{"gene":"NAA60","run_date":"2026-04-29T11:37:56","timeline":{"discoveries":[{"year":2011,"finding":"NAA60 (NatF/Naa60p) is an N-terminal acetyltransferase (NAT) that targets Met-Lys- and other Met-starting protein N-termini. In vitro peptide library acetylation assays with purified recombinant human and Drosophila homologues established its NAT activity. Ectopic expression in yeast followed by N-terminal COFRADIC confirmed in vivo acetylation of Met-starting yeast protein N-termini. Knockdown in Drosophila cells induced chromosomal segregation defects.","method":"In vitro peptide library acetylation assays; N-terminal COFRADIC proteomics; yeast ectopic expression; RNAi knockdown with chromosomal segregation phenotype readout","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods (in vitro enzymatic assay, in vivo proteomics, cellular phenotype), replicated across organisms","pmids":["21750686"],"is_preprint":false},{"year":2015,"finding":"NAA60 localizes to the cytosolic face of Golgi membranes (not the lumen), as established by a new membrane topology assay (PROMPT) and selective membrane permeabilization. Nt-acetylome analysis of NAA60-knockdown cells showed that NAA60 specifically acetylates transmembrane proteins with N-termini facing the cytosol. NAA60 knockdown causes Golgi fragmentation, indicating a role in maintaining Golgi structural integrity.","method":"PROMPT membrane topology assay; selective membrane permeabilization; Nt-acetylome mass spectrometry after siRNA knockdown; fluorescence microscopy of Golgi morphology","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods (novel topology assay, quantitative proteomics, cellular phenotype) in a single rigorous study","pmids":["25732826"],"is_preprint":false},{"year":2016,"finding":"Crystal structures of human NAA60 in complex with Acetyl-CoA or CoA were solved. The structures revealed that Tyr97 and His138 are key catalytic residues, Phe34 influences coenzyme positioning (a new regulatory mechanism), and a non-conserved β3-β4 long loop participates in activity regulation. NAA60 also harbors lysine Nε-acetyltransferase (KAT) activity toward lysine ε-amines. The amphipathic helix following the GNAT domain contributes to Golgi localization.","method":"X-ray crystallography; biochemical acetyltransferase assays; active-site mutagenesis (Tyr97, His138, Phe34)","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 — crystal structures with functional validation by mutagenesis and enzymatic assays","pmids":["27550639"],"is_preprint":false},{"year":2017,"finding":"The C-terminal tail of NAA60 contains two amphipathic helices that anchor the protein to the cytosolic face of Golgi membranes as a peripheral membrane protein. The helices are unstructured in solution and fold into α-helical conformations only in the presence of liposomes. NAA60 shows strong and specific binding preference for membranes containing PI(4)P, explaining its primary Golgi residency. Mutational analysis of the hydrophobic face of the two α-helices abolished membranous localization. Anchoring likely occurs post-translationally.","method":"Computational modeling; in vitro liposome-binding assays; cellular mutational/localization studies; lipid-binding assays with PI(4)P-containing membranes","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods (modeling, in vitro lipid binding, cellular mutagenesis) establishing the membrane anchoring mechanism","pmids":["28196861"],"is_preprint":false},{"year":2024,"finding":"Biallelic loss-of-function variants in NAA60 cause autosomal recessive primary familial brain calcification (PFBC). NAA60 directly acetylates the N-terminus of the phosphate importer SLC20A2 (PiT2) in vitro. Loss of NAA60 in cells reduces surface levels of SLC20A2 and decreases extracellular phosphate uptake, providing a biochemical mechanism linking NAA60 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 loss-of-function cells; patient variant functional analysis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro substrate identification combined with cellular loss-of-function and functional transport assay","pmids":["38480682"],"is_preprint":false},{"year":2024,"finding":"A homozygous frameshift variant (p.D154Lfs*113) in NAA60 disrupts NAA60 protein localization to the Golgi and accelerates protein degradation. The mutant NAA60 alters its interaction with PFBC-related proteins PiT2 (SLC20A2) and XPR1, affecting intracellular phosphate homeostasis. Mass spectrometry of NAA60 KO cells revealed decreased expression of multiple brain calcification-associated membrane proteins including RFC (reduced folate carrier).","method":"Western blot; immunofluorescence; co-immunoprecipitation; mass spectrometry in NAA60 KO cell lines","journal":"Movement disorders","confidence":"Medium","confidence_rationale":"Tier 