{"gene":"TG_297970","gene_symbol":"AAP","run_date":"2026-09-10T21:09:19","timeline":{"discoveries":[{"year":2016,"finding":"Recombinant TgAAP expressed in E. coli requires metal ions for activity and shows substrate preference for N-terminal acidic amino acids glutamate (Glu) and aspartate (Asp), establishing it as a metal-dependent aspartyl aminopeptidase.","method":"In vitro enzymatic assay of recombinant protein expressed in E. coli","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro enzymatic characterization of recombinant protein with substrate specificity determination, single lab","pmids":["27678060"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"finding":"TgAAP localizes to the cytoplasm of T. gondii tachyzoites, as determined by indirect immunofluorescence of wild-type parasites.","method":"Indirect immunofluorescence analysis","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct localization experiment with confirmation by absence in knockout strain, single lab","pmids":["27678060"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"finding":"CRISPR/Cas9-mediated knockout of TgAAP inhibits parasite attachment/invasion, replication, and substrate-specific aminopeptidase activity, but does not completely abolish parasite growth, indicating TgAAP contributes to but is not essential for these processes.","method":"CRISPR/Cas9 knockout, Western blotting, phenotype analysis (attachment/invasion and replication assays)","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic knockout with multiple phenotypic readouts (invasion, replication, enzymatic activity), single lab","pmids":["27678060"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"finding":"AAP7 (a novel apical annuli protein) is required to traffic LMBD3 to the plasma membrane via a novel route; in stable AAP7-depleted parasites, LMBD3 no longer localizes to the annuli but instead accumulates among secretory organelles, indicating AAP7 connects plasma membrane-embedded LMBD3 to IMC suture-embedded AAP proteins.","method":"Auxin-inducible degron (ATc-resistant) depletion of AAP7, fluorescence localization of LMBD3","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional depletion with direct localization readout of a binding partner, single lab, preprint","pmids":["41256620"],"is_preprint":true,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"finding":"AAP7 depletion leads to accumulation of polyglutamylated subpellicular microtubules (SPMTs) at the basal end of the parasite, indicating AAP7 influences SPMT turnover.","method":"Conditional depletion of AAP7, immunofluorescence detection of polyglutamylated tubulin","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single indirect readout (polyglutamylation marker accumulation) in a depletion background, single lab, preprint, no direct mechanistic link established","pmids":["41256620"],"is_preprint":true,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"Toxoplasma gondii encodes two distinct 'AAP' proteins: TgAAP (aspartyl aminopeptidase), a cytoplasmic metal-dependent enzyme with preference for N-terminal acidic residues that contributes to parasite attachment/invasion and replication; and AAP7, a novel apical annuli protein that connects IMC suture-embedded annuli components to the plasma membrane via LMBD3 trafficking and is required for GRA17-dependent dense granule secretion through the parasitophorous vacuole membrane pore."},"narrative":{"mechanistic_narrative":"The 'AAP' designation in Toxoplasma gondii spans two mechanistically distinct proteins. TgAAP is a cytoplasmic, metal-dependent aspartyl aminopeptidase that cleaves N-terminal acidic residues, preferring glutamate and aspartate [PMID:27678060]; CRISPR/Cas9 knockout reduces parasite attachment/invasion, replication, and substrate-specific aminopeptidase activity without abolishing growth, marking it a contributory but non-essential enzyme [PMID:27678060]. AAP7 is a separate apical annuli protein that traffics LMBD3 to the plasma membrane, connecting plasma-membrane-embedded LMBD3 to IMC suture-embedded AAP proteins; its depletion causes LMBD3 to mislocalize among secretory organelles [PMID:41256620]. AAP7 depletion also leads to accumulation of polyglutamylated subpellicular microtubules (SPMTs) at the basal end of the parasite, indicating AAP7 influences SPMT turnover [PMID:41256620].","teleology":[{"year":2016,"claim":"Establishing the enzymatic identity of TgAAP answered whether the protein is a functional aminopeptidase and defined its substrate preference, positioning it as a metal-dependent aspartyl aminopeptidase acting on N-terminal acidic residues.","evidence":"In vitro enzymatic assay of recombinant protein expressed in E. coli","pmids":["27678060"],"confidence":"High","gaps":["Physiological substrates in the parasite not identified","Metal-ion identity and catalytic mechanism not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"claim":"Localizing TgAAP to the tachyzoite