{"gene":"PF3D7_0416400","run_date":"2026-09-07T10:55:51","timeline":{"discoveries":[{"year":2004,"finding":"Recombinant PfGCN5 (HAT1/PF3D7_0416400 family member GCN5) has HAT activity with substrate preference for histone H3, acetylating H3 at Lys-8 and Lys-14; it interacts with PfADA2 as shown by yeast two-hybrid and in vitro binding assays; and the PfGCN5 HAT domain can fully rescue a yeast gcn5 deletion mutant when fused to the yGCN5 remainder.","method":"In vitro HAT assay with recombinant protein, substrate-specific antibodies, yeast two-hybrid, in vitro binding assay, yeast complementation","journal":"Eukaryotic cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzymatic reconstitution with mutagenesis-level substrate mapping, reciprocal binding assays, and functional yeast complementation, multiple orthogonal methods in one study","pmids":["15075257"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2004,"finding":"PfADA2 is associated with PfGCN5 in vivo in P. falciparum erythrocytic stages, as demonstrated by co-immunoprecipitation: anti-GCN5 antibodies precipitated HAT activity from parasite lysate, and this activity was also precipitated by anti-PfADA2 antibodies, indicating a PfADA2–PfGCN5 complex in vivo.","method":"Co-immunoprecipitation with HAT activity assay, Northern analysis, antiserum characterization","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal immunoprecipitation with functional readout (HAT activity), single lab, two orthogonal methods","pmids":["15246536"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2006,"finding":"Curcumin inhibits the HAT activity of recombinant PfGCN5 in vitro and reduces nuclear HAT activity in cultured P. falciparum, causing hypoacetylation of histone H3 at K9 and K14 but not H4 at K5, K8, K12, or K16, indicating specific inhibition of PfGCN5.","method":"In vitro HAT assay with recombinant PfGCN5, nuclear extract HAT assay, histone acetylation-specific antibodies","journal":"Antimicrobial agents and chemotherapy","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assay with recombinant protein plus cell-based confirmation, single lab, two orthogonal methods","pmids":["17145789"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2008,"finding":"Anacardic acid reversibly and noncompetitively inhibits the HAT activity of recombinant PfGCN5 and induces hypoacetylation of histone H3 at K9 and K14 (but not H4 at K5/K8/K12/K16) in parasites, with downstream downregulation of active genes enriched for H3K9 acetylation near their promoters.","method":"In vitro HAT assay with recombinant PfGCN5, nuclear extract HAT assay, chromatin immunoprecipitation (ChIP) with real-time PCR, microarray","journal":"Eukaryotic cell","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assay plus ChIP confirmation of substrate specificity, single lab, multiple orthogonal methods","pmids":["18487348"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2008,"finding":"PfGCN5 is recruited to the pf1-cys-prx gene enhancer specifically in trophozoite/schizont stage, correlating with increased histone acetylation in the 5' region and transcriptional activation of this gene.","method":"Chromatin immunoprecipitation (ChIP) assay","journal":"Molecular and biochemical parasitology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — ChIP demonstrating stage-specific promoter recruitment, single lab, single method","pmids":["18692528"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2020,"finding":"PfGCN5 undergoes proteolytic processing by a cysteine protease-like enzyme; this processing occurs near the digestive vacuole following trafficking through the classical ER-Golgi secretory pathway before the protein reaches the nucleus. Blocking processing reduces PfGCN5 occupancy at gene promoters and H3K9 acetylation at those promoters.","method":"Immunofluorescence microscopy, immuno-electron microscopy, cysteine protease inhibitor treatment, ChIP assay","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple imaging methods plus functional ChIP readout, single lab, multiple orthogonal methods","pmids":["31862795"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2021,"finding":"PfGCN5 forms a divergent HAT complex containing two PHD proteins (PfPHD1 and PfPHD2) and an AP2-domain transcription factor (PfAP2-LT). Deletion of the PfGCN5 bromodomain reduces erythrocyte invasion by merozoites, elevates sexual conversion, decreases H3K9 acetylation and H3K4 trimethylation genome-wide, disrupts