2-3 — co-IP and KO proteomics support interaction and downstream effects, but mechanistic depth is limited compared to dedicated biochemical studies","pmids":["39229657"],"is_preprint":false},{"year":2025,"finding":"BioID proximity labeling of NAA60 identified over 100 proximal partners enriched at the trans-side of the Golgi, including golgins and GRASP proteins essential for Golgi integrity. Sub-organellar localization analysis refined NAA60 localization to the medial/trans-Golgi. Biotinylated peptide mapping from transmembrane interactors provided topology data for NAA60 substrates within the secretory pathway.","method":"BioID proximity labeling; streptavidin affinity purification; mass spectrometry; sub-organellar localization imaging","journal":"Open biology","confidence":"Medium","confidence_rationale":"Tier 2 — proximity interactomics with sub-organellar localization analysis, single study","pmids":["39965656"],"is_preprint":false},{"year":2025,"finding":"NAA60 acetylates the N-termini of LRRC8A and LRRC8D (volume-regulated anion channel subunits), facilitating cis- and carboplatin uptake. Loss of NAA60 decreases platinum drug uptake and confers drug resistance in BRCA1;p53-deficient cells and tumors. Introduction of positively charged amino acids at LRRC8A/D N-termini (mimicking loss of the acetyl moiety) decreased platinum drug sensitivity, functionally validating the role of Nt-acetylation.","method":"NAA60 knockout; N-terminal mutagenesis of LRRC8A/D; platinum drug uptake and cytotoxicity assays; in vivo tumor models","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 2 — KO with functional rescue and mutagenesis mimicry, single study with in vitro and in vivo readouts","pmids":["41053424"],"is_preprint":false},{"year":2011,"finding":"Human NAA60 (also referred to as HAT4) localizes to the Golgi apparatus and displays substrate preference for lysine residues in the globular domain of free histone H4 (H4K79 and H4K91). HAT4 depletion impaired nucleosome assembly, inhibited cell proliferation, sensitized cells to DNA damage, and induced apoptosis.","method":"Subcellular fractionation/immunofluorescence for Golgi localization; in vitro HAT assay with free histone H4; siRNA depletion with phenotypic readouts (nucleosome assembly, proliferation, DNA damage sensitivity, apoptosis)","journal":"Molecular cell","confidence":"Medium","confidence_rationale":"Tier 1-2 — in vitro enzymatic assay with substrate specificity plus KD phenotypes, but NAA60 as a KAT for histone H4 is not yet widely replicated","pmids":["21981917"],"is_preprint":false}],"current_model":"NAA60 (NatF) is a Golgi-localized, peripheral membrane N-terminal acetyltransferase anchored to the cytosolic face of the Golgi via two C-terminal amphipathic helices that bind PI(4)P-enriched membranes; it co-translationally or post-translationally acetylates the Met-starting N-termini of transmembrane proteins (including SLC20A2/PiT2, LRRC8A, and LRRC8D) that face the cytosol, thereby regulating their surface trafficking and function, maintaining Golgi structural integrity, and ensuring normal chromosome segregation, while loss-of-function variants cause primary familial brain calcification by impairing phosphate homeostasis."},"narrative":{"teleology":[{"year":2011,"claim":"Identification of NAA60 as a novel NAT with Met-starting substrate specificity and a role in chromosome segregation established a previously unrecognized acetyltransferase activity distinct from known NatA–E complexes.","evidence":"In vitro peptide library acetylation, yeast ectopic expression with N-terminal COFRADIC, and Drosophila RNAi showing segregation defects","pmids":["21750686"],"confidence":"High","gaps":["Endogenous substrates in metazoan cells were not identified","Mechanism linking Nt-acetylation to chromosome segregation was unresolved","Subcellular localization in mammalian cells was not yet characterized"]},{"year":2011,"claim":"An independent study reported NAA60 (HAT4) as a Golgi-localized lysine acetyltransferase for histone H4 (K79, K91), linking it to nucleosome assembly and DNA damage sensitivity, though its KAT activity has not been widely replicated.","evidence":"In vitro HAT assay with free histone H4; siRNA depletion with proliferation, apoptosis, and DNA damage readouts","pmids":["21981917"],"confidence":"Medium","gaps":["KAT activity toward histone H4 has not been independently confirmed by other groups","Whether Golgi-localized NAA60 encounters free histone H4 in a physiological context is unclear","Relationship between KAT and NAT activities remains unresolved"]},{"year":2015,"claim":"Demonstrating that NAA60 resides on the cytosolic face of Golgi membranes and specifically acetylates transmembrane proteins with cytosol-facing N-termini resolved its substrate class and explained why its loss