{"gene":"NAA35","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2021,"finding":"Cryo-EM structure of S. pombe NatC with a NatE/C-type bisubstrate analog and inositol hexaphosphate (IP6) revealed that all three subunits (Naa30, Naa35, Naa38) are required for normal NatC acetylation activity in yeast, and that IP6 binds tightly to NatC to stabilize the complex; the molecular basis for IP6-mediated NatC complex stabilization was described.","method":"Cryo-EM structure determination, biochemical activity assays, IP6 binding studies","journal":"Structure (London, England : 1993)","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure with bisubstrate analog plus biochemical validation, multiple orthogonal methods in one rigorous study","pmids":["34019809"],"is_preprint":false},{"year":2023,"finding":"Cryo-EM structures of human NatC with and without NAA38 showed that NAA38 increases the thermostability and broadens the substrate-specificity profile of NatC by ordering an N-terminal segment of NAA35 and reorienting an NAA30 N-terminal peptide-binding loop for optimal catalysis; human NatC engages the stabilizing inositol hexaphosphate differently from yeast NatC.","method":"Cryo-EM structure determination with and without NAA38, biochemical thermostability and substrate-specificity assays","journal":"Structure (London, England : 1993)","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structures plus biochemical functional validation, multiple orthogonal methods in one rigorous study","pmids":["36638802"],"is_preprint":false},{"year":2003,"finding":"NAA35 (Mak10) was established as an auxiliary subunit of the NatC N-terminal acetyltransferase complex in yeast, alongside catalytic subunit Mak3p and auxiliary subunit Mak31p; the complex acetylates N-termini of proteins where the initiator methionine is followed by a bulky hydrophobic/amphipathic residue.","method":"Biochemical complex composition analysis, enzymatic activity characterization","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — complex composition established by biochemical methods, single review/synthesis paper but based on accumulated yeast data","pmids":["12890471"],"is_preprint":false},{"year":2016,"finding":"Human NAA35 (hNaa35) is required for human NatC catalytic activity in vivo: hNaa30 (catalytic subunit) could restore NatC-dependent Arl3 Golgi localization in yeast lacking yNaa30 only when either yeast or human Naa35 was co-expressed; hNaa35 alone could not replace its yeast orthologue without co-expression of hNaa30, indicating co-evolution of the two NatC subunits.","method":"Yeast complementation assay using microscopy-based Arl3-Golgi localization readout, co-expression experiments","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic complementation with functional phenotypic readout, single lab, two experimental approaches","pmids":["27555049"],"is_preprint":false},{"year":2017,"finding":"NAA35 is an auxiliary subunit of the human NatC complex (NAA30-NAA35-NAA38); the NatC complex acetylates cytoplasmic proteins co-translationally when the initiator methionine is followed by a bulky hydrophobic/amphipathic residue at position 2, as confirmed by in vitro acetyltransferase activity of NatC on a classical NatC substrate peptide.","method":"In vitro acetyltransferase activity assay on substrate peptides, subcellular localization by imaging","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro enzymatic assay confirming complex composition and activity, single lab","pmids":["29247799"],"is_preprint":false},{"year":1992,"finding":"The yeast MAK10 gene (ortholog of NAA35) encodes a 733-amino acid protein that is essential for maintenance of the L-A dsRNA virus-like particles; mak10 mutants show temperature-dependent loss of L-A replication and MAK10 is also required for optimal growth on non-fermentable carbon sources independent of its effect on L-A, suggesting competition between the mitochondrial genome and L-A dsRNA for the MAK10 protein. MAK10 expression is glucose-repressed and regulated by TUP1 and CYC8.","method":"Gene cloning, lacZ fusion reporter assays, genetic analysis of mak10 deletion and point mutants, growth assays on nonfermentable carbon sources","journal":"Genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct gene characterization with multiple genetic and biochemical methods, single lab","pmids":["1398065"],"is_preprint":false},{"year":1987,"finding":"Yeast mak10 mutations cause instability of L-A dsRNA-containing (major class) virus-like