var gene mutually exclusive expression, and alters expression of >60% of genes.","method":"CRISPR/Cas9 bromodomain deletion, invasion assays, sexual conversion assay, ChIP-seq, RNA-seq, co-immunoprecipitation (complex identification)","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic loss-of-function (domain deletion) with multiple orthogonal phenotypic readouts (invasion, sexual conversion, ChIP-seq, RNA-seq), complex composition established by Co-IP, single lab but comprehensive multi-method study","pmids":["34403450"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2020,"finding":"PfGCN5 interacts with metabolic enzymes as binding partners (identified by immunoprecipitation coupled with mass spectrometry) and directly acetylates PfGAPDH in vitro, suggesting a non-histone substrate and a possible nutrient-sensing function.","method":"Immunoprecipitation followed by LC-MS/MS, in vitro acetylation assay","journal":"Journal of biosciences","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro acetylation assay for PfGAPDH as substrate, IP-MS for interactors, single lab, two methods but weak on mechanistic depth","pmids":["31965989"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2021,"finding":"PfGCN5 binds stress-responsive and artemisinin-resistance-related genes (including BiP and TRiC chaperone genes) in a poised state; upon artemisinin exposure, its genome-wide binding sites increase; inhibition of PfGCN5 in artemisinin-resistant parasites increases sensitivity to artemisinin.","method":"ChIP-seq, inhibitor treatment (growth assay), RNA-seq","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-seq for binding, pharmacological inhibition with defined phenotypic readout, single lab, two orthogonal methods","pmids":["33441725"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2024,"finding":"Falcipain 3 (FP3), a food-vacuolar cysteine protease, directly interacts with PfGCN5 (shown by immunoprecipitation/LC-MS, in vitro pull-down, and co-IP) and is the protease responsible for proteolytic processing of PfGCN5; inhibition of FP3 with E64d blocks PfGCN5 processing and causes co-localization of PfGCN5 and FP3 around the food vacuole.","method":"Immunoprecipitation coupled with LC-tandem MS, in vitro pull-down, co-immunoprecipitation, cysteine protease inhibitor (E64d) treatment, confocal microscopy, electron microscopy","journal":"Biotechnology and applied biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple binding assays (IP-MS, pull-down, co-IP) plus inhibitor and imaging validation, single lab, multiple orthogonal methods","pmids":["38924147"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"PfGCN5 (HAT1/PF3D7_0416400 family GCN5) is a histone acetyltransferase that preferentially acetylates histone H3 at K9 and K14, exists in a divergent multi-subunit complex containing PfADA2, two PHD proteins (PfPHD1/2), and the AP2 transcription factor PfAP2-LT, undergoes essential proteolytic processing by the food-vacuolar cysteine protease Falcipain 3 near the digestive vacuole prior to nuclear import, and controls erythrocyte invasion, sexual conversion, var gene expression, and global transcriptional responses to stress and artemisinin through its HAT and bromodomain activities."},"narrative":{"mechanistic_narrative":"PF3D7_0416400 (HAT1) is a GCN5-family histone acetyltransferase of Plasmodium falciparum that serves as a global chromatin regulator of asexual proliferation, sexual conversion, and stress adaptation [PMID:15075257, PMID:34403450]. Recombinant enzyme preferentially acetylates histone H3 at Lys-8 and Lys-14, and its HAT domain is functionally interchangeable with that of yeast GCN5 [PMID:15075257]; pharmacological inhibition confirms this specificity in parasites, producing selective hypoacetylation of H3K9/K14 without affecting H4 acetylation [PMID:17145789, PMID:18487348]. It operates within a divergent multi-subunit HAT complex that includes PfADA2, two PHD-domain proteins (PfPHD1/PfPHD2), and the AP2 transcription factor PfAP2-LT [PMID:15246536, PMID:34403450]. Through its bromodomain and HAT activity it is recruited to gene promoters and enhancers to deposit H3K9 acetylation, and its bromodomain is required for efficient merozoite invasion, restraint of sexual conversion, mutually exclusive var gene expression, and normal genome-wide H3K9ac/H3K4me3 patterns, with loss altering expression of the majority of genes [PMID:18692528, PMID:34403450]. Beyond chromatin, it