fragments the Golgi.","evidence":"PROMPT topology assay, selective permeabilization, Nt-acetylome proteomics after siRNA knockdown, and Golgi morphology imaging","pmids":["25732826"],"confidence":"High","gaps":["The molecular mechanism by which loss of Nt-acetylation leads to Golgi fragmentation was not determined","How NAA60 is recruited to and retained at the Golgi membrane was unknown"]},{"year":2016,"claim":"Crystal structures of NAA60 with Acetyl-CoA/CoA revealed the catalytic mechanism (Tyr97/His138) and an amphipathic helix contributing to Golgi targeting, providing an atomic-level framework for understanding substrate recognition and membrane association.","evidence":"X-ray crystallography with active-site mutagenesis and enzymatic assays","pmids":["27550639"],"confidence":"High","gaps":["No co-crystal with a peptide substrate to define the substrate-binding groove","Full membrane-anchoring mechanism was not yet established from the structure alone"]},{"year":2017,"claim":"Identification of two C-terminal amphipathic helices that fold on PI(4)P-containing membranes explained the selective Golgi residency of NAA60 and established it as a peripheral rather than integral membrane protein.","evidence":"Liposome-binding assays, PI(4)P-specificity lipid binding, and cellular mutagenesis of hydrophobic helix faces","pmids":["28196861"],"confidence":"High","gaps":["Whether additional protein–protein interactions contribute to Golgi retention was not tested","Regulation of NAA60 membrane association under signaling or stress conditions was not examined"]},{"year":2024,"claim":"Linking NAA60 to primary familial brain calcification through biallelic loss-of-function variants and showing that NAA60 directly Nt-acetylates SLC20A2 to promote its surface expression and phosphate uptake provided the first disease mechanism and a defined physiological substrate–function axis.","evidence":"In vitro Nt-acetylation of SLC20A2, surface biotinylation, phosphate uptake assays in NAA60 KO cells, and patient variant analysis","pmids":["38480682"],"confidence":"High","gaps":["Whether Nt-acetylation affects SLC20A2 folding, stability, or trafficking specifically is unresolved","Animal model confirmation of the PFBC phenotype was not reported in this study"]},{"year":2024,"claim":"An independent study on a patient frameshift variant confirmed that disease-causing NAA60 mutations mislocalise from the Golgi and undergo accelerated degradation, and proteomics of NAA60 KO cells revealed broader changes in brain calcification-associated membrane proteins.","evidence":"Immunofluorescence, co-immunoprecipitation, and mass spectrometry in NAA60 KO cell lines","pmids":["39229657"],"confidence":"Medium","gaps":["Co-IP interactions with PiT2 and XPR1 lack reciprocal validation","Downstream proteomic changes could be indirect consequences of Golgi dysfunction"]},{"year":2025,"claim":"BioID proximity labeling refined NAA60 to the medial/trans-Golgi and identified golgins and GRASP proteins as proximal partners, providing a molecular context for how NAA60 loss disrupts Golgi architecture.","evidence":"BioID with streptavidin purification, mass spectrometry, and sub-organellar imaging","pmids":["39965656"],"confidence":"Medium","gaps":["Proximity partners have not been validated as direct physical interactors or substrates","Whether NAA60 acetylates golgins/GRASPs or merely co-resides with them is unknown"]},{"year":2025,"claim":"Demonstrating that NAA60 Nt-acetylates LRRC8A/D to facilitate platinum drug uptake through VRAC channels extended its functional repertoire beyond phosphate homeostasis to chemoresistance and ion channel regulation.","evidence":"NAA60 KO cells, N-terminal mutagenesis of LRRC8A/D, platinum uptake/cytotoxicity assays, and in vivo tumor models","pmids":["41053424"],"confidence":"Medium","gaps":["Structural basis for how Nt-acetylation alters VRAC channel permeability is unresolved","Whether this mechanism operates in non-BRCA1 contexts has not been tested"]},{"year":null,"claim":"How Nt-acetylation by NAA60 mechanistically governs the trafficking, stability, or activity of its transmembrane substrates — and the full catalogue of in vivo substrates — remains incompletely defined.","evidence":"","pmids":[],"confidence":"Low","gaps":["No substrate-bound co-crystal structure exists to explain selectivity","Whether NAA60 KAT activity toward histone H4 is physiologically relevant remains unresolved","Animal models recapitulating PFBC or Golgi fragmentation phenotypes have not been reported"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,2,4,7]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,4,8]}],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[1,2,3,6,8]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,4]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[4,7]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[1,6]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[4,5]}],"complexes":[],"partners":["SLC20A2","LRRC8A","LRRC8D","XPR1"],"other_free_text":[]},"mechanistic_narrative":"NAA60 (NatF) is a Golgi-localized N-terminal acetyltransferase that co- and post-translationally acetylates Met-starting N-termini of transmembrane proteins whose cytosolic tails face the cytoplasm, thereby controlling their surface trafficking, Golgi structural integrity, and chromosome segregation [PMID:21750686, PMID:25732826]. The enzyme is anchored to the cytosolic face of medial/trans-Golgi membranes as a peripheral membrane protein via two C-terminal amphipathic helices that fold upon contact with PI(4)P-enriched lipid bilayers, and its GNAT-domain catalytic mechanism depends on Tyr97 and His138 [PMID:28196861, PMID:27550639]. Functionally validated substrates include the phosphate importer SLC20A2 and the volume-regulated anion channel subunits LRRC8A/LRRC8D, whose Nt-acetylation by NAA60 promotes surface expression and channel-mediated drug uptake, respectively [PMID:38480682, PMID:41053424]. Biallelic loss-of-function variants in NAA60 cause autosomal recessive primary familial brain calcification through impaired SLC20A2 surface delivery and phosphate homeostasis [PMID:38480682]."},"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":160,"is_preprint":false},{"pmid":"8449400","id":"PMC_8449400","title":"The HAT4 gene of Arabidopsis encodes a developmental regulator.","date":"1993","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/8449400","citation_count":107,"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":"32548857","id":"PMC_32548857","title":"The Arabidopsis Nα -acetyltransferase NAA60 locates to the plasma membrane and is vital for the high salt stress response.","date":"2020","source":"The New phytologist","url":"https://pubmed.ncbi.nlm.nih.gov/32548857","citation_count":41,"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":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":"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":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":16,"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":8811,"output_tokens":2648,"usd":0.033077},"stage2":{"model":"claude-opus-4-6","input_tokens":5956,"output_tokens":2657,"usd":0.144308},"total_usd":0.177385,"stage1_batch_id":"msgbatch_012ZiiCUBfB62NCTQd4Bk1Jo","stage2_batch_id":"msgbatch_01Xuqq9aUHoQouS7UVGZNStZ","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2011,\n      \"finding\": \"NAA60 (NatF/Naa60p) is an N-terminal acetyltransferase (NAT) that targets Met-Lys- and other Met-starting protein N-termini. In vitro peptide library acetylation assays with purified recombinant human and Drosophila homologues established its NAT activity. Ectopic expression in yeast followed by N-terminal COFRADIC confirmed in vivo acetylation of Met-starting yeast protein N-termini. Knockdown in Drosophila cells induced chromosomal segregation defects.\",\n      \"method\": \"In vitro peptide library acetylation assays; N-terminal COFRADIC proteomics; yeast ectopic expression; RNAi knockdown with chromosomal segregation phenotype readout\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods (in vitro enzymatic assay, in vivo proteomics, cellular phenotype), replicated across organisms\",\n      \"pmids\": [\"21750686\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NAA60 localizes to the cytosolic face of Golgi membranes (not the lumen), as established by a new membrane topology assay (PROMPT) and selective membrane permeabilization. Nt-acetylome analysis of NAA60-knockdown cells showed that NAA60 specifically acetylates transmembrane proteins with N-termini facing the cytosol. NAA60 knockdown causes Golgi fragmentation, indicating a role in maintaining Golgi structural integrity.\",\n      \"method\": \"PROMPT membrane topology assay; selective membrane permeabilization; Nt-acetylome mass spectrometry after siRNA knockdown; fluorescence microscopy of Golgi morphology\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods (novel topology assay, quantitative proteomics, cellular phenotype) in a single rigorous study\",\n      \"pmids\": [\"25732826\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Crystal structures of human NAA60 in complex with Acetyl-CoA or CoA were solved. The structures revealed that Tyr97 and His138 are key catalytic residues, Phe34 influences coenzyme positioning (a new regulatory mechanism), and a non-conserved β3-β4 long loop participates in activity regulation. NAA60 also harbors lysine Nε-acetyltransferase (KAT) activity toward lysine ε-amines. The amphipathic helix following the GNAT domain contributes to Golgi localization.