particles in vitro, demonstrating that Mak10 (NAA35 ortholog) is required for structural stability of mature L-A dsRNA-containing particles but not for particles containing L-A plus-strand ssRNA.","method":"In vitro RNA polymerase assay, particle stability assay after CsCl density gradient centrifugation, temperature-sensitive mutant analysis","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro particle stability assay with defined mutants, replicated across multiple conditions","pmids":["3550421"],"is_preprint":false},{"year":2026,"finding":"CRISPR screening identified NAA35 (along with Naa38 and Naa30) as a key mediator of ER stress resistance in muscle cells. In cancer cachexia, ATF6-branch UPR upregulates Naa35 expression; Naa35 knockdown in LLC tumor-bearing mice reduced cathepsin K (CTSK) protein levels, prevented CTSK-mediated proteolysis of insulin receptor substrate 1, preserved AKT and S6K phosphorylation, suppressed MuRF1 and MAFbx1 expression, and restored muscle mass and grip strength.","method":"Genome-wide CRISPR screen, shRNA knockdown in vivo (AAV delivery), western blotting, ATF6 inhibitor/activator pharmacology, muscle function assays","journal":"Journal of cachexia, sarcopenia and muscle","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR screen plus in vivo KD with multiple functional readouts, single lab, mechanistic pathway partially delineated","pmids":["41852114"],"is_preprint":false},{"year":2021,"finding":"Human NAA30 can functionally replace yeast MAK3/NAA30 in mak3∆ mutant growth phenotypes (non-fermentable carbon sources and stress conditions), but this rescue depends on the genetic background of the yeast strain, indicating evolutionary conservation of the NatC (NAA30-NAA35-NAA38) complex function.","method":"Comparative viability and growth assays in yeast complementation experiments, two yeast strain backgrounds","journal":"Bioscience reports","confidence":"Low","confidence_rationale":"Tier 3 / Moderate — functional complementation assay in yeast, multiple strains but single lab and no direct biochemical readout for NAA35 specifically","pmids":["33600573"],"is_preprint":false},{"year":2025,"finding":"RNAi knockdown of NAA35 in Tribolium castaneum caused a significant reduction in eggs laid by females, indicating a required role for NAA35 in female reproduction in this insect model.","method":"RNA interference (RNAi) knockdown, reproductive phenotype quantification","journal":"Insect molecular biology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single RNAi phenotype in insect model, no molecular mechanism established for NAA35 specifically beyond general NAT activity","pmids":["40437965"],"is_preprint":false}],"current_model":"NAA35 is the large auxiliary subunit of the NatC N-terminal acetyltransferase complex (NAA30-NAA35-NAA38), which co-translationally acetylates the N-termini of proteins bearing a bulky hydrophobic/amphipathic residue after the initiator methionine; structural studies show NAA35 is required for NatC catalytic activity, interacts with the smaller auxiliary subunit NAA38 to order its own N-terminal segment and reorient NAA30's substrate-binding loop, and participates in binding inositol hexaphosphate (IP6) to stabilize the complex, while in muscle cells NAA35 expression is induced via the ATF6-UPR branch and promotes muscle wasting in cancer cachexia through upregulation of cathepsin K and suppression of anabolic signaling."},"narrative":{"mechanistic_narrative":"NAA35 is the large auxiliary subunit of the NatC N-terminal acetyltransferase complex (NAA30-NAA35-NAA38), which co-translationally acetylates the N-termini of cytoplasmic proteins bearing a bulky hydrophobic or amphipathic residue after the initiator methionine [PMID:12890471, PMID:29247799]. Cryo-EM structures of the complex show that all three subunits are required for normal NatC activity and that inositol hexaphosphate (IP6) binds tightly to stabilize the assembly [PMID:34019809]; within this architecture, the smaller auxiliary subunit NAA38 orders an N-terminal segment of NAA35 and reorients the NAA30 peptide-binding loop to broaden substrate specificity and increase thermostability [PMID:36638802]. NAA35 is functionally indispensable for catalysis: human NAA30 restores NatC-dependent substrate acetylation in yeast only when a Naa35 orthologue is co-expressed, reflecting tight co-evolution of the catalytic and auxiliary subunits [PMID:27555049]. Beyond its core acetyltransferase role, NAA35 is induced through the ATF6 