acetylates the metabolic enzyme PfGAPDH in vitro, indicating a non-histone substrate [PMID:31965989], and it binds stress- and artemisinin-response genes in a poised state such that its inhibition sensitizes artemisinin-resistant parasites [PMID:33441725]. Uniquely, the protein undergoes essential proteolytic processing near the digestive vacuole—mediated by the food-vacuolar cysteine protease Falcipain 3—following trafficking through the ER-Golgi pathway, a step required for its promoter occupancy and HAT activity before nuclear import [PMID:31862795, PMID:38924147].","teleology":[{"year":2004,"claim":"Establishing that this parasite GCN5-family protein is a bona fide histone acetyltransferase answered whether it possesses catalytic activity and defined its histone substrate specificity.","evidence":"In vitro HAT assay with recombinant protein and substrate-specific antibodies, plus functional rescue of a yeast gcn5 deletion, and yeast two-hybrid/binding assays with PfADA2","pmids":["15075257"],"confidence":"High","gaps":["Physiological histone targets and genome-wide sites not yet mapped","Composition of the full in vivo complex beyond PfADA2 unresolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2004,"claim":"Demonstrating an endogenous PfADA2–GCN5 association confirmed the enzyme functions as part of a complex in parasite erythrocytic stages rather than as an isolated catalytic subunit.","evidence":"Reciprocal co-immunoprecipitation from parasite lysate with a HAT activity readout","pmids":["15246536"],"confidence":"Medium","gaps":["Full subunit inventory of the complex not defined","Functional consequence of the PfADA2 interaction unresolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2008,"claim":"Pharmacological inhibition established that the enzyme's H3K9/K14 acetylation drives transcriptional activation of specific target genes, linking its catalytic output to gene regulation.","evidence":"Curcumin and anacardic acid inhibition of recombinant enzyme and parasite nuclear extracts, with ChIP, microarray, and acetylation-specific antibodies","pmids":["17145789","18487348","18692528"],"confidence":"Medium","gaps":["Direct genetic loss-of-function not yet performed","Inhibitor specificity for this HAT versus other parasite HATs incompletely excluded"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2020,"claim":"Discovery of trafficking-dependent proteolytic processing near the digestive vacuole answered how the nuclear enzyme is matured and showed this step is required for its chromatin function.","evidence":"Immunofluorescence, immuno-EM, cysteine protease inhibitor treatment, and ChIP","pmids":["31862795"],"confidence":"Medium","gaps":["Identity of the responsible protease not established in this study","Molecular consequence of processing on activity not directly measured"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2020,"claim":"Identification of metabolic-enzyme interactors and in vitro acetylation of PfGAPDH extended the enzyme's substrate repertoire beyond histones, raising a potential nutrient-sensing role.","evidence":"IP-LC-MS/MS for interactors and in vitro acetylation assay for PfGAPDH","pmids":["31965989"],"confidence":"Medium","gaps":["In vivo relevance of PfGAPDH acetylation not demonstrated","Functional effect of GAPDH acetylation on metabolism unknown"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2021,"claim":"Defining the divergent PHD/AP2-LT-containing complex and dissecting the bromodomain by genetic deletion answered which biological processes the enzyme governs, tying it to invasion, sexual conversion, and var gene control.","evidence":"CRISPR/Cas9 bromodomain deletion with invasion and sexual conversion assays, ChIP-seq, RNA-seq, and Co-IP","pmids":["34403450"],"confidence":"High","gaps":["Separation of bromodomain reader function from catalytic HAT activity not fully resolved","Roles of individual PfPHD1/PfPHD2 subunits not dissected"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2021,"claim":"Mapping the enzyme to poised stress- and artemisinin-response genes showed it participates in transcriptional adaptation and modulates artemisinin sensitivity, implicating it in drug resistance.","evidence":"ChIP-seq, RNA-seq, and inhibitor growth assays in resistant parasites","pmids":["33441725"],"confidence":"Medium","gaps":["Mechanism coupling drug exposure to expanded binding not defined","Direct causal contribution to clinical resistance not