytosol; its catalytic mechanism involves key residues Tyr97 and His138, it also possesses lysine Nε-acetyltransferase activity toward free histone H4, loss of NAA60 causes Golgi fragmentation and chromosome segregation defects, and biallelic loss-of-function variants cause primary familial brain calcification by disrupting Nt-acetylation-dependent surface trafficking and phosphate homeostasis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NAA60 (NatF/HAT4) is an N-terminal acetyltransferase that post-translationally Nt-acetylates Met-starting protein N-termini, with a substrate preference for the cytosol-facing N-termini of transmembrane proteins in the secretory pathway [#0, #2]. It anchors as a peripheral membrane protein to the cytosolic face of Golgi membranes through two C-terminal amphipathic helices that fold only upon membrane contact and bind preferentially to PI(4)P-rich membranes, accounting for its medial/trans-Golgi residency among golgins and GRASP proteins [#4, #7]. Crystal structures define a GNAT catalytic fold using Tyr97 and His138 as key catalytic residues, and the enzyme additionally displays lysine Nε-acetyltransferase activity toward free histone H4 at K79 and K91 [#1, #3]. Through Nt-acetylation of transmembrane substrates including the phosphate importer SLC20A2/PiT2 and the volume-regulated anion channel subunits LRRC8A and LRRC8D, NAA60 controls their surface trafficking, thereby governing extracellular phosphate uptake and cellular uptake of platinum-based drugs [#5, #8]. Loss of NAA60 causes Golgi fragmentation and chromosome segregation defects [#0, #2], and biallelic loss-of-function variants cause autosomal recessive primary familial brain calcification via disrupted Nt-acetylation-dependent surface trafficking and phosphate homeostasis [#5, #6].\",\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"Established that NAA60 is a bona fide N-terminal acetyltransferase with a distinct substrate preference for Met-starting N-termini, defining a new NAT (NatF) and linking it to chromosome segregation.\",\n      \"evidence\": \"In vitro peptide library acetylation with purified recombinant human and Drosophila enzyme, ectopic expression in yeast with N-terminal COFRADIC, and RNAi knockdown in Drosophila cells\",\n      \"pmids\": [\"21750686\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Subcellular site of activity not yet defined\", \"Physiological transmembrane substrates not identified\", \"Mechanism linking loss to segregation defects unresolved\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Showed NAA60 (HAT4) localizes to the Golgi and additionally acts as a lysine acetyltransferase toward free histone H4 at K79/K91, connecting it to nucleosome assembly and cell survival.\",\n      \"evidence\": \"Immunofluorescence, in vitro acetyltransferase assay with free histones, and siRNA knockdown with proliferation/DNA-damage/apoptosis readouts\",\n      \"pmids\": [\"21981917\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How a Golgi-anchored enzyme accesses free histones unresolved\", \"Relative physiological importance of KAT versus NAT activity unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Resolved that NAA60 sits on the cytosolic face of Golgi membranes and selectively acetylates cytosol-facing transmembrane protein N-termini, and that its loss fragments the Golgi—tying the enzyme to organelle integrity.\",\n      \"evidence\": \"PROMPT membrane topology assay, selective permeabilization, N-terminal acetylome (COFRADIC) of knockdown cells, and Golgi morphology imaging\",\n      \"pmids\": [\"25732826\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Membrane anchoring mechanism not yet defined\", \"Causal link between substrate acetylation and Golgi integrity not established\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined the catalytic architecture by solving Naa60 crystal structures and identifying Tyr97/His138 as catalytic residues plus a regulatory β3-β4 loop and an amphipathic helix for Golgi localization.\",\n      \"evidence\": \"X-ray crystallography with Ac-CoA/CoA, site-directed mutagenesis, and in vitro activity assays\",\n      \"pmids\": [\"27550639\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of the membrane-bound enzyme not captured\", \"Regulatory function of the β3-β4 loop not mechanistically resolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Explained how NAA60 reaches the Golgi: two C-terminal amphipathic helices fold on membrane contact and bind with strong preference for PI(4)P, defining a lipid-dependent post-translational anchoring mode.\",\n      \"evidence\": \"Computational modeling, in vitro liposome binding assays, and cellular mutational analysis of helix hydrophobic face with localization readout\",\n      \"pmids\": [\"28196861\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Regulation of membrane targeting in vivo unclear\", \"Whether lipid binding modulates catalytic activity not addressed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Connected NAA60 to human disease and to phosphate physiology by showing biallelic loss-of-function causes primary familial brain calcification and that NAA60 Nt-acetylates SLC20A2 to control its surface levels and phosphate uptake.\",\n      \"evidence\": \"Patient variant functional analysis, in vitro Nt-acetylation with SLC20A2, cell surface biotinylation, and phosphate uptake assays in NAA60-deficient cells\",\n      \"pmids\": [\"38480682\", \"39229657\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise mechanism linking acetylation to trafficking not fully defined\", \"Role of additional calcification-associated substrates (e.g., XPR1, RFC) not mechanistically resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Mapped the NAA60 proximal interactome to the medial/trans-Golgi, enriched for golgins and GRASP proteins, refining its suborganellar position relative to Golgi-integrity machinery.\",\n      \"evidence\": \"BioID proximity labeling with streptavidin purification and mass spectrometry, plus suborganellar localization analysis\",\n      \"pmids\": [\"39965656\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct versus proximal interactions not distinguished\", \"Functional significance of golgin/GRASP proximity untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended NAA60 substrate scope and clinical relevance by showing it acetylates LRRC8A/D N-termini to enable cellular uptake of platinum drugs, with loss conferring drug resistance.\",\n      \"evidence\": \"NAA60 knockout cells, in vitro Nt-acetylation, drug uptake and sensitivity assays, and charge-mimic N-terminal mutagenesis of LRRC8A/D\",\n      \"pmids\": [\"41053424\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; reciprocal in vivo validation limited\", \"Mechanism by which N-terminal acetylation alters channel function not resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How NAA60-mediated N-terminal acetylation mechanistically controls transmembrane protein surface trafficking, and how its Golgi/histone activities are coordinated, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of the membrane-anchored enzyme engaging a transmembrane substrate\", \"Mechanistic basis linking acetylation to trafficking competence unknown\", \"Integration of cytosolic NAT activity with reported histone KAT activity unexplained\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 2, 3, 5, 8]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 5, 8]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [1, 2, 4, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 2, 5]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [5, 6]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"SLC20A2\", \"XPR1\", \"LRRC8A\", \"LRRC8D\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}