branch of the unfolded protein response in muscle and drives cancer-cachexia muscle wasting: its knockdown lowers cathepsin K, preserves insulin receptor substrate 1 and downstream AKT/S6K signaling, suppresses the atrophy genes MuRF1 and MAFbx1, and restores muscle mass [PMID:41852114].","teleology":[{"year":1987,"claim":"Before its enzymatic identity was known, the yeast orthologue Mak10 was found to be required for the structural stability of L-A double-stranded RNA virus-like particles, providing the first functional handle on the gene.","evidence":"In vitro RNA polymerase and particle stability assays on CsCl gradients with temperature-sensitive mutants in yeast","pmids":["3550421"],"confidence":"Medium","gaps":["No molecular mechanism linking Mak10 to particle stability","No connection yet to acetyltransferase activity"]},{"year":1992,"claim":"Cloning of MAK10 defined it as an essential single gene whose loss destabilizes L-A replication and impairs growth on non-fermentable carbon sources, establishing pleiotropic cellular roles and glucose-repressed regulation.","evidence":"Gene cloning, lacZ reporter fusions, deletion and point-mutant genetics, and growth assays in yeast","pmids":["1398065"],"confidence":"Medium","gaps":["Biochemical activity of the encoded protein not yet defined","Relationship between L-A and carbon-source phenotypes unresolved"]},{"year":2003,"claim":"The gene was reassigned as an auxiliary subunit of the NatC N-terminal acetyltransferase complex, explaining its activity as part of a Met-X(bulky hydrophobic) substrate-acetylating machine.","evidence":"Biochemical complex composition and enzymatic activity characterization in yeast","pmids":["12890471"],"confidence":"Medium","gaps":["Catalytic contribution of the auxiliary subunit versus Mak3 not separated","Human complex not yet characterized"]},{"year":2016,"claim":"Cross-species complementation showed human NAA35 is required for human NatC catalytic activity in vivo and co-evolves with its catalytic partner NAA30.","evidence":"Yeast complementation using Arl3-Golgi localization readout with human/yeast subunit co-expression","pmids":["27555049"],"confidence":"Medium","gaps":["Direct structural basis of the subunit interdependence not shown","No in vitro reconstitution of the human complex"]},{"year":2017,"claim":"The human NatC complex (NAA30-NAA35-NAA38) was reconstituted and shown to acetylate a classical NatC substrate peptide in vitro, confirming subunit composition and substrate logic in human cells.","evidence":"In vitro acetyltransferase activity assay and subcellular imaging","pmids":["29247799"],"confidence":"Medium","gaps":["Quantitative contribution of each subunit to catalysis not resolved","Substrate repertoire beyond model peptides undefined"]},{"year":2021,"claim":"A cryo-EM structure of the NatC complex with a bisubstrate analog and IP6 established the structural requirement for all three subunits and revealed IP6 as a complex-stabilizing cofactor.","evidence":"Cryo-EM structure determination, biochemical activity assays, and IP6 binding studies in S. pombe NatC","pmids":["34019809"],"confidence":"High","gaps":["Species-specific differences in cofactor engagement not yet addressed","Functional consequences of IP6 in cells not tested"]},{"year":2023,"claim":"Human NatC cryo-EM structures defined the mechanistic role of NAA38 in ordering an NAA35 N-terminal segment and reorienting the NAA30 peptide-binding loop, accounting for thermostability and broadened substrate specificity.","evidence":"Cryo-EM structures with and without NAA38 plus thermostability and substrate-specificity assays of human NatC","pmids":["36638802"],"confidence":"High","gaps":["Catalytic mechanism of acetyl transfer not fully kinetically dissected","In-cell substrate consequences of NAA38-dependent specificity not mapped"]},{"year":2026,"claim":"NAA35 was identified as a UPR-induced mediator of muscle wasting, connecting its acetyltransferase-complex role to a disease pathway in cancer cachexia via cathepsin K and IRS1/AKT signaling.","evidence":"Genome-wide CRISPR screen, in vivo shRNA knockdown in tumor-bearing mice, ATF6 pharmacology, western blotting, and muscle function assays","pmids":["41852114"],"confidence":"Medium","gaps":["Whether NatC acetyltransferase activity per se drives CTSK regulation not established","Direct NAA35 substrates in the cachexia pathway unidentified"]},{"year":null,"claim":"The specific substrate repertoire of NAA35-containing NatC in human physiology and how its acetyltransferase function connects mechanistically to its disease and reproductive phenotypes remain open.