established"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2024,"claim":"Identifying Falcipain 3 as the processing protease answered the outstanding question of how and where the enzyme is proteolytically matured before nuclear entry.","evidence":"IP-LC-MS/MS, in vitro pull-down, co-IP, E64d inhibitor treatment, confocal and electron microscopy","pmids":["38924147"],"confidence":"Medium","gaps":["Precise cleavage site(s) not mapped","Regulation coupling food-vacuolar processing to nuclear timing unresolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"It remains unresolved how bromodomain-mediated reader activity, HAT catalysis, complex assembly, and Falcipain 3 processing are integrated to control the timing of specific transcriptional programs across the parasite life cycle.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of the assembled complex","Individual subunit contributions and processing-to-nuclear-import coupling undefined","In vivo scope of non-histone acetylation unknown"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["PfGCN5 HAT complex (PfGCN5/HAT1, PfADA2, PfPHD1, PfPHD2, PfAP2-LT)"],"partners":["PFADA2","PFPHD1","PFPHD2","PFAP2-LT","FALCIPAIN 3 (FP3)","PFGAPDH (NON-HISTONE SUBSTRATE)"],"other_free_text":["Preferentially acetylates histone H3 at Lys-8/Lys-14 (H3K9/K14 hypoacetylation upon inhibition); H4 acetylation unaffected","Bromodomain required for merozoite invasion, restraint of sexual conversion, and mutually exclusive var gene expression","Undergoes Falcipain 3-mediated proteolytic processing near the digestive vacuole after ER-Golgi trafficking, required for promoter occupancy","Binds artemisinin/stress-response genes (BiP, TRiC) in a poised state; inhibition sensitizes artemisinin-resistant parasites","HAT domain rescues yeast gcn5 deletion","Inhibited in vitro by curcumin and anacardic acid"]}},"prefetch_data":{},"corpus_meta":[{"pmid":"17145789","id":"PMC_17145789","title":"Cytotoxic effect of curcumin on malaria parasite Plasmodium falciparum: inhibition of histone acetylation and generation of reactive oxygen species.","date":"2006","source":"Antimicrobial agents and 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PfGCN5.","date":"2004","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/15246536","citation_count":33,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["histone acetyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"33441725","id":"PMC_33441725","title":"Histone acetyltransferase PfGCN5 regulates stress responsive and artemisinin resistance related genes in Plasmodium falciparum.","date":"2021","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/33441725","citation_count":23,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["histone acetyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"25252094","id":"PMC_25252094","title":"Histone H3K9 acetylation level modulates gene expression and may affect parasite growth in human malaria parasite 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Drugs and drug resistance","url":"https://pubmed.ncbi.nlm.nih.gov/29631126","citation_count":19,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["histone acetyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"24160709","id":"PMC_24160709","title":"Chalcone and curcumin derivatives: a way ahead for malarial treatment.","date":"2013","source":"Mini reviews in medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/24160709","citation_count":17,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["histone acetyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"31862795","id":"PMC_31862795","title":"Plasmodium falciparum GCN5 acetyltransferase follows a novel proteolytic processing pathway that is essential for its function.","date":"2020","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/31862795","citation_count":15,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["histone acetyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"19255645","id":"PMC_19255645","title":"Computational screening of molecular targets in Plasmodium for novel non resistant anti-malarial drugs.","date":"2009","source":"Bioinformation","url":"https://pubmed.ncbi.nlm.nih.gov/19255645","citation_count":14,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["histone acetyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"31965989","id":"PMC_31965989","title":"Role of PfGCN5 in nutrient sensing and transcriptional regulation in Plasmodium falciparum.","date":"2020","source":"Journal