cytoplasm placed its aminopeptidase activity in the cytosolic compartment rather than a secretory or membrane site.","evidence":"Indirect immunofluorescence of wild-type parasites","pmids":["27678060"],"confidence":"Medium","gaps":["Cytoplasmic interactors and downstream targets not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"claim":"Genetic knockout tested whether TgAAP activity is functionally required, showing it contributes to attachment/invasion and replication but is dispensable for viability.","evidence":"CRISPR/Cas9 knockout with invasion, replication, and aminopeptidase activity assays","pmids":["27678060"],"confidence":"Medium","gaps":["Molecular link between aminopeptidase activity and invasion/replication phenotype unresolved","Compensating peptidases not excluded"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"claim":"Characterizing AAP7 defined a distinct apical annuli protein whose role is trafficking LMBD3 to the plasma membrane, linking the annuli architecture to plasma-membrane cargo delivery.","evidence":"Auxin-inducible degron depletion of AAP7 with fluorescence localization of LMBD3 (preprint)","pmids":["41256620"],"confidence":"Medium","gaps":["Direct physical interaction between AAP7 and LMBD3 not demonstrated","Molecular basis of the novel trafficking route unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"claim":"Connecting AAP7 depletion to a secretion defect identified impaired GRA17 dense granule secretion as the critical functional consequence, causally validated by GRA17 overexpression rescue of the bubble vacuole phenotype.","evidence":"Conditional depletion, dense granule secretion assays, and GRA17 overexpression rescue (preprint)","pmids":["41256620"],"confidence":"Medium","gaps":["Mechanism by which AAP7 promotes GRA17 secretion not defined","Whether the effect is direct or through LMBD3 trafficking unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"claim":"Observing polyglutamylated microtubule accumulation upon AAP7 depletion raised a possible role in SPMT turnover.","evidence":"Conditional depletion with immunofluorescence of polyglutamylated tubulin (preprint)","pmids":["41256620"],"confidence":"Low","gaps":["Single indirect marker readout with no direct mechanistic link to AAP7","Not independently confirmed","Causality versus secondary consequence of secretion defect unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["LMBD3 (TRAFFICKED BY AAP7)","GRA17 (SECRETION DEPENDENT ON AAP7)"],"other_free_text":["TgAAP: metal-dependent aspartyl aminopeptidase preferring N-terminal Glu/Asp; cytoplasmic; contributes to but not essential for attachment/invasion and replication.","AAP7: apical annuli protein required for LMBD3 plasma membrane trafficking and GRA17-dependent dense granule secretion across the parasitophorous vacuole membrane.","AAP7 depletion causes accumulation of polyglutamylated subpellicular microtubules at the basal end (Low confidence)."]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"27678060","id":"PMC_27678060","title":"Characterization of aspartyl aminopeptidase from Toxoplasma gondii.","date":"2016","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/27678060","citation_count":20,"is_preprint":false},{"pmid":"36825701","id":"PMC_36825701","title":"Parasite Metalo-aminopeptidases as Targets in Human Infectious Diseases.","date":"2023","source":"Current drug targets","url":"https://pubmed.ncbi.nlm.nih.gov/36825701","citation_count":3,"is_preprint":false},{"pmid":"41256620","id":"PMC_41256620","title":"Toxoplasma gondii AAP7 is essential for functionally connecting the IMC embedded apical annuli to the plasma membrane.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/41256620","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":4057,"output_tokens":1494,"usd":0.017291,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":6631,"output_tokens":2801,"usd":0.05159,"stage2_stop_reason":"end_turn"},"total_usd":0.072395,"stage1_batch_id":"msgbatch_01S6GjoYQm8LTPUsdhASdqag","stage2_batch_id":"msgbatch_01VX63RTMdBJqsG6gQMqoCjz","note":"batch pricing = 50% of standard","corrective_rerun":"2026-09-10 wrong-gene rerun (round-4 rules)","corrective_reason":"summary amended"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2016,\n      \"finding\": \"Recombinant TgAAP expressed in E. coli requires metal ions for activity and shows substrate preference for N-terminal acidic amino acids glutamate (Glu) and aspartate (Asp), establishing it as a metal-dependent aspartyl aminopeptidase.\",\n      \"method\": \"In vitro enzymatic assay of recombinant protein expressed in E. coli\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro enzymatic characterization of recombinant protein with substrate specificity determination, single lab\",\n      \"pmids\": [\"27678060\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TgAAP localizes to the cytoplasm of T. gondii tachyzoites, as determined by indirect immunofluorescence of wild-type parasites.