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No catalog of endogenous NatC substrates tied to specific phenotypes","Causal link between acetylation activity and cachexia/reproduction phenotypes untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,2,4]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[2,4]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,3]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[2,4]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[7]}],"complexes":["NatC N-terminal acetyltransferase complex (NAA30-NAA35-NAA38)"],"partners":["NAA30","NAA38"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q5VZE5","full_name":"N-alpha-acetyltransferase 35, NatC auxiliary subunit","aliases":["Embryonic growth-associated protein homolog","Protein 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N-terminal acetylation protects proteins from ubiquitination and degradation by the N-end rule pathway (PubMed:37891180). Involved in regulation of apoptosis and proliferation of smooth muscle cells (PubMed:19398576)","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q5VZE5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/NAA35","classification":"Common Essential","n_dependent_lines":725,"n_total_lines":1208,"dependency_fraction":0.6001655629139073},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/NAA35","total_profiled":1310},"omim":[{"mim_id":"619438","title":"N-ALPHA-ACETYLTRANSFERASE 35, NatC AUXILIARY SUBUNIT; NAA35","url":"https://www.omim.org/entry/619438"},{"mim_id":"617990","title":"N-ALPHA-ACETYLTRANSFERASE 38, NatC AUXILIARY SUBUNIT; NAA38","url":"https://www.omim.org/entry/617990"},{"mim_id":"617989","title":"N-ALPHA-ACETYLTRANSFERASE 30, NatC CATALYTIC SUBUNIT; NAA30","url":"https://www.omim.org/entry/617989"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Plasma membrane","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/NAA35"},"hgnc":{"alias_symbol":["FLJ21613","FLJ22643","bA379P1.1"],"prev_symbol":["MAK10"]},"alphafold":{"accession":"Q5VZE5","domains":[{"cath_id":"-","chopping":"85-342","consensus_level":"medium","plddt":91.1122,"start":85,"end":342},{"cath_id":"-","chopping":"346-512","consensus_level":"medium","plddt":95.0487,"start":346,"end":512}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5VZE5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q5VZE5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q5VZE5-F1-predicted_aligned_error_v6.png","plddt_mean":89.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NAA35","jax_strain_url":"https://www.jax.org/strain/search?query=NAA35"},"sequence":{"accession":"Q5VZE5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q5VZE5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q5VZE5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5VZE5"}},"corpus_meta":[{"pmid":"25666757","id":"PMC_25666757","title":"Whole-exome 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NatE/C-type bisubstrate analog and inositol hexaphosphate (IP6) revealed that all three subunits (Naa30, Naa35, Naa38) are required for normal NatC acetylation activity in yeast, and that IP6 binds tightly to NatC to stabilize the complex; the molecular basis for IP6-mediated NatC complex stabilization was described.\",\n      \"method\": \"Cryo-EM structure determination, biochemical activity assays, IP6 binding studies\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure with bisubstrate analog plus biochemical validation, multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"34019809\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Cryo-EM structures of human NatC with and without NAA38 showed that NAA38 increases the thermostability and broadens the substrate-specificity profile of NatC by ordering an N-terminal segment of NAA35 and reorienting an NAA30 N-terminal peptide-binding loop for optimal catalysis; human NatC engages the stabilizing inositol hexaphosphate differently from yeast NatC.\",\n      \"method\": \"Cryo-EM structure determination with and without NAA38, biochemical thermostability and substrate-specificity assays\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structures plus biochemical functional validation, multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"36638802\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"NAA35 (Mak10) was established as an auxiliary subunit of the NatC N-terminal acetyltransferase complex in yeast, alongside catalytic subunit Mak3p and auxiliary subunit Mak31p; the complex acetylates N-termini of proteins where the initiator methionine is followed by a bulky hydrophobic/amphipathic residue.