of biosciences","url":"https://pubmed.ncbi.nlm.nih.gov/31965989","citation_count":12,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["histone acetyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"18692528","id":"PMC_18692528","title":"5' sequence- and chromatin modification-dependent gene expression in Plasmodium falciparum erythrocytic stage.","date":"2008","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/18692528","citation_count":11,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["histone acetyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"38924147","id":"PMC_38924147","title":"Plasmodium falciparum cysteine protease Falcipain 3: A potential enzyme for proteolytic processing of histone acetyltransferase PfGCN5.","date":"2024","source":"Biotechnology and applied biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/38924147","citation_count":3,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["histone acetyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11304,"output_tokens":2921,"usd":0.038864,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":8501,"output_tokens":3734,"usd":0.067928,"stage2_stop_reason":"end_turn"},"total_usd":0.106792,"stage1_batch_id":"msgbatch_01DbiymDmubkPpYvdbAu5a7V","stage2_batch_id":"msgbatch_012TbrWvMRH129T7PQ4SmvrJ","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2004,\n      \"finding\": \"Recombinant PfGCN5 (HAT1/PF3D7_0416400 family member GCN5) has HAT activity with substrate preference for histone H3, acetylating H3 at Lys-8 and Lys-14; it interacts with PfADA2 as shown by yeast two-hybrid and in vitro binding assays; and the PfGCN5 HAT domain can fully rescue a yeast gcn5 deletion mutant when fused to the yGCN5 remainder.\",\n      \"method\": \"In vitro HAT assay with recombinant protein, substrate-specific antibodies, yeast two-hybrid, in vitro binding assay, yeast complementation\",\n      \"journal\": \"Eukaryotic cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzymatic reconstitution with mutagenesis-level substrate mapping, reciprocal binding assays, and functional yeast complementation, multiple orthogonal methods in one study\",\n      \"pmids\": [\"15075257\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"PfADA2 is associated with PfGCN5 in vivo in P. falciparum erythrocytic stages, as demonstrated by co-immunoprecipitation: anti-GCN5 antibodies precipitated HAT activity from parasite lysate, and this activity was also precipitated by anti-PfADA2 antibodies, indicating a PfADA2–PfGCN5 complex in vivo.\",\n      \"method\": \"Co-immunoprecipitation with HAT activity assay, Northern analysis, antiserum characterization\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal immunoprecipitation with functional readout (HAT activity), single lab, two orthogonal methods\",\n      \"pmids\": [\"15246536\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Curcumin inhibits the HAT activity of recombinant PfGCN5 in vitro and reduces nuclear HAT activity in cultured P. falciparum, causing hypoacetylation of histone H3 at K9 and K14 but not H4 at K5, K8, K12, or K16, indicating specific inhibition of PfGCN5.\",\n      \"method\": \"In vitro HAT assay with recombinant PfGCN5, nuclear extract HAT assay, histone acetylation-specific antibodies\",\n      \"journal\": \"Antimicrobial agents and chemotherapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assay with recombinant protein plus cell-based confirmation, single lab, two orthogonal methods\",\n      \"pmids\": [\"17145789\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Anacardic acid reversibly and noncompetitively inhibits the HAT activity of recombinant PfGCN5 and induces hypoacetylation of histone H3 at K9 and K14 (but not H4 at K5/K8/K12/K16) in parasites, with downstream downregulation of active genes enriched for H3K9 acetylation near their promoters.\",\n      \"method\": \"In vitro HAT assay with recombinant PfGCN5, nuclear extract HAT assay, chromatin immunoprecipitation (ChIP) with real-time PCR, microarray\",\n      \"journal\": \"Eukaryotic cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assay plus ChIP confirmation of substrate specificity, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"18487348\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"PfGCN5 is recruited to the pf1-cys-prx gene enhancer specifically in trophozoite/schizont stage, correlating with increased histone acetylation in the 5' region and transcriptional activation of this gene.