\",\n      \"method\": \"X-ray crystallography; biochemical acetyltransferase assays; active-site mutagenesis (Tyr97, His138, Phe34)\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structures with functional validation by mutagenesis and enzymatic assays\",\n      \"pmids\": [\"27550639\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The C-terminal tail of NAA60 contains two amphipathic helices that anchor the protein to the cytosolic face of Golgi membranes as a peripheral membrane protein. The helices are unstructured in solution and fold into α-helical conformations only in the presence of liposomes. NAA60 shows strong and specific binding preference for membranes containing PI(4)P, explaining its primary Golgi residency. Mutational analysis of the hydrophobic face of the two α-helices abolished membranous localization. Anchoring likely occurs post-translationally.\",\n      \"method\": \"Computational modeling; in vitro liposome-binding assays; cellular mutational/localization studies; lipid-binding assays with PI(4)P-containing membranes\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods (modeling, in vitro lipid binding, cellular mutagenesis) establishing the membrane anchoring mechanism\",\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). NAA60 directly acetylates the N-terminus of the phosphate importer SLC20A2 (PiT2) in vitro. Loss of NAA60 in cells reduces surface levels of SLC20A2 and decreases extracellular phosphate uptake, providing a biochemical mechanism linking NAA60 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 loss-of-function cells; patient variant functional analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro substrate identification combined with cellular loss-of-function and functional transport assay\",\n      \"pmids\": [\"38480682\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"A homozygous frameshift variant (p.D154Lfs*113) in NAA60 disrupts NAA60 protein localization to the Golgi and accelerates protein degradation. The mutant NAA60 alters its interaction with PFBC-related proteins PiT2 (SLC20A2) and XPR1, affecting intracellular phosphate homeostasis. Mass spectrometry of NAA60 KO cells revealed decreased expression of multiple brain calcification-associated membrane proteins including RFC (reduced folate carrier).\",\n      \"method\": \"Western blot; immunofluorescence; co-immunoprecipitation; mass spectrometry in NAA60 KO cell lines\",\n      \"journal\": \"Movement disorders\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — co-IP and KO proteomics support interaction and downstream effects, but mechanistic depth is limited compared to dedicated biochemical studies\",\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 at the trans-side of the Golgi, including golgins and GRASP proteins essential for Golgi integrity. Sub-organellar localization analysis refined NAA60 localization to the medial/trans-Golgi. Biotinylated peptide mapping from transmembrane interactors provided topology data for NAA60 substrates within the secretory pathway.\",\n      \"method\": \"BioID proximity labeling; streptavidin affinity purification; mass spectrometry; sub-organellar localization imaging\",\n      \"journal\": \"Open biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — proximity interactomics with sub-organellar localization analysis, single study\",\n      \"pmids\": [\"39965656\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NAA60 acetylates the N-termini of LRRC8A and LRRC8D (volume-regulated anion channel subunits), facilitating cis- and carboplatin uptake. Loss of NAA60 decreases platinum drug uptake and confers drug resistance in BRCA1;p53-deficient cells and tumors. Introduction of positively charged amino acids at LRRC8A/D N-termini (mimicking loss of the acetyl moiety) decreased platinum drug sensitivity, functionally validating the role of Nt-acetylation.\",\n      \"method\": \"NAA60 knockout; N-terminal mutagenesis of LRRC8A/D; platinum drug uptake and cytotoxicity assays; in vivo tumor models\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO with functional rescue and mutagenesis mimicry, single study with in vitro and in vivo readouts\",\n      \"pmids\": [\"41053424\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Human NAA60 (also referred to as HAT4) localizes to the Golgi apparatus and displays substrate preference for lysine residues in the globular domain of free histone H4 (H4K79 and H4K91). HAT4 depletion impaired nucleosome assembly, inhibited cell proliferation, sensitized cells to DNA damage, and induced apoptosis.