\",\n      \"method\": \"Biochemical complex composition analysis, enzymatic activity characterization\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — complex composition established by biochemical methods, single review/synthesis paper but based on accumulated yeast data\",\n      \"pmids\": [\"12890471\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Human NAA35 (hNaa35) is required for human NatC catalytic activity in vivo: hNaa30 (catalytic subunit) could restore NatC-dependent Arl3 Golgi localization in yeast lacking yNaa30 only when either yeast or human Naa35 was co-expressed; hNaa35 alone could not replace its yeast orthologue without co-expression of hNaa30, indicating co-evolution of the two NatC subunits.\",\n      \"method\": \"Yeast complementation assay using microscopy-based Arl3-Golgi localization readout, co-expression experiments\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic complementation with functional phenotypic readout, single lab, two experimental approaches\",\n      \"pmids\": [\"27555049\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NAA35 is an auxiliary subunit of the human NatC complex (NAA30-NAA35-NAA38); the NatC complex acetylates cytoplasmic proteins co-translationally when the initiator methionine is followed by a bulky hydrophobic/amphipathic residue at position 2, as confirmed by in vitro acetyltransferase activity of NatC on a classical NatC substrate peptide.\",\n      \"method\": \"In vitro acetyltransferase activity assay on substrate peptides, subcellular localization by imaging\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro enzymatic assay confirming complex composition and activity, single lab\",\n      \"pmids\": [\"29247799\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"The yeast MAK10 gene (ortholog of NAA35) encodes a 733-amino acid protein that is essential for maintenance of the L-A dsRNA virus-like particles; mak10 mutants show temperature-dependent loss of L-A replication and MAK10 is also required for optimal growth on non-fermentable carbon sources independent of its effect on L-A, suggesting competition between the mitochondrial genome and L-A dsRNA for the MAK10 protein. MAK10 expression is glucose-repressed and regulated by TUP1 and CYC8.\",\n      \"method\": \"Gene cloning, lacZ fusion reporter assays, genetic analysis of mak10 deletion and point mutants, growth assays on nonfermentable carbon sources\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct gene characterization with multiple genetic and biochemical methods, single lab\",\n      \"pmids\": [\"1398065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1987,\n      \"finding\": \"Yeast mak10 mutations cause instability of L-A dsRNA-containing (major class) virus-like particles in vitro, demonstrating that Mak10 (NAA35 ortholog) is required for structural stability of mature L-A dsRNA-containing particles but not for particles containing L-A plus-strand ssRNA.\",\n      \"method\": \"In vitro RNA polymerase assay, particle stability assay after CsCl density gradient centrifugation, temperature-sensitive mutant analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro particle stability assay with defined mutants, replicated across multiple conditions\",\n      \"pmids\": [\"3550421\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"CRISPR screening identified NAA35 (along with Naa38 and Naa30) as a key mediator of ER stress resistance in muscle cells. In cancer cachexia, ATF6-branch UPR upregulates Naa35 expression; Naa35 knockdown in LLC tumor-bearing mice reduced cathepsin K (CTSK) protein levels, prevented CTSK-mediated proteolysis of insulin receptor substrate 1, preserved AKT and S6K phosphorylation, suppressed MuRF1 and MAFbx1 expression, and restored muscle mass and grip strength.\",\n      \"method\": \"Genome-wide CRISPR screen, shRNA knockdown in vivo (AAV delivery), western blotting, ATF6 inhibitor/activator pharmacology, muscle function assays\",\n      \"journal\": \"Journal of cachexia, sarcopenia and muscle\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR screen plus in vivo KD with multiple functional readouts, single lab, mechanistic pathway partially delineated\",\n      \"pmids\": [\"41852114\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Human NAA30 can functionally replace yeast MAK3/NAA30 in mak3∆ mutant growth phenotypes (non-fermentable carbon sources and stress conditions), but this rescue depends on the genetic background of the yeast strain, indicating evolutionary conservation of the NatC (NAA30-NAA35-NAA38) complex function.