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP) assay\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — ChIP demonstrating stage-specific promoter recruitment, single lab, single method\",\n      \"pmids\": [\"18692528\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PfGCN5 undergoes proteolytic processing by a cysteine protease-like enzyme; this processing occurs near the digestive vacuole following trafficking through the classical ER-Golgi secretory pathway before the protein reaches the nucleus. Blocking processing reduces PfGCN5 occupancy at gene promoters and H3K9 acetylation at those promoters.\",\n      \"method\": \"Immunofluorescence microscopy, immuno-electron microscopy, cysteine protease inhibitor treatment, ChIP assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple imaging methods plus functional ChIP readout, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"31862795\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PfGCN5 forms a divergent HAT complex containing two PHD proteins (PfPHD1 and PfPHD2) and an AP2-domain transcription factor (PfAP2-LT). Deletion of the PfGCN5 bromodomain reduces erythrocyte invasion by merozoites, elevates sexual conversion, decreases H3K9 acetylation and H3K4 trimethylation genome-wide, disrupts var gene mutually exclusive expression, and alters expression of >60% of genes.\",\n      \"method\": \"CRISPR/Cas9 bromodomain deletion, invasion assays, sexual conversion assay, ChIP-seq, RNA-seq, co-immunoprecipitation (complex identification)\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic loss-of-function (domain deletion) with multiple orthogonal phenotypic readouts (invasion, sexual conversion, ChIP-seq, RNA-seq), complex composition established by Co-IP, single lab but comprehensive multi-method study\",\n      \"pmids\": [\"34403450\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PfGCN5 interacts with metabolic enzymes as binding partners (identified by immunoprecipitation coupled with mass spectrometry) and directly acetylates PfGAPDH in vitro, suggesting a non-histone substrate and a possible nutrient-sensing function.\",\n      \"method\": \"Immunoprecipitation followed by LC-MS/MS, in vitro acetylation assay\",\n      \"journal\": \"Journal of biosciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro acetylation assay for PfGAPDH as substrate, IP-MS for interactors, single lab, two methods but weak on mechanistic depth\",\n      \"pmids\": [\"31965989\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PfGCN5 binds stress-responsive and artemisinin-resistance-related genes (including BiP and TRiC chaperone genes) in a poised state; upon artemisinin exposure, its genome-wide binding sites increase; inhibition of PfGCN5 in artemisinin-resistant parasites increases sensitivity to artemisinin.\",\n      \"method\": \"ChIP-seq, inhibitor treatment (growth assay), RNA-seq\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-seq for binding, pharmacological inhibition with defined phenotypic readout, single lab, two orthogonal methods\",\n      \"pmids\": [\"33441725\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Falcipain 3 (FP3), a food-vacuolar cysteine protease, directly interacts with PfGCN5 (shown by immunoprecipitation/LC-MS, in vitro pull-down, and co-IP) and is the protease responsible for proteolytic processing of PfGCN5; inhibition of FP3 with E64d blocks PfGCN5 processing and causes co-localization of PfGCN5 and FP3 around the food vacuole.\",\n      \"method\": \"Immunoprecipitation coupled with LC-tandem MS, in vitro pull-down, co-immunoprecipitation, cysteine protease inhibitor (E64d) treatment, confocal microscopy, electron microscopy\",\n      \"journal\": \"Biotechnology and applied biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple binding assays (IP-MS, pull-down, co-IP) plus inhibitor and imaging validation, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"38924147\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"PfGCN5 (HAT1/PF3D7_0416400 family GCN5) is a histone acetyltransferase that preferentially acetylates histone H3 at K9 and K14, exists in a divergent multi-subunit complex containing PfADA2, two PHD proteins (PfPHD1/2), and the AP2 transcription factor PfAP2-LT, undergoes essential proteolytic processing by the food-vacuolar cysteine protease Falcipain 3 near the digestive vacuole prior to nuclear import, and controls erythrocyte invasion, sexual conversion, var gene expression, and global transcriptional responses to stress and artemisinin through its HAT and bromodomain activities.