\",\n      \"method\": \"Subcellular fractionation/immunofluorescence for Golgi localization; in vitro HAT assay with free histone H4; siRNA depletion with phenotypic readouts (nucleosome assembly, proliferation, DNA damage sensitivity, apoptosis)\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro enzymatic assay with substrate specificity plus KD phenotypes, but NAA60 as a KAT for histone H4 is not yet widely replicated\",\n      \"pmids\": [\"21981917\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NAA60 (NatF) is a Golgi-localized, peripheral membrane N-terminal acetyltransferase anchored to the cytosolic face of the Golgi via two C-terminal amphipathic helices that bind PI(4)P-enriched membranes; it co-translationally or post-translationally acetylates the Met-starting N-termini of transmembrane proteins (including SLC20A2/PiT2, LRRC8A, and LRRC8D) that face the cytosol, thereby regulating their surface trafficking and function, maintaining Golgi structural integrity, and ensuring normal chromosome segregation, while loss-of-function variants cause primary familial brain calcification by impairing phosphate homeostasis.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"NAA60 (NatF) is a Golgi-localized N-terminal acetyltransferase that co- and post-translationally acetylates Met-starting N-termini of transmembrane proteins whose cytosolic tails face the cytoplasm, thereby controlling their surface trafficking, Golgi structural integrity, and chromosome segregation [PMID:21750686, PMID:25732826]. The enzyme is anchored to the cytosolic face of medial/trans-Golgi membranes as a peripheral membrane protein via two C-terminal amphipathic helices that fold upon contact with PI(4)P-enriched lipid bilayers, and its GNAT-domain catalytic mechanism depends on Tyr97 and His138 [PMID:28196861, PMID:27550639]. Functionally validated substrates include the phosphate importer SLC20A2 and the volume-regulated anion channel subunits LRRC8A/LRRC8D, whose Nt-acetylation by NAA60 promotes surface expression and channel-mediated drug uptake, respectively [PMID:38480682, PMID:41053424]. Biallelic loss-of-function variants in NAA60 cause autosomal recessive primary familial brain calcification through impaired SLC20A2 surface delivery and phosphate homeostasis [PMID:38480682].\",\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"Identification of NAA60 as a novel NAT with Met-starting substrate specificity and a role in chromosome segregation established a previously unrecognized acetyltransferase activity distinct from known NatA–E complexes.\",\n      \"evidence\": \"In vitro peptide library acetylation, yeast ectopic expression with N-terminal COFRADIC, and Drosophila RNAi showing segregation defects\",\n      \"pmids\": [\"21750686\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Endogenous substrates in metazoan cells were not identified\",\n        \"Mechanism linking Nt-acetylation to chromosome segregation was unresolved\",\n        \"Subcellular localization in mammalian cells was not yet characterized\"\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"An independent study reported NAA60 (HAT4) as a Golgi-localized lysine acetyltransferase for histone H4 (K79, K91), linking it to nucleosome assembly and DNA damage sensitivity, though its KAT activity has not been widely replicated.\",\n      \"evidence\": \"In vitro HAT assay with free histone H4; siRNA depletion with proliferation, apoptosis, and DNA damage readouts\",\n      \"pmids\": [\"21981917\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"KAT activity toward histone H4 has not been independently confirmed by other groups\",\n        \"Whether Golgi-localized NAA60 encounters free histone H4 in a physiological context is unclear\",\n        \"Relationship between KAT and NAT activities remains unresolved\"\n      ]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Demonstrating that NAA60 resides on the cytosolic face of Golgi membranes and specifically acetylates transmembrane proteins with cytosol-facing N-termini resolved its substrate class and explained why its loss fragments the Golgi.\",\n      \"evidence\": \"PROMPT topology assay, selective permeabilization, Nt-acetylome proteomics after siRNA knockdown, and Golgi morphology imaging\",\n      \"pmids\": [\"25732826\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The molecular mechanism by which loss of Nt-acetylation leads to Golgi fragmentation was not determined\",\n        \"How NAA60 is recruited to and retained at the Golgi membrane was unknown\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Crystal structures of NAA60 with Acetyl-CoA/CoA revealed the catalytic mechanism (Tyr97/His138) and an amphipathic helix contributing to Golgi targeting, providing an atomic-level framework for understanding substrate recognition and membrane association.