\",\n      \"method\": \"Indirect immunofluorescence analysis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct localization experiment with confirmation by absence in knockout strain, single lab\",\n      \"pmids\": [\"27678060\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CRISPR/Cas9-mediated knockout of TgAAP inhibits parasite attachment/invasion, replication, and substrate-specific aminopeptidase activity, but does not completely abolish parasite growth, indicating TgAAP contributes to but is not essential for these processes.\",\n      \"method\": \"CRISPR/Cas9 knockout, Western blotting, phenotype analysis (attachment/invasion and replication assays)\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic knockout with multiple phenotypic readouts (invasion, replication, enzymatic activity), single lab\",\n      \"pmids\": [\"27678060\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"AAP7 (a novel apical annuli protein) is required to traffic LMBD3 to the plasma membrane via a novel route; in stable AAP7-depleted parasites, LMBD3 no longer localizes to the annuli but instead accumulates among secretory organelles, indicating AAP7 connects plasma membrane-embedded LMBD3 to IMC suture-embedded AAP proteins.\",\n      \"method\": \"Auxin-inducible degron (ATc-resistant) depletion of AAP7, fluorescence localization of LMBD3\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional depletion with direct localization readout of a binding partner, single lab, preprint\",\n      \"pmids\": [\"41256620\"],\n      \"is_preprint\": true,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"AAP7 depletion results in reduced secretion of dense granule proteins, specifically reduced secretion of the parasitophorous vacuole membrane pore-forming protein GRA17, causing 'bubble' vacuoles; overexpression of GRA17 rescues bubble vacuoles in AAP7-depleted parasites, identifying impaired GRA17 secretion as the critical functional defect.\",\n      \"method\": \"Conditional depletion (ATc-resistant stable parasites), dense granule secretion assays, GRA17 overexpression rescue experiment\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — depletion phenotype validated by genetic rescue with overexpression, single lab, preprint\",\n      \"pmids\": [\"41256620\"],\n      \"is_preprint\": true,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"AAP7 depletion leads to accumulation of polyglutamylated subpellicular microtubules (SPMTs) at the basal end of the parasite, indicating AAP7 influences SPMT turnover.\",\n      \"method\": \"Conditional depletion of AAP7, immunofluorescence detection of polyglutamylated tubulin\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single indirect readout (polyglutamylation marker accumulation) in a depletion background, single lab, preprint, no direct mechanistic link established\",\n      \"pmids\": [\"41256620\"],\n      \"is_preprint\": true,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"Toxoplasma gondii encodes two distinct 'AAP' proteins: TgAAP (aspartyl aminopeptidase), a cytoplasmic metal-dependent enzyme with preference for N-terminal acidic residues that contributes to parasite attachment/invasion and replication; and AAP7, a novel apical annuli protein that connects IMC suture-embedded annuli components to the plasma membrane via LMBD3 trafficking and is required for GRA17-dependent dense granule secretion through the parasitophorous vacuole membrane pore.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"The 'AAP' designation in Toxoplasma gondii spans two mechanistically distinct proteins. TgAAP is a cytoplasmic, metal-dependent aspartyl aminopeptidase that cleaves N-terminal acidic residues, preferring glutamate and aspartate [#0, #1]; CRISPR/Cas9 knockout reduces parasite attachment/invasion, replication, and substrate-specific aminopeptidase activity without abolishing growth, marking it a contributory but non-essential enzyme [#2]. AAP7 is a separate apical annuli protein that traffics LMBD3 to the plasma membrane, connecting plasma-membrane-embedded LMBD3 to IMC suture-embedded AAP proteins; its depletion causes LMBD3 to mislocalize among secretory organelles [#3]. AAP7 is required for dense granule secretion of the parasitophorous vacuole membrane pore-forming protein GRA17, and the resulting 'bubble' vacuole phenotype is rescued by GRA17 overexpression, identifying impaired GRA17 secretion as the critical defect [#4].\",\n  \"teleology\": [\n    {\n      \"year\": 2016,\n      \"claim\": \"Establishing the enzymatic identity of TgAAP answered whether the protein is a functional aminopeptidase and defined its substrate preference, positioning it as a metal-dependent aspartyl aminopeptidase acting on N-terminal acidic residues.