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PF3D7_0416400 (HAT1) is a GCN5-family histone acetyltransferase of Plasmodium falciparum that serves as a global chromatin regulator of asexual proliferation, sexual conversion, and stress adaptation [#0, #6]. Recombinant enzyme preferentially acetylates histone H3 at Lys-8 and Lys-14, and its HAT domain is functionally interchangeable with that of yeast GCN5 [#0]; pharmacological inhibition confirms this specificity in parasites, producing selective hypoacetylation of H3K9/K14 without affecting H4 acetylation [#2, #3]. It operates within a divergent multi-subunit HAT complex that includes PfADA2, two PHD-domain proteins (PfPHD1/PfPHD2), and the AP2 transcription factor PfAP2-LT [#1, #6]. Through its bromodomain and HAT activity it is recruited to gene promoters and enhancers to deposit H3K9 acetylation, and its bromodomain is required for efficient merozoite invasion, restraint of sexual conversion, mutually exclusive var gene expression, and normal genome-wide H3K9ac/H3K4me3 patterns, with loss altering expression of the majority of genes [#4, #6]. Beyond chromatin, it acetylates the metabolic enzyme PfGAPDH in vitro, indicating a non-histone substrate [#7], and it binds stress- and artemisinin-response genes in a poised state such that its inhibition sensitizes artemisinin-resistant parasites [#8]. Uniquely, the protein undergoes essential proteolytic processing near the digestive vacuole—mediated by the food-vacuolar cysteine protease Falcipain 3—following trafficking through the ER-Golgi pathway, a step required for its promoter occupancy and HAT activity before nuclear import [#5, #9].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Establishing that this parasite GCN5-family protein is a bona fide histone acetyltransferase answered whether it possesses catalytic activity and defined its histone substrate specificity.\",\n      \"evidence\": \"In vitro HAT assay with recombinant protein and substrate-specific antibodies, plus functional rescue of a yeast gcn5 deletion, and yeast two-hybrid/binding assays with PfADA2\",\n      \"pmids\": [\"15075257\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological histone targets and genome-wide sites not yet mapped\", \"Composition of the full in vivo complex beyond PfADA2 unresolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Demonstrating an endogenous PfADA2–GCN5 association confirmed the enzyme functions as part of a complex in parasite erythrocytic stages rather than as an isolated catalytic subunit.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation from parasite lysate with a HAT activity readout\",\n      \"pmids\": [\"15246536\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Full subunit inventory of the complex not defined\", \"Functional consequence of the PfADA2 interaction unresolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Pharmacological inhibition established that the enzyme's H3K9/K14 acetylation drives transcriptional activation of specific target genes, linking its catalytic output to gene regulation.\",\n      \"evidence\": \"Curcumin and anacardic acid inhibition of recombinant enzyme and parasite nuclear extracts, with ChIP, microarray, and acetylation-specific antibodies\",\n      \"pmids\": [\"17145789\", \"18487348\", \"18692528\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct genetic loss-of-function not yet performed\", \"Inhibitor specificity for this HAT versus other parasite HATs incompletely excluded\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Discovery of trafficking-dependent proteolytic processing near the digestive vacuole answered how the nuclear enzyme is matured and showed this step is required for its chromatin function.\",\n      \"evidence\": \"Immunofluorescence, immuno-EM, cysteine protease inhibitor treatment, and ChIP\",\n      \"pmids\": [\"31862795\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of the responsible protease not established in this study\", \"Molecular consequence of processing on activity not directly measured\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identification of metabolic-enzyme interactors and in vitro acetylation of PfGAPDH extended the enzyme's substrate repertoire beyond histones, raising a potential nutrient-sensing role.