\",\n      \"evidence\": \"X-ray crystallography with active-site mutagenesis and enzymatic assays\",\n      \"pmids\": [\"27550639\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No co-crystal with a peptide substrate to define the substrate-binding groove\",\n        \"Full membrane-anchoring mechanism was not yet established from the structure alone\"\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identification of two C-terminal amphipathic helices that fold on PI(4)P-containing membranes explained the selective Golgi residency of NAA60 and established it as a peripheral rather than integral membrane protein.\",\n      \"evidence\": \"Liposome-binding assays, PI(4)P-specificity lipid binding, and cellular mutagenesis of hydrophobic helix faces\",\n      \"pmids\": [\"28196861\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether additional protein–protein interactions contribute to Golgi retention was not tested\",\n        \"Regulation of NAA60 membrane association under signaling or stress conditions was not examined\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Linking NAA60 to primary familial brain calcification through biallelic loss-of-function variants and showing that NAA60 directly Nt-acetylates SLC20A2 to promote its surface expression and phosphate uptake provided the first disease mechanism and a defined physiological substrate–function axis.\",\n      \"evidence\": \"In vitro Nt-acetylation of SLC20A2, surface biotinylation, phosphate uptake assays in NAA60 KO cells, and patient variant analysis\",\n      \"pmids\": [\"38480682\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether Nt-acetylation affects SLC20A2 folding, stability, or trafficking specifically is unresolved\",\n        \"Animal model confirmation of the PFBC phenotype was not reported in this study\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"An independent study on a patient frameshift variant confirmed that disease-causing NAA60 mutations mislocalise from the Golgi and undergo accelerated degradation, and proteomics of NAA60 KO cells revealed broader changes in brain calcification-associated membrane proteins.\",\n      \"evidence\": \"Immunofluorescence, co-immunoprecipitation, and mass spectrometry in NAA60 KO cell lines\",\n      \"pmids\": [\"39229657\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Co-IP interactions with PiT2 and XPR1 lack reciprocal validation\",\n        \"Downstream proteomic changes could be indirect consequences of Golgi dysfunction\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"BioID proximity labeling refined NAA60 to the medial/trans-Golgi and identified golgins and GRASP proteins as proximal partners, providing a molecular context for how NAA60 loss disrupts Golgi architecture.\",\n      \"evidence\": \"BioID with streptavidin purification, mass spectrometry, and sub-organellar imaging\",\n      \"pmids\": [\"39965656\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Proximity partners have not been validated as direct physical interactors or substrates\",\n        \"Whether NAA60 acetylates golgins/GRASPs or merely co-resides with them is unknown\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrating that NAA60 Nt-acetylates LRRC8A/D to facilitate platinum drug uptake through VRAC channels extended its functional repertoire beyond phosphate homeostasis to chemoresistance and ion channel regulation.\",\n      \"evidence\": \"NAA60 KO cells, N-terminal mutagenesis of LRRC8A/D, platinum uptake/cytotoxicity assays, and in vivo tumor models\",\n      \"pmids\": [\"41053424\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Structural basis for how Nt-acetylation alters VRAC channel permeability is unresolved\",\n        \"Whether this mechanism operates in non-BRCA1 contexts has not been tested\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How Nt-acetylation by NAA60 mechanistically governs the trafficking, stability, or activity of its transmembrane substrates — and the full catalogue of in vivo substrates — remains incompletely defined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No substrate-bound co-crystal structure exists to explain selectivity\",\n        \"Whether NAA60 KAT activity toward histone H4 is physiologically relevant remains unresolved\",\n        \"Animal models recapitulating PFBC or Golgi fragmentation phenotypes have not been reported\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 2, 4, 7]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 4, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [1, 2, 3, 6, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": []},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 4]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [4, 7]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [1, 6]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [4, 5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"SLC20A2\",\n      \"LRRC8A\",\n      \"LRRC8D\",\n      \"XPR1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}