\",\n      \"method\": \"Comparative viability and growth assays in yeast complementation experiments, two yeast strain backgrounds\",\n      \"journal\": \"Bioscience reports\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — functional complementation assay in yeast, multiple strains but single lab and no direct biochemical readout for NAA35 specifically\",\n      \"pmids\": [\"33600573\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RNAi knockdown of NAA35 in Tribolium castaneum caused a significant reduction in eggs laid by females, indicating a required role for NAA35 in female reproduction in this insect model.\",\n      \"method\": \"RNA interference (RNAi) knockdown, reproductive phenotype quantification\",\n      \"journal\": \"Insect molecular biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single RNAi phenotype in insect model, no molecular mechanism established for NAA35 specifically beyond general NAT activity\",\n      \"pmids\": [\"40437965\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NAA35 is the large auxiliary subunit of the NatC N-terminal acetyltransferase complex (NAA30-NAA35-NAA38), which co-translationally acetylates the N-termini of proteins bearing a bulky hydrophobic/amphipathic residue after the initiator methionine; structural studies show NAA35 is required for NatC catalytic activity, interacts with the smaller auxiliary subunit NAA38 to order its own N-terminal segment and reorient NAA30's substrate-binding loop, and participates in binding inositol hexaphosphate (IP6) to stabilize the complex, while in muscle cells NAA35 expression is induced via the ATF6-UPR branch and promotes muscle wasting in cancer cachexia through upregulation of cathepsin K and suppression of anabolic signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NAA35 is the large auxiliary subunit of the NatC N-terminal acetyltransferase complex (NAA30-NAA35-NAA38), which co-translationally acetylates the N-termini of cytoplasmic proteins bearing a bulky hydrophobic or amphipathic residue after the initiator methionine [#2, #4]. Cryo-EM structures of the complex show that all three subunits are required for normal NatC activity and that inositol hexaphosphate (IP6) binds tightly to stabilize the assembly [#0]; within this architecture, the smaller auxiliary subunit NAA38 orders an N-terminal segment of NAA35 and reorients the NAA30 peptide-binding loop to broaden substrate specificity and increase thermostability [#1]. NAA35 is functionally indispensable for catalysis: human NAA30 restores NatC-dependent substrate acetylation in yeast only when a Naa35 orthologue is co-expressed, reflecting tight co-evolution of the catalytic and auxiliary subunits [#3]. Beyond its core acetyltransferase role, NAA35 is induced through the ATF6 branch of the unfolded protein response in muscle and drives cancer-cachexia muscle wasting: its knockdown lowers cathepsin K, preserves insulin receptor substrate 1 and downstream AKT/S6K signaling, suppresses the atrophy genes MuRF1 and MAFbx1, and restores muscle mass [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 1987,\n      \"claim\": \"Before its enzymatic identity was known, the yeast orthologue Mak10 was found to be required for the structural stability of L-A double-stranded RNA virus-like particles, providing the first functional handle on the gene.\",\n      \"evidence\": \"In vitro RNA polymerase and particle stability assays on CsCl gradients with temperature-sensitive mutants in yeast\",\n      \"pmids\": [\"3550421\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No molecular mechanism linking Mak10 to particle stability\", \"No connection yet to acetyltransferase activity\"]\n    },\n    {\n      \"year\": 1992,\n      \"claim\": \"Cloning of MAK10 defined it as an essential single gene whose loss destabilizes L-A replication and impairs growth on non-fermentable carbon sources, establishing pleiotropic cellular roles and glucose-repressed regulation.