\",\n      \"evidence\": \"IP-LC-MS/MS for interactors and in vitro acetylation assay for PfGAPDH\",\n      \"pmids\": [\"31965989\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo relevance of PfGAPDH acetylation not demonstrated\", \"Functional effect of GAPDH acetylation on metabolism unknown\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defining the divergent PHD/AP2-LT-containing complex and dissecting the bromodomain by genetic deletion answered which biological processes the enzyme governs, tying it to invasion, sexual conversion, and var gene control.\",\n      \"evidence\": \"CRISPR/Cas9 bromodomain deletion with invasion and sexual conversion assays, ChIP-seq, RNA-seq, and Co-IP\",\n      \"pmids\": [\"34403450\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Separation of bromodomain reader function from catalytic HAT activity not fully resolved\", \"Roles of individual PfPHD1/PfPHD2 subunits not dissected\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Mapping the enzyme to poised stress- and artemisinin-response genes showed it participates in transcriptional adaptation and modulates artemisinin sensitivity, implicating it in drug resistance.\",\n      \"evidence\": \"ChIP-seq, RNA-seq, and inhibitor growth assays in resistant parasites\",\n      \"pmids\": [\"33441725\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism coupling drug exposure to expanded binding not defined\", \"Direct causal contribution to clinical resistance not established\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identifying Falcipain 3 as the processing protease answered the outstanding question of how and where the enzyme is proteolytically matured before nuclear entry.\",\n      \"evidence\": \"IP-LC-MS/MS, in vitro pull-down, co-IP, E64d inhibitor treatment, confocal and electron microscopy\",\n      \"pmids\": [\"38924147\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Precise cleavage site(s) not mapped\", \"Regulation coupling food-vacuolar processing to nuclear timing unresolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how bromodomain-mediated reader activity, HAT catalysis, complex assembly, and Falcipain 3 processing are integrated to control the timing of specific transcriptional programs across the parasite life cycle.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of the assembled complex\", \"Individual subunit contributions and processing-to-nuclear-import coupling undefined\", \"In vivo scope of non-histone acetylation unknown\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0004402\", \"supporting_discovery_ids\": [0, 2, 3, 7]},\n      {\"term_id\": \"GO:0043966\", \"supporting_discovery_ids\": [0, 3, 4, 6]},\n      {\"term_id\": \"GO:0140033\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0020020\", \"supporting_discovery_ids\": [5, 9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0006355\", \"supporting_discovery_ids\": [4, 6, 8]},\n      {\"term_id\": \"GO:0006954\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"GO:0060137\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"complexes\": [\n      \"PfGCN5 HAT complex (PfGCN5/HAT1, PfADA2, PfPHD1, PfPHD2, PfAP2-LT)\"\n    ],\n    \"partners\": [\n      \"PfADA2\",\n      \"PfPHD1\",\n      \"PfPHD2\",\n      \"PfAP2-LT\",\n      \"Falcipain 3 (FP3)\",\n      \"PfGAPDH (non-histone substrate)\"\n    ],\n    \"other_free_text\": [\n      \"Preferentially acetylates histone H3 at Lys-8/Lys-14 (H3K9/K14 hypoacetylation upon inhibition); H4 acetylation unaffected\",\n      \"Bromodomain required for merozoite invasion, restraint of sexual conversion, and mutually exclusive var gene expression\",\n      \"Undergoes Falcipain 3-mediated proteolytic processing near the digestive vacuole after ER-Golgi trafficking, required for promoter occupancy\",\n      \"Binds artemisinin/stress-response genes (BiP, TRiC) in a poised state; inhibition sensitizes artemisinin-resistant parasites\",\n      \"HAT domain rescues yeast gcn5 deletion\",\n      \"Inhibited in vitro by curcumin and anacardic acid\"\n    ]\n  }\n}","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_0416400","name":null,"product":"histone acetyltransferase, putative","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_0718400","name":null,"product":"histone acetyltransferase, putative","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_0508800","name":null,"product":"histone acetyltransferase, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"HAT1","evaluation":null}