\",\n      \"evidence\": \"In vitro enzymatic assay of recombinant protein expressed in E. coli\",\n      \"pmids\": [\"27678060\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological substrates in the parasite not identified\", \"Metal-ion identity and catalytic mechanism not resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Localizing TgAAP to the tachyzoite cytoplasm placed its aminopeptidase activity in the cytosolic compartment rather than a secretory or membrane site.\",\n      \"evidence\": \"Indirect immunofluorescence of wild-type parasites\",\n      \"pmids\": [\"27678060\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cytoplasmic interactors and downstream targets not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Genetic knockout tested whether TgAAP activity is functionally required, showing it contributes to attachment/invasion and replication but is dispensable for viability.\",\n      \"evidence\": \"CRISPR/Cas9 knockout with invasion, replication, and aminopeptidase activity assays\",\n      \"pmids\": [\"27678060\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular link between aminopeptidase activity and invasion/replication phenotype unresolved\", \"Compensating peptidases not excluded\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Characterizing AAP7 defined a distinct apical annuli protein whose role is trafficking LMBD3 to the plasma membrane, linking the annuli architecture to plasma-membrane cargo delivery.\",\n      \"evidence\": \"Auxin-inducible degron depletion of AAP7 with fluorescence localization of LMBD3 (preprint)\",\n      \"pmids\": [\"41256620\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct physical interaction between AAP7 and LMBD3 not demonstrated\", \"Molecular basis of the novel trafficking route unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Connecting AAP7 depletion to a secretion defect identified impaired GRA17 dense granule secretion as the critical functional consequence, causally validated by GRA17 overexpression rescue of the bubble vacuole phenotype.\",\n      \"evidence\": \"Conditional depletion, dense granule secretion assays, and GRA17 overexpression rescue (preprint)\",\n      \"pmids\": [\"41256620\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which AAP7 promotes GRA17 secretion not defined\", \"Whether the effect is direct or through LMBD3 trafficking unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Observing polyglutamylated microtubule accumulation upon AAP7 depletion raised a possible role in SPMT turnover.\",\n      \"evidence\": \"Conditional depletion with immunofluorescence of polyglutamylated tubulin (preprint)\",\n      \"pmids\": [\"41256620\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single indirect marker readout with no direct mechanistic link to AAP7\", \"Not independently confirmed\", \"Causality versus secondary consequence of secretion defect unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Whether TgAAP and AAP7 share any functional relationship, and the physiological substrates of TgAAP and the direct molecular partners of AAP7, remain undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural models for either protein\", \"TgAAP endogenous substrates unidentified\", \"Direct AAP7 binding partners not biochemically confirmed\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008238\", \"supporting_discovery_ids\": [0, 2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0046872\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005737\", \"supporting_discovery_ids\": [1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0020008\", \"supporting_discovery_ids\": [4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0046718\", \"supporting_discovery_ids\": [2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0140029\", \"supporting_discovery_ids\": [4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"LMBD3 (trafficked by AAP7)\", \"GRA17 (secretion dependent on AAP7)\"],\n    \"other_free_text\": [\n      \"TgAAP: metal-dependent aspartyl aminopeptidase preferring N-terminal Glu/Asp; cytoplasmic; contributes to but not essential for attachment/invasion and replication.\",\n      \"AAP7: apical annuli protein required for LMBD3 plasma membrane trafficking and GRA17-dependent dense granule secretion across the parasitophorous vacuole membrane.\",\n      \"AAP7 depletion causes accumulation of polyglutamylated subpellicular microtubules at the basal end (Low confidence).\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_297970","name":"AAP","product":"M18 aspartyl aminopeptidase","uniprot_acc":"A0A125YW18","chromosome":"TGME49_chrII","start_pos":2146311},{"strain":"GT1","gene_id":"TGGT1_297970","name":null,"product":"aspartyl aminopeptidase","uniprot_acc":null,"chromosome":"TGGT1_chrII","start_pos":2122080},{"strain":"RH88","gene_id":"TGRH88_002740","name":null,"product":"aspartyl aminopeptidase","uniprot_acc":null,"chromosome":"CM023083","start_pos":2321693},{"strain":"VEG","gene_id":"TGVEG_297970","name":null,"product":"aspartyl aminopeptidase","uniprot_acc":"A0A125YW20","chromosome":"TGVEG_chrII","start_pos":2156519}]}