\",\n      \"evidence\": \"Gene cloning, lacZ reporter fusions, deletion and point-mutant genetics, and growth assays in yeast\",\n      \"pmids\": [\"1398065\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Biochemical activity of the encoded protein not yet defined\", \"Relationship between L-A and carbon-source phenotypes unresolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"The gene was reassigned as an auxiliary subunit of the NatC N-terminal acetyltransferase complex, explaining its activity as part of a Met-X(bulky hydrophobic) substrate-acetylating machine.\",\n      \"evidence\": \"Biochemical complex composition and enzymatic activity characterization in yeast\",\n      \"pmids\": [\"12890471\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Catalytic contribution of the auxiliary subunit versus Mak3 not separated\", \"Human complex not yet characterized\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Cross-species complementation showed human NAA35 is required for human NatC catalytic activity in vivo and co-evolves with its catalytic partner NAA30.\",\n      \"evidence\": \"Yeast complementation using Arl3-Golgi localization readout with human/yeast subunit co-expression\",\n      \"pmids\": [\"27555049\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct structural basis of the subunit interdependence not shown\", \"No in vitro reconstitution of the human complex\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"The human NatC complex (NAA30-NAA35-NAA38) was reconstituted and shown to acetylate a classical NatC substrate peptide in vitro, confirming subunit composition and substrate logic in human cells.\",\n      \"evidence\": \"In vitro acetyltransferase activity assay and subcellular imaging\",\n      \"pmids\": [\"29247799\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Quantitative contribution of each subunit to catalysis not resolved\", \"Substrate repertoire beyond model peptides undefined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"A cryo-EM structure of the NatC complex with a bisubstrate analog and IP6 established the structural requirement for all three subunits and revealed IP6 as a complex-stabilizing cofactor.\",\n      \"evidence\": \"Cryo-EM structure determination, biochemical activity assays, and IP6 binding studies in S. pombe NatC\",\n      \"pmids\": [\"34019809\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Species-specific differences in cofactor engagement not yet addressed\", \"Functional consequences of IP6 in cells not tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Human NatC cryo-EM structures defined the mechanistic role of NAA38 in ordering an NAA35 N-terminal segment and reorienting the NAA30 peptide-binding loop, accounting for thermostability and broadened substrate specificity.\",\n      \"evidence\": \"Cryo-EM structures with and without NAA38 plus thermostability and substrate-specificity assays of human NatC\",\n      \"pmids\": [\"36638802\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Catalytic mechanism of acetyl transfer not fully kinetically dissected\", \"In-cell substrate consequences of NAA38-dependent specificity not mapped\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"NAA35 was identified as a UPR-induced mediator of muscle wasting, connecting its acetyltransferase-complex role to a disease pathway in cancer cachexia via cathepsin K and IRS1/AKT signaling.\",\n      \"evidence\": \"Genome-wide CRISPR screen, in vivo shRNA knockdown in tumor-bearing mice, ATF6 pharmacology, western blotting, and muscle function assays\",\n      \"pmids\": [\"41852114\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether NatC acetyltransferase activity per se drives CTSK regulation not established\", \"Direct NAA35 substrates in the cachexia pathway unidentified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The specific substrate repertoire of NAA35-containing NatC in human physiology and how its acetyltransferase function connects mechanistically to its disease and reproductive phenotypes remain open.\",\n      \"evidence\": null,\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No catalog of endogenous NatC substrates tied to specific phenotypes\", \"Causal link between acetylation activity and cachexia/reproduction phenotypes untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 2, 4]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [2, 4]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [2, 4]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"complexes\": [\"NatC N-terminal acetyltransferase complex (NAA30-NAA35-NAA38)\"],\n    \"partners\": [\"NAA30\", \"NAA38\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":4